WO2014150914A2 - Phi-4 polypeptides and methods for their use - Google Patents

Phi-4 polypeptides and methods for their use Download PDF

Info

Publication number
WO2014150914A2
WO2014150914A2 PCT/US2014/024524 US2014024524W WO2014150914A2 WO 2014150914 A2 WO2014150914 A2 WO 2014150914A2 US 2014024524 W US2014024524 W US 2014024524W WO 2014150914 A2 WO2014150914 A2 WO 2014150914A2
Authority
WO
WIPO (PCT)
Prior art keywords
xaa
ser
ala
gin
glu
Prior art date
Application number
PCT/US2014/024524
Other languages
French (fr)
Other versions
WO2014150914A8 (en
WO2014150914A3 (en
Inventor
Ruth Cong
Jingtong Hou
Zhenglin Hou
Phillip A. Patten
Takashi Yamamoto
Original Assignee
Pioneer Hi-Bred International, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN201480027131.7A priority Critical patent/CN105473605A/en
Priority to RU2015143825A priority patent/RU2015143825A/en
Priority to CA2901316A priority patent/CA2901316A1/en
Priority to US14/776,212 priority patent/US10023877B2/en
Priority to EP14768504.4A priority patent/EP2971000A4/en
Priority to MX2015013269A priority patent/MX360160B/en
Priority to BR112015023709A priority patent/BR112015023709A2/en
Application filed by Pioneer Hi-Bred International, Inc. filed Critical Pioneer Hi-Bred International, Inc.
Publication of WO2014150914A2 publication Critical patent/WO2014150914A2/en
Publication of WO2014150914A3 publication Critical patent/WO2014150914A3/en
Priority to ZA2015/05705A priority patent/ZA201505705B/en
Publication of WO2014150914A8 publication Critical patent/WO2014150914A8/en
Priority to US16/013,580 priority patent/US10577626B2/en
Priority to US16/741,271 priority patent/US11180775B2/en
Priority to US17/451,031 priority patent/US11697821B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "5414WOPCT_Sequence_Listing" created on March 04, 2014, and having a size of 5,935 kilobytes and is filed concurrently with the specification.
  • sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences encoding them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants.
  • biopesticides Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides.
  • a microbial agent such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides.
  • biopesticides present a lower risk of pollution and environmental hazards, and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides.
  • biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops.
  • Bacillus Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others.
  • Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus.
  • Microbial insecticides particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control.
  • Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus.
  • corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bt.
  • These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for pest control.
  • compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include the pesticidal polypeptide sequences and antibodies to those polypeptides.
  • the nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants.
  • the nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant.
  • Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds.
  • isolated or recombinant nucleic acid molecules are provided encoding PHI-4 polypeptides including amino acid substitutions, amino acid deletions, amino acid insertions, and fragments thereof, and combinations thereof. Additionally, amino acid sequences corresponding to the PHI-4 polypeptides are encompassed. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed.
  • transgenic plants of the embodiments express one or more of the pesticidal sequences disclosed herein.
  • the transgenic plant further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling coleopteran, lepidopteran, hemipteran or nematode pests. It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.
  • kits for detecting the presence of a PHI-4 polypeptide or detecting the presence of a nucleotide sequence encoding a PHI-4 polypeptide in a sample is provided.
  • the kit is provided along with all reagents and control samples necessary for carrying out a method for detecting the intended agent, as well as instructions for use.
  • compositions and methods of the embodiments are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes.
  • compositions of the embodiments are also useful for generating altered or improved proteins that have pesticidal activity or for detecting the presence of PHI-4 polypeptides or nucleic acids in products or organisms.
  • Embodiment 1 is a PHI-4 polypeptide having improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 2 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 1.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 3 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 2 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 4 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 2.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 5 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 3 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 6 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 7 is the PHI-4 polypeptide of any one of embodiments 1-6, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
  • WCRW Western Corn Root Worm
  • Embodiment 8 is the PHI-4 polypeptide of any one of embodiments 1-7, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
  • Embodiment 9 is the PHI-4 polypeptide of any one of embodiments 1-7, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
  • Embodiment 10 is the PHI-4 polypeptide of any one of embodiments 1 -7, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
  • Embodiment 1 1 is the PHI-4 polypeptide of any one of embodiments 1-10, wherein the PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
  • Embodiment 12 is the PHI-4 polypeptide of embodiment 1 1 , wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acid at position 99 is Lys or Leu; the amino acid at position 145 is Leu or Val; the amino acid at position 150 is Arg or Gin; the amino acid at position 151 is Asp or Ser; the amino acid at position 153 is Leu or lie; the amino acid at position 163 is Leu or Val; the amino acid at position 171 is Tyr or Phe; the amino acid at position 172 is lie or Leu; the amino acid at position 182 is Asp or Gin; the amino acid at position 196 is Gin or Asn; the amino acid at position 206 is Tyr
  • Embodiment 13 is the PHI-4 polypeptide of embodiment 1 1 or 12, further comprising one or more amino acid substitutions at position 86, 359, 464, 465, 466, 467, 468, 499 or 517.
  • Embodiment 14 is the PHI-4 polypeptide of embodiment 13, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
  • Embodiment 15 is the PHI-4 polypeptide of any one of embodiments 1-12, having 1 to 54 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 16 is the PHI-4 polypeptide of any one of embodiments 1-12, having
  • Embodiment 17 is the PHI-4 polypeptide of any one of embodiments 1-12, having 1 to 20 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 18 is the PHI-4 polypeptide of any one of embodiments 1-12, having 1 to 15 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 19 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 54 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 20 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 27 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 21 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 20 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 22 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 15 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 23 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO: 35.
  • Embodiment 24 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 90% identity to SEQ ID NO: 35.
  • Embodiment 25 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 95% identity to SEQ ID NO: 35.
  • Embodiment 26 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 97% identity to SEQ ID NO: 35.
  • Embodiment 27 is a PHI-4 polypeptide, comprising at least one amino acid substitution at a residue relative to SEQ ID NO: 35 in a structural domain selected from:
  • PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
  • Embodiment 28 is the PHI-4 polypeptide of embodiment 27, wherein the hydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys.
  • Embodiment 29 is the PHI-4 polypeptide of embodiment 27 or 28, wherein the membrane insertion loops are between about the amino acid at position 92 (Val) and about the amino acid at position 101 (Ala) and between about the amino acid at position 21 1 (Gly) and about the amino acid at position 220 (Glu), relative to SEQ ID NO: 35.
  • Embodiment 30 is the PHI-4 polypeptide of embodiment 29, wherein the membrane insertion initiation loop residue is selected from position 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219, 220 of SEQ ID NO: 35.
  • Embodiment 31 is the PHI-4 polypeptide of any one of embodiments 27-30, wherein the receptor binding loops are between about the amino acid at position 332 (Asp) and about the amino acid at position 340 (Asp), between about the amino acid at position 395 (Asp) and about the amino acid at position 403 (Asp), between amino acid at position 458 (Asp) and about the amino acid at position 466 (Asp) relative to SEQ ID NO: 35.
  • the receptor binding loops are between about the amino acid at position 332 (Asp) and about the amino acid at position 340 (Asp), between about the amino acid at position 395 (Asp) and about the amino acid at position 403 (Asp), between amino acid at position 458 (Asp) and about the amino acid at position 466 (Asp) relative to SEQ ID NO: 35.
  • Embodiment 32 is the PHI-4 polypeptide of embodiment 31 , wherein the receptor binding loop residue is selected from positions 332, 333, 334, 335, 336, 337, 338, 339, 340, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 458, 459, 460, 461 , 462, 463, 464, 465, 466 of SEQ ID NO: 35.
  • Embodiment 33 is the PHI-4 polypeptide of any one of embodiments 27-32, wherein the protease sensitive region residue is selected from between about the amino acid at position 305 (Lys) and about the amino acid at position 316 (Lys) and from about the amino acid at position 500 (Arg) and about the amino acid at position 535 (Lys) relative to SEQ ID NO: 35.
  • Embodiment 34 is the PHI-4 polypeptide of embodiment 27, wherein the protease is trypsin.
  • Embodiment 35 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQ ID NO: 35.
  • Embodiment 36 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 90% sequence identity to SEQ ID NO: 35.
  • Embodiment 37 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 95% sequence identity to SEQ ID NO: 35.
  • Embodiment 38 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 97% sequence identity to SEQ ID NO: 35.
  • Embodiment 39 is a PHI-4 polypeptide, comprising an amino acid sequence of the formula,
  • Gly Xaa lie Xaa Xaa Leu Xaa
  • Gly Ser lie Ala Cys Val Xaa Thr
  • Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa at position 61 is Arg, Ly
  • Embodiment 40 is a PHI-4 polypeptide, comprising an amino acid sequence of the formula
  • Xaa Tyr Gly Pro Tyr Xaa lie Ser Xaa Ala Ala Val Gly Gly Arg
  • Ser Ala lie Ala Ser Met Gin Glu Met Xaa Ser Met Leu Ser Gin 530 535
  • Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position
  • Embodiment 41 is the PHI-4 polypeptide of embodiment 39 or 40, further comprising one or more amino acid substitutions at position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 3 or SEQ ID NO: 4.
  • Embodiment 42 is the PHI-4 polypeptide of embodiment 41 , wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
  • Embodiment 43 is the PHI-4 polypeptide of embodiment 39-42, further comprising one or more conservative amino acid substitution, insertion of one or more amino acids, deletion of one or more amino acids, and combinations thereof.
  • Embodiment 44 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 1.5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 45 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 2 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 46 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 2.5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 47 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 3 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 48 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 49 is the PHI-4 polypeptide of any one of embodiments 39-48, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
  • Embodiment 50 is the PHI-4 polypeptide of any one of embodiments 39-49, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
  • Embodiment 51 is the PHI-4 polypeptide of any one of embodiments 39-49, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
  • Embodiment 52 is the PHI-4 polypeptide of any one of embodiments 39-49, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
  • Embodiment 53 is the PHI-4 polypeptide of any one of embodiments 39-49, having
  • Embodiment 54 is the PHI-4 polypeptide of any one of embodiments 39-49, having 1 to 27 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
  • Embodiment 55 is the PHI-4 polypeptide of any one of embodiments 39-49, having 1 to 20 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
  • Embodiment 56 is the PHI-4 polypeptide of any one of embodiments 39-49, having 1 to 15 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
  • Embodiment 57 is the PHI-4 polypeptide of any one of embodiments 1-56, wherein 1 -25 amino acids are deleted from the N-terminus of the PHI-4 polypeptide and/or C- terminus of the PHI-4 polypeptide.
  • Embodiment 58 is the PHI-4 polypeptide of any one of embodiments 1-53, wherein 1 -20 amino acids are deleted from the C-terminus of the PHI-4 polypeptide.
  • Embodiment 59 is a polynucleotide encoding a PHI-4 polypeptide, wherein the PHI-4 polypeptide has improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 60 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 1 .5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 61 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 62 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2.5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 63 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 3 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 64 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
  • Embodiment 65 is the polynucleotide of any one of embodiments 59-64, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
  • WCRW Western Corn Root Worm
  • Embodiment 66 is the polynucleotide of any one of embodiments 59-64, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
  • Embodiment 67 is the polynucleotide of any one of embodiments 59-64, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
  • Embodiment 68 is the polynucleotide of any one of embodiments 59-64, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
  • Embodiment 69 is the polynucleotide of any one of embodiments 59-68, wherein the PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
  • Embodiment 70 is the polynucleotide of any one of embodiments 69, wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acid at position 99 is Lys or Leu; the amino acid at position 145 is Leu or Val; the amino acid at position 150 is Arg or Gin; the amino acid at position 151 is Asp or Ser; the amino acid at position 153 is Leu or lie; the amino acid at position 163 is Leu or Val; the amino acid at position 171 is Tyr or Phe; the amino acid at position 172 is lie or Leu; the amino acid at position 182 is Asp or Gin; the amino acid at position 196 is Gin or Asn; the amino acid at position
  • Embodiment 71 is the polynucleotide of embodiment 69 or 70, wherein the PHI-4 polypeptide further comprises one or more amino acid substitutions compared to the native amino acid at position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 35.
  • Embodiment 72 is the polynucleotide of embodiment 71 , wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
  • Embodiment 73 is the polynucleotide of any one of embodiments 59-69 and 72, wherein the PHI-4 polypeptide has 1 to 54 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 74 is the polynucleotide of any one of embodiments 59-70, wherein the PHI-4 polypeptide has 1 to 27 amino acid substitutions compared to SEQ ID NO: 2.
  • Embodiment 75 is the polynucleotide of any one of embodiments 59-70, wherein the PHI-4 polypeptide has 1 to 20 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 76 is the polynucleotide of any one of embodiments 59-70, wherein the PHI-4 polypeptide has 1 to 15 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 77 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 54 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 78 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 27 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 79 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 20 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 80 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 15 amino acid substitutions compared to SEQ ID NO: 35.
  • Embodiment 81 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO: 35.
  • Embodiment 82 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 90% identity to SEQ ID NO: 35.
  • Embodiment 83 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 95% identity to SEQ ID NO: 35.
  • Embodiment 84 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 97% identity to SEQ ID NO: 35.
  • Embodiment 85 is a polynucleotide encoding a PHI-4 polypeptide, wherein the PHI-4 polypeptide has at least one amino acid substitution at a residue relative to SEQ ID NO: 35 in a structural domain selected from:
  • PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
  • Embodiment 86 is the polynucleotide of embodiment 85, wherein the hydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys.
  • Embodiment 87 is the polynucleotide of embodiment 85 or 86, wherein the membrane insertion loops are between about the amino acid at position 92 (Val) and about the amino acid at position 101 (Ala) and between about the amino acid 21 1 (Gly) and about the amino acid 220 (Glu), relative to SEQ ID NO: 35.
  • Embodiment 88 is the polynucleotide of embodiment 87, wherein the PHI-4 polypeptide has one or more amino substitution compared to the native amino acid at position 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219, and 220 of SEQ ID NO: 35.
  • Embodiment 89 is the polynucleotide of any one of embodiments 85, 86, 87 or 88, wherein the receptor binding loops are between about the amino acid at position 332 (Asp) and about the amino acid at position 340 (Asp), between about the amino acid at position 395 (Asp) and about the amino acid at position 403 (Asp), and between about the amino acid at position 458 (Asp) and about the amino acid 466 (Asp), relative to SEQ ID NO: 35.
  • Embodiment 90 is the polynucleotide of embodiment 89, wherein the PHI-4 polypeptide has one or more amino substitution compared to the native amino acid at position 332, 333, 334, 335, 336, 337, 338, 339, 340, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 458, 459, 460, 461 , 462, 463, 464, 465, 466 of SEQ ID NO: 35.
  • Embodiment 91 is the polynucleotide of any one of embodiments 85, 86, 87, 88,
  • protease sensitive region residue is selected from between about the amino acid at position 305 (Lys) and about the amino acid at position 316 (Lys) and/or between about the amino acid at position 500 (Arg) and about the amino acid at position 535 (Lys) relative to SEQ ID NO: 35.
  • Embodiment 92 is the polynucleotide of any one of embodiments 85, 86, 87, 88,
  • protease is trypsin
  • Embodiment 93 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQ ID NO: 35.
  • Embodiment 94 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 90% sequence identity to SEQ ID NO: 35.
  • Embodiment 95 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 95% sequence identity to SEQ ID NO: 35.
  • Embodiment 96 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 97% sequence identity to SEQ ID NO: 35.
  • Embodiment 97 is a polynucleotide encoding a PHI-4 polypeptide, wherein the
  • PHI-4 polypeptide comprises an amino acid sequence of the formula
  • Gly Xaa lie Xaa Xaa Leu Xaa
  • Gly Ser lie Ala Cys Val Xaa Thr
  • Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa at position 61 is Arg, Ly
  • Embodiment 98 is a polynucleotide encoding a PHI-4 polypeptide, wherein the
  • PHI-4 polypeptide comprises an amino acid sequence of the formula
  • Gly Asp lie Glu Xaa Leu Arg
  • Gly Ser lie Ala Cys Val Xaa Thr
  • Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position
  • Embodiment 99 is the polynucleotide of embodiments 97 or 98, wherein the PHI-4 polypeptide further comprises one or more amino acid substitutions at position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 3 or SEQ ID NO: 4.
  • Embodiment 100 is the polynucleotide of embodiment 99, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
  • Embodiment 101 is the polynucleotide of any one of embodiments 97-100, wherein the PHI-4 polypeptide further comprises one or more more conservative amino acid substitution, insertion of one or more amino acids, deletion of one or more amino acids, and combinations thereof.
  • Embodiment 102 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 1.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 103 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 104 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 105 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 3 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 106 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
  • Embodiment 107 is the polynucleotide of any one of embodiments 97-106, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
  • WCRW Western Corn Root Worm
  • Embodiment 108 is the polynucleotide of any one of embodiments 97-107, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
  • Embodiment 109 is the polynucleotide of any one of embodiments 97-107, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
  • Embodiment 1 10 is the polynucleotide of any one of embodiments 97-107, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
  • Embodiment 1 1 1 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 54 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
  • Embodiment 1 12 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 27 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
  • Embodiment 1 13 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 20 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
  • Embodiment 1 14 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 15 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
  • Embodiment 1 15 is the polynucleotide of any one of embodiments 97-1 14, wherein 1 -25 amino acids are deleted from the N-terminus of the PHI-4 polypeptide and/or C-terminus of the PHI-4 polypeptide.
  • Embodiment 1 16 is the polynucleotide of any one of embodiments 97-1 14, wherein 1-20 amino acids are deleted from the C-terminus of the PHI-4 polypeptide.
  • Embodiment 1 17 is a composition, comprising an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1 -58.
  • Embodiment 1 18 is a method of inhibiting growth or killing an insect pest, comprising contacting the insect pest with the composition of embodiment 1 17.
  • Embodiment 1 19 is a method for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the resistant insect pest population with the composition of embodiment 1 17.
  • Embodiment 120 is the method of controlling an insect pest population resistant to an pesticidal protein of embodiment 1 19, wherein the pesticidal protein is selected from CrylAc, CrylAb, Cry1A.105, CrylAc, Cry1 F, Cry1 Fa2, Cryl F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1 , Cry34Ab1 , Cry35Ab1 , Vip3A, Cry9c, eCry3.1Ab and CBI-Bt.
  • the pesticidal protein is selected from CrylAc, CrylAb, Cry1A.105, CrylAc, Cry1 F, Cry1 Fa2, Cryl F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1 , Cry34Ab1 , Cry35Ab1 , Vip3A, Cry9c, eCry3.1Ab and CBI-Bt.
  • Embodiment 121 is a transgenic plant or progeny thereof, comprising the polynucleotide of any one of embodiments 59-1 16.
  • Embodiment 122 is the transgenic plant or progeny thereof of embodiment 121 , wherein the transgenic plant is a monocotyledon.
  • Embodiment 123 is the transgenic plant or progeny thereof of embodiment 122, wherein the plant is selected from barley, corn, oat, rice, rye, sorghum, turf grass, sugarcane, wheat, alfalfa, banana, broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower, safflower, soybean, strawberry, sugar beet, sweet potato, tobacco, tomato ornamental, shrub, nut, chickpea, pigeon pea, millets, hops and pasture grasses.
  • the plant is selected from barley, corn, oat, rice, rye, sorghum, turf grass, sugarcane, wheat, alfalfa, banana, broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, celery, citrus, cotton,
  • Embodiment 124 is the transgenic plant or progeny thereof of embodiment 123, further comprising one or more additional transgenic traits.
  • Embodiment 125 is the transgenic plant of embodiment 124, wherein the one or more additional transgenic trait is selected from insect resistance, herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stalk strength, increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, salt resistance or tolerance and increased yield under stress.
  • the one or more additional transgenic trait is selected from insect resistance, herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stalk strength, increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, salt resistance or tolerance and increased yield under stress.
  • Embodiment 126 is seed, grain or processed product thereof of the transgenic plant of any one of embodiments 121-126, wherein the seed, grain or processed product thereof comprises the polynucleotide of any one of embodiments 121-125.
  • Embodiment 127 is an expression cassette, comprising the polynucleotide of any one of embodiments 59-1 16 operably linked to one or more regulatory sequences directing expression of the PHI-4 polypeptide.
  • Embodiment 128 is a transgenic plant or plant cell, comprising the expression cassette of embodiment 127.
  • Embodiment 129 is seed, grain or processed product thereof of the transgenic plant of embodiment 128, wherein the seed, grain or processed product thereof comprises the recombinant nucleic acid molecule of embodiment of 1 and the recombinant nucleic acid molecule of 24.
  • Embodiment 130 is the seed of embodiment 129, wherein one or more seed treatment has been applied to the seed.
  • Embodiment 131 is a method for expressing in a plant a polynucleotide encoding an insecticidal protein, comprising
  • step (b) obtaining a transformed plant cell comprising the nucleic acid sequence of step (a);
  • Embodiment 132 is a method for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof, an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1-58.
  • Embodiment 133 is a method for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1-58.
  • Embodiment 134 is a method of inhibiting growth or killing an insect pest, comprising contacting the insect pest with a composition comprising an insecticidally- effective amount of the the PHI-4 polypeptide of any one of embodiments 1-58.
  • Embodiment 135 is a method for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1-58.
  • Embodiment 136 is a fusion protein comprising the PHI-4 polypeptide of any one of embodiments 1-58.
  • Embodiment 137 is a fusion protein comprising the PHI-4 polypeptide of any one of embodiments 1-58 and a maltose binding protein.
  • Embodiment 138 is the fusion protein of embodiment 137, wherein the maltose binding protein has an amino acid sequence of SEQ I D NO: 1516 or SEQ ID NO: 1517.
  • Figure 1 shows FAE analysis of MPB::PHI-4-SFR12-004 (SEQ ID NO: 31 ).
  • the reference protein is MBP::PHI-4 polypeptide of (SEQ ID NO: 6).
  • the % Response is given on the y axis.
  • the dose of the toxin fragment is given on the x axis in parts per million (ppm).
  • Half dose (+) and full dose ( * ) indicate a mean response from six replicate wells at the indicated concentration. All data are from a single experiment.
  • Figure 2 shows EC50 analysis of MBP::PHI-4 polypeptide of SEQ ID NO: 6 and MPB::PHI-4-SFR12-004 (SEQ ID NO: 35).
  • the % Response is given on the y axis.
  • the dose of the toxin fragment is given on the x axis in parts per million (ppm).
  • the % Response is given on the y axis.
  • the dose of the toxin fragment is given on the x axis in parts per million (ppm).
  • the protein concentration (toxin portion of the protein only) is given on the x axis.
  • Each symbol indicates a mean response from twenty-four replicate wells at the indicated concentration. All data are from a single experiment, triangles: MBP::PHI-4 (SEQ ID NO: 6); circles: MBP::PHI-4-SFR12-004 (SEQ ID NO: 31 ).
  • Figure 3 shows the amino acids sequence of the C-terminal portion of the PHI-4 polypeptide (SEQ ID NO: 2) is given. Three stretches of sequence corresponding to nine amino acid motifs that align to the putative sugar binding loop motif D-X-G-(S/T)-G-X 3 -D (SEQ ID NO: 40) are highlighted in grey.
  • Figure 4 shows EC50 data for SEQ ID NO: 610, SEQ ID NO: 595, SEQ ID NO: 584, SEQ ID NO: 591 , SEQ I D NO: 576, SEQ ID NO: 73, SEQ I D NO: 74, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 150, SEQ ID NO: 150, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 167, SEQ ID NO: 164, SEQ ID NO: 164, SEQ ID NO: 170, SEQ ID NO: 170, SEQ ID NO: 795, SEQ ID NO: 794, SEQ ID NO: 784, SEQ ID NO: 799, SEQ ID NO: 785, SEQ ID NO: 788, SEQ ID NO: 786, SEQ ID NO: 796, SEQ ID NO: 787.
  • the x- axis corresponds to the mean fold improvement in EC50, relative to MBP::PHI-4 fusion (SEQ ID NO: 6) from at least three independent EC50 measurements.
  • the y-axis corresponds to the mean fold improvement of the mean FAE Index from at least three independent measurements.
  • compositions and methods for controlling pests involve transforming organisms with a nucleic acid sequence encoding a PHI-4 polypeptide.
  • the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms possessing pesticidal activity.
  • transformed bacteria, plants, plant cells, plant tissues and seeds are provided.
  • Compositions are pesticidal nucleic acids and proteins of bacterial species.
  • the nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered PHI-4 polypeptides by methods known in the art, such as site directed mutagenesis, domain swapping or DNA shuffling.
  • the PHI-4 polypeptides find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran, and nematode pest populations and for producing compositions with pesticidal activity.
  • Insect pests of interest include, but are not limited to, the superfamily of stink bugs and other related insects including, but not limited to, species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guild ini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria - Bean plataspid), and the family Cydnidae ⁇ Scaptocoris castanea - Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includen
  • Pesticidal toxin or “pesticidal protein” is intended a toxin that has toxic activity against one or more pests, including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera and Coleoptera orders or the Nematoda phylum or a protein that has homology to such a protein.
  • Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.
  • Pesticidal proteins include but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (201 1 ) PLoS Pathogens, 7:1 -13) from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et ai, (2010) J. Agric. Food Chem.
  • Pseudomonas sp. such as PSEEN3174 (Monalysin, (201 1 ) PLoS Pathogens, 7:1 -13) from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-
  • B. thuringiensis insecticidal proteins include, but are not limited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8 (Accession # 126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa1 1 (Accession # CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession
  • Examples of ⁇ -endotoxins also include but are not limited to CrylA proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-1 1 toxin (N-terminal deletion of a-helix 1 and/or a-helix 2 variants of cry proteins such as CrylA, Cry3A) of US Patent Numbers 8,304,604 and 8.304,605, Cryl B of US Patent Application Serial Number 10/525,318; Cryl C of US Patent Number 6,033,874; Cryl F of US Patent Numbers 5,188,960, 6,218,188; Cry1A/F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry
  • Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature” (201 1 ), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the "www" prefix).
  • the insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101 :1-16).
  • Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to CrylAc, Cry1Ac+Cry2Ab, CrylAb, Cry1A.105, Cryl F, Cry1 Fa2, Cry1 F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1 , Cry34Ab1 , Cry35Ab1 , Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (201 1 ) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C.
  • More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cryl Fa (US2012/0317682), Cryl BE & Cryl F (US2012/031 1746), CryI CA & CrylAB (US2012/031 1745), Cryl F & CryCa (US2012/0317681 ), Cryl DA & Cryl BE (US2012/0331590), Cryl DA & Cryl Fa (US2012/0331589), CrylAB & Cryl BE (US2012/0324606), and Cryl Fa & Cry2Aa, Cryl l or Cryl E (US2012/0324605).
  • Vip3Ab & Cryl Fa US2012/0317682
  • Cryl BE & Cryl F US2012/031 1746
  • CryI CA & CrylAB US2012/031 1745
  • Cryl F & CryCa US2012/0317681
  • Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491 ,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like.
  • VIP vegetable insecticidal proteins
  • Pesticidal proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can be accessed on the world-wide web using the "www" prefix).
  • Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491 ,698 and 8,084,418).
  • Some TC proteins have "stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism.
  • TC protein from Photorhabdus, Xenorhabdus or Paenibacillus, for example
  • TC protein TC protein "potentiators” derived from a source organism of a different genus.
  • TC protein TC protein "potentiators" derived from a source organism of a different genus.
  • TC proteins There are three main types of TC proteins. As referred to herein, Class A proteins ("Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptBIXb and XptCIWi.
  • Class C proteins are TccC, XptCIXb and XptBIWi.
  • Pesticidal proteins also include spider, snake and scorpion venom proteins.
  • spider venom peptides include but not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366).
  • the PHI-4 polypeptides include amino acid sequences deduced from the full-length nucleic acid sequences disclosed herein, and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in or in the pest after ingestion of the protein.
  • nucleic Acid Molecules that confer pesticidal activity.
  • amino acid sequences of PHI-4 polypeptides The protein resulting from translation of these PHI-4 polypeptide genes allows cells to control or kill pests that ingest it.
  • nucleic acid molecules comprising nucleic acid sequences encoding PHI-4 polypeptides and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology.
  • nucleic acid molecule is intended to include DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • nucleic acid molecule or DNA
  • isolated nucleic acid molecule or DNA
  • recombinant nucleic acid molecule or DNA
  • an isolated or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • isolated or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes.
  • the recombinant nucleic acid molecule encoding a PHI- 4 polypeptide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a nucleic acid molecule encoding the PHI-4 polypeptide is a non-genomic nucleic acid sequence.
  • a “non-genomic nucleic acid sequence "or “non-genomic nucleic acid molecule” refers to a nucleic acid molecule that has one or more change in the nucleic acid sequence compared to a native or genomic nucleic acid sequence.
  • the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5' and/or 3' untranslated region; and modification of a polyadenylation site.
  • the non-genomic nucleic acid molecule is a cDNA.
  • the non-genomic nucleic acid molecule is a cDNA
  • polynucleotides encoding a PHI-4 polypeptide(s) or related proteins are contemplated. Such polynucleotides are useful for production of PHI-4 polypeptides in host cells when operably linked to suitable promoter, transcription termination and/or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides encoding PHI-4 polypeptides or related proteins.
  • the present disclosure provides isolated or recombinant polynucleotides that encode any of the PHI-4 polypeptides disclosed herein.
  • Table 1 is a Codon Table that provides the synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
  • the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence. Table 1
  • each codon in a nucleic acid can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence.
  • the disclosure contemplates and provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the disclosure that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (set forth in Table 1 ), as applied to the polynucleotide sequences of the present disclosure.
  • a group of two or more different codons that, when translated in the same context, all encode the same amino acid, are referred to herein as "synonymous codons.”
  • Polynucleotides encoding PHI-4 polypeptides of the present disclosure may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform with the optimum codon usage of the desired host organism.
  • Polynucleotides encoding a PHI-4 polypeptide can also be synthesized de novo from a PHI-4 polypeptide sequence.
  • the sequence of the polynucleotide gene can be deduced from a PHI-4 polypeptide sequence through use of the genetic code.
  • Computer programs such as "BackTranslate” (GCGTM Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide.
  • PHI-4 polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to, the PHI-4 polypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526.
  • synthetic PHI-4 polynucleotide sequences of the disclosure can be designed so that they will be expressed in plants.
  • US Patent Number 5,500,365 describes a method for synthesizing plant genes to improve the expression level of the protein encoded by the synthesized gene. This method relates to the modification of the structural gene sequences of the exogenous transgene, to cause them to be more efficiently transcribed, processed, translated and expressed by the plant.
  • genes that are expressed well in plants include elimination of sequences that can cause undesired intron splicing or polyadenylation in the coding region of a gene transcript while retaining substantially the amino acid sequence of the toxic portion of the insecticidal protein.
  • a similar method for obtaining enhanced expression of transgenes in monocotyledonous plants is disclosed in US Patent Number 5,689,052.
  • the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30, SEQ ID NOs: 1163-1505 and variants, fragments and complements thereof.
  • complement is intended a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex.
  • the nucleic acid molecule encoding a PHI-4 polypeptide is a nucleic acid molecule having the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30 and SEQ ID NOs: 1 163-1505.
  • the corresponding amino acid sequences for the insecticidal protein encoded by these nucleic acid sequences are set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30 and SEQ ID NOs: 1 163-1505.
  • the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 35, wherein the polypeptide has pesticidal activity.
  • the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 80% and 95% identity, to the amino acid sequence of SEQ ID NO: 35, wherein the polypeptide has pesticidal activity.
  • the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% identity, to any one of the amino acid sequence of SEQ ID NOs: 51-1 162 SEQ ID NOs: 1518-1526 wherein the polypeptide has pesticidal activity.
  • the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 51 is
  • exemplary nucleic acid molecules encode a PHI-4 of any one of SEQ ID NOs: 51 -1 162 and SEQ ID NOs: 1518-1526 as well as amino acid substitutions, amino acid deletions, insertions and fragments thereof and combinations thereof.
  • nucleic acid molecules encode a PHI-4 polypeptide of Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9 and combinations of the amino acid substitutions thereof and amino acid deletions and/or insertions thereof.
  • nucleic acid molecules that encode transcription and/or translation products that are subsequently spliced to ultimately produce functional PHI-4.
  • Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans-splicing.
  • the substrate for splicing can be polynucleotides (e.g., RNA transcripts) or polypeptides.
  • An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a PHI-4 encoding sequence.
  • trans splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full-length pesticidal encoding sequence.
  • the use of a splicing enhancer sequence which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides (US Patent Numbers 6,365,377 and 6,531 ,316).
  • the polynucleotides do not directly encode a full-length PHI-4, but rather encode a fragment or fragments of a PHI-4.
  • polynucleotides can be used to express a functional PHI-4 through a mechanism involving splicing, where splicing can occur at the level of polynucleotide (e.g., intron/exon) and/or polypeptide (e.g., intein/extein).
  • splicing can occur at the level of polynucleotide (e.g., intron/exon) and/or polypeptide (e.g., intein/extein).
  • This can be useful, for example, in controlling expression of pesticidal activity, since functional pesticidal polypeptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product.
  • introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide; use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant.
  • Nucleic acid molecules that are fragments of these nucleic acid sequences encoding PHI-4 are also encompassed by the embodiments.
  • fragment is intended a portion of the nucleic acid sequence encoding a PHI-4.
  • a fragment of a nucleic acid sequence may encode a biologically active portion of a PHI-4 polypeptide or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a PHI-4 comprise at least about 50, 100, 200, 300, 400, 500, 600 or 700, contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding a PHI-4 disclosed herein, depending upon the intended use.
  • contiguous nucleotides is intended nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the PHI-4 and, hence, retain insecticidal activity.
  • the fragment will encode a polypeptide having at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the insecticidal activity of the full-length PHI-4.
  • the insecticidal activity is Lepodoptera activity.
  • the insecticidal activity is Hemiptera activity.
  • a fragment of a nucleic acid sequence encoding a PHI-4 encoding a biologically active portion of a protein will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200 or 250, contiguous amino acids or up to the total number of amino acids present in a full-length PHI-4 of the embodiments.
  • the fragment is an N-terminal or a C-terminal truncation of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino acids relative to SEQ ID NO: 35, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526 or variants thereof, e.g., by proteolysis, insertion of a start codon, deletion of the codons encoding the deleted amino acids with the concomitant insertion of a stop codon or by insertion of a stop codon in the coding sequence.
  • the fragments encompassed herein result from the removal of the N-terminal 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or more amino acids relative to SEQ ID NO: 35, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526 or variants thereof, e.g., by proteolysis or by insertion of a start codon in the coding sequence.
  • the PHI-4 are encoded by a nucleic acid sequence sufficiently identical to the nucleic acid sequence of SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOS: 24-30.
  • “sufficiently identical” is intended an amino acid or nucleic acid sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters.
  • sequence homology identity is against the full length sequence of the polynucleotide encoding a PHI-4 or against the full length sequence of a PHI-4 polypeptide.
  • the polynucleotide encoding the PHI-4 has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOS: 24-30.
  • SEQ ID NO: 7 SEQ ID NO: 7
  • SEQ ID NOS: 24-30 One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleic acid sequences by taking into account codon degeneracy, amino acid similarity, reading frame
  • the sequences are aligned for optimal comparison purposes.
  • the two sequences are the same length.
  • the comparison is across the entirety of the reference sequence (e.g., across the entirety of SEQ ID NO: 35 or across the entirety of one of SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOS: 24-30).
  • the percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et ai, (1990) J. Mol. Biol. 215:403.
  • Gapped BLAST in BLAST 2.0
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra.
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • Alignment may also be performed manually by inspection.
  • ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence and thus can provide data about the sequence conservation of the entire amino acid sequence.
  • the ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed.
  • GENEDOCTM A non-limiting example of a software program useful for analysis of ClustalW alignments.
  • GENEDOCTM (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins.
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA).
  • ALIGN program version 2.0
  • a gap length penalty of 12 and a gap penalty of 4 can be used.
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software to determine sequence identity or similarity using the following default parameters: % identity and % similarity for a nucleic acid sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmpii scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used.
  • Equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • PHI-4 polypeptide "Variants" of the PHI-4 polypeptide encoding nucleic acid sequences include those sequences that encode the PHI-4 polypeptides disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the PHI-4 polypeptides disclosed as discussed below.
  • PCR polymerase chain reaction
  • variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, nucleotide additions and/or nucleotide deletions into the corresponding nucleic acid sequence disclosed herein, such that one or more amino acid substitutions, amino acid additions or amino acid deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.
  • variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis and the resultant mutants can be screened for ability to confer pesticidal activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
  • polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional pesticidal polypeptide homologues and fragments thereof with desired properties.
  • standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional pesticidal polypeptide homologues and fragments thereof with desired properties.
  • a variety of such reactions are known, including those developed by the inventors and their co-workers.
  • Methods for producing a variant of any nucleic acid listed herein comprising recursively recombining such polynucleotide with a second (or more) polynucleotide, thus forming a library of variant polynucleotides are also embodiments of the disclosure, as are the libraries produced, the cells comprising the libraries, and any recombinant polynucleotide produces by such methods. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on pesticidal activity, as is wherein such recursive recombination is done in vitro or in vivo.
  • a variety of diversity generating protocols including nucleic acid recursive recombination protocols are available and fully described in the art.
  • the procedures can be used separately, and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and/or organisms with new and/or improved characteristics.
  • any of the diversity generating procedures described herein can be the generation of one or more nucleic acids, which can be selected or screened for nucleic acids with or which confer desirable properties or that encode proteins with or which confer desirable properties.
  • any nucleic acids that are produced can be selected for a desired activity or property, e.g. pesticidal activity or such activity at a desired pH, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable format, by any of the assays in the art, see, e.g., discussion of screening of insecticidal activity, infra.
  • a variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner.
  • Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2): 157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith, (1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 229:1 193-1201 ; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass, et al., (1988) Science 242:240-245); oligon
  • Additional suitable methods include point mismatch repair (Kramer, et al. , (1984) Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter, et al., (1985) NucI Acids Res 13:4431 -4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) NucI Acids Res 14:51 15), restriction-selection and restriction-purification (Wells, et al., (1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total gene synthesis (Nambiar, et al.
  • nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from other organisms, particularly other bacteria. In this manner, methods such as PCR, hybridization and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments. Such sequences include sequences that are orthologs of the disclosed sequences.
  • the term "orthologs" refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest.
  • Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook”. See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds.
  • PCR PCR Strategies
  • nested primers single specific primers
  • degenerate primers gene-specific primers
  • vector- specific primers partially-mismatched primers
  • the bacterial cell lysates can be screened with antibodies generated against a PHI-4 polypeptide using Western blotting and/or ELISA methods. This type of assays can be performed in a high throughput fashion. Positive samples can be further analyzed by various techniques such as antibody based protein purification and identification. Methods of generating antibodies are well known in the art as discussed infra.
  • mass spectrometry based protein identification method can be used to identify homologs of a PHI-4 polypeptide using protocols in the literatures (Patterson, (1998), 10(22): 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc).
  • LC-MS/MS based protein identification method is used to associate the MS data of given cell lysate or desired molecular weight enriched samples (excised from SDS-PAGE gel of relevant molecular weight bands to a PHI-4 polypeptide) with sequence information of a PHI-4 polypeptide and its homologs. Any match in peptide sequences indicates the potential of having the homologous proteins in the samples. Additional techniques (protein purification and molecular biology) can be used to isolate the protein and identify the sequences of the homologs.
  • hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides, and may be labeled with a detectable group such as 32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor.
  • Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known PHI-4 polypeptide-encoding nucleic acid sequence disclosed herein.
  • the probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequence encoding a PHI-4 polypeptide of the disclosure or a fragment or variant thereof.
  • Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001 ), supra, herein incorporated by reference.
  • an entire nucleic acid sequence, encoding a PHI-4 polypeptide, disclosed herein or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding nucleic acid sequences encoding PHI-4 polypeptide-like sequences and messenger RNAs.
  • probes include sequences that are unique and are preferably at least about 10 nucleotides in length or at least about 20 nucleotides in length.
  • probes may be used to amplify corresponding pesticidal sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1 .0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5x to 1 *SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at 37°C, and a wash in O.l xSSC at 60 to 65°C.
  • wash buffers may comprise about 0.1 % to about 1 % SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 °C for each 1 % of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ⁇ 90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • Tm thermal melting point
  • PHI-4 polypeptides are encompassed by the disclosure.
  • PHI-4 polypeptide or "PHI-4 protein” as used herein interchangeably is intended a polypeptide that has increased insecticidal activity against one or more insect pests of the Lepidoptera and/or Coleoptera orders compared to the protein of SEQ ID NO: 35, and is sufficiently identical to the protein of SEQ ID NO: 35.
  • a variety of PHI-4 polypeptides are contemplated.
  • the Western Corn Rootworm active protein AXMI-205 (SEQ ID NO: 35) encoded by the polynucleotide of SEQ ID NO: 34 was identified from the Chromobacterium Strain ATX 2024 (US201 10023184). Synthetic genes encoding AXMI-205; AXMI-205 variants having a truncation of the last 10 and 20 amino acids from the C-terminus (SEQ ID NO: 36 and SEQ ID NO: 37); and alanine scanning at every other residue from residue 307- 536 of AXMI-205 (SEQ ID NO: 35), with S307A, D315A, V317A, S349A, G351A, K353A, V355A, D395A, G399A, W407A, G419A, P435A, S443A, K465A, V467A, F483A, P487A, S495A, D497A, E499A, K509A, and
  • AXMI-205 variants evo 24 (E499A); evo25 (V467A - SEQ ID NO: 41 ); evo30 (V467L - SEQ ID NO: 42); PMIibl PoollG2_p2al 1 (S468L - SEQ ID NO: 45); PMIibl PoollG2_plcl (V467T - SEQ ID NO: 46), PMIibl PoollG2_pla4 (R464N - SEQ ID NO: 47), evo34 (E86T - SEQ ID NO: 43) and evo35 (Q517R - SEQ ID NO: 44) having increased insecticidal activity are disclosed in WO2013/016617.
  • protein As used herein, the terms “protein,” “peptide molecule” or “polypeptide” includes any molecule that comprises five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules may undergo modification, including post- translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation or oligomerization. Thus, as used herein, the terms “protein,” “peptide molecule” or “polypeptide” includes any protein that is modified by any biological or non-biological process.
  • amino acid and “amino acids” refer to all naturally occurring L-amino acids.
  • a "recombinant protein” is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • a PHI-4 polypeptide that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10% or 5% (by dry weight) of non-pesticidal protein (also referred to herein as a "contaminating protein").
  • improved activity or “increased activity” is intended an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210% at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least
  • the improvement consists of a decrease in the EC50 of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210% at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about
  • the EC50 of the PHI-4 polypeptide is ⁇ 100 ppm, ⁇ 90 ppm, ⁇ 80 ppm, ⁇ 70 ppm, ⁇ 60 ppm, ⁇ 50 ppm, ⁇ 45 ppm, ⁇ 40 ppm, ⁇ 35 ppm, ⁇ 30 ppm, ⁇ 25 ppm, ⁇ 20 ppm, ⁇ 19 ppm, ⁇ 18 ppm, ⁇ 17 ppm, ⁇ 16 ppm, ⁇ 15 ppm, ⁇ 14 ppm, ⁇ 13 ppm, ⁇ 12 ppm, ⁇ 1 1 ppm, ⁇ 10 ppm, ⁇ 9 ppm, ⁇ 8 ppm, ⁇ 7 ppm, ⁇ 6 ppm, ⁇ 5 ppm, ⁇ 4 ppm, ⁇ 3 ppm, ⁇ 2 ppm, ⁇ 1 ppm, ⁇ 0.9 ppm, ⁇ 0.8 ppm, ⁇ 0.7 ppm
  • the improvement consists of an increase in the Mean FAE Index of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about
  • the improvement consists of an increase in the Mean Deviation Score of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210% at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least
  • the improved activity of the PHI-4 polypeptide is relative to the pesticidal activity of AXMI-205(evo25) (SEQ ID NO: 41 ), AXMI-205(evo30) (SEQ ID NO: 42), Axmi205 PMIibl PoollG2_p2al 1 (mutation S468L; SEQ ID NO: 45), Axmi205 PMIibl PoollG2_plcl (mutation V467T; SEQ ID NO: 46), Axmi205 PMIibl PoollG2_pla4 (mutation R464N; SEQ ID NO: 47), AXMI-205(evo34) (SEQ ID NO: 43) or AXMI- 205(evo35) (SEQ ID NO: 44).
  • MFI Mean FAE Index
  • MFI mean of multiple FAEGN an arithmetic mean of FAEGN.
  • Mean Deviation Score refers to the arithmetic mean of multiple Deviation Scores.
  • the PHI-4 polypeptides have increased insecticidal activity against one or more insect pests of the order Coleoptera. In some embodiments the PHI-4 polypeptides have increased insecticidal activity against one or more insect pests of the corn rootworm complex (western corn rootworm, Diabrotica virgifera; northern corn rootworm, D. barberi: and the Mexican corn rootworm, D. virgifera zeae.
  • corn rootworm complex western corn rootworm, Diabrotica virgifera; northern corn rootworm, D. barberi: and the Mexican corn rootworm, D. virgifera zeae.
  • the PHI-4 polypeptides have increased insecticidal activity against Diabrotica virgifera larvae - Western Corn Root Worm (WCRW).
  • “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to a PHI-4 polypeptide and that exhibit insecticidal activity. “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 and that exhibit insecticidal activity.
  • a biologically active portion of a PHI-4 polypeptide can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 516, 517, 518, 519, 520, 521 , 522, 523, 524, 525, 526, 527, 528, 529, 530, 531 , 532, 533, 534 or 535 amino acids in length.
  • Such biologically active portions can be prepared by recombinant techniques and evaluated for insecticidal activity.
  • a fragment comprises at least 8 contiguous amino acids of a PHI-4 polypeptide.
  • a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 2, SEQ ID NOs: 51 -1 162, or SEQ ID NOs: 1518-1526. In some embodiments a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NOs: 51-1 162, or SEQ ID NOs: 1518-1526. The embodiments encompass other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250 or more amino acids.
  • the fragment is an N-terminal and/or a C-terminal truncation of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 35, SEQ ID NOs: 51- 1 162, SEQ ID NOs: 1518-1526 or variants thereof e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon and/or insertion of a stop codon.
  • the fragments encompassed herein result from the removal of the C-terminal 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 amino acids relative to SEQ ID NO: 35, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526, and variants thereof by proteolysis or by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon.
  • the proteolytic cleavage site is between Lys at 520 and Ser at 521 Ser or Lys at 313 and Val at 314 of SEQ ID NO: 35 or variants thereof.
  • polynucleotide encoding the truncated PHI-4 polypeptide can be engineered to add a start codon at the N-terminus such as ATG encoding methionine or methionine followed by an alanine. It is also well known in the art that depending on what host the PHI-4 polypeptide is expressed in the methionine may be partially of completed processed off.
  • fragments, biologically active portions of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526 as well as amino acid substitutions, amino acid deletions and/or insertions thereof are also provided, and may be used to practice the methods of the disclosure.
  • PHI-4 polypeptides are provided having one or more amino acid substitution compared to AXMI-205 (SEQ ID NO: 35). In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at solvent exposed surface residues to modify the protein characteristics of AXMI-205 (SEQ ID NO: 35), including but not limited to the ionic polarity of the protein surface. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at hydrophilic residues such as Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys.
  • the PHI-4 polypeptides are provided having amino acid substitutions changing a Lysine or Arginine to a Glutamine, Glutamic Acid, Asparagine or Glutamic Acid; changing a Glutamic Acid or Aspartic Acid to a Lysine, Asparagine or Glutamine; and changing a Glutamine to a Asparagine or Lysine.
  • PHI-4 polypeptides are provided having amino acid substitutions at residues in a membrane insertion loop. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions in a membrane insertion loop between about amino acid at position 92 (Val) and 101 (Ala) and/or at position 21 1 (Gly) and 220 (Glu) relative to SEQ ID NO: 35.
  • PHI-4 polypeptides are provided having amino acid substitutions at residues and receptor binding loops. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues and receptor binding loops between about amino acid 332 (Asp) and 340 (Asp), 395 (Asp) and 403 (Asp) , 458 (Asp) and 466 (Asp) relative to SEQ ID NO: 35.
  • PHI-4 polypeptides are provided having amino acid substitutions at residues in a protease sensitive region. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues in a protease sensitive region from about amino acid residues between 305 (Lys) and 316 (Lys) and 500 (Arg) and 535 (Lys) relative to SEQ ID NO: 35.
  • proteins or polypeptides having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the parental amino acid sequence.
  • a PHI-4 polypeptide has at least about 60%, 65%, about 70%, 75%, at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater identity across the entire length of the amino acid sequence of SEQ ID NO: 2, SEQ ID NOs: 51 -1 162, SEQ ID NOs: 1518-1526.
  • a PHI-4 polypeptide has at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater identity across the entire length of the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NOs: 51- 1 162, or SEQ ID NOs: 1518-1526.
  • a PHI-4 polypeptide comprises an amino acid sequence having at least 80% identity, to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162 or SEQ ID NO: 1518-1526, wherein the polypeptide has insecticidal activity.
  • a PHI-4 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162 or SEQ ID NO: 1518-1526, wherein the polypeptide has insecticidal activity.
  • a PHI-4 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162 or SEQ ID NO: 1518-1526 wherein the polypeptide has insecticidal activity.
  • a PHI-4 polypeptide comprises an amino acid sequence having at least 97% identity to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162 or SEQ ID NO: 1518-1526, wherein the polypeptide has insecticidal activity.
  • a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 3, wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; X
  • a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 4 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 4 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
  • a PHI-4 polypeptide comprises an amino acid sequence of
  • SEQ ID NO: 4 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 4 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
  • a PHI-4 polypeptide comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 4.
  • a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 4, wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is
  • a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75 or 76 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 3 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
  • PHI-4 polypeptide comprises an amino acid sequence of
  • SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53 or 54 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 3 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
  • PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14 or 15 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 3 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
  • a PHI-4 polypeptide comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • a PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
  • a PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35 wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val
  • the PHI-4 polypeptide comprising one or more amino acid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468, 499 or 517.
  • the PHI-4 polypeptide comprising one or more amino acid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468, 499 or 517, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 399 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
  • exemplary PHI-4 polypeptides are encoded by the polynucleotide sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30, SEQ ID NOs. 1 163-1505, and SEQ ID NOs. 1527-1535.
  • a PHI-4 polypeptide includes variants where an amino acid that is part of a proteolytic cleavage site is changed to another amino acid to eliminate or alter the proteolytic cleavage at that site.
  • the proteolytic cleavage is by a protease in the insect gut. In other embodiments the proteolytic cleavage is by a plant protease in the transgenic plant.
  • exemplary PHI-4 polypeptides are the polypeptides shown in Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9 and combinations of the amino substitutions thereof as well as amino acid deletions, and or insertions and fragments thereof.
  • the PHI-4 polypeptide is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid molecule of SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30, SEQ ID NOs. 1 163-1505, and SEQ ID NOs. 1527-1535.
  • Variants include polypeptides that differ in amino acid sequence due to mutagenesis.
  • Variant proteins encompassed by the disclosure are biologically active, that is they continue to possess the desired biological activity (i.e. pesticidal activity) of the native protein.
  • By "retains activity” is intended that the variant will have at least about 30%, at least about 50%, at least about 70% or at least about 80% of the insecticidal activity of the native protein.
  • the variants may have improved activity over the native protein.
  • Bacterial genes quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can initiate at a TTG codon, though in this event the TTG encodes a methionine. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium.
  • the PHI-4 polypeptide may be expressed as a precursor protein with an intervening sequence that catalyzes multi-step, post translational protein splicing.
  • Protein splicing involves the excision of an intervening sequence from a polypeptide with the concomitant joining of the flanking sequences to yield a new polypeptide (Chong, et al., (1996) J. Biol. Chem. 271 :22159-22168).
  • inteins This intervening sequence or protein splicing element, referred to as inteins, which catalyze their own excision through three coordinated reactions at the N-terminal and C-terminal splice junctions: an acyl rearrangement of the N-terminal cysteine or Serine; a transesterfication reaction between the two termini to form a branched ester or thioester intermediate and peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein (Evans, et al., (2000) J. Biol. Chem. 275:9091-9094.
  • the PHI-4 polypeptide may be encoded by two separate genes where the intein of the precursor protein comes from the two genes, referred to as a split- intein and the two portions of the precursor are joined by a peptide bond formation.
  • This peptide bond formation is accomplished by intein-mediated trans-splicing.
  • a first and a second expression cassette comprising the two separate genes further code for inteins capable of mediating protein trans-splicing.
  • trans-splicing the proteins and polypeptides encoded by the first and second fragments may be linked by peptide bond formation.
  • Trans-splicing inteins may be selected from the nucleolar and organellar genomes of different organisms including eukaryotes, archaebacteria and eubacteria. Inteins that may be used for are listed at neb.com/neb/inteins.html, which can be accessed on the world-wide web using the "www" prefix).
  • the nucleotide sequence coding for an intein may be split into a 5' and a 3' part that code for the 5' and the 3' part of the intein, respectively. Sequence portions not necessary for intein splicing (e.g., homing endonuclease domain) may be deleted.
  • the intein coding sequence is split such that the 5' and the 3' parts are capable of trans-splicing.
  • a suitable splitting site of the intein coding sequence the considerations published by Southworth, et al., (1998) EMBO J. 17:918-926 may be followed.
  • the 5' intein coding sequence is linked to the 3' end of the first fragment coding for the N-terminal part of the PHI-4 polypeptide and the 3' intein coding sequence is linked to the 5' end of the second fragment coding for the C-terminal part of the PHI-4 polypeptide.
  • the trans-splicing partners can be designed using any split intein, including any naturally-occurring or artificially-split split intein.
  • split inteins are known, for example: the split intein of the DnaE gene of Synechocystis sp. PCC6803 (see, Wu, et al., (1998) Proc Natl Acad Sci USA 95(16):9226-31 and Evans, et al., (2000) J Biol Chem 275(13):9091-4 and of the DnaE gene from Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett 580(7): 1853-8).
  • Non-split inteins have been artificially split in the laboratory to create new split inteins, for example: the artificially split Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta 1387:422-32) and split See VMA intein (see, Brenzel, et al., (2006) Biochemistry 45(6): 1571 -8) and an artificially split fungal mini-intein (see, Elleuche, et al., (2007) Biochem Biophys Res Commun 355(3):830-4).
  • Naturally-occurring non-split inteins may have endonuclease or other enzymatic activities that can typically be removed when designing an artificially-split split intein.
  • Such mini-inteins or minimized split inteins are well known in the art and are typically less than 200 amino acid residues long (see, Wu, et al., (1998) Biochim Biophys Acta 1387:422-32).
  • Suitable split inteins may have other purification enabling polypeptide elements added to their structure, provided that such elements do not inhibit the splicing of the split intein or are added in a manner that allows them to be removed prior to splicing.
  • Protein splicing has been reported using proteins that comprise bacterial intein- like (BIL) domains (see, Amitai, et al., (2003) Mol Microbiol 47:61 -73) and hedgehog (Hog) auto-processing domains (the latter is combined with inteins when referred to as the Hog/intein superfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem. 279 32001- 7) and domains such as these may also be used to prepare artificially-split inteins.
  • non-splicing members of such families may be modified by molecular biology methodologies to introduce or restore splicing activity in such related species.
  • the PHI-4 polypeptide is a circular permuted variant.
  • the PHI-4 polypeptide is a circular permuted variant of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526.
  • a new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus.
  • the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C- terminus of the chain.
  • the length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. 20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157:105-132; solvent exposed surface area, Lee and Richards, (1971 ) J. Mol. Biol.
  • linkers with a range of number of residues (calculated using 2 to 3.8 A per residue) are then selected. These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly- Gly-Ser cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
  • Sequences of pesticidal polypeptides capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above.
  • Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below.
  • a novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus.
  • Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch and Sander, (1983) Biopolymers 22:2577-2637; the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol. 154:51 1 -533).
  • additional information is known about solvent exposure of residues; one example is a site of post-translational attachment of carbohydrate which is necessarily on the surface of the protein.
  • methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops.
  • Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile and Salvatore, (1993) Eur. J. Biochem. 218:603- 621 ).
  • the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure.
  • the occurrence of sequences within regions that are known to be involved in periodic secondary structure are regions that should be avoided.
  • regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini.
  • PCR polymerase chain reaction
  • fusion proteins include within its amino acid sequence an amino acid sequence comprising a PHI-4 polypeptide including but not limited to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162, and SEQ ID NOs: 1518-1526 and active fragments thereof.
  • fusion proteins comprising a PHI-4 polypeptide and a second pesticidal polypeptide such a Cry protein.
  • Methods for design and construction of fusion proteins are known to those of skill in the art.
  • Polynucleotides encoding a PHI-4 polypeptide may be fused to signal sequences which will direct the localization of the PHI-4 polypeptide to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of the PHI-4 polypeptide of the embodiments from a prokaryotic or eukaryotic cell.
  • E. coli one may wish to direct the expression of the protein to the periplasmic space.
  • Examples of signal sequences or proteins (or fragments thereof) to which the PHI-4 polypeptide may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase.
  • MBP maltose binding protein
  • ompA signal sequence the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit
  • alkaline phosphatase Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs.
  • the PHI-4 polypeptide may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria (see, US Patent Numbers 5,576,195 and 5,846,818).
  • Plant plastid transit peptide/polypeptide fusions are well known in the art (see, US Patent Number 7,193,133).
  • Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also well known in the art.
  • the plastid transit peptide is generally fused N-terminal to the polypeptide to be targeted (e.g., the fusion partner).
  • the fusion protein consists essentially of the peptide transit plastid and the PHI-4 polypeptide to be targeted.
  • the fusion protein comprises the peptide transit plastid and the polypeptide to be targeted.
  • the plastid transit peptide is preferably at the N-terminus of the fusion protein.
  • additional amino acid residues may be N-terminal to the plastid transit peptide providing that the fusion protein is at least partially targeted to a plastid.
  • the plastid transit peptide is in the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein.
  • the plastid transit peptide is generally cleaved from the fusion protein upon insertion into the plastid.
  • the position of cleavage may vary slightly between plant species, at different plant developmental stages, as a result of specific intercellular conditions or the particular combination of transit peptide/fusion partner used.
  • the plastid transit peptide cleavage is homogenous such that the cleavage site is identical in a population of fusion proteins.
  • the plastid transit peptide is not homogenous, such that the cleavage site varies by 1-10 amino acids in a population of fusion proteins.
  • the plastid transit peptide can be recombinantly fused to a second protein in one of several ways.
  • a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at a position corresponding to its C-terminal end and the same or a compatible site can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminal end. Care must be taken in designing these sites to ensure that the coding sequences of the transit peptide and the second protein are kept "in frame" to allow the synthesis of the desired fusion protein. In some cases, it may be preferable to remove the initiator methionine codon of the second protein when the new restriction site is introduced.
  • restriction endonuclease recognition sites on both parent molecules and their subsequent joining through recombinant DNA techniques may result in the addition of one or more extra amino acids between the transit peptide and the second protein. This generally does not affect targeting activity as long as the transit peptide cleavage site remains accessible and the function of the second protein is not altered by the addition of these extra amino acids at its N-terminus.
  • one skilled in the art can create a precise cleavage site between the transit peptide and the second protein (with or without its initiator methionine) using gene synthesis (Stemmer, et a/. , (1995) Gene 164:49-53) or similar methods.
  • the transit peptide fusion can intentionally include amino acids downstream of the cleavage site.
  • the amino acids at the N-terminus of the mature protein can affect the ability of the transit peptide to target proteins to plastids and/or the efficiency of cleavage following protein import. This may be dependent on the protein to be targeted. See, e.g., Comai, et al. , (1988) J. Biol. Chem. 263(29):15104-9.
  • fusion proteins comprising a PHI-4 polypeptide, a pesticidal protein such as a cry protein, and an amino acid linker.
  • R 1 is a PHI-4 polypeptide
  • R 2 is a pesticidal protein with a different but complementary activity to the PHI-4 polypeptide, including but not limited to cry proteins; a polypeptide that increases the solubility and/or stability of the PHI-4 polypeptide; or a transit peptide or leader sequence.
  • the R 1 polypeptide is fused either directly or through a linker segment to the R 2 polypeptide.
  • the term "directly" defines fusions in which the polypeptides are joined without a peptide linker.
  • L represents a chemical bound or polypeptide segment to which both R 1 and R 2 are fused in frame
  • L is a linear peptide to which R 1 and R 2 are bound by amide bonds linking the carboxy terminus of R 1 to the amino terminus of L and carboxy terminus of L to the amino terminus of R 2 .
  • fused in frame is meant that there is no translation termination or disruption between the reading frames of R 1 and R 2 .
  • the linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length.
  • the linkers joining the two molecules are preferably designed to (1 ) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, (3) have minimal hydrophobic or charged characteristic which could interact with the functional protein domains and (4) provide steric separation of R 1 and R 2 such that R 1 and R 2 could interact simultaneously with their corresponding receptors on a single cell.
  • surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence.
  • Other neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions.
  • the linkers comprise sequences selected from the group of formulas: (Gly 3 Ser) n , (Gly 4 Ser) n , (Gly 5 Ser) n , (Gly n Ser) n or (AlaGlySer) n where n is an integer.
  • a highly-flexible linker is the (GlySer)-rich spacer region present within the pill protein of the filamentous bacteriophages, e.g., bacteriophages M13 or fd (Schaller, et al., 1975). This region provides a long, flexible spacer region between two domains of the pill surface protein.
  • linkers in which an endopeptidase recognition sequence is included.
  • Such a cleavage site may be valuable to separate the individual components of the fusion to determine if they are properly folded and active in vitro.
  • various endopeptidases include, but are not limited to, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue Plasminogen activator, clostripain, Chymosin, Collagenase, Russell's Viper Venom Protease, Postproline cleavage enzyme, V8 protease, Thrombin and factor Xa.
  • the linker comprises the amino acids EEKKN from the multi-gene expression vehicle (MGEV), which is cleaved by vacuolar proteases as disclosed in US 2007/0277263.
  • MGEV multi-gene expression vehicle
  • peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides.
  • those hinge regions where the cysteines are replaced with serines are particularly useful.
  • Preferred linkers of the present disclosure include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines.
  • the fusion proteins are not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere adversely with the folding and function of the individual molecules of the fusion.
  • chimeric PHI-4 polypeptide are provided that are created through joining two or more portions of genes, which originally encoded separate insecticidal proteins from different species, to create a chimeric gene.
  • the translation of the chimeric gene results in a single chimeric pesticidal polypeptide with regions, motifs or domains derived from each of the original polypeptides.
  • the chimeric protein comprises portions, motifs or domains of PHI-4 polypeptides in any combination.
  • the chimeric insecticidal polypeptide includes but not limited to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526.
  • DNA sequences may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by the wild-type (or native) pesticidal protein.
  • proteins may be altered in various ways including amino acid substitutions, amino acid deletions, amino acid truncations, and insertions of one or more amino acids, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155 or more amino acid substitutions, amino acid deletions and/or insertions or combinations thereof compared to any one of SEQ ID NO: 35, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526.
  • a PHI-4 polypeptide comprises the deletion of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or more amino acids from the C-terminus of the PHI-4 polypeptide relative to the amino acid position of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526.
  • Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a PHI-4 polypeptide can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution.
  • the changes encoded in the amino acid sequence will not substantially affect the function of the protein.
  • Such variants will possess the desired pesticidal activity.
  • the ability of a PHI-4 polypeptide to confer pesticidal activity may be improved by the use of such techniques upon the compositions of this disclosure.
  • conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues.
  • a “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a PHI-4 polypeptide without altering the biological activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • polar, negatively charged residues and their amides e.g., aspartic acid, asparagine, glutamic, acid, glutamine
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • small aliphatic, nonpolar or slightly polar residues e.g., Alanine, serine, threonine, proline, glycine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • large aliphatic, nonpolar residues e.g., methionine, leucine, isoleucine, va
  • amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologous proteins).
  • residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins).
  • residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
  • residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
  • amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference.
  • the hydropathic index of amino acids may be considered.
  • hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1 ): 105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and the like. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, ibid). These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1 .9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1 .3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).
  • the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred and those within +0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1 ); glutamate (+3.0.+0.1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+0.1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1 .3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity.
  • This can include amino acid insertions, amino acid deletions or amino acid alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification.
  • the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions.
  • Such fusion proteins are often used to (1 ) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity or epitope to facilitate either protein purification, protein detection or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, mitochondria or chloroplasts of plants or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.
  • the substitution is an alanine for the native amino acid at the recited position(s).
  • nucleic acid sequence(s) encoding the variant protein or polypeptide are also encompassed.
  • Variant nucleotide and amino acid sequences of the disclosure also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different PHI-4 polypeptide coding regions can be used to create a new PHI-4 polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91 :10747-10751 ; Stemmer, (1994) Nature 370:389-391 ; Crameri, et al. , (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.
  • Domain swapping or shuffling is another mechanism for generating altered PHI-4 polypeptides. Domains may be swapped between PHI-4 polypeptides, resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov, et al., (2001 ) Appl. Environ. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al. , (1991 ) J. Biol. Chem.
  • Antibodies to a PHI-4 polypeptide of the embodiments or to variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; US Patent Number 4,196,265).
  • kits for detecting the presence of a PHI-4 polypeptide or detecting the presence of a nucleotide sequence encoding a PHI-4 polypeptide, in a sample are provided.
  • the kit provides antibody-based reagents for detecting the presence of a PHI-4 polypeptide in a tissue sample.
  • the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding PHI-4 polypeptide(s).
  • the kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit.”
  • Receptors to the PHI-4 polypeptide of the embodiments or to variants or fragments thereof are also encompassed.
  • Methods for identifying receptors are well known in the art (see, Hofmann, et. al., (1988) Eur. J. Biochem. 173:85-91 ; Gill, et al., (1995) J. Biol. Chem. 27277-27282) can be employed to identify and isolate the receptor that recognizes the PHI-4 polypeptides using the brush-border membrane vesicles from susceptible insects.
  • PHI-4 polypeptide can be labeled with fluorescent dye and other common labels such as streptavidin.
  • BBMV Brush-border membrane vesicles
  • susceptible insects such as soybean looper and stink bugs
  • BBMV Brush-border membrane vesicles
  • Labeled PHI-4 polypeptides can be incubated with blotted membrane of BBMV and labeled the PHI-4 polypeptides can be identified with the labeled reporters.
  • Identification of protein band(s) that interact with the PHI-4 polypeptides can be detected by N-terminal amino acid gas phase sequencing or mass spectrometry based protein identification method (Patterson, (1998) 10(22): 1 -24, Current Protocol in Molecular Biology published by John Wiley & Son Inc).
  • the corresponding gene can be cloned from genomic DNA or cDNA library of the susceptible insects and binding affinity can be measured directly with the PHI-4 polypeptides. Receptor function for insecticidal activity by the PHI-4 polypeptides can be verified by accomplished by RNAi type of gene knock out method (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849-46851 ).
  • nucleotide constructs are not intended to limit the embodiments to nucleotide constructs comprising DNA.
  • nucleotide constructs particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein.
  • the nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences.
  • nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides and combinations thereof.
  • deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
  • a further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae.
  • the transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.
  • the sequences of the embodiments are provided in DNA constructs for expression in the organism of interest.
  • the construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments.
  • operably linked refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and where necessary to join two protein coding regions in the same reading frame.
  • the construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.
  • Such a DNA construct is provided with a plurality of restriction sites for insertion of the PHI-4 polypeptide gene sequence to be under the transcriptional regulation of the regulatory regions.
  • the DNA construct may additionally contain selectable marker genes.
  • the DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host.
  • the transcriptional initiation region i.e., the promoter
  • the transcriptional initiation region may be native, analogous, foreign or heterologous to the host organism and/or to the sequence of the embodiments.
  • the promoter may be the natural sequence or alternatively a synthetic sequence.
  • the term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.
  • the DNA construct may also include a transcriptional enhancer sequence.
  • an "enhancer” refers to a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
  • Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al. , (1989) Molecular Biology of RNA ed.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al. , (1991 ) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991 ) Cell 64:671-674; Sanfacon, et al., (1991 ) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261 -1272; Munroe, et al., (1990) Gene 91 : 151 -158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891 -7903 and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.
  • a nucleic acid may be optimized for increased expression in the host organism.
  • the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-1 1 for a discussion of host- preferred codon usage.
  • nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res.
  • the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize.
  • Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant- preferred genes. See, for example, US Patent Numbers 5,380,831 , and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • a nucleic acid may be optimized for increased expression in the host organism.
  • the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-1 1 for a discussion of host- preferred codon usage.
  • nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res.
  • the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize.
  • Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant- preferred genes. See, for example, US Patent Numbers 5,380,831 , and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep 37:677-684, 2010, herein incorporated by reference.
  • Table 2 shows a maize optimal codon analysis (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).
  • Val GUU 182 0.07 2, 595 1.51 GGC* 7, 842 2.91 1,282 0.75
  • Codon usage was compared using Chi squared contingency test to identify optimal codons. Codons that occur significantly more often (P ⁇ 0.01 ) are indicated with an asterisk.
  • the recombinant nucleic acid molecule encoding a PHI-4 polypeptide has maize optimized codons.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well- characterized sequences that may be deleterious to gene expression.
  • the GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.
  • host cell refers to a cell which contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E.
  • coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells.
  • An example of a monocotyledonous host cell is a maize host cell.
  • the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci.
  • TEV leader tobacco Etch Virus
  • MDMV leader Maize Dwarf Mosaic Virus
  • human immunoglobulin heavy-chain binding protein BiP
  • untranslated leader from the coat protein mRNA of alfalfa mosaic virus AMV RNA 4
  • tobacco mosaic virus leader TMV (Gallie, et al. , (1989) in Molecular Biology of RNA, ed.
  • Such constructs may also contain a "signal sequence" or "leader sequence” to facilitate co-translational or post- translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus.
  • signal sequence is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are protolytically activated in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments.
  • leader sequence is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle.
  • this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria and the like.
  • Nuclear-encoded proteins targeted to the chloroplast thylakoid lumen compartment have a characteristic bipartite transit peptide, composed of a stromal targeting signal peptide and a lumen targeting signal peptide.
  • the stromal targeting information is in the amino-proximal portion of the transit peptide.
  • the lumen targeting signal peptide is in the carboxyl-proximal portion of the transit peptide, and contains all the information for targeting to the lumen.
  • Recent research in proteomics of the higher plant chloroplast has achieved in the identification of numerous nuclear- encoded lumen proteins (Kieselbach et al. FEBS LETT 480:271 -276, 2000; Peltier et al. Plant Cell 12:319-341 , 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001 ), the lumen targeting signal peptide of which can potentially be used in accordance with the present disclosure.
  • CTP chloroplast transit peptides
  • chimeric CTPs comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza sativa 1-deoxy-D xyulose-5- Phosphate Synthase, Oryza saf/Va-Superoxide dismutase, Oryza sai/Va-soluble starch synthase, Oryza sai/Va-NADP-dependent Malic acid enzyme, Oryza sai/Va-Phospho-2- dehydro-3-deoxyheptonate Aldolase 2, Oryza sai/Va-L-Ascorbate peroxidase 5, Oryza sai/Va-Phosphoglucan water dikinase, Zea Mays sRUBISCO, Zea Mays-beta- glucosi
  • the PHI-4 polypeptide gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle.
  • the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, US Patent Number 5,380,831 , herein incorporated by reference.
  • the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the embodiments.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism.
  • Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999/43838 and US Patent Number 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al.
  • constitutive promoters include, for example, those discussed in US Patent Numbers 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,61 1.
  • wound-inducible promoters are wound-inducible promoters.
  • Such wound-inducible promoters may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. 28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wunl and wun2, US Patent Number 5,428,148; winl and win2 (Stanford, et al., (1989) Mol. Gen. Genet.
  • pin II potato proteinase inhibitor
  • pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments.
  • pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1 ,3-glucanase, chitinase, etc.
  • PR proteins pathogenesis-related proteins
  • SAR proteins SAR proteins
  • beta-1 ,3-glucanase chitinase, etc.
  • PR proteins pathogenesis-related proteins
  • SAR proteins SAR proteins
  • beta-1 ,3-glucanase chitinase
  • chitinase etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4:645-656 and Van Loon, (1985)
  • promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331 ; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen, et al., (1996) Plant J.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid.
  • promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991 ) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991 ) Mol. Gen. Genet. 227:229-237 and US Patent Numbers 5,814,618 and 5,789,156), herein incorporated by reference.
  • steroid-responsive promoters see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991 ) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al.,
  • Tissue-preferred promoters can be utilized to target enhanced PHI-4 polypeptide expression within a particular plant tissue.
  • Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7)792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al. , (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 1 12(3):1331-1341 ; Van Camp, et al., (1996) Plant Physiol.
  • Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al. , (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1 129-1 138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
  • Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al. , (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991 ) Plant Cell 3(10): 1051 -1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol.
  • Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350).
  • the TR1 ' gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics.
  • Additional root-preferred promoters include the VfENOD- GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.
  • seed-specific promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108, herein incorporated by reference.
  • seed-preferred promoters include, but are not limited to, Cim1 (cytokinin- induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1 -phosphate synthase) (see, US Patent Number 6,225,529, herein incorporated by reference).
  • Gamma-zein and Glb-1 are endosperm-specific promoters.
  • seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku, K.D. and Goldberg, R.B. Plant Cell 1 :1079-1093, 1989), bean ⁇ -phaseolin, napin, ⁇ - conglycinin, glycinin 1 , soybean lectin, cruciferin, and the like.
  • seed- specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1 , shrunken 2, globulin 1 , etc. See also, WO 2000/12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.
  • seed specific promoters include but are not limited to seed coat promoter from Arabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr (US Patent Numbers 7,294,760 and 7,847,153).
  • a promoter that has "preferred" expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue.
  • weak promoters will be used.
  • the term "weak promoter” as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts is intended. Alternatively, it is recognized that the term “weak promoters” also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.
  • Such weak constitutive promoters include, for example the core promoter of the
  • Rsyn7 promoter (WO 1999/43838 and US Patent Number 6,072,050), the core 35S CaMV promoter, and the like.
  • Other constitutive promoters include, for example, those disclosed in US Patent Numbers 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,61 1 , herein incorporated by reference.
  • the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D).
  • selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213 and Meijer, et al., (1991 ) Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91 ); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.
  • selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the embodiments.
  • the methods of the embodiments involve introducing a polypeptide or polynucleotide into a plant.
  • "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant.
  • the methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant.
  • Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods and virus-mediated methods.
  • “Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof.
  • Transient transformation is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
  • plant is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),
  • the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods.
  • transient transformation methods include, but are not limited to, the introduction of the PHI-4 polypeptide or variants and fragments thereof directly into the plant or the introduction of the PHI-4 polypeptide transcript into the plant.
  • Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al. , (1986) Mol Gen. Genet. 202:179-185; Nomura, et al. , (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci.
  • the PHI-4 polypeptide polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).
  • the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system.
  • a site-specific recombination system See, for example, WO 1999/25821 , WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are herein incorporated by reference.
  • the polynucleotide of the embodiments can be contained in transfer cassette flanked by two non-identical recombination sites.
  • the transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
  • Plant transformation vectors may be comprised of one or more DNA vectors needed for achieving plant transformation.
  • DNA vectors needed for achieving plant transformation.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium- mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "gene of interest" (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders.
  • a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5:446-451 ).
  • Several types of Agrobacterium strains e.g. LBA4404, GV3101 , EHA101 , EHA105, etc.
  • the second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
  • plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
  • target plant cells e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.
  • a maximum threshold level of appropriate selection depending on the selectable marker gene
  • Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely.
  • fertile seeds e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750.
  • the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81- 84. These plants may then be grown, and either pollinated with the same transformed strain or different strains and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.
  • the nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired PHI-4 polypeptide. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a PHI-4 polypeptide of the embodiments, may have the desired pesticidal activity.
  • Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments.
  • Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules are known in the art. See, for example, US Patent Numbers 5,889,191 ; 5,889,190; 5,866,785; 5,589,367 and 5,316,931 , herein incorporated by reference.
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid- directed RNA polymerase.
  • tissue-preferred expression of a nuclear-encoded and plastid- directed RNA polymerase Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :7301 -7305.
  • the embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots.
  • the embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa ⁇ Medicago sativa), rice ⁇ Oryza sativa), rye ⁇ Secale cereale), sorghum ⁇ Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet ⁇ Pennisetum glaucum), proso millet ⁇ Panicum miliaceum), foxtail millet ⁇ Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton ⁇ Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cass
  • Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Locopersicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseolus vulgaris
  • lima beans Phaseolus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Conifers that may be employed in practicing the embodiments include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta) and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean
  • Turf grasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewing's fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (
  • Augustine grass (Stenotaphrum secundatum); zoysia grass ⁇ Zoysia spp.); Bahia grass ⁇ Paspalum notatum); carpet grass ⁇ Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue gramma ⁇ Bouteloua gracilis); buffalo grass ⁇ Buchloe dactyloids); sideoats gramma ⁇ Bouteloua curtipendula).
  • Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants.
  • Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.
  • Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc.
  • Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • heterologous foreign DNA Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
  • PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell, (2001 ) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
  • Plant transformation may be confirmed by Southern blot analysis of genomic DNA
  • RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, (2001 ) supra). Expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, (2001 ) supra).
  • Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001 , supra) using antibodies that bind to one or more epitopes present on the PHI-4 polypeptide.
  • Transgenic plants may comprise a stack of one or more insecticidal polynucleotides disclosed herein with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences.
  • Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene, and co- transformation of genes into a single plant cell.
  • stacked includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid).
  • stacked traits comprise a molecular stack where the sequences are physically adjacent to each other.
  • a trait refers to the phenotype derived from a particular sequence or groups of sequences. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors.
  • the polynucleotide sequences of interest can be combined at any time and in any order.
  • the traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes.
  • the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis).
  • Expression of the sequences can be driven by the same promoter or by different promoters.
  • polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 1999/25821 , WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are herein incorporated by reference.
  • the polynucleotides encoding the PHI-4 polypeptides disclosed herein, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance or stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like).
  • additional input traits e.g., herbicide resistance, fungal resistance, virus resistance or stress tolerance, disease resistance, male sterility, stalk strength, and the like
  • output traits e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like.
  • Transgenes useful for stacking include but are not limited to:
  • a Plant disease resistance genes Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen.
  • R disease resistance gene
  • Avr avirulence
  • a plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example, Jones, et al., (1994) Science 266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene for resistance to Pseudomonas syringae pv.
  • a plant resistant to a disease is one that is more resistant to a pathogen as compared to the wild type plant.
  • DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Numbers 40098, 67136, 31995 and 31998.
  • Genes encoding pesticidal proteins may also be stacked including but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (201 1 ) PLoS Pathogens, 7:1-13), from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et al, (2010) J. Agric. Food Chem.
  • Pseudomonas sp. such as PSEEN3174 (Monalysin, (201 1 ) PLoS Pathogens, 7:1-13), from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbio
  • B. thuringiensis insecticidal proteins include, but are not limited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8 (Accession # 126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa1 1 (Accession # CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession # AAM44
  • Examples of ⁇ -endotoxins also include but are not limited to CrylA proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-1 1 toxin (N-terminal deletion of a-helix 1 and/or a-helix 2 variants of Cry proteins such as CrylA) of US Patent Numbers 8,304,604 and 8.304,605, Cryl B of US Patent Application Serial Number 10/525,318; Cryl C of US Patent Number 6,033,874; Cryl F of US Patent Numbers 5,188,960, 6,218,188; Cry1A/F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent
  • Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature” (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the "www" prefix).
  • the insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16).
  • Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to CrylAc, Cry1Ac+Cry2Ab, CrylAb, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C.
  • Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491 ,869
  • Pesticidal proteins also include VI P (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279,
  • Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491 ,698 and 8,084,418).
  • TC toxin complex
  • TC proteins have "stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism.
  • the toxicity of a "stand-alone" TC protein can be enhanced by one or more TC protein "potentiators" derived from a source organism of a different genus.
  • TC protein A There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins.
  • Class A proteins are TcbA, TcdA, XptA1 and XptA2.
  • Class B proteins are TcaC, TcdB, XptBIXb and XptCIWi.
  • Class C proteins are TccC, XptCIXb and XptBIWi.
  • Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but are not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366).
  • (C) A polynucleotide encoding an insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon or an antagonist or agonist thereof. See, for example, the disclosure by Hammock, et al., (1990) Nature 344:458, of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
  • (E) A polynucleotide encoding an enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
  • a polynucleotide encoding an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic.
  • a glycolytic enzyme for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase,
  • G A polynucleotide encoding a molecule that stimulates signal transduction.
  • Botella et al., (1994) Plant Molec. Biol. 24:757, of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol. 104:1467, who provide the nucleotide sequence of a maize calmodulin cDNA clone.
  • (J) A gene encoding a viral-invasive protein or a complex toxin derived therefrom.
  • the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See, Beachy, et al. , (1990) Ann. Rev. Phytopathol. 28:451.
  • Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
  • (M) A polynucleotide encoding a developmental-arrestive protein produced in nature by a pathogen or a parasite.
  • fungal endo alpha-1 ,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo- alpha-1 ,4-D-galacturonase.
  • the cloning and characterization of a gene which encodes a bean endopolygalacturonase- inhibiting protein is described by Toubart, et al., (1992) Plant J. 2:367.
  • N A polynucleotide encoding a developmental-arrestive protein produced in nature by a plant. For example, Logemann, et al., (1992) Bio/Technology 10:305, have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.
  • (Q) Detoxification genes such as for fumonisin, beauvericin, moniliformin and zearalenone and their structurally related derivatives. For example, see, US Patent Numbers 5,716,820; 5,792,931 ; 5,798,255; 5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.
  • Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes See, for example, Shoemaker, et al., Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif. (1995).
  • Patent Number 5,689,035 and incorporated by reference for this purpose.
  • a herbicide that inhibits the growing point or meristem
  • Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively.
  • a polynucleotide encoding a protein for resistance to Glyphosate resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes, respectively
  • Glyphosate resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes, respectively
  • other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes).
  • PAT phosphinothricin acetyl transferase
  • bar Streptomyces hygroscopicus phosphinothricin acety
  • Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido- reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose.
  • glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase.
  • a DNA molecule encoding a mutant aroA gene can be obtained under ATCC Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Patent Number 4,769,061 to Comai.
  • EP Application Number 0 333 033 to Kumada, et al., and US Patent Number 4,975,374 to Goodman, et al. disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L- phosphinothricin.
  • nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans, et al.,; De Greef, et al., (1989) Bio/Technology 7:61 , describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity.
  • C A polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene).
  • psbA and gs+genes triazine
  • nitrilase gene a benzonitrile gene.
  • Przibilla, et al., (1991 ) Plant Cell 3:169 describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes.
  • Nucleotide sequences for nitrilase genes are disclosed in US Patent Number 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Bioc em. J
  • genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).
  • the protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in US Patent Numbers 6,288,306 B1 ; 6,282,837 B1 and 5,767,373 and International Publication WO 2001/12825.
  • the aad-1 gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-1 ) protein.
  • AAD-1 aryloxyalkanoate dioxygenase
  • the trait confers tolerance to 2,4- dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as "fop" herbicides such as quizalofop) herbicides.
  • the aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005/107437 (see also, US 2009/0093366).
  • the aad- 12 gene derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).
  • phenoxy auxin e.g., 2,4-D, MCPA
  • pyridyloxy auxins e.g., fluroxypyr, triclopyr
  • LMP lipid metabolism protein
  • HSI2 Sugar-lnducible 2
  • Altered carbohydrates affected for example, by altering a gene for an enzyme that affects the branching pattern of starch or, a gene altering thioredoxin such as NTR and/or TRX (see, US Patent Number 6,531 ,648. which is incorporated by reference for this purpose) and/or a gamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, US Patent Number 6,858,778 and US Patent Application Publication Number 2005/0160488, US Patent Application Publication Number 2005/0204418, which are incorporated by reference for this purpose). See, Shiroza, et al., (1988) J. Bacteriol.
  • D Altered antioxidant content or composition, such as alteration of tocopherol or tocotrienols.
  • tocopherol or tocotrienols For example, see, US Patent Number 6,787,683, US Patent Application Publication Number 2004/0034886 and WO 2000/68393 involving the manipulation of antioxidant levels and WO 2003/082899 through alteration of a homogentisate geranyl geranyl transferase (hggt).
  • FRT sites that may be used in the FLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.
  • Lox sites that may be used in the Cre/Loxp system.
  • Other systems that may be used include the Gin recombinase of phage Mu (Maeser, et al., (1991 ) Vicki Chandler, The Maize Handbook ch. 1 18 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and the R/RS system of the pSRi plasmid (Araki, et al., 1992).
  • G Genes that increase expression of vacuolar pyrophosphatase such as AVP1 (US Patent Number 8,058,515) for increased yield; nucleic acid encoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5) polypeptides, an oligopeptide transporter protein (OPT4-like) polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschel related homeobox 1 -like (WOX1 -like) polypeptide (U. Patent Application Publication Number US 201 1/0283420).
  • J Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins for modulating development, modulating response to stress, and modulating stress tolerance (US Patent Application Publication Number US 2010/0287669).
  • K Polynucleotides that encode proteins that confer a drought tolerance phenotype (DTP) for conferring drought resistance (WO 2012/058528).
  • TPP Trehalose Phosphate Phosphatase
  • genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth and/or plant structure, can be introduced or introgressed into plants, see e.g., WO 1997/4981 1 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and US Patent Number 6,573,430 (TFL), US Patent Number 6,713,663 (FT), WO 1996/14414 (CON), WO 1996/38560, WO 2001/21822 (VRN1 ), WO 2000/44918 (VRN2), WO 1999/49064 (Gl), WO 2000/46358 (FR1 ), WO 1997/29123, US Patent Number 6,794,560, US Patent Number 6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO 2004/031349 (transcription factors).
  • ACCDP 1 -AminoCyclopropane-1 - Carboxylate Deaminase-like Polypeptide
  • VIM1 Variariant in Methylation 1
  • NDK nucleoside diphosphatase kinase
  • the stacked trait may be a trait or event that has received regulatory approval including but not limited to the events in Table 2A -1 F.
  • Table 2A Heliant us annuus Sunflower
  • EMS ethyl methanesulfonate
  • IMINTA-1 IMINTA-4 BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using sodium azide. Event Company Description
  • LLRICE06 LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerant rice produced by inserting a modified
  • phosphinothricin acetyltransferase encoding gene from the soil bacterium
  • EMS ethyl methanesulfonate
  • the introduced csr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacid synthase protein that confers tolerance to imidazolinone herbicides due to a point mutation that results in a single amino acid substitution in which the serine residue at position 653 is replaced by asparagine (S653N).
  • glyphosate N-acetlytransferase which detoxifies glyphosate
  • a modified acetolactate synthase (ALS) gene which is tolerant to ALS-inhibiting herbicides.
  • GTS 40-3-2 Monsanto Company Glyphosate tolerant soybean variety produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.
  • EPSPS modified 5-enolpyruvylshikimate-3- phosphate synthase
  • tumefaciens strain CP4 and resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens) via expression of the Cry1 Ac encoding gene from B. thuringiensis.
  • EPSPS modified 5-enolpyruvylshikimate-3- phosphate synthase
  • EEB European corn borer
  • CEW corn earworm
  • FAW fall army worm
  • BCW black cutworm
  • glyphosate and glufosinate-ammonium containing herbicides include European corn borer (ECB, Ostrinia nubilalis), corn earworm (CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), and black cutworm (BCW, Agrotis ipsilon).
  • PAT phosphinothricin N-acetyltransferase
  • Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacillus thuringiensis.
  • Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT1 1 , which contains the crylAb gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.
  • PAT phosphinothricin N-acetyltransferase
  • Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacillus thuringiensis.
  • Tolerance to glyphosate herbicide is derived from GA21 which contains a modified EPSPS gene from maize.
  • CBH-351 Aventis CropScience Insect-resistant and glufosinate ammonium herbicide tolerant maize developed by inserting genes encoding Cry9C protein from Bacillus thuringiensis subsp tolworthi and
  • PAT phosphinothricin acetyltransferase
  • crylF gene from Bacillus thuringiensis var aizawai and the phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus.
  • PAT phosphinothricin acetyltransferase
  • cry34Ab1 and cry35Ab1 genes from Bred International Inc. Bacillus thuringiensis strain PS149B1.
  • DBT418 Dekalb Genetics Insect-resistant and glufosinate ammonium
  • Somaclonal variants with a modified acetyl-CoA- carboxylase were selected by culture of embryos on sethoxydim enriched medium.
  • EMS ethyl methanesulfonate
  • EPSPS (formerly Zeneca modified 5-enolpyruvyl shikimate-3-phosphate Seeds) synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids.
  • MON863 (OECD identifier: MON-00863-5) and MON810 (OECD identifier: MON-00810-6)
  • NK603 (OECD identifier:MON-00603-6).

Abstract

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling, inhibiting growth or killing lepidopteran, coleopteran, dipteran, fungal, hemipteran, and nematode pest populations and for producing compositions with insecticidal activity.

Description

PHI-4 POLYPEPTIDES AND METHODS FOR THEIR USE
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "5414WOPCT_Sequence_Listing" created on March 04, 2014, and having a size of 5,935 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD
This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences encoding them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants.
BACKGROUND
Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards, and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops.
Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control.
Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bt. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for pest control.
Accordingly, there remains a need for new pesticidal proteins with different ranges of insecticidal activity against insect pests, e.g., insecticidal proteins which are active against a variety of insects in the order Coleoptera. In addition, there remains a need for biopesticides having activity against a variety of insect pests that have developed resistance to existing pesticides.
SUMMARY
In one aspect compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds.
In particular, isolated or recombinant nucleic acid molecules are provided encoding PHI-4 polypeptides including amino acid substitutions, amino acid deletions, amino acid insertions, and fragments thereof, and combinations thereof. Additionally, amino acid sequences corresponding to the PHI-4 polypeptides are encompassed. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed.
In another aspect methods are provided for producing the polypeptides and for using those polypeptides for controlling, inhibiting growth or killing a Lepidopteran, Coleopteran, nematode, fungi, Hemipteran and/or Dipteran pests. The transgenic plants of the embodiments express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling coleopteran, lepidopteran, hemipteran or nematode pests. It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.
In another aspect methods for detecting the nucleic acids and polypeptides of the embodiments in a sample are also included. A kit for detecting the presence of a PHI-4 polypeptide or detecting the presence of a nucleotide sequence encoding a PHI-4 polypeptide in a sample is provided. The kit is provided along with all reagents and control samples necessary for carrying out a method for detecting the intended agent, as well as instructions for use.
In another aspect the compositions and methods of the embodiments are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes. The compositions of the embodiments are also useful for generating altered or improved proteins that have pesticidal activity or for detecting the presence of PHI-4 polypeptides or nucleic acids in products or organisms.
The following embodiments are encompassed by the present disclosure.
Embodiment 1 is a PHI-4 polypeptide having improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 2 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 1.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 3 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 2 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 4 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 2.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 5 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 3 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 6 is the PHI-4 polypeptide of embodiment 1 , wherein the insecticidal activity is increased about 5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 7 is the PHI-4 polypeptide of any one of embodiments 1-6, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
Embodiment 8 is the PHI-4 polypeptide of any one of embodiments 1-7, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index. Embodiment 9 is the PHI-4 polypeptide of any one of embodiments 1-7, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
Embodiment 10 is the PHI-4 polypeptide of any one of embodiments 1 -7, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
Embodiment 1 1 is the PHI-4 polypeptide of any one of embodiments 1-10, wherein the PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
Embodiment 12 is the PHI-4 polypeptide of embodiment 1 1 , wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acid at position 99 is Lys or Leu; the amino acid at position 145 is Leu or Val; the amino acid at position 150 is Arg or Gin; the amino acid at position 151 is Asp or Ser; the amino acid at position 153 is Leu or lie; the amino acid at position 163 is Leu or Val; the amino acid at position 171 is Tyr or Phe; the amino acid at position 172 is lie or Leu; the amino acid at position 182 is Asp or Gin; the amino acid at position 196 is Gin or Asn; the amino acid at position 206 is Tyr or Phe; the amino acid at position 210 is Val or lie; the amino acid at position 216 is Glu or Gin; the amino acid at position 220 is Glu, Gin, His or Asp; the amino acid at position 278 is Glu or Asn; the amino acid at position 283 is lie or Val; the amino acid at position 289 is Lys, Gin or Leu; the amino acid at position 293 is Arg, Gin or Glu; the amino acid at position 328 is Lys or Glu; the amino acid at position 333 is Ser, Lys or Val; the amino acid at position 334 is Gly, Lys or Arg; the amino acid at position 336 is Gly or Ala; the amino acid at position 338 is Ser or Val; the amino acid at position 339 is Glu, Asn or Gin; the amino acid at position 342 is Ala or Ser; the amino acid at position 346 is Pro or Ala; the amino acid at position 354 is Met or Leu; the amino acid at position 355 is Val or lie; the amino acid at position 370 is His or Arg; the amino acid at position 389 is Trp or Leu; the amino acid at position 393 is Trp or Leu; the amino acid at position 396 is Ala, Leu, Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly, Lys or Pro; the amino acid at position 402 is Lys, His, Gly or Trp; the amino acid at position 403 is Asp or Tyr; the amino acid at position 410 is lie or Val; the amino acid at position 412 is Pro or Ala; the amino acid at position 416 is Arg or Glu; the amino acid at position 417 is Ala or Ser; the amino acid at position 426 is Thr or Ser; the amino acid at position 442 is Gin or Glu; the amino acid at position 447 is Asp or Lys; the amino acid at position 452 is Gin or Lys; the amino acid at position 454 is Arg or Gin; the amino acid at position 455 is Val or lie; the amino acid at position 457 is Trp or Asn; the amino acid at position 461 is Thr or Ser; the amino acid at position 462 is Gly or Ala; the amino acid at position 500 is Arg or Gin; the amino acid at position 509 is Lys or Gin; the amino acid at position 520 is Lys, Glu or Gin; and the amino acid at position 527 is Gin or Lys.
Embodiment 13 is the PHI-4 polypeptide of embodiment 1 1 or 12, further comprising one or more amino acid substitutions at position 86, 359, 464, 465, 466, 467, 468, 499 or 517.
Embodiment 14 is the PHI-4 polypeptide of embodiment 13, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
Embodiment 15 is the PHI-4 polypeptide of any one of embodiments 1-12, having 1 to 54 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 16 is the PHI-4 polypeptide of any one of embodiments 1-12, having
1 to 27 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 17 is the PHI-4 polypeptide of any one of embodiments 1-12, having 1 to 20 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 18 is the PHI-4 polypeptide of any one of embodiments 1-12, having 1 to 15 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 19 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 54 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 20 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 27 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 21 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 20 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 22 is the PHI-4 polypeptide of any one of embodiments 13 or 14, comprising 2 to 15 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 23 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO: 35. Embodiment 24 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 90% identity to SEQ ID NO: 35.
Embodiment 25 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 95% identity to SEQ ID NO: 35.
Embodiment 26 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at least 97% identity to SEQ ID NO: 35.
Embodiment 27 is a PHI-4 polypeptide, comprising at least one amino acid substitution at a residue relative to SEQ ID NO: 35 in a structural domain selected from:
a hydrophilic residue;
a residue in a membrane insertion initiation loop;
a residue in a receptor binding loop; and
a residue in a protease sensitive region,
wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
Embodiment 28 is the PHI-4 polypeptide of embodiment 27, wherein the hydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys.
Embodiment 29 is the PHI-4 polypeptide of embodiment 27 or 28, wherein the membrane insertion loops are between about the amino acid at position 92 (Val) and about the amino acid at position 101 (Ala) and between about the amino acid at position 21 1 (Gly) and about the amino acid at position 220 (Glu), relative to SEQ ID NO: 35.
Embodiment 30 is the PHI-4 polypeptide of embodiment 29, wherein the membrane insertion initiation loop residue is selected from position 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219, 220 of SEQ ID NO: 35.
Embodiment 31 is the PHI-4 polypeptide of any one of embodiments 27-30, wherein the receptor binding loops are between about the amino acid at position 332 (Asp) and about the amino acid at position 340 (Asp), between about the amino acid at position 395 (Asp) and about the amino acid at position 403 (Asp), between amino acid at position 458 (Asp) and about the amino acid at position 466 (Asp) relative to SEQ ID NO: 35.
Embodiment 32 is the PHI-4 polypeptide of embodiment 31 , wherein the receptor binding loop residue is selected from positions 332, 333, 334, 335, 336, 337, 338, 339, 340, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 458, 459, 460, 461 , 462, 463, 464, 465, 466 of SEQ ID NO: 35.
Embodiment 33 is the PHI-4 polypeptide of any one of embodiments 27-32, wherein the protease sensitive region residue is selected from between about the amino acid at position 305 (Lys) and about the amino acid at position 316 (Lys) and from about the amino acid at position 500 (Arg) and about the amino acid at position 535 (Lys) relative to SEQ ID NO: 35.
Embodiment 34 is the PHI-4 polypeptide of embodiment 27, wherein the protease is trypsin.
Embodiment 35 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQ ID NO: 35.
Embodiment 36 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 90% sequence identity to SEQ ID NO: 35.
Embodiment 37 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 95% sequence identity to SEQ ID NO: 35.
Embodiment 38 is the PHI-4 polypeptide of any one of embodiments 27-34, wherein the PHI-4 polypeptide has at least 97% sequence identity to SEQ ID NO: 35.
Embodiment 39 is a PHI-4 polypeptide, comprising an amino acid sequence of the formula,
5 10 15
Met Xaa Ser Ala Ala Asn Ala Gly Xaa Leu Gly Asn Leu Xaa Gly
20 25 30
Xaa Thr Ser Xaa Gly Met Xaa Tyr Xaa Val Asn Gly Leu Tyr Ala
35 40 45
Ser Pro Glu Ser Leu Xaa Gly Gin Pro Leu Phe Xaa Xaa Gly Gly
50 55 60
Xaa Leu Asp Ser Xaa Xaa lie Glu Gly Xaa Xaa Xaa Xaa Phe Pro
65 70 75
Xaa Ser Met His Val His Thr Tyr Phe His Ser Asp Xaa Xaa Gin
80 85 90
Xaa Val Ser Xaa Xaa lie Xaa Xaa Xaa Arg Xaa Xaa Xaa Ser Xaa
95 100 105
His Val Gly Xaa Ser Gly Xaa Xaa Xaa Leu Phe Ser Xaa Ser Xaa
110 115 120
Ser Val Asp Xaa Thr Thr Xaa Xaa Gin Gin Leu Xaa Glu lie Thr
125 130 135
Xaa Ser Ser Thr Arg Glu Xaa His Val Leu Trp Tyr lie Ser Leu
140 145 150
Pro Gly Ala Ala Thr Leu Xaa Ser Met Leu Xaa Xaa Xaa Xaa Xaa
155 160 165
Xaa Asp Xaa Xaa Xaa Pro Asn Met Xaa Ala Met Xaa Leu Phe Xaa 170 175 180
Xaa Xaa Gly Pro XXaaaa Xaa Xaa Xaa Xaa Ala Ala Val Gly Gly Arg
185 190 195
Leu Xaa Xaa Xaa XXaaaa Ala Ser Lys Xaa Leu Xaa Met Xaa Ser Ser
200 205 210
Xaa Ser Leu Ser TThhrr Thr Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Xaa
215 220 225
Gly Glu lie Xaa lHiee Xaa His Gly Ser Xaa Met Glu Lys Gin Val
230 235 240 Asn Ser Phe Xaa XXaaaa Xaa Ser Thr lie Arg Xaa Thr Ala Thr Gly
245 250 255
Gly Lys Pro Gly XXaaaa Thr Xaa Arg lie Leu His Gly Pro Asp Ser
260 265 270
Xaa Xaa Ala Phe SSeerr Xaa Trp Ala Xaa Ser Leu Leu Xaa Tyr Ala
275 280 285
Thr Leu Met Asp PPhhee Xaa Thr Xaa Ser Leu Xaa Xaa He Xaa Ala
290 295 300
Leu Xaa Asp Xaa PPrroo Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Ala Xaa
305 310 315 Pro Xaa Xaa Met XXaaaa Xaa Ser Gin Xaa Ser He Pro Xaa Val Asp
320 325 330
Xaa Val Leu Leu MMeett Asp Ala Arg Pro Pro Met Val Xaa Ala Gly
335 340 345
Xaa Asp Xaa Xaa XXaaaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa
335500 355 360
Xaa Ser Thr Ser XXaaaa Xaa Tyr Lys Xaa Xaa Gly Gin Phe Xaa Gin
336655 370 375
Arg Xaa His Xaa SSeerr Val Ala Asp Gly His Xaa Pro He Xaa Xaa
338800 385 390
Asp Leu Phe Asp XXaaaa Gly Xaa Xaa Xaa Xaa Pro Val Gly Xaa Gin
339955 400 405
Xaa Val Trp Asp XXaaaa Xaa Xaa Xaa Gly Lys Xaa Xaa Xaa Tyr Xaa
441100 415 420
Cys Trp Arg Xaa XXaaaa Xaa Xaa Gin Gly Tyr Xaa Xaa Xaa Gly Asp
425 430 435 Val Xaa Met Leu AAllaa Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro
440 445 450 Xaa Xaa Xaa Cys Xaa His Xaa Ser Leu Xaa Ala Xaa Xaa Xaa Thr
455 460 465
Leu Xaa Xaa Xaa Xaa Trp Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa
470 475 480
Xaa Val Ser Leu Trp Xaa Xaa Gly Ala Ala Gly Ala Val Ala Ser
485 490 495
Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser
500 505 510
Gly Xaa lie Xaa Xaa Leu Xaa Gly Ser lie Ala Cys Val Xaa Thr
515 520 525
Ser Ala lie Ala Ser Met Xaa Xaa Met Xaa Ser Met Leu Ser Xaa
530 535
His Xaa Gly Met Glu Ala Met Met Ser Lys Leu (SEQ ID NO: 3 ) ,
wherein
Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position 79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, lie, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa at position 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa at position 87 is Lys or Gin; Xaa at position 88 is Met, lie or Leu; Xaa at position 90 is Gin or Glu; Xaa at position 94 is Val or lie; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie, Met, Phe, Cys, Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 is Leu or lie; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn, Glu, Cys, Ala or Arg; Xaa at position 1 12 is Thr or Ser; Xaa at position 1 13 is Asp, Glu or Met; Xaa at position 1 17 is Thr or Ser; Xaa at position 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa at position 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa at position 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149 is Phe or Val; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaa at position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa at position 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu, Gin, Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg; Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp or Cys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser or Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lys or lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp or Asn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg; Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr or His; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr or Phe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu or Pro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Ser or Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met, Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaa at position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaa at position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 264 is Glu or Gin; Xaa at position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa at position 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292 is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position 297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position 303 is Phe or Tyr; Xaa at position 305 is Lys, Gin, Ala, lie, Met, Asn, Thr or Val; Xaa at position 306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa at position 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn or Gin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin, Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys, lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338 is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala, Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu or Val; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Val or lie; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn or His; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met or Leu; Xaa at position 355 is Val, lie or Leu; Xaa at position 359 is Gly or Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Phe or lie; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu, lie or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385 is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 395 is Asp or Cys; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin, His or Thr; Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Gin or Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie, Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys, Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is lie or Val; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala, Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 is Met or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 is Asp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 is Val or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 is Gin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu, lie, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa at position 452 is Gin, Lys or Glu, Ala; Xaa at position 453 is Asn or Asp; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp, Gin, Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or lie; Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaa at position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaa at position 464 is Arg, Gly, His, Gin, Thr, Phe, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu; Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu or Gin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg, Glu or Gin; Xaa at position 509 is Lys, Gin, Glu or Ala; Xaa at position 517 is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa at position 520 is Lys, Gin, Glu, His or Ala; Xaa at position 525 is Gin or Lys; and Xaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp; wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 3 is an amino acid different from the corresponding amino acid of SEQ ID NO: 35and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
Embodiment 40 is a PHI-4 polypeptide, comprising an amino acid sequence of the formula
5 10 15
Met Xaa Ser Ala Ala Asn Ala Gly Gin Leu Gly Asn Leu Pro Gly
20 25 30
Val Thr Ser Met Gly Met Gly Tyr Xaa Val Asn Gly Leu Tyr Ala
35 40 45
Ser Pro Glu Ser Leu Leu Gly Gin Pro Leu Phe Xaa Xaa Gly Gly
50 55 60
Xaa Leu Asp Ser lie Glu lie Glu Gly Arg Ser Tyr Thr Phe Pro
65 70 75
Arg Ser Met His Val His Thr Tyr Phe His Ser Asp Phe Xaa Gin
80 85 90
Asp Val Ser Xaa Glu lie Xaa Glu Tyr Arg Glu Lys Met Ser Gin
95 100 105
His Val Gly Val Ser Gly Xaa Xaa Xaa Leu Phe Ser Ala Ser Leu
110 115 120
Ser Val Asp Xaa Thr Thr Thr Asp Gin Gin Leu Thr Glu lie Thr
125 130 135
Tyr Ser Ser Thr Arg Glu Ala His Val Leu Trp Tyr lie Ser Leu
140 145 150
Pro Gly Ala Ala Thr Leu Arg Ser Met Leu Arg Xaa Xaa Phe Xaa
155 160 165
Xaa Asp Xaa Asn Asn Pro Asn Met Pro Ala Met Xaa Leu Phe Xaa
170 175 180
Xaa Tyr Gly Pro Tyr Xaa lie Ser Xaa Ala Ala Val Gly Gly Arg
185 190 195
Leu Xaa Tyr Ser Ala Ala Ser Lys Thr Leu Lys Met Asp Ser Ser
200 205 210
Xaa Ser Leu Ser Thr Thr Ala Xaa Met Ser Xaa Lys Ala Leu Val
215 220 225
Gly Glu lie Lys lie Xaa His Gly Ser Xaa Met Glu Lys Gin Val
230 235 240
Asn Ser Phe Arg Ser Asn Ser Thr lie Arg Leu Thr Ala Thr Gly
245 250 255 Gly Lys Pro Gly Met Thr Xaa Arg He Leu His Gly Pro Asp Ser 260 265 270 Xaa Xaa Ala Phe Ser Xaa Trp Ala Glu Ser Leu Leu Asp Tyr Ala
275 280 285 Thr Leu Met Asp Phe Ser Thr Xaa Ser Leu Xaa Pro He Trp Ala
290 295 300 Leu Ala Asp Xaa Pro Glu Arg Xaa Val Glu Leu Glu Asp Ala Phe
305 310 315
Pro Glu Phe Met Lys Gin Ser Gin Gin Ser He Pro Xaa Val Asp
320 325 330
Lys Val Leu Leu MMeett Asp Ala Arg Pro Met Val Xaa Ala Gly
335 340 345 Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Leu Ala Xaa Phe Asn
350 355 360 Xaa Ser Thr Ser Asn Gly Tyr Lys Met Xaa Gly Gin Phe Xaa Gin
365 370 375
Arg Asn His Ala SSeerr Val Ala Asp Gly His Ala Pro He Phe Lys
380 385 390 Asp Leu Phe Asp LLeeuu Gly Val Leu Lys Ala Pro Val Gly Trp Gin
395 400 405 Xaa Val Trp Asp AAsspp Xaa Gly Ser Gly Lys Xaa Xaa Xaa Tyr Ala
410 415 420 Cys Trp Arg Ala lHiee Xaa Xaa Gin Gly Tyr Xaa Xaa Xaa Gly Asp
442255 430 435 Val Met Met Leu AAllaa Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro
444400 445 450
Asp Tyr Val Cys XXaaaa His Gin Ser Leu Cys Ala Xaa Val Gin Thr
455 460 465
Leu Xaa Asn Xaa Xaa Trp Trp Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa
470 475 480
Asp Val Ser Leu TTrrpp Xaa Xaa Gly Ala Ala Gly Ala Val Ala Ser
448855 490 495
Ser Cys Phe Ala GGllyy Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser
550000 505 510
Gly Asp He Glu XXaaaa Leu Arg Gly Ser He Ala Cys Val Xaa Thr
515 520 525
Ser Ala lie Ala Ser Met Gin Glu Met Xaa Ser Met Leu Ser Gin 530 535
His Xaa Gly Met Glu Ala Met Met Ser Lys Leu (SEQ ID NO: 4), wherein
Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 147 is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or lie; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu or Gin; Xaa at position 166 is Arg or Gin; Xaa at position 171 is Tyr or Phe; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 196 is Gin, Lys, Asn or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is Tyr or Phe; Xaa at position 216 is Glu or Gin; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin or Lys; Xaa at position 257 is Gin or lie; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 278 is Glu or Asn; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 313 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa at position 334 is Gly, Arg, Lys or lie; Xaa at position 336 is Gly or Ala; Xaa at position 338 is Ser, His, Val, Lys or Ala; Xaa at position 339 is Glu, Asn, lie or Pro; Xaa at position 343 is Val or lie; Xaa at position 346 is Pro or Ala; Xaa at position 355 is Val or lie; Xaa at position 359 is Gly or Ala; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly or Thr; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaa at position 403 is Asp or Tyr; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg or Glu; Xaa at position 417 is Ala or Ser; Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu or lie; Xaa at position 452 is Gin, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp or Gin; Xaa at position 455 is Val or lie; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie or Tyr; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala or Ser; Xaa at position 464 is Arg, Gly, His, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 500 is Arg or Gin; Xaa at position 509 is Lys, Gin or Ala; Xaa at position 520 is Lys, Gin, Glu, His or Ala; and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu; wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 4 is an amino acid different from the corresponding amino acid of SEQ ID NO: 35 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
Embodiment 41 is the PHI-4 polypeptide of embodiment 39 or 40, further comprising one or more amino acid substitutions at position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 3 or SEQ ID NO: 4.
Embodiment 42 is the PHI-4 polypeptide of embodiment 41 , wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
Embodiment 43 is the PHI-4 polypeptide of embodiment 39-42, further comprising one or more conservative amino acid substitution, insertion of one or more amino acids, deletion of one or more amino acids, and combinations thereof.
Embodiment 44 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 1.5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 45 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 2 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 46 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 2.5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 47 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 3 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 48 is the PHI-4 polypeptide of any one of embodiments 39 - 43, wherein the insecticidal activity is increased about 5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 49 is the PHI-4 polypeptide of any one of embodiments 39-48, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae. Embodiment 50 is the PHI-4 polypeptide of any one of embodiments 39-49, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
Embodiment 51 is the PHI-4 polypeptide of any one of embodiments 39-49, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
Embodiment 52 is the PHI-4 polypeptide of any one of embodiments 39-49, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
Embodiment 53 is the PHI-4 polypeptide of any one of embodiments 39-49, having
1 to 54 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
Embodiment 54 is the PHI-4 polypeptide of any one of embodiments 39-49, having 1 to 27 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
Embodiment 55 is the PHI-4 polypeptide of any one of embodiments 39-49, having 1 to 20 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
Embodiment 56 is the PHI-4 polypeptide of any one of embodiments 39-49, having 1 to 15 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or 4.
Embodiment 57 is the PHI-4 polypeptide of any one of embodiments 1-56, wherein 1 -25 amino acids are deleted from the N-terminus of the PHI-4 polypeptide and/or C- terminus of the PHI-4 polypeptide.
Embodiment 58 is the PHI-4 polypeptide of any one of embodiments 1-53, wherein 1 -20 amino acids are deleted from the C-terminus of the PHI-4 polypeptide.
Embodiment 59 is a polynucleotide encoding a PHI-4 polypeptide, wherein the PHI-4 polypeptide has improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 60 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 1 .5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 61 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 62 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2.5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35). Embodiment 63 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 3 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 64 is the polynucleotide of embodiment 59, wherein the insecticidal activity of the PHI-4 polypeptide is increased about 5 fold or greater compared to AXMI- 205 (SEQ ID NO: 35).
Embodiment 65 is the polynucleotide of any one of embodiments 59-64, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
Embodiment 66 is the polynucleotide of any one of embodiments 59-64, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
Embodiment 67 is the polynucleotide of any one of embodiments 59-64, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
Embodiment 68 is the polynucleotide of any one of embodiments 59-64, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
Embodiment 69 is the polynucleotide of any one of embodiments 59-68, wherein the PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
Embodiment 70 is the polynucleotide of any one of embodiments 69, wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acid at position 99 is Lys or Leu; the amino acid at position 145 is Leu or Val; the amino acid at position 150 is Arg or Gin; the amino acid at position 151 is Asp or Ser; the amino acid at position 153 is Leu or lie; the amino acid at position 163 is Leu or Val; the amino acid at position 171 is Tyr or Phe; the amino acid at position 172 is lie or Leu; the amino acid at position 182 is Asp or Gin; the amino acid at position 196 is Gin or Asn; the amino acid at position 206 is Tyr or Phe; the amino acid at position 210 is Val or lie; the amino acid at position 216 is Glu or Gin; the amino acid at position 220 is Glu, Gin, His or Asp; the amino acid at position 278 is Glu or Asn; the amino acid at position 283 is lie or Val; the amino acid at position 289 is Lys, Gin or Leu; the amino acid at position 293 is Arg, Gin or Glu; the amino acid at position 328 is Lys or Glu; the amino acid at position 333 is Ser, Lys or Val; the amino acid at position 334 is Gly, Lys or Arg; the amino acid at position 336 is Gly or Ala; the amino acid at position 338 is Ser or Val; the amino acid at position 339 is Glu, Asn or Gin; the amino acid at position 342 is Ala or Ser; the amino acid at position 346 is Pro or Ala; the amino acid at position 354 is Met or Leu; the amino acid at position 355 is Val or lie; the amino acid at position 370 is His or Arg; the amino acid at position 389 is Trp or Leu; the amino acid at position 393 is Trp or Leu; the amino acid at position 396 is Ala, Leu, Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly, Lys or Pro; the amino acid at position 402 is Lys, His, Gly or Trp; the amino acid at position 403 is Asp or Tyr; the amino acid at position 410 is lie or Val; the amino acid at position 412 is Pro or Ala; the amino acid at position 416 is Arg or Glu; the amino acid at position 417 is Ala or Ser; the amino acid at position 426 is Thr or Ser; the amino acid at position 442 is Gin or Glu; the amino acid at position 447 is Asp or Lys; the amino acid at position 452 is Gin or Lys; the amino acid at position 454 is Arg or Gin; the amino acid at position 455 is Val or lie; the amino acid at position 457 is Trp or Asn; the amino acid at position 461 is Thr or Ser; the amino acid at position 462 is Gly or Ala; the amino acid at position 500 is Arg or Gin; the amino acid at position 509 is Lys or Gin; the amino acid at position 520 is Lys, Glu or Gin; and the amino acid at position 527 is Gin or Lys.
Embodiment 71 is the polynucleotide of embodiment 69 or 70, wherein the PHI-4 polypeptide further comprises one or more amino acid substitutions compared to the native amino acid at position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 35.
Embodiment 72 is the polynucleotide of embodiment 71 , wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
Embodiment 73 is the polynucleotide of any one of embodiments 59-69 and 72, wherein the PHI-4 polypeptide has 1 to 54 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 74 is the polynucleotide of any one of embodiments 59-70, wherein the PHI-4 polypeptide has 1 to 27 amino acid substitutions compared to SEQ ID NO: 2. Embodiment 75 is the polynucleotide of any one of embodiments 59-70, wherein the PHI-4 polypeptide has 1 to 20 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 76 is the polynucleotide of any one of embodiments 59-70, wherein the PHI-4 polypeptide has 1 to 15 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 77 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 54 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 78 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 27 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 79 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 20 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 80 is the polynucleotide of embodiments 71 or 72, wherein the PHI-4 polypeptide has 2 to 15 amino acid substitutions compared to SEQ ID NO: 35.
Embodiment 81 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO: 35.
Embodiment 82 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 90% identity to SEQ ID NO: 35.
Embodiment 83 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 95% identity to SEQ ID NO: 35.
Embodiment 84 is the polynucleotide of any one of embodiments 59-80, wherein the PHI-4 polypeptide has at least 97% identity to SEQ ID NO: 35.
Embodiment 85 is a polynucleotide encoding a PHI-4 polypeptide, wherein the PHI-4 polypeptide has at least one amino acid substitution at a residue relative to SEQ ID NO: 35 in a structural domain selected from:
a hydrophilic residue;
a residue in a membrane insertion initiation loop;
a residue in a receptor binding loop; and
a residue in a protease sensitive region,
wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
Embodiment 86 is the polynucleotide of embodiment 85, wherein the hydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys.
Embodiment 87 is the polynucleotide of embodiment 85 or 86, wherein the membrane insertion loops are between about the amino acid at position 92 (Val) and about the amino acid at position 101 (Ala) and between about the amino acid 21 1 (Gly) and about the amino acid 220 (Glu), relative to SEQ ID NO: 35. Embodiment 88 is the polynucleotide of embodiment 87, wherein the PHI-4 polypeptide has one or more amino substitution compared to the native amino acid at position 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219, and 220 of SEQ ID NO: 35.
Embodiment 89 is the polynucleotide of any one of embodiments 85, 86, 87 or 88, wherein the receptor binding loops are between about the amino acid at position 332 (Asp) and about the amino acid at position 340 (Asp), between about the amino acid at position 395 (Asp) and about the amino acid at position 403 (Asp), and between about the amino acid at position 458 (Asp) and about the amino acid 466 (Asp), relative to SEQ ID NO: 35.
Embodiment 90 is the polynucleotide of embodiment 89, wherein the PHI-4 polypeptide has one or more amino substitution compared to the native amino acid at position 332, 333, 334, 335, 336, 337, 338, 339, 340, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 458, 459, 460, 461 , 462, 463, 464, 465, 466 of SEQ ID NO: 35.
Embodiment 91 is the polynucleotide of any one of embodiments 85, 86, 87, 88,
89 or 90, wherein the protease sensitive region residue is selected from between about the amino acid at position 305 (Lys) and about the amino acid at position 316 (Lys) and/or between about the amino acid at position 500 (Arg) and about the amino acid at position 535 (Lys) relative to SEQ ID NO: 35.
Embodiment 92 is the polynucleotide of any one of embodiments 85, 86, 87, 88,
89, 90 or 91 , wherein the protease is trypsin.
Embodiment 93 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQ ID NO: 35.
Embodiment 94 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 90% sequence identity to SEQ ID NO: 35.
Embodiment 95 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 95% sequence identity to SEQ ID NO: 35.
Embodiment 96 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at least 97% sequence identity to SEQ ID NO: 35.
Embodiment 97 is a polynucleotide encoding a PHI-4 polypeptide, wherein the
PHI-4 polypeptide comprises an amino acid sequence of the formula,
5 10 15
Met Xaa Ser Ala Ala Asn Ala Gly Xaa Leu Gly Asn Leu Xaa Gly
20 25 30
Xaa Thr Ser Xaa Gly Met Xaa Tyr Xaa Val Asn Gly Leu Tyr Ala
35 40 45 Ser Pro Glu Ser Leu Xaa Gly Gin Pro Leu Phe Xaa Xaa Gly Gly 50 55 60 Xaa Leu Asp Ser Xaa Xaa He Glu Gly Xaa Xaa Xaa Xaa Phe Pro
65 70 75 Xaa Ser Met His Val His Thr Tyr Phe His Ser Asp Xaa Xaa Gin
80 85 90 Xaa Val Ser Xaa Xaa He Xaa Xaa Xaa Arg Xaa Xaa Xaa Ser Xaa
95 100 105 His Val Gly Xaa Ser Gly Xaa Xaa Xaa Leu Phe Ser Xaa Ser Xaa
110 115 120 Ser Val Asp Xaa Thr Thr Xaa Xaa Gin Gin Leu Xaa Glu He Thr
125 130 135 Xaa Ser Ser Thr Arg Glu Xaa His Val Leu Trp Tyr He Ser Leu
140 145 150 Pro Gly Ala Ala Thr Leu Xaa Ser Met Leu Xaa Xaa Xaa Xaa Xaa
155 160 165 Xaa Asp Xaa Xaa Xaa Pro Asn Met Xaa Ala Met Xaa Leu Phe Xaa
170 175 180 Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa Ala Ala Val Gly Gly Arg
185 190 195 Leu Xaa Xaa Xaa Xaa Ala Ser Lys Xaa Leu Xaa Met Xaa Ser Ser
200 205 210 Xaa Ser Leu Ser Thr Thr Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Xaa
215 220 225 Gly Glu He Xaa He Xaa His Gly Ser Xaa Met Glu Lys Gin Val
230 235 240 Asn Ser Phe Xaa Xaa Xaa Ser Thr He Arg Xaa Thr Ala Thr Gly
245 250 255 Gly Lys Pro Gly Xaa Thr Xaa Arg He Leu His Gly Pro Asp Ser
260 265 270 Xaa Xaa Ala Phe Ser Xaa Trp Ala Xaa Ser Leu Leu Xaa Tyr Ala
275 280 285 Thr Leu Met Asp Phe Xaa Thr Xaa Ser Leu Xaa Xaa He Xaa Ala
290 295 300 Leu Xaa Asp Xaa Pro Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Ala Xaa
305 310 315 Pro Xaa Xaa Met Xaa Xaa Ser Gin Xaa Ser He Pro Xaa Val Asp 320 325 330
Xaa Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly
335 340 345
Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa
350 355 360
Xaa Ser Thr Ser Xaa Xaa Tyr Lys Xaa Xaa Gly Gin Phe Xaa Gin
365 370 375
Arg Xaa His Xaa Ser Val Ala Asp Gly His Xaa Pro lie Xaa Xaa
380 385 390
Asp Leu Phe Asp Xaa Gly Xaa Xaa Xaa Xaa Pro Val Gly Xaa Gin
395 400 405
Xaa Val Trp Asp Xaa Xaa Xaa Xaa Gly Lys Xaa Xaa Xaa Tyr Xaa
410 415 420
Cys Trp Arg Xaa Xaa Xaa Xaa Gin Gly Tyr Xaa Xaa Xaa Gly Asp
425 430 435
Val Xaa Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro
440 445 450
Xaa Xaa Xaa Cys Xaa His Xaa Ser Leu Xaa Ala Xaa Xaa Xaa Thr
455 460 465
Leu Xaa Xaa Xaa Xaa Trp Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa
470 475 480
Xaa Val Ser Leu Trp Xaa Xaa Gly Ala Ala Gly Ala Val Ala Ser
485 490 495
Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser
500 505 510
Gly Xaa lie Xaa Xaa Leu Xaa Gly Ser lie Ala Cys Val Xaa Thr
515 520 525
Ser Ala lie Ala Ser Met Xaa Xaa Met Xaa Ser Met Leu Ser Xaa
530 535
His Xaa Gly Met Glu Ala Met Met Ser Lys Leu (SEQ ID NO: 3), wherein
Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position 79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, lie, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa at position 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa at position 87 is Lys or Gin; Xaa at position 88 is Met, lie or Leu; Xaa at position 90 is Gin or Glu; Xaa at position 94 is Val or lie; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie, Met, Phe, Cys, Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 is Leu or lie; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn, Glu, Cys, Ala or Arg; Xaa at position 1 12 is Thr or Ser; Xaa at position 1 13 is Asp, Glu or Met; Xaa at position 1 17 is Thr or Ser; Xaa at position 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa at position 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa at position 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149 is Phe or Val; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaa at position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa at position 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu, Gin, Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg; Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp or Cys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser or Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lys or lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp or Asn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg; Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr or His; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr or Phe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu or Pro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Ser or Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met, Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaa at position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaa at position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 264 is Glu or Gin; Xaa at position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa at position 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292 is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position 297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position 303 is Phe or Tyr; Xaa at position 305 is Lys, Gin, Ala, lie, Met, Asn, Thr or Val; Xaa at position 306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa at position 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn or Gin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin, Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys, lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338 is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala, Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu or Val; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Val or lie; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn or His; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met or Leu; Xaa at position 355 is Val, lie or Leu; Xaa at position 359 is Gly or Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Phe or lie; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu, lie or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385 is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 395 is Asp or Cys; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin, His or Thr; Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Gin or Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie, Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys, Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is lie or Val; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala, Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 is Met or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 is Asp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 is Val or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 is Gin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu, lie, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa at position 452 is Gin, Lys or Glu, Ala; Xaa at position 453 is Asn or Asp; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp, Gin, Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or lie; Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaa at position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaa at position 464 is Arg, Gly, His, Gin, Thr, Phe, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu; Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu or Gin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg, Glu or Gin; Xaa at position 509 is Lys, Gin, Glu or Ala; Xaa at position 517 is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa at position 520 is Lys, Gin, Glu, His or Ala; Xaa at position 525 is Gin or Lys; and Xaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp; wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 3 is an amino acid different from the corresponding amino acid of SEQ ID NO: 35 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
Embodiment 98 is a polynucleotide encoding a PHI-4 polypeptide, wherein the
PHI-4 polypeptide comprises an amino acid sequence of the formula,
5 10 15
Met Xaa Ser Ala Ala Asn Ala Gly Gin Leu Gly Asn Leu Pro Gly
20 25 30
Val Thr Ser Met Gly Met Gly Tyr Xaa Val Asn Gly Leu Tyr Ala
35 40 45
Ser Pro Glu Ser Leu Leu Gly Gin Pro Leu Phe Xaa Xaa Gly Gly
50 55 60
Xaa Leu Asp Ser lie Glu lie Glu Gly Arg Ser Tyr Thr Phe Pro
65 70 75
Arg Ser Met His Val His Thr Tyr Phe His Ser Asp Phe Xaa Gin
80 85 90
Asp Val Ser Xaa Glu lie Xaa Glu Tyr Arg Glu Lys Met Ser Gin
95 100 105
His Val Gly Val Ser Gly Xaa Xaa Xaa Leu Phe Ser Ala Ser Leu
110 115 120
Ser Val Asp Xaa Thr Thr Thr Asp Gin Gin Leu Thr Glu lie Thr 125 130 135
Tyr Ser Ser Thr Arg Glu Ala His Val Leu Trp Tyr He Ser Leu
140 145 150
Pro Gly Ala Ala Thr Leu Arg Ser Met Leu Arg Xaa Xaa Phe Xaa
155 160 165
Xaa Asp Xaa Asn Asn Pro Asn Met Pro Ala Met Xaa Leu Phe Xaa
170 175 180
Xaa Tyr Gly Pro Tyr Xaa He Ser Xaa Ala Ala Val Gly Gly Arg
185 190 195 Leu Xaa Tyr Ser Ala Ala Ser Lys Thr Leu Lys Met Asp Ser Ser
200 205 210
Xaa Ser Leu Ser Thr Thr Ala Xaa Met Ser Xaa Lys Ala Leu Val
215 220 225
Gly Glu He Lys He Xaa His Gly Ser Xaa Met Glu Lys Gin Val
230 235 240
Asn Ser Phe Arg Ser Asn Ser Thr He Arg Leu Thr Ala Thr Gly
245 250 255
Gly Lys Pro Gly Met Thr Xaa Arg He Leu His Gly Pro Asp Ser
260 265 270 Xaa Xaa Ala Phe Ser Xaa Trp Ala Glu Ser Leu Leu Asp Tyr Ala
275 280 285
Thr Leu Met Asp Phe Ser Thr Xaa Ser Leu Xaa Pro He Trp Ala
290 295 300
Leu Ala Asp Xaa Pro Glu Arg Xaa Val Glu Leu Glu Asp Ala Phe
305 310 315
Pro Glu Phe Met Lys Gin Ser Gin Gin Ser He Pro Xaa Val Asp
320 325 330
Lys Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly
335 340 345 Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Leu Ala Xaa Phe Asn
350 355 360
Xaa Ser Thr Ser Asn Gly Tyr Lys Met Xaa Gly Gin Phe Xaa Gin
365 370 375
Arg Asn His Ala Ser Val Ala Asp Gly His Ala Pro He Phe Lys
380 385 390
Asp Leu Phe Asp Leu Gly Val Leu Lys Ala Pro Val Gly Trp Gin
395 400 405 Xaa Val Trp Asp Asp Xaa Gly Ser Gly Lys Xaa Xaa Xaa Tyr Ala
410 415 420
Cys Trp Arg Ala lie Xaa Xaa Gin Gly Tyr Xaa Xaa Xaa Gly Asp
425 430 435
Val Met Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro
440 445 450
Asp Tyr Val Cys Xaa His Gin Ser Leu Cys Ala Xaa Val Gin Thr
455 460 465
Leu Xaa Asn Xaa Xaa Trp Trp Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa
470 475 480
Asp Val Ser Leu Trp Xaa Xaa Gly Ala Ala Gly Ala Val Ala Ser
485 490 495
Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser
500 505 510
Gly Asp lie Glu Xaa Leu Arg Gly Ser lie Ala Cys Val Xaa Thr
515 520 525
Ser Ala lie Ala Ser Met Gin Glu Met Xaa Ser Met Leu Ser Gin
530 535
His Xaa Gly Met Glu Ala Met Met Ser Lys Leu (SEQ ID NO: 4), wherein
Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 147 is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or lie; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu or Gin; Xaa at position 166 is Arg or Gin; Xaa at position 171 is Tyr or Phe; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 196 is Gin, Lys, Asn or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is Tyr or Phe; Xaa at position 216 is Glu or Gin; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin or Lys; Xaa at position 257 is Gin or lie; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 278 is Glu or Asn; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 313 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa at position 334 is Gly, Arg, Lys or lie; Xaa at position 336 is Gly or Ala; Xaa at position 338 is Ser, His, Val, Lys or Ala; Xaa at position 339 is Glu, Asn, lie or Pro; Xaa at position 343 is Val or lie; Xaa at position 346 is Pro or Ala; Xaa at position 355 is Val or lie; Xaa at position 359 is Gly or Ala; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly or Thr; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaa at position 403 is Asp or Tyr; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg or Glu; Xaa at position 417 is Ala or Ser; Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu or lie; Xaa at position 452 is Gin, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp or Gin; Xaa at position 455 is Val or lie; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie or Tyr; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala or Ser; Xaa at position 464 is Arg, Gly, His, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 500 is Arg or Gin; Xaa at position 509 is Lys, Gin or Ala; Xaa at position 520 is Lys, Gin, Glu, His or Ala; and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu; wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 4 is an amino acid different from the corresponding amino acid of SEQ ID NO: 35 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
Embodiment 99 is the polynucleotide of embodiments 97 or 98, wherein the PHI-4 polypeptide further comprises one or more amino acid substitutions at position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 3 or SEQ ID NO: 4.
Embodiment 100 is the polynucleotide of embodiment 99, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
Embodiment 101 is the polynucleotide of any one of embodiments 97-100, wherein the PHI-4 polypeptide further comprises one or more more conservative amino acid substitution, insertion of one or more amino acids, deletion of one or more amino acids, and combinations thereof. Embodiment 102 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 1.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 103 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 104 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 2.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 105 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 3 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 106 is the polynucleotide of any one of embodiments 97-101 , wherein the insecticidal activity of the PHI-4 polypeptide is increased about 5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
Embodiment 107 is the polynucleotide of any one of embodiments 97-106, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
Embodiment 108 is the polynucleotide of any one of embodiments 97-107, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean FAE Index.
Embodiment 109 is the polynucleotide of any one of embodiments 97-107, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as an EC50 value.
Embodiment 1 10 is the polynucleotide of any one of embodiments 97-107, wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) is quantified as a Mean Deviation Score.
Embodiment 1 1 1 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 54 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
Embodiment 1 12 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 27 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
Embodiment 1 13 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 20 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4. Embodiment 1 14 is the polynucleotide of any one of embodiments 97-1 10, wherein the PHI-4 polypeptide has 1 to 15 amino acid substitutions at a position(s) designated as Xaa in SEQ ID NO: 3 or SEQ ID NO: 4.
Embodiment 1 15 is the polynucleotide of any one of embodiments 97-1 14, wherein 1 -25 amino acids are deleted from the N-terminus of the PHI-4 polypeptide and/or C-terminus of the PHI-4 polypeptide.
Embodiment 1 16 is the polynucleotide of any one of embodiments 97-1 14, wherein 1-20 amino acids are deleted from the C-terminus of the PHI-4 polypeptide.
Embodiment 1 17 is a composition, comprising an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1 -58.
Embodiment 1 18 is a method of inhibiting growth or killing an insect pest, comprising contacting the insect pest with the composition of embodiment 1 17.
Embodiment 1 19 is a method for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the resistant insect pest population with the composition of embodiment 1 17.
Embodiment 120 is the method of controlling an insect pest population resistant to an pesticidal protein of embodiment 1 19, wherein the pesticidal protein is selected from CrylAc, CrylAb, Cry1A.105, CrylAc, Cry1 F, Cry1 Fa2, Cryl F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1 , Cry34Ab1 , Cry35Ab1 , Vip3A, Cry9c, eCry3.1Ab and CBI-Bt.
Embodiment 121 is a transgenic plant or progeny thereof, comprising the polynucleotide of any one of embodiments 59-1 16. Embodiment 122 is the transgenic plant or progeny thereof of embodiment 121 , wherein the transgenic plant is a monocotyledon.
Embodiment 123 is the transgenic plant or progeny thereof of embodiment 122, wherein the plant is selected from barley, corn, oat, rice, rye, sorghum, turf grass, sugarcane, wheat, alfalfa, banana, broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower, safflower, soybean, strawberry, sugar beet, sweet potato, tobacco, tomato ornamental, shrub, nut, chickpea, pigeon pea, millets, hops and pasture grasses.
Embodiment 124 is the transgenic plant or progeny thereof of embodiment 123, further comprising one or more additional transgenic traits.
Embodiment 125 is the transgenic plant of embodiment 124, wherein the one or more additional transgenic trait is selected from insect resistance, herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stalk strength, increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, salt resistance or tolerance and increased yield under stress.
Embodiment 126 is seed, grain or processed product thereof of the transgenic plant of any one of embodiments 121-126, wherein the seed, grain or processed product thereof comprises the polynucleotide of any one of embodiments 121-125.
Embodiment 127 is an expression cassette, comprising the polynucleotide of any one of embodiments 59-1 16 operably linked to one or more regulatory sequences directing expression of the PHI-4 polypeptide.
Embodiment 128 is a transgenic plant or plant cell, comprising the expression cassette of embodiment 127.
Embodiment 129 is seed, grain or processed product thereof of the transgenic plant of embodiment 128, wherein the seed, grain or processed product thereof comprises the recombinant nucleic acid molecule of embodiment of 1 and the recombinant nucleic acid molecule of 24.
Embodiment 130 is the seed of embodiment 129, wherein one or more seed treatment has been applied to the seed.
Embodiment 131 is a method for expressing in a plant a polynucleotide encoding an insecticidal protein, comprising
(a) inserting into a plant cell the polynucleotide of any one of embodiment 59- 1 16 encoding the PHI-4 polypeptide;
(b) obtaining a transformed plant cell comprising the nucleic acid sequence of step (a); and
(c) generating from the transformed plant cell a plant capable of expressing the polynucleotide encoding the PHI-4 polypeptide.
Embodiment 132 is a method for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof, an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1-58.
Embodiment 133 is a method for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1-58.
Embodiment 134 is a method of inhibiting growth or killing an insect pest, comprising contacting the insect pest with a composition comprising an insecticidally- effective amount of the the PHI-4 polypeptide of any one of embodiments 1-58. Embodiment 135 is a method for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of the PHI-4 polypeptide of any one of embodiments 1-58.
Embodiment 136 is a fusion protein comprising the PHI-4 polypeptide of any one of embodiments 1-58.
Embodiment 137 is a fusion protein comprising the PHI-4 polypeptide of any one of embodiments 1-58 and a maltose binding protein.
Embodiment 138 is the fusion protein of embodiment 137, wherein the maltose binding protein has an amino acid sequence of SEQ I D NO: 1516 or SEQ ID NO: 1517.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows FAE analysis of MPB::PHI-4-SFR12-004 (SEQ ID NO: 31 ). The reference protein is MBP::PHI-4 polypeptide of (SEQ ID NO: 6). The % Response is given on the y axis. The dose of the toxin fragment is given on the x axis in parts per million (ppm). Half dose (+) and full dose (*) indicate a mean response from six replicate wells at the indicated concentration. All data are from a single experiment.
Figure 2 shows EC50 analysis of MBP::PHI-4 polypeptide of SEQ ID NO: 6 and MPB::PHI-4-SFR12-004 (SEQ ID NO: 35). The % Response is given on the y axis. The dose of the toxin fragment is given on the x axis in parts per million (ppm). The % Response is given on the y axis. The dose of the toxin fragment is given on the x axis in parts per million (ppm). The protein concentration (toxin portion of the protein only) is given on the x axis. Each symbol indicates a mean response from twenty-four replicate wells at the indicated concentration. All data are from a single experiment, triangles: MBP::PHI-4 (SEQ ID NO: 6); circles: MBP::PHI-4-SFR12-004 (SEQ ID NO: 31 ).
Figure 3 shows the amino acids sequence of the C-terminal portion of the PHI-4 polypeptide (SEQ ID NO: 2) is given. Three stretches of sequence corresponding to nine amino acid motifs that align to the putative sugar binding loop motif D-X-G-(S/T)-G-X3-D (SEQ ID NO: 40) are highlighted in grey.
Figure 4 shows EC50 data for SEQ ID NO: 610, SEQ ID NO: 595, SEQ ID NO: 584, SEQ ID NO: 591 , SEQ I D NO: 576, SEQ ID NO: 73, SEQ I D NO: 74, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 150, SEQ ID NO: 150, SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 167, SEQ ID NO: 164, SEQ ID NO: 164, SEQ ID NO: 170, SEQ ID NO: 170, SEQ ID NO: 795, SEQ ID NO: 794, SEQ ID NO: 784, SEQ ID NO: 799, SEQ ID NO: 785, SEQ ID NO: 788, SEQ ID NO: 786, SEQ ID NO: 796, SEQ ID NO: 787. The x- axis corresponds to the mean fold improvement in EC50, relative to MBP::PHI-4 fusion (SEQ ID NO: 6) from at least three independent EC50 measurements. The y-axis corresponds to the mean fold improvement of the mean FAE Index from at least three independent measurements.
Figure 5. TO seedlings in the V3 - V4 growth stage were challenged as described (Oleson J. et al J. Economic Entomology 98:1 -8; 2005) and root nodal injury scores were recorded. The groups indicated along the x axis were either controls or were transformed with a plant vector of Example 18: 1 - untransformed control; 2 - positive control transgenic expressing positive control protein; 3 - AXMI-205 polypeptide (SEQ ID NO: 22); 4 - PHI-4-B09 polypeptide (SEQ ID NO: 23); 5 - PHI-4-D09 polypeptide (SEQ ID NO: 24); 6 - PHI-4-H08 polypeptide (SEQ ID NO: 25). DETAILED DESCRIPTION
It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.
As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.
The present disclosure is drawn to compositions and methods for controlling pests. The methods involve transforming organisms with a nucleic acid sequence encoding a PHI-4 polypeptide. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms possessing pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are pesticidal nucleic acids and proteins of bacterial species. The nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered PHI-4 polypeptides by methods known in the art, such as site directed mutagenesis, domain swapping or DNA shuffling. The PHI-4 polypeptides find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran, and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, the superfamily of stink bugs and other related insects including, but not limited to, species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guild ini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria - Bean plataspid), and the family Cydnidae {Scaptocoris castanea - Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalis Hubner.
By "pesticidal toxin" or "pesticidal protein" is intended a toxin that has toxic activity against one or more pests, including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera and Coleoptera orders or the Nematoda phylum or a protein that has homology to such a protein. Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins include but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (201 1 ) PLoS Pathogens, 7:1 -13) from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et ai, (2010) J. Agric. Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al , (2009) Annals of Microbiology 59:45-50 and Li, et al, (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal 3:101- 1 18 and Morgan, et al., (2001 ) Applied and Envir. Micro. 67:2062-2069), US Patent Number 6,048,838, and US Patent Number 6,379,946; and δ-endotoxins including, but not limited to, the cry1 , cry2, cry3, cry4, cry5, cry6, cry7, cry8, cry9, cry10, cry1 1 , cry12, cry13, cry14, cry15, cry16, cry17, cry18, cry19, cry20, cry21 , cry22, cry23, cry24, cry25, cry26, cry27, cry 28, cry 29, cry 30, cry31 , cry32, cry33, cry34, cry35,cry36, cry37, cry38, cry39, cry40, cry41 , cry42, cry43, cry44, cry45, cry 46, cry47, cry49, cry 51 , cry 52, cry 53, cry54, cry55, cry56, cry57, cry58, cry59, cry60, cry61 , cry62, cry63, cry64, cry65, cry66, cry67, cry68, cry69, cry70, cry71 , cry72, and cry73 classes of δ-endotoxin genes and the B. thuringiensis cytolytic cytl and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins include, but are not limited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8 (Accession # 126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa1 1 (Accession # CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession # AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession # HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession # HQ439790); Cry1Aa19 (Accession # HQ685121 ); Cry1Aa20 (Accession # JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession # KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession # AAA22613); Cry1Ab3 (Accession # AAA22561 ); Cry1Ab4 (Accession # BAA00071 ); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession # AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession # AAA22551 ); Cry1Ab9 (Accession # CAA38701 ); Cry1Ab10 (Accession # A29125); Cry1Ab1 1 (Accession # 112419); Cry1Ab12 (Accession # AAC64003); Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877); Cry1Ab15 (Accession # AAO13302); Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259); Cry1Ab19 (Accession # AAW31761 ); Cry1Ab20 (Accession # ABB72460); Cry1Ab21 (Accession # ABS 18384); Cry1Ab22 (Accession # ABW87320); Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778); Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729); Cry1Ab27 (Accession # JN 135249); Cry1Ab28 (Accession # JN 135250); Cry1Ab29 (Accession # JN 135251 ); Cry1Ab30 (Accession # JN135252); Cry1Ab31 (Accession # JN135253); Cry1Ab32 (Accession # JN135254); Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668); Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337); Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858); Cry1Ac1 (Accession # AAA22331 ); Cry1Ac2 (Accession # AAA22338); Cry1Ac3 (Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5 (Accession # AAA22339); Cry1Ac6 (Accession # AAA86266); Cry1Ac7 (Accession # AAB46989); Cry1Ac8 (Accession # AAC44841 ); Cry1Ac9 (Accession # AAB49768); Cry1Ac10 (Accession # CAA05505 ); Cry1Ac1 1 (Accession # CAA10270); Cry1Ac12 (Accession # 112418); Cry1Ac13 (Accession # AAD38701 ); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15 (Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17 (Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19 (Accession # ABD37053); Cry1Ac20 (Accession # ABB89046 ); Cry1Ac21 (Accession # AAY66992 ); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23 (Accession # CAQ30431); Cry1Ac24 (Accession # ABL01535); Cry1Ac25 (Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27 (Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29 (Accession # DQ438941); Cry1Ac30 (Accession # GQ227507); Cry1Ac31 (Accession # GU446674); Cry1Ac32 (Accession # HM061081); Cry1Ac33 (Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35 (Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37 (Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2 (Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1 (Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1 (Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3 (Accession # HQ439779); Cry1Ai1 (Accession # AA039719); Cry1Ai2 (Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1 (Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3 (Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5 (Accession # ABO20894); Cry1Ba6 (Accession # ABL60921); Cry1Ba7 (Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2 (Accession # HQ439782); Cry1Bc1 (Accession # CAA86568); Cry1Bd1 (Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1 (Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3 (Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1 (Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1 (Accession # AAO39720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1 (Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2 (Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4 (Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6 [1] (Accession # AAF37224 ); Cry1Ca7 (Accession # AAG50438); Cry1Ca8 (Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10 (Accession # AAN16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12 (Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14 (Accession # JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession # AAG35409); Cry1Cb3 (Accession # ACD50894 ); Cry1Cb-like (Accession # AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession # 176415); Cry1Da3 (Accession # HQ439784); Cry1Db1 (Accession # CAA80234 ); Cry1Db2 (Accession # AAK48937 ); Cry1Dc1 (Accession # ABK35074); Cry1Ea1 (Accession # CAA37933); Cry1Ea2 (Accession # CAA39609); Cry1Ea3 (Accession # AAA22345); Cry1Ea4 (Accession # AAD04732); Cry1Ea5 (Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession # AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession # HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession # JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession # AAA22348); Cry1Fa2 (Accession # AAA22347); Cry1Fa3 (Accession # HM070028); Cry1Fa4 (Accession # HM439638); Cry1Fb1 (Accession # CAA80235); Cry1Fb2 (Accession # BAA25298); Cry1Fb3 (Accession # AAF21767); Cry1Fb4 (Accession # AAC10641); Cry1Fb5 (Accession # AA013295); Cry1Fb6 (Accession # ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1 (Accession # CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1 (Accession # AAD10291); Cry1Gb2 (Accession # AA013756); Cry1Gc1 (Accession # AAQ52381); Cry1Ha1 (Accession # CAA80236); Cry1Hb1 (Accession # AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like (Accession # AAF01213); Cry1la1 (Accession # CAA44633); Cry1la2 (Accession # AAA22354); Cry1la3 (Accession # AAC36999); Cry1la4 (Accession # AAB00958); Cry1la5 (Accession # CAA70124); Cry1la6 (Accession # AAC26910); Cry1la7 (Accession # AAM73516); Cry1la8 (Accession
# AAK66742); Cry1la9 (Accession # AAQ08616); Cry1la10 (Accession # AAP86782); Cry1la11 (Accession # CAC85964 ); Cry1la12 (Accession # AAV53390); Cry1la13 (Accession # ABF83202); Cry1la14 (Accession # ACG63871); Cry1la15 (Accession # FJ617445); Cry1la16 (Accession # FJ617448); Cry1la17 (Accession # GU989199); Cry1la18 (Accession # ADK23801); Cry1la19 (Accession # HQ439787); Cry1la20 (Accession # JQ228426); Cry1la21 (Accession # JQ228424); Cry1la22 (Accession # JQ228427); Cry1la23 (Accession # JQ228428); Cry1la24 (Accession # JQ228429); Cry1la25 (Accession # JQ228430); Cry1la26 (Accession # JQ228431); Cry1la27 (Accession # JQ228432); Cry1la28 (Accession # JQ228433); Cry1la29 (Accession # JQ228434); Cry1la30 (Accession # JQ317686); Cry1la31 (Accession # JX944038); Cry1la32 (Accession # JX944039); Cry1la33 (Accession # JX944040); Cry1lb1 (Accession # AAA82114); Cry1lb2 (Accession # ABW88019); Cry1lb3 (Accession # ACD75515); Cry1lb4 (Accession # HM051227); Cry1lb5 (Accession # HM070028); Cry1lb6 (Accession # ADK38579); Cry1lb7 (Accession # JN571740); Cry1lb8 (Accession
# JN675714); Cry1lb9 (Accession # JN675715); Cry1lb10 (Accession # JN675716); Cry1lb11 (Accession # JQ228423); Crylld (Accession # AAC62933); Cry1lc2 (Accession # AAE71691); Cry1ld1 (Accession # AAD44366); Cry1ld2 (Accession # JQ228422); Cry1le1 (Accession # AAG43526); Cry1le2 (Accession # HM439636); Cry1le3 (Accession # KC156647); Cry1le4 (Accession # KC156681); Cryllfl (Accession
# AAQ52382); Cry1lg1 (Accession # KC156701); Cry1 l-like (Accession # AAC31094); Cry1 l-like (Accession # ABG88859); CryUal (Accession # AAA22341); Cry1Ja2 (Accession # HM070030); Cry1Ja3 (Accession # JQ228425); CryUbl (Accession # AAA98959); CryUd (Accession # AAC31092); Cry1Jc2 (Accession # AAQ52372); CryUdl (Accession # CAC50779); Cry1Ka1 (Accession # AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1 (Accession # AAS60191); Cry1La2 (Accession # HM070031 ); Cry1 Ma1 (Accession # FJ884067); Cry1 Ma2 (Accession # KC156659); Cry1 Na1 (Accession # KC156648); Cry1 Nb1 (Accession # KC156678); Cry1- like (Accession # AAC31091 ); Cry2Aa1 (Accession # AAA22335); Cry2Aa2 (Accession # AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession # AAC04867); Cry2Aa5 (Accession # CAA10671 ); Cry2Aa6 (Accession # CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8 (Accession # AA013734); Cry2Aa9 (Accession # AAO13750 ); Cry2Aa10 (Accession # AAQ04263); Cry2Aa1 1 (Accession # AAQ52384); Cry2Aa12 (Accession # ABI83671 ); Cry2Aa13 (Accession # ABL01536); Cry2Aa14 (Accession # ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1 (Accession # AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3 (Accession # AAG36762); Cry2Ab4 (Accession # AA013296 ); Cry2Ab5 (Accession # AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7 (Accession # AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9 (Accession # ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab1 1 (Accession # CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13 (Accession # ACG76120); Cry2Ab14 (Accession # ACG76121 ); Cry2Ab15 (Accession # HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17 (Accession # HQ439789); Cry2Ab18 (Accession # JN 135255); Cry2Ab19 (Accession # JN 135256); Cry2Ab20 (Accession # JN 135257); Cry2Ab21 (Accession # JN 135258); Cry2Ab22 (Accession # JN 135259); Cry2Ab23 (Accession # JN 135260); Cry2Ab24 (Accession # JN 135261 ); Cry2Ab25 (Accession # J N415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27 (Accession # JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1 (Accession # CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3 (Accession # AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5 (Accession # ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7 (Accession # CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9 (Accession # CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac1 1 (Accession # CAM83895); Cry2Ac12 (Accession # CAM83896); Cry2Ad1 (Accession # AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3 (Accession # CAK29504); Cry2Ad4 (Accession # CAM32331 ); Cry2Ad5 (Accession # CA078739 ); Cry2Ae1 (Accession # AAQ52362); Cry2Af1 (Accession # ABO30519); Cry2Af2 (Accession # GQ866915); Cry2Ag1 (Accession # ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2 (Accession # ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4 (Accession # KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj (Accession # ); Cry2Ak1 (Accession # KC156660); Cry2Ba1 (Accession # KC156658); Cry3Aa1 (Accession # AAA22336); Cry3Aa2 (Accession # AAA22541 ); Cry3Aa3 (Accession # CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5 (Accession # AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7 (Accession # CAB4141 1 ); Cry3Aa8 (Accession # AAS79487); Cry3Aa9 (Accession # AAW05659); Cry3Aa10 (Accession # AAU2941 1 ); Cry3Aa1 1 (Accession # AAW82872); Cry3Aa12 (Accession # ABY49136 ); Cry3Ba1 (Accession # CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3 (Accession # JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2 (Accession # AAA74198); Cry3Bb3 (Accession # 115475); Cry3Ca1 (Accession # CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession # BAA00179); Cry4Aa3 (Accession # CAD30148); Cry4Aa4 (Accession # AFB18317); Cry4A-like (Accession # AAY96321 ); Cry4Ba1 (Accession # CAA30312); Cry4Ba2 (Accession # CAA301 14); Cry4Ba3 (Accession # AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5 (Accession # CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1 (Accession # EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2 (Accession # FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1 (Accession # AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1 (Accession # I34543); Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2 (Accession # ABW88931 ); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1 (Accession # HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1 (Accession # HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1 (Accession # HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1 (Accession # AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3 (Accession # ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7Aa1 (Accession # AAA22351 ); Cry7Ab1 (Accession # AAA21 120); Cry7Ab2 (Accession # AAA21 121 ); Cry7Ab3 (Accession # ABX24522); Cry7Ab4 (Accession # EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6 (Accession # ACI44005); Cry7Ab7 (Accession # ADB89216); Cry7Ab8 (Accession # GU 145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession # ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession # ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession # ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession # KC156679); Cry7Ea1 (Accession # HM035086); Cry7Ea2 (Accession # HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession # HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession # HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession # HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession # KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession # KC156697); Cry7Ha1 (Accession # KC156651 ); Cry7la1 (Accession # KC156665); Cry7Ja1 (Accession # KC156671 ); Cry7Ka1 (Accession # KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession # BAM99307); Cry8Aa1 (Accession # AAA21 1 17); Cry8Ab1 (Accession # EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession # KC156684); Cry8Ba1 (Accession # AAA21 1 18); Cry8Bb1 (Accession # CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession # AAA21 1 19); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession # EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession # BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession # BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession # AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession # KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession # HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession # AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession # FJ 198072); Cry8Ha1 (Accession # AAW81032); Cry8la1 (Accession # EU381044); Cry8la2 (Accession # GU073381 ); Cry8la3 (Accession # HM044664); Cry8la4 (Accession # KC156674); Cry8lb1 (Accession # GU325772); Cry8lb2 (Accession # KC156677); Cry8Ja1 (Accession # EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession # ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession # KC156675); Cry8La1 (Accession # GU325771 ); Cry8Ma1 (Accession # HM044665); Cry8Ma2 (Accession # EEM86551 ); Cry8Ma3 (Accession # HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession # HQ388415); Cry8Qa1 (Accession # HQ441 166); Cry8Qa2 (Accession # KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession # JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession # FJ770571 ); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession # CAA41 122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession # GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession # JX1741 10); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession # CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession # AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession # AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession # AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession # GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession # KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession # AAO12908); Cry9Ea3 (Accession # ABM21765); Cry9Ea4 (Accession # ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession # ACG63872 ); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession # GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession # CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession # KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession # AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession # KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession # KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession # AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession # CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession # DQ167578); Cry1 1Aa1 (Accession # AAA22352); Cry1 1Aa2 (Accession # AAA2261 1 ); Cry1 1Aa3 (Accession # CAD30081 ); Cry1 1Aa4 (Accession # AFB18319); Cry1 1Aa-like (Accession # DQ166531 ); Cry1 1 Ba1 (Accession # CAA60504); Cry1 1 Bb1 (Accession # AAC97162); Cry1 1 Bb2 (Accession # HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession # AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession # KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession # CAA63860); Cry17Aa1 (Accession # CAA67841 ); Cry18Aa1 (Accession # CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession # AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession # BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession # AAB93476); Cry20Ba1 (Accession # ACS93601 ); Cry20Ba2 (Accession # KC156694); Cry20-like (Accession # GQ144333); Cry21Aa1 (Accession # I32932); Cry21Aa2 (Accession # I66477); Cry21 Ba1 (Accession # BAC06484); Cry21 Ca1 (Accession # JF521577); Cry21 Ca2 (Accession # KC156687); Cry21 Da1 (Accession # JF521578); Cry22Aa1 (Accession # I34547); Cry22Aa2 (Accession # CAD43579); Cry22Aa3 (Accession # ACD9321 1 ); Cry22Ab1 (Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1 (Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1 (Accession # AAF76375); Cry24Aa1 (Accession # AAC61891 ); Cry24Ba1 (Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1 (Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1 (Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2 (Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1 (Accession # CAC80986); Cry30Ba1 (Accession # BAD00052); Cry30Ca1 (Accession # BAD67157); Cry30Ca2 (Accession # ACU24781 ); Cry30Da1 (Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1 (Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1 (Accession # ACI22625 ); Cry30Ga1 (Accession # ACG60020); Cry30Ga2 (Accession # HQ638217); Cry31Aa1 (Accession # BAB1 1757); Cry31Aa2 (Accession # AAL87458); Cry31Aa3 (Accession # BAE79808); Cry31Aa4 (Accession # BAF32571 ); Cry31Aa5 (Accession # BAF32572); Cry31Aa6 (Accession # BAI44026); Cry31Ab1 (Accession # BAE79809); Cry31Ab2 (Accession # BAF32570); Cry31Ac1 (Accession # BAF34368); Cry31Ac2 (Accession # AB731600); Cry31Ad1 (Accession # BAI44022); Cry32Aa1 (Accession # AAG3671 1 ); Cry32Aa2 (Accession # GU063849); Cry32Ab1 (Accession # GU063850); Cry32Ba1 (Accession # BAB78601 ); Cry32Ca1 (Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32Da1 (Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2 (Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1 (Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1 (Accession # KC156661 ); Cry32Hb1 (Accession # KC156666); Cry32la1 (Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1 (Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1 (Accession # KC156690); Cry32Mb1 (Accession # KC156704); Cry32Na1 (Accession # KC156691 ); Cry320a1 (Accession # KC156703); Cry32Pa1 (Accession # KC156705); Cry32Qa1 (Accession # KC156706); Cry32Ra1 (Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1 (Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1 (Accession # AAL26871 ); Cry34Aa1 (Accession # AAG50341 ); Cry34Aa2 (Accession # AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4 (Accession # AAT29030); Cry34Ab1 (Accession # AAG41671 ); Cry34Ac1 (Accession # AAG501 18); Cry34Ac2 (Accession # AAK64562); Cry34Ac3 (Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2 (Accession # AAT29033); Cry34Ba3 (Accession # AAT29031 ); Cry35Aa1 (Accession # AAG50342); Cry35Aa2 (Accession # AAK64561 ); Cry35Aa3 (Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1 (Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3 (Accession # AY536891 ); Cry35Ac1 (Accession # AAG501 17); Cry35Ba1 (Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3 (Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1 (Accession # AAF76376 ); Cry38Aa1 (Accession # AAK64559); Cry39Aa1 (Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1 (Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1 (Accession # ACF15199); Cry41Aa1 (Accession # BAD35157); Cry41Ab1 (Accession # BAD35163); Cry41 Ba1 (Accession # HM461871 ); Cry41 Ba2 (Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1 (Accession # BAD15301 ); Cry43Aa2 (Accession # BAD95474 ); Cry43Ba1 (Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1 (Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like (Accession # BAD15305); Cry44Aa (Accession # BAD08532); Cry45Aa (Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2 (Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47Aa (Accession # AAY24695); Cry48Aa (Accession # CAJ 18351 ); Cry48Aa2 (Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546 ); Cry48Ab (Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa (Accession # CAH56541 ); Cry49Aa2 (Accession # CAJ86541 ); Cry49Aa3 (Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1 (Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1 (Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1 (Accession # ABI 14444); Cry51Aa2 (Accession # GU570697); Cry52Aa1 (Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1 (Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1 (Accession # ACA52194); Cry54Aa2 (Accession # GQ140349); Cry54Ba1 (Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1 (Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1 (Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3 (Accession # JX025567); Cry57Aa1 (Accession # ANC87261 ); Cry58Aa1 (Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1 (Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2 (Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1 (Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3 (Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2 (Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1 (Accession # HM054509); Cry63Aa1 (Accession # BAI44028); Cry64Aa1 (Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2 (Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581 ); Cry66Aa2 (Accession # ZP_04099945); Cry67Aa1 (Accession # HM485582); Cry67Aa2 (Accession # ZP_04148882); Cry68Aa1 (Accession # HQ1 131 14); Cry69Aa1 (Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1 (Accession # JN209957); Cry70Aa1 (Accession # JN646781 ); Cry70Ba1 (Accession # ADO51070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1 (Accession # JX025568); Cry72Aa1 (Accession # JX025569); and Cry73Aa (Accession # AEH76822); CytlAa (GenBank Accession # X03182), CytlAb (GenBank Accession # X98793), CytI B (GenBank Accession # U37196), Cyt2A (GenBank Accession # Z14147), Cyt2B (GenBank Accession # U52043).
Examples of δ-endotoxins also include but are not limited to CrylA proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-1 1 toxin (N-terminal deletion of a-helix 1 and/or a-helix 2 variants of cry proteins such as CrylA, Cry3A) of US Patent Numbers 8,304,604 and 8.304,605, Cryl B of US Patent Application Serial Number 10/525,318; Cryl C of US Patent Number 6,033,874; Cryl F of US Patent Numbers 5,188,960, 6,218,188; Cry1A/F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of US Patent Numbers 7,329,736, 7,449,552, 7,803,943, 7,476,781 , 7, 105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology 74:7145-7151 ; a Cry22, a Cry34Ab1 protein of US Patent Numbers 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 protein of US Patent Numbers 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006/0191034, 2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1 protein of US Patent Numbers 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI-027, AXMI-036, and AXMI-038 of US Patent Number 8,236,757; AXMI-031, AXMI-039, AXMI- 040, AXMI-049 of US7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US 2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965; AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462; AXMI-150 of US Patent Number 8,084,416; AXMI-205 of US20110023184; AXMI-011, AXMI-012, AXMI- 013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI- R1 and related proteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224Z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 of W011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of US Patent Number 8,334,431; AXMI- 001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211; AXMI-066 and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of US Patent Number 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543; cry proteins such as CrylA and Cry3A having modified proteolytic sites of US Patent Number 8,319,019; a CrylAc, Cry2Aa and CryICa toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011/0064710. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (201 1 ), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the "www" prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101 :1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to CrylAc, Cry1Ac+Cry2Ab, CrylAb, Cry1A.105, Cryl F, Cry1 Fa2, Cry1 F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1 , Cry34Ab1 , Cry35Ab1 , Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (201 1 ) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera- gmc.org/index. php?action=gm_crop_database which can be accessed on the world-wide web using the "www" prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cryl Fa (US2012/0317682), Cryl BE & Cryl F (US2012/031 1746), CryI CA & CrylAB (US2012/031 1745), Cryl F & CryCa (US2012/0317681 ), Cryl DA & Cryl BE (US2012/0331590), Cryl DA & Cryl Fa (US2012/0331589), CrylAB & Cryl BE (US2012/0324606), and Cryl Fa & Cry2Aa, Cryl l or Cryl E (US2012/0324605). Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491 ,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can be accessed on the world-wide web using the "www" prefix). Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491 ,698 and 8,084,418). Some TC proteins have "stand alone" insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a "stand-alone" TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein "potentiators" derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins ("Protein A") are stand-alone toxins. Class B proteins ("Protein B") and Class C proteins ("Protein C") enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptBIXb and XptCIWi. Examples of Class C proteins are TccC, XptCIXb and XptBIWi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366).
In some embodiments the PHI-4 polypeptides include amino acid sequences deduced from the full-length nucleic acid sequences disclosed herein, and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in or in the pest after ingestion of the protein.
Thus, provided herein are novel isolated or recombinant nucleic acid sequences that confer pesticidal activity. Also provided are the amino acid sequences of PHI-4 polypeptides. The protein resulting from translation of these PHI-4 polypeptide genes allows cells to control or kill pests that ingest it. Nucleic Acid Molecules, and Variants and Fragments Thereof
One aspect pertains to isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding PHI-4 polypeptides and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in vitro. A "recombinant" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an "isolated" or "recombinant" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, "isolated" or "recombinant" when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecule encoding a PHI- 4 polypeptide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
In some embodiments a nucleic acid molecule encoding the PHI-4 polypeptide is a non-genomic nucleic acid sequence. As used herein a "non-genomic nucleic acid sequence "or "non-genomic nucleic acid molecule" refers to a nucleic acid molecule that has one or more change in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5' and/or 3' untranslated region; and modification of a polyadenylation site. In some embodiments the non-genomic nucleic acid molecule is a cDNA. In some embodiments the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.
A variety of polynucleotides encoding a PHI-4 polypeptide(s) or related proteins are contemplated. Such polynucleotides are useful for production of PHI-4 polypeptides in host cells when operably linked to suitable promoter, transcription termination and/or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides encoding PHI-4 polypeptides or related proteins.
The present disclosure provides isolated or recombinant polynucleotides that encode any of the PHI-4 polypeptides disclosed herein. Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding β-glucosidase polypeptides of the present disclosure exist. Table 1 is a Codon Table that provides the synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the disclosure where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence. Table 1
Figure imgf000050_0001
Such "silent variations" are one species of "conservative" variation. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence. The disclosure contemplates and provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the disclosure that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (set forth in Table 1 ), as applied to the polynucleotide sequences of the present disclosure.
A group of two or more different codons that, when translated in the same context, all encode the same amino acid, are referred to herein as "synonymous codons." Polynucleotides encoding PHI-4 polypeptides of the present disclosure may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform with the optimum codon usage of the desired host organism. Those having ordinary skill in the art will recognize that tables and other references providing preference information for a wide range of organisms are readily available.
Polynucleotides encoding a PHI-4 polypeptide can also be synthesized de novo from a PHI-4 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from a PHI-4 polypeptide sequence through use of the genetic code. Computer programs such as "BackTranslate" (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide. Examples of PHI-4 polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to, the PHI-4 polypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526. Furthermore, synthetic PHI-4 polynucleotide sequences of the disclosure can be designed so that they will be expressed in plants. US Patent Number 5,500,365 describes a method for synthesizing plant genes to improve the expression level of the protein encoded by the synthesized gene. This method relates to the modification of the structural gene sequences of the exogenous transgene, to cause them to be more efficiently transcribed, processed, translated and expressed by the plant. Features of genes that are expressed well in plants include elimination of sequences that can cause undesired intron splicing or polyadenylation in the coding region of a gene transcript while retaining substantially the amino acid sequence of the toxic portion of the insecticidal protein. A similar method for obtaining enhanced expression of transgenes in monocotyledonous plants is disclosed in US Patent Number 5,689,052.
In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30, SEQ ID NOs: 1163-1505 and variants, fragments and complements thereof. By "complement" is intended a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a nucleic acid molecule having the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30 and SEQ ID NOs: 1 163-1505. The corresponding amino acid sequences for the insecticidal protein encoded by these nucleic acid sequences are set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30 and SEQ ID NOs: 1 163-1505.
In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 35, wherein the polypeptide has pesticidal activity. In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 80% and 95% identity, to the amino acid sequence of SEQ ID NO: 35, wherein the polypeptide has pesticidal activity.
In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% identity, to any one of the amino acid sequence of SEQ ID NOs: 51-1 162 SEQ ID NOs: 1518-1526 wherein the polypeptide has pesticidal activity.
In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position 79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, lie, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa at position 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa at position 87 is Lys or Gin; Xaa at position 88 is Met, lie or Leu; Xaa at position 90 is Gin or Glu; Xaa at position 94 is Val or lie; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie, Met, Phe, Cys, Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 is Leu or lie; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn, Glu, Cys, Ala or Arg; Xaa at position 1 12 is Thr or Ser; Xaa at position 1 13 is Asp, Glu or Met; Xaa at position 1 17 is Thr or Ser; Xaa at position 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa at position 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa at position 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149 is Phe or Val; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaa at position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa at position 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu, Gin, Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg; Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp or Cys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser or Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lys or lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp or Asn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg; Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr or His; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr or Phe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu or Pro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Ser or Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met, Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaa at position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaa at position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 264 is Glu or Gin; Xaa at position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa at position 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292 is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position 297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position 303 is Phe or Tyr; Xaa at position 305 is Lys, Gin, Ala, lie, Met, Asn, Thr or Val; Xaa at position 306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa at position 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn or Gin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin, Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys, lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338 is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala, Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu or Val; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Val or lie; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn or His; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met or Leu; Xaa at position 355 is Val, lie or Leu; Xaa at position 359 is Gly or Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Phe or lie; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu, lie or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385 is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 395 is Asp or Cys; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin, His or Thr; Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Gin or Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie, Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys, Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is lie or Val; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala, Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 is Met or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 is Asp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 is Val or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 is Gin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu, lie, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa at position 452 is Gin, Lys or Glu, Ala; Xaa at position 453 is Asn or Asp; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp, Gin, Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or lie; Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaa at position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaa at position 464 is Arg, Gly, His, Gin, Thr, Phe, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu; Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu or Gin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg, Glu or Gin; Xaa at position 509 is Lys, Gin, Glu or Ala; Xaa at position 517 is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa at position 520 is Lys, Gin, Glu, His or Ala; Xaa at position 525 is Gin or Lys; and Xaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp; and having one or more amino acid substitutions at positions designated as Xaa in SEQ ID NO: 3 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
In some embodiments the nucleic acid molecule encoding a PHI-4 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 4, wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 147 is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or lie; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu or Gin; Xaa at position 166 is Arg or Gin; Xaa at position 171 is Tyr or Phe; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 196 is Gin, Lys, Asn or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is Tyr or Phe; Xaa at position 216 is Glu or Gin; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin or Lys; Xaa at position 257 is Gin or lie; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 278 is Glu or Asn; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 313 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa at position 334 is Gly, Arg, Lys or lie; Xaa at position 336 is Gly or Ala; Xaa at position 338 is Ser, His, Val, Lys or Ala; Xaa at position 339 is Glu, Asn, lie or Pro; Xaa at position 343 is Val or lie; Xaa at position 346 is Pro or Ala; Xaa at position 355 is Val or lie; Xaa at position 359 is Gly or Ala; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly or Thr; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaa at position 403 is Asp or Tyr; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg or Glu; Xaa at position 417 is Ala or Ser; Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu or lie; Xaa at position 452 is Gin, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp or Gin; Xaa at position 455 is Val or lie; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie or Tyr; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala or Ser; Xaa at position 464 is Arg, Gly, His, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 500 is Arg or Gin; Xaa at position 509 is Lys, Gin or Ala; Xaa at position 520 is Lys, Gin, Glu, His or Ala; and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu; and having one or more amino acid substitutions at positions designated as Xaa in SEQ ID NO: 4 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
In some embodiments exemplary nucleic acid molecules encode a PHI-4 of any one of SEQ ID NOs: 51 -1 162 and SEQ ID NOs: 1518-1526 as well as amino acid substitutions, amino acid deletions, insertions and fragments thereof and combinations thereof.
In some embodiments the nucleic acid molecules encode a PHI-4 polypeptide of Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9 and combinations of the amino acid substitutions thereof and amino acid deletions and/or insertions thereof.
Also provided are nucleic acid molecules that encode transcription and/or translation products that are subsequently spliced to ultimately produce functional PHI-4. Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans-splicing. The substrate for splicing can be polynucleotides (e.g., RNA transcripts) or polypeptides. An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a PHI-4 encoding sequence. An example of trans splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full-length pesticidal encoding sequence. The use of a splicing enhancer sequence, which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides (US Patent Numbers 6,365,377 and 6,531 ,316). Thus, in some embodiments the polynucleotides do not directly encode a full-length PHI-4, but rather encode a fragment or fragments of a PHI-4. These polynucleotides can be used to express a functional PHI-4 through a mechanism involving splicing, where splicing can occur at the level of polynucleotide (e.g., intron/exon) and/or polypeptide (e.g., intein/extein). This can be useful, for example, in controlling expression of pesticidal activity, since functional pesticidal polypeptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product. In another example, introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide; use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant.
Nucleic acid molecules that are fragments of these nucleic acid sequences encoding PHI-4 are also encompassed by the embodiments. By "fragment" is intended a portion of the nucleic acid sequence encoding a PHI-4. A fragment of a nucleic acid sequence may encode a biologically active portion of a PHI-4 polypeptide or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a PHI-4 comprise at least about 50, 100, 200, 300, 400, 500, 600 or 700, contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding a PHI-4 disclosed herein, depending upon the intended use. By "contiguous" nucleotides is intended nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the PHI-4 and, hence, retain insecticidal activity. By "retains activity" is intended that the fragment will encode a polypeptide having at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the insecticidal activity of the full-length PHI-4. In one embodiment, the insecticidal activity is Lepodoptera activity. In another embodiment, the insecticidal activity is Hemiptera activity.
In some embodiments a fragment of a nucleic acid sequence encoding a PHI-4 encoding a biologically active portion of a protein will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200 or 250, contiguous amino acids or up to the total number of amino acids present in a full-length PHI-4 of the embodiments. In some embodiments, the fragment is an N-terminal or a C-terminal truncation of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino acids relative to SEQ ID NO: 35, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526 or variants thereof, e.g., by proteolysis, insertion of a start codon, deletion of the codons encoding the deleted amino acids with the concomitant insertion of a stop codon or by insertion of a stop codon in the coding sequence. In some embodiments, the fragments encompassed herein result from the removal of the N-terminal 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or more amino acids relative to SEQ ID NO: 35, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526 or variants thereof, e.g., by proteolysis or by insertion of a start codon in the coding sequence.
In some embodiments the PHI-4 are encoded by a nucleic acid sequence sufficiently identical to the nucleic acid sequence of SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOS: 24-30. By "sufficiently identical" is intended an amino acid or nucleic acid sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. In some embodiments the sequence homology identity is against the full length sequence of the polynucleotide encoding a PHI-4 or against the full length sequence of a PHI-4 polypeptide. In some embodiments the polynucleotide encoding the PHI-4 has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOS: 24-30. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleic acid sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions) x100). In one embodiment, the two sequences are the same length. In another embodiment, the comparison is across the entirety of the reference sequence (e.g., across the entirety of SEQ ID NO: 35 or across the entirety of one of SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOS: 24-30). The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et ai, (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleic acid sequences homologous to pesticidal-like nucleic acid molecules. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to pesticidal protein molecules. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection.
Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins, et al., (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used.
Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software to determine sequence identity or similarity using the following default parameters: % identity and % similarity for a nucleic acid sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmpii scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
The embodiments also encompass nucleic acid molecules encoding variants of
PHI-4 polypeptide. "Variants" of the PHI-4 polypeptide encoding nucleic acid sequences include those sequences that encode the PHI-4 polypeptides disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the PHI-4 polypeptides disclosed as discussed below.
The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequences thereby leading to changes in the amino acid sequence of the encoded PHI-4 polypeptides, without altering the biological activity of the proteins. Thus, variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, nucleotide additions and/or nucleotide deletions into the corresponding nucleic acid sequence disclosed herein, such that one or more amino acid substitutions, amino acid additions or amino acid deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.
Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis and the resultant mutants can be screened for ability to confer pesticidal activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional pesticidal polypeptide homologues and fragments thereof with desired properties. A variety of such reactions are known, including those developed by the inventors and their co-workers. Methods for producing a variant of any nucleic acid listed herein comprising recursively recombining such polynucleotide with a second (or more) polynucleotide, thus forming a library of variant polynucleotides are also embodiments of the disclosure, as are the libraries produced, the cells comprising the libraries, and any recombinant polynucleotide produces by such methods. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on pesticidal activity, as is wherein such recursive recombination is done in vitro or in vivo.
A variety of diversity generating protocols, including nucleic acid recursive recombination protocols are available and fully described in the art. The procedures can be used separately, and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and/or organisms with new and/or improved characteristics.
While distinctions and classifications are made in the course of the ensuing discussion for clarity, it will be appreciated that the techniques are often not mutually exclusive. Indeed, the various methods can be used singly or in combination, in parallel or in series, to access diverse sequence variants.
The result of any of the diversity generating procedures described herein can be the generation of one or more nucleic acids, which can be selected or screened for nucleic acids with or which confer desirable properties or that encode proteins with or which confer desirable properties. Following diversification by one or more of the methods herein or otherwise available to one of skill, any nucleic acids that are produced can be selected for a desired activity or property, e.g. pesticidal activity or such activity at a desired pH, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable format, by any of the assays in the art, see, e.g., discussion of screening of insecticidal activity, infra. A variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner.
Descriptions of a variety of diversity generating procedures for generating modified nucleic acid sequences, e.g., those coding for polypeptides having pesticidal activity or fragments thereof, are found in the following publications and the references cited therein: Soong, et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Targeting 4: 1-4; Ness et al. (1999) Nat Biotechnol 17:893-896; Chang et al. (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999) Curr Opin Chem Biol 3:284-290; Christians, et al. , (1999) Nat Biotechnol 17:259-264; Crameri, et al., (1998) Nature 391 :288-291 ; Crameri, et al., (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA 94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733; Crameri, et al., (1996) Nat Med 2: 100-103; Crameri, et al., (1996) Nat Biotechnol 14:315-319; Gates, et al. , (1996) J Mol Biol 255:373-386; Stemmer, (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, et al. , (1995) Gene, 164:49- 53; Stemmer, (1995) Science 270:1510; Stemmer, (1995) Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91 :10747-10751.
Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2): 157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith, (1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 229:1 193-1201 ; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass, et al., (1988) Science 242:240-245); oligonucleotide-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350; Zoller and Smith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985) NucI Acids Res 13:8749-8764; Taylor, et al. , (1985) NucI Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein (1986) NucI Acids Res 14:9679-9698; Sayers, et al., (1988) NucI Acids Res 16:791-802 and Sayers, et al., (1988) NucI Acids Res 16: 803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984) NucI Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) NucI Acids Res 16:7207 and Fritz, et al. , (1988) NucI Acids Res 16:6987-6999).
Additional suitable methods include point mismatch repair (Kramer, et al. , (1984) Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter, et al., (1985) NucI Acids Res 13:4431 -4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) NucI Acids Res 14:51 15), restriction-selection and restriction-purification (Wells, et al., (1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total gene synthesis (Nambiar, et al. , (1984) Science 223:1299-1301 ; Sakamar and Khorana, (1988) NucI Acids Res 14:6361-6372; Wells, et al. , (1985) Gene 34:315-323 and Grundstrom, et al., (1985) NucI Acids Res 13:3305- 3316), double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455). Additional details on many of the above methods can be found in Methods Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.
Additional details regarding various diversity generating methods can be found in the following US Patents, PCT Publications and Applications and EPO Publications: US Patent Number 5,723,323, US Patent Number 5,763,192, US Patent Number 5,814,476, US Patent Number 5,817,483, US Patent Number 5,824,514, US Patent Number 5,976,862, US Patent Number 5,605,793, US Patent Number 5,81 1 ,238, US Patent Number 5,830,721 , US Patent Number 5,834,252, US Patent Number 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO 1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368, EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO 1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832, WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO 2000/18906, WO 2000/04190, WO 2000/42561 , WO 2000/42559, WO 2000/42560, WO 2001/23401 , and PCT/US01/06775.
The nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from other organisms, particularly other bacteria. In this manner, methods such as PCR, hybridization and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments. Such sequences include sequences that are orthologs of the disclosed sequences. The term "orthologs" refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.
In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook". See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector- specific primers, partially-mismatched primers, and the like.
To identify potential PHI-4 polypeptides from bacterial collections, the bacterial cell lysates can be screened with antibodies generated against a PHI-4 polypeptide using Western blotting and/or ELISA methods. This type of assays can be performed in a high throughput fashion. Positive samples can be further analyzed by various techniques such as antibody based protein purification and identification. Methods of generating antibodies are well known in the art as discussed infra.
Alternatively, mass spectrometry based protein identification method can be used to identify homologs of a PHI-4 polypeptide using protocols in the literatures (Patterson, (1998), 10(22): 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Specifically, LC-MS/MS based protein identification method is used to associate the MS data of given cell lysate or desired molecular weight enriched samples (excised from SDS-PAGE gel of relevant molecular weight bands to a PHI-4 polypeptide) with sequence information of a PHI-4 polypeptide and its homologs. Any match in peptide sequences indicates the potential of having the homologous proteins in the samples. Additional techniques (protein purification and molecular biology) can be used to isolate the protein and identify the sequences of the homologs.
In hybridization methods, all or part of the pesticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001 ), supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides, and may be labeled with a detectable group such as 32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known PHI-4 polypeptide-encoding nucleic acid sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequence encoding a PHI-4 polypeptide of the disclosure or a fragment or variant thereof. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001 ), supra, herein incorporated by reference.
For example, an entire nucleic acid sequence, encoding a PHI-4 polypeptide, disclosed herein or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding nucleic acid sequences encoding PHI-4 polypeptide-like sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length or at least about 20 nucleotides in length. Such probes may be used to amplify corresponding pesticidal sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1 .0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1 % SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1 * to 2*SSC (20xSSC=3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5x to 1 *SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at 37°C, and a wash in O.l xSSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1 % to about 1 % SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: Tm=81.5°C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)- 500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 °C for each 1 % of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with≥90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1 , 2, 3 or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 1 1 , 12, 13, 14, 15 or 20°C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel, et al. , eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley- Interscience, New York). See, Sambrook, et al. , (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Proteins and Variants and Fragments Thereof
PHI-4 polypeptides are encompassed by the disclosure. By "PHI-4 polypeptide" or "PHI-4 protein" as used herein interchangeably is intended a polypeptide that has increased insecticidal activity against one or more insect pests of the Lepidoptera and/or Coleoptera orders compared to the protein of SEQ ID NO: 35, and is sufficiently identical to the protein of SEQ ID NO: 35. A variety of PHI-4 polypeptides are contemplated.
The Western Corn Rootworm active protein AXMI-205 (SEQ ID NO: 35) encoded by the polynucleotide of SEQ ID NO: 34 was identified from the Chromobacterium Strain ATX 2024 (US201 10023184). Synthetic genes encoding AXMI-205; AXMI-205 variants having a truncation of the last 10 and 20 amino acids from the C-terminus (SEQ ID NO: 36 and SEQ ID NO: 37); and alanine scanning at every other residue from residue 307- 536 of AXMI-205 (SEQ ID NO: 35), with S307A, D315A, V317A, S349A, G351A, K353A, V355A, D395A, G399A, W407A, G419A, P435A, S443A, K465A, V467A, F483A, P487A, S495A, D497A, E499A, K509A, and 1513A identified as having WCRW activity; are disclosed in US201 10023184. AXMI-205 variants evo 24 (E499A); evo25 (V467A - SEQ ID NO: 41 ); evo30 (V467L - SEQ ID NO: 42); PMIibl PoollG2_p2al 1 (S468L - SEQ ID NO: 45); PMIibl PoollG2_plcl (V467T - SEQ ID NO: 46), PMIibl PoollG2_pla4 (R464N - SEQ ID NO: 47), evo34 (E86T - SEQ ID NO: 43) and evo35 (Q517R - SEQ ID NO: 44) having increased insecticidal activity are disclosed in WO2013/016617.
As used herein, the terms "protein," "peptide molecule" or "polypeptide" includes any molecule that comprises five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules may undergo modification, including post- translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation or oligomerization. Thus, as used herein, the terms "protein," "peptide molecule" or "polypeptide" includes any protein that is modified by any biological or non-biological process. The terms "amino acid" and "amino acids" refer to all naturally occurring L-amino acids.
A "recombinant protein" is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. A PHI-4 polypeptide that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10% or 5% (by dry weight) of non-pesticidal protein (also referred to herein as a "contaminating protein").
By "improved activity" or "increased activity" is intended an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210% at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or higher or at least about 1.1 -fold, at least about 1 .2- fold, at least about 1.3-fold, at least about 1.4-fold or at least about 1.5-fold, at least about 1.6-fold, at least about 1 .7-fold, at least about 1 .8-fold, at least about 1 .9-fold, at least about 2-fold, at least about 2.1 -fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3 -fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5- fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at least about 4-fold, at least about 4.1-fold, at least about 4.2-fold, at least about
4.3- fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5-fold, at least about 5.1-fold, at least about 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, at least about 5.5-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.1-fold, at least about 6.2-fold, at least about 6.3-fold, at least about 6.4-fold, at least about 6.5-fold, at least about 6.6- fold, at least about 6.7-fold, at least about 6.8-fold, at least about 6.9-fold, at least about 7-fold, at least about 7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at least about
7.4- fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1 -fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at least about 9.7- fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or higher increase in the pesticidal activity of the variant protein relative to the pesticidal activity of AXMI-205 (SEQ ID NO: 35).
In some embodiments, the improvement consists of a decrease in the EC50 of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210% at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or higher or at least about 1.1 -fold, at least about 1 .2- fold, at least about 1.3-fold, at least about 1.4-fold or at least about 1.5-fold, at least about 1.6-fold, at least about 1 .7-fold, at least about 1 .8-fold, at least about 1 .9-fold, at least about 2-fold, at least about 2.1 -fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3 -fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5- fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at least about 4-fold, at least about 4.1-fold, at least about 4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5-fold, at least about 5.1-fold, at least about 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, at least about 5.5-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.1-fold, at least about 6.2-fold, at least about 6.3-fold, at least about 6.4-fold, at least about 6.5-fold, at least about 6.6- fold, at least about 6.7-fold, at least about 6.8-fold, at least about 6.9-fold, at least about 7-fold, at least about 7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at least about 7.4-fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1 -fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at least about 9.7- fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or greater reduction in the EC50 of the PHI-4 polypeptide relative to the pesticidal activity of AXMI- 205 (SEQ ID NO: 35).
In some embodiments the EC50 of the PHI-4 polypeptide is <100 ppm, <90 ppm, <80 ppm, <70 ppm, <60 ppm, <50 ppm, <45 ppm, <40 ppm, <35 ppm, <30 ppm, <25 ppm, <20 ppm, <19 ppm, <18 ppm, <17 ppm, <16 ppm, <15 ppm, <14 ppm, <13 ppm, <12 ppm, <1 1 ppm, <10 ppm, <9 ppm, <8 ppm, <7 ppm, <6 ppm, <5 ppm, <4 ppm, <3 ppm, <2 ppm, <1 ppm, <0.9 ppm, <0.8 ppm, <0.7 ppm, <0.6 ppm, <0.5 ppm, <0.4 ppm, <0.3 ppm, <0.2 ppm or <0.1 ppm.
In some embodiments, the improvement consists of an increase in the Mean FAE Index of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about
100%, at least about 1 10%, at least about 120%, at least about 130%, at least about
140%, at least about 150%, at least about 160%, at least about 170%, at least about
180%, at least about 190%, at least about 200%, at least about 210% at least about
220%, at least about 230%, at least about 240%, at least about 250%, at least about
260%, at least about 270%, at least about 280%, at least about 290%, at least about
300%, at least about 310%, at least about 320%, at least about 330%, at least about
340%, at least about 350%, at least about 360%, at least about 370%, at least about
380%, at least about 390%, at least about 400%, at least about 410%, at least about
420%, at least about 430%, at least about 440%, at least about 450%, at least about
460%, at least about 470%, at least about 480%, at least about 490%, at least about
500%, at least about 510%, at least about 520%, at least about 530%, at least about
540%, at least about 550%, at least about 560%, at least about 570%, at least about
580%, at least about 590%, at least about 600%, at least about 650%, at least about
700%, at least about 750%, at least about 800%, at least about 850%, at least about
900%, at least about 950%, at least about 1000% or higher or at least about 1 .1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold or at least about 1.5- fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3 -fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8- fold, at least about 3.9-fold, at least about 4-fold, at least about 4.1 -fold, at least about 4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5-fold, at least about 5.1 -fold, at least about 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, at least about 5.5-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.1-fold, at least about 6.2-fold, at least about 6.3-fold, at least about 6.4-fold, at least about 6.5- fold, at least about 6.6-fold, at least about 6.7-fold, at least about 6.8-fold, at least about 6.9-fold, at least about 7-fold, at least about 7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at least about 7.4-fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6- fold, at least about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or higher increase in the Mean FAE Index of the PHI-4 polypeptide relative to the pesticidal activity of AXMI-205 (SEQ ID NO: 35).
In some embodiments, the improvement consists of an increase in the Mean Deviation Score of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210% at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or higher or at least about 1.1 - fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1 .4-fold or at least about 1.5-fold, at least about 1 .6-fold, at least about 1 .7-fold, at least about 1 .8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3 -fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4- fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at least about 4-fold, at least about 4.1 -fold, at least about
4.2- fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5-fold, at least about 5.1 -fold, at least about 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, at least about 5.5-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.1-fold, at least about 6.2-fold, at least about 6.3-fold, at least about 6.4-fold, at least about 6.5- fold, at least about 6.6-fold, at least about 6.7-fold, at least about 6.8-fold, at least about 6.9-fold, at least about 7-fold, at least about 7.1-fold, at least about 7.2-fold, at least about
7.3- fold, at least about 7.4-fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6- fold, at least about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or higher increase in the Mean Deviation Score of the PHI-4 polypeptide relative to the pesticidal activity of AXMI-205 (SEQ ID NO: 35).
In some embodiments the improved activity of the PHI-4 polypeptide is relative to the pesticidal activity of AXMI-205(evo25) (SEQ ID NO: 41 ), AXMI-205(evo30) (SEQ ID NO: 42), Axmi205 PMIibl PoollG2_p2al 1 (mutation S468L; SEQ ID NO: 45), Axmi205 PMIibl PoollG2_plcl (mutation V467T; SEQ ID NO: 46), Axmi205 PMIibl PoollG2_pla4 (mutation R464N; SEQ ID NO: 47), AXMI-205(evo34) (SEQ ID NO: 43) or AXMI- 205(evo35) (SEQ ID NO: 44).
"Mean FAE Index" (MFI) refers to the mean of multiple FAEGN an arithmetic mean of FAEGN. As used herein, the "Mean Deviation Score" refers to the arithmetic mean of multiple Deviation Scores.
In some embodiments the PHI-4 polypeptides have increased insecticidal activity against one or more insect pests of the order Coleoptera. In some embodiments the PHI-4 polypeptides have increased insecticidal activity against one or more insect pests of the corn rootworm complex (western corn rootworm, Diabrotica virgifera; northern corn rootworm, D. barberi: and the Mexican corn rootworm, D. virgifera zeae.
In some embodiments the PHI-4 polypeptides have increased insecticidal activity against Diabrotica virgifera larvae - Western Corn Root Worm (WCRW).
"Fragments" or "biologically active portions" include polypeptide fragments comprising amino acid sequences sufficiently identical to a PHI-4 polypeptide and that exhibit insecticidal activity. "Fragments" or "biologically active portions" include polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 and that exhibit insecticidal activity. A biologically active portion of a PHI-4 polypeptide can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 516, 517, 518, 519, 520, 521 , 522, 523, 524, 525, 526, 527, 528, 529, 530, 531 , 532, 533, 534 or 535 amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for insecticidal activity. As used here, a fragment comprises at least 8 contiguous amino acids of a PHI-4 polypeptide. In some embodiments a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 2, SEQ ID NOs: 51 -1 162, or SEQ ID NOs: 1518-1526. In some embodiments a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NOs: 51-1 162, or SEQ ID NOs: 1518-1526. The embodiments encompass other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250 or more amino acids.
In some embodiments, the fragment is an N-terminal and/or a C-terminal truncation of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 35, SEQ ID NOs: 51- 1 162, SEQ ID NOs: 1518-1526 or variants thereof e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon and/or insertion of a stop codon. In some embodiments, the fragments encompassed herein result from the removal of the C-terminal 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 amino acids relative to SEQ ID NO: 35, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526, and variants thereof by proteolysis or by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon. In particular embodiments the proteolytic cleavage site is between Lys at 520 and Ser at 521 Ser or Lys at 313 and Val at 314 of SEQ ID NO: 35 or variants thereof. It is well known in the art that polynucleotide encoding the truncated PHI-4 polypeptide can be engineered to add a start codon at the N-terminus such as ATG encoding methionine or methionine followed by an alanine. It is also well known in the art that depending on what host the PHI-4 polypeptide is expressed in the methionine may be partially of completed processed off.
In some embodiments fragments, biologically active portions, of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, SEQ ID NOs: 1518-1526 as well as amino acid substitutions, amino acid deletions and/or insertions thereof are also provided, and may be used to practice the methods of the disclosure.
In some embodiments PHI-4 polypeptides are provided having one or more amino acid substitution compared to AXMI-205 (SEQ ID NO: 35). In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at solvent exposed surface residues to modify the protein characteristics of AXMI-205 (SEQ ID NO: 35), including but not limited to the ionic polarity of the protein surface. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at hydrophilic residues such as Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys. In some embodiments the PHI-4 polypeptides are provided having amino acid substitutions changing a Lysine or Arginine to a Glutamine, Glutamic Acid, Asparagine or Glutamic Acid; changing a Glutamic Acid or Aspartic Acid to a Lysine, Asparagine or Glutamine; and changing a Glutamine to a Asparagine or Lysine.
In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues in a membrane insertion loop. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions in a membrane insertion loop between about amino acid at position 92 (Val) and 101 (Ala) and/or at position 21 1 (Gly) and 220 (Glu) relative to SEQ ID NO: 35.
In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues and receptor binding loops. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues and receptor binding loops between about amino acid 332 (Asp) and 340 (Asp), 395 (Asp) and 403 (Asp) , 458 (Asp) and 466 (Asp) relative to SEQ ID NO: 35.
In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues in a protease sensitive region. In some embodiments PHI-4 polypeptides are provided having amino acid substitutions at residues in a protease sensitive region from about amino acid residues between 305 (Lys) and 316 (Lys) and 500 (Arg) and 535 (Lys) relative to SEQ ID NO: 35. By variants is intended proteins or polypeptides having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the parental amino acid sequence. In some embodiments a PHI-4 polypeptide has at least about 60%, 65%, about 70%, 75%, at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater identity across the entire length of the amino acid sequence of SEQ ID NO: 2, SEQ ID NOs: 51 -1 162, SEQ ID NOs: 1518-1526. In some embodiments a PHI-4 polypeptide has at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater identity across the entire length of the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NOs: 51- 1 162, or SEQ ID NOs: 1518-1526.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence having at least 80% identity, to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162 or SEQ ID NO: 1518-1526, wherein the polypeptide has insecticidal activity.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162 or SEQ ID NO: 1518-1526, wherein the polypeptide has insecticidal activity.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162 or SEQ ID NO: 1518-1526 wherein the polypeptide has insecticidal activity.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence having at least 97% identity to the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162 or SEQ ID NO: 1518-1526, wherein the polypeptide has insecticidal activity.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 3, wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie or Val; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position 79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, lie, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa at position 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa at position 87 is Lys or Gin; Xaa at position 88 is Met, lie or Leu; Xaa at position 90 is Gin or Glu; Xaa at position 94 is Val or lie; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie, Met, Phe, Cys, Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 is Leu or lie; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn, Glu, Cys, Ala or Arg; Xaa at position 1 12 is Thr or Ser; Xaa at position 1 13 is Asp, Glu or Met; Xaa at position 1 17 is Thr or Ser; Xaa at position 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa at position 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa at position 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149 is Phe or Val; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaa at position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa at position 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu, Gin, Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg; Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp or Cys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser or Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lys or lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp or Asn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg; Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr or His; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr or Phe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu or Pro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Ser or Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met, Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaa at position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaa at position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 264 is Glu or Gin; Xaa at position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa at position 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292 is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position 297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position 303 is Phe or Tyr; Xaa at position 305 is Lys, Gin, Ala, lie, Met, Asn, Thr or Val; Xaa at position 306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa at position 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn or Gin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin, Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys, lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338 is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala, Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu or Val; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Val or lie; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn or His; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met or Leu; Xaa at position 355 is Val, lie or Leu; Xaa at position 359 is Gly or Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Phe or lie; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu, lie or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385 is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 395 is Asp or Cys; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin, His or Thr; Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Gin or Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie, Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys, Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is lie or Val; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala, Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 is Met or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 is Asp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 is Val or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 is Gin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu, lie, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa at position 452 is Gin, Lys or Glu, Ala; Xaa at position 453 is Asn or Asp; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp, Gin, Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or lie; Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaa at position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaa at position 464 is Arg, Gly, His, Gin, Thr, Phe, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu; Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu or Gin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg, Glu or Gin; Xaa at position 509 is Lys, Gin, Glu or Ala; Xaa at position 517 is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa at position 520 is Lys, Gin, Glu, His or Ala; Xaa at position 525 is Gin or Lys; and Xaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp; and having one or more amino acid substitutions at positions designated as Xaa in SEQ ID NO: 3; and amino acid substitutions, deletions, insertions, and combinations thereof and fragments thereof; and wherein the PHI-4 polypeptide has increased insecticidal activity compared to SEQ ID NO: 2.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 4 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 4 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence of
SEQ ID NO: 4 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 4 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 4.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 4, wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu or Tyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser, lie, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met, Val or Leu; Xaa at position 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 147 is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaa at position 150 is Arg, Gin, Glu or Asn; Xaa at position 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or lie; Xaa at position 162 is Glu, Asp, Gin, Asn or Leu; Xaa at position 165 is Lys, Glu or Gin; Xaa at position 166 is Arg or Gin; Xaa at position 171 is Tyr or Phe; Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser; Xaa at position 182 is Asp or Gin; Xaa at position 196 is Gin, Lys, Asn or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is Tyr or Phe; Xaa at position 216 is Glu or Gin; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin or Lys; Xaa at position 257 is Gin or lie; Xaa at position 261 is Gin, Glu, Lys or Ala; Xaa at position 278 is Glu or Asn; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 313 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa at position 334 is Gly, Arg, Lys or lie; Xaa at position 336 is Gly or Ala; Xaa at position 338 is Ser, His, Val, Lys or Ala; Xaa at position 339 is Glu, Asn, lie or Pro; Xaa at position 343 is Val or lie; Xaa at position 346 is Pro or Ala; Xaa at position 355 is Val or lie; Xaa at position 359 is Gly or Ala; Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His; Xaa at position 396 is Ala, Leu, Lys, Asn, Gly or Thr; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaa at position 403 is Asp or Tyr; Xaa at position 41 1 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg or Glu; Xaa at position 417 is Ala or Ser; Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu or lie; Xaa at position 452 is Gin, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp or Gin; Xaa at position 455 is Val or lie; Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie or Tyr; Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala or Ser; Xaa at position 464 is Arg, Gly, His, Ala, Asp, Ser or Lys; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly or Arg; Xaa at position 471 is Gin, Lys, Glu or Met; Xaa at position 472 is Pro or Ser; Xaa at position 500 is Arg or Gin; Xaa at position 509 is Lys, Gin or Ala; Xaa at position 520 is Lys, Gin, Glu, His or Ala; and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu; and having one or more amino acid substitutions at positions designated as Xaa in SEQ ID NO: 4; and amino acid substitutions, deletions, insertions, and combinations thereof and fragments thereof; and wherein the PHI-4 polypeptide has increased insecticidal activity compared to SEQ ID NO: 2.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75 or 76 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 3 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
In some embodiments PHI-4 polypeptide comprises an amino acid sequence of
SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53 or 54 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 3 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
In some embodiments PHI-4 polypeptide comprises an amino acid sequence of SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 1 1 , 12, 13, 14 or 15 amino acid substitutions, in any combination, at residues designated by Xaa in SEQ ID NO: 3 compared to the native amino acid at the corresponding position of SEQ ID NO: 2.
In some embodiments a PHI-4 polypeptide comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 3.
In some embodiments a PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
In some embodiments a PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35 wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acid at position 99 is Lys or Leu; the amino acid at position 145 is Leu or Val; the amino acid at position 150 is Arg or Gin; the amino acid at position 151 is Asp or Ser; the amino acid at position 153 is Leu or lie; the amino acid at position 163 is Leu or Val; the amino acid at position 171 is Tyr or Phe; the amino acid at position 172 is lie or Leu; the amino acid at position 182 is Asp or Gin; the amino acid at position 196 is Gin or Asn; the amino acid at position 206 is Tyr or Phe; the amino acid at position 210 is Val or lie; the amino acid at position 216 is Glu or Gin; the amino acid at position 220 is Glu, Gin, His or Asp; the amino acid at position 278 is Glu or Asn; the amino acid at position 283 is lie or Val; the amino acid at position 289 is Lys, Gin or Leu; the amino acid at position 293 is Arg, Gin or Glu; the amino acid at position 328 is Lys or Glu; the amino acid at position 333 is Ser, Lys or Val; the amino acid at position 334 is Gly, Lys or Arg; the amino acid at position 336 is Gly or Ala; the amino acid at position 338 is Ser or Val; the amino acid at position 339 is Glu, Asn or Gin; the amino acid at position 342 is Ala or Ser; the amino acid at position 346 is Pro or Ala; the amino acid at position 354 is Met or Leu; the amino acid at position 355 is Val or lie; the amino acid at position 370 is His or Arg; the amino acid at position 389 is Trp or Leu; the amino acid at position 393 is Trp or Leu; the amino acid at position 396 is Ala, Leu, Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly, Lys or Pro; the amino acid at position 402 is Lys, His, Gly or Trp; the amino acid at position 403 is Asp or Tyr; the amino acid at position 410 is lie or Val; the amino acid at position 412 is Pro or Ala; the amino acid at position 416 is Arg or Glu; the amino acid at position 417 is Ala or Ser; the amino acid at position 426 is Thr or Ser; the amino acid at position 442 is Gin or Glu; the amino acid at position 447 is Asp or Lys; the amino acid at position 452 is Gin or Lys; the amino acid at position 454 is Arg or Gin; the amino acid at position 455 is Val or lie; the amino acid at position 457 is Trp or Asn; the amino acid at position 461 is Thr or Ser; the amino acid at position 462 is Gly or Ala; the amino acid at position 500 is Arg or Gin; the amino acid at position 509 is Lys or Gin; the amino acid at position 520 is Lys, Glu or Gin; and the amino acid at position 527 is Gin or Lys., and amino acid deletions, insertions, and combinations thereof and fragments thereof.
In some embodiments the PHI-4 polypeptide comprising one or more amino acid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468, 499 or 517.
In some embodiments the PHI-4 polypeptide comprising one or more amino acid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468, 499 or 517, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 399 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
In some embodiments exemplary PHI-4 polypeptides are encoded by the polynucleotide sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30, SEQ ID NOs. 1 163-1505, and SEQ ID NOs. 1527-1535.
In some embodiments a PHI-4 polypeptide includes variants where an amino acid that is part of a proteolytic cleavage site is changed to another amino acid to eliminate or alter the proteolytic cleavage at that site. In some embodiments the proteolytic cleavage is by a protease in the insect gut. In other embodiments the proteolytic cleavage is by a plant protease in the transgenic plant.
In some embodiments exemplary PHI-4 polypeptides are the polypeptides shown in Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, and Table 9 and combinations of the amino substitutions thereof as well as amino acid deletions, and or insertions and fragments thereof.
In some embodiments the PHI-4 polypeptide is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid molecule of SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 1 1 , SEQ ID NOs: 24-30, SEQ ID NOs. 1 163-1505, and SEQ ID NOs. 1527-1535. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the disclosure are biologically active, that is they continue to possess the desired biological activity (i.e. pesticidal activity) of the native protein. By "retains activity" is intended that the variant will have at least about 30%, at least about 50%, at least about 70% or at least about 80% of the insecticidal activity of the native protein. In some embodiments, the variants may have improved activity over the native protein.
Bacterial genes quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can initiate at a TTG codon, though in this event the TTG encodes a methionine. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that use of one of the alternate methionine codons may also lead to generation of pesticidal proteins. These pesticidal proteins are encompassed in the present disclosure and may be used in the methods of the present disclosure. It will be understood that, when expressed in plants, it will be necessary to alter the alternate start codon to ATG for proper translation.
In another aspect the PHI-4 polypeptide may be expressed as a precursor protein with an intervening sequence that catalyzes multi-step, post translational protein splicing. Protein splicing involves the excision of an intervening sequence from a polypeptide with the concomitant joining of the flanking sequences to yield a new polypeptide (Chong, et al., (1996) J. Biol. Chem. 271 :22159-22168). This intervening sequence or protein splicing element, referred to as inteins, which catalyze their own excision through three coordinated reactions at the N-terminal and C-terminal splice junctions: an acyl rearrangement of the N-terminal cysteine or Serine; a transesterfication reaction between the two termini to form a branched ester or thioester intermediate and peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein (Evans, et al., (2000) J. Biol. Chem. 275:9091-9094. The elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., US Patent Number 5,496,714; Comb, et al., US Patent Number 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc. 121 :5597-5598; Chong, et al., (1997) Gene 192:271-281 , Chong, et al., (1998) Nucleic Acids Res. 26:5109-51 15; Chong, et al., (1998) J. Biol. Chem. 273:10567-10577; Cotton, et al., (1999) J. Am. Chem. Soc. 121 :1 100-1 101 ; Evans, et al., (1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459:166-172; Mathys, et al., (1999) Gene 231 :1 -13; Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543- 3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. Biolmol. NMR 14:105-1 14; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem. 273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, et al., (1999) Biotechniques 27:1 10-120; Wood, et al., (1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231 ; Wu, et al., (1998b) Biochim Biophys Acta 1387:422- 432; Xu, et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al., (1998) J. Am. Chem. Soc. 120:5591 -5592). For the application of inteins in plant transgenes see Yang, J, et al., {Transgene Res 15:583-593 (2006)) and Evans, et al., {Annu. Rev. Plant Biol. 56:375-392, (2005)).
In another aspect the PHI-4 polypeptide may be encoded by two separate genes where the intein of the precursor protein comes from the two genes, referred to as a split- intein and the two portions of the precursor are joined by a peptide bond formation. This peptide bond formation is accomplished by intein-mediated trans-splicing. For this purpose, a first and a second expression cassette comprising the two separate genes further code for inteins capable of mediating protein trans-splicing. By trans-splicing, the proteins and polypeptides encoded by the first and second fragments may be linked by peptide bond formation. Trans-splicing inteins may be selected from the nucleolar and organellar genomes of different organisms including eukaryotes, archaebacteria and eubacteria. Inteins that may be used for are listed at neb.com/neb/inteins.html, which can be accessed on the world-wide web using the "www" prefix). The nucleotide sequence coding for an intein may be split into a 5' and a 3' part that code for the 5' and the 3' part of the intein, respectively. Sequence portions not necessary for intein splicing (e.g., homing endonuclease domain) may be deleted. The intein coding sequence is split such that the 5' and the 3' parts are capable of trans-splicing. For selecting a suitable splitting site of the intein coding sequence, the considerations published by Southworth, et al., (1998) EMBO J. 17:918-926 may be followed. In constructing the first and the second expression cassette, the 5' intein coding sequence is linked to the 3' end of the first fragment coding for the N-terminal part of the PHI-4 polypeptide and the 3' intein coding sequence is linked to the 5' end of the second fragment coding for the C-terminal part of the PHI-4 polypeptide.
In general, the trans-splicing partners can be designed using any split intein, including any naturally-occurring or artificially-split split intein. Several naturally-occurring split inteins are known, for example: the split intein of the DnaE gene of Synechocystis sp. PCC6803 (see, Wu, et al., (1998) Proc Natl Acad Sci USA 95(16):9226-31 and Evans, et al., (2000) J Biol Chem 275(13):9091-4 and of the DnaE gene from Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett 580(7): 1853-8). Non-split inteins have been artificially split in the laboratory to create new split inteins, for example: the artificially split Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta 1387:422-32) and split See VMA intein (see, Brenzel, et al., (2006) Biochemistry 45(6): 1571 -8) and an artificially split fungal mini-intein (see, Elleuche, et al., (2007) Biochem Biophys Res Commun 355(3):830-4). There are also intein databases available that catalogue known inteins (see, for example the online-database available at: bioinformatics.weizmann.ac.il/~pietro/inteins/lnteinstable.html, which can be accessed on the world-wide web using the "www" prefix).
Naturally-occurring non-split inteins may have endonuclease or other enzymatic activities that can typically be removed when designing an artificially-split split intein. Such mini-inteins or minimized split inteins are well known in the art and are typically less than 200 amino acid residues long (see, Wu, et al., (1998) Biochim Biophys Acta 1387:422-32). Suitable split inteins may have other purification enabling polypeptide elements added to their structure, provided that such elements do not inhibit the splicing of the split intein or are added in a manner that allows them to be removed prior to splicing. Protein splicing has been reported using proteins that comprise bacterial intein- like (BIL) domains (see, Amitai, et al., (2003) Mol Microbiol 47:61 -73) and hedgehog (Hog) auto-processing domains (the latter is combined with inteins when referred to as the Hog/intein superfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem. 279 32001- 7) and domains such as these may also be used to prepare artificially-split inteins. In particular, non-splicing members of such families may be modified by molecular biology methodologies to introduce or restore splicing activity in such related species. Recent studies demonstrate that splicing can be observed when a N-terminal split intein component is allowed to react with a C-terminal split intein component not found in nature to be its "partner"; for example, splicing has been observed utilizing partners that have as little as 30 to 50% homology with the "natural" splicing partner (see, Dassa, et al., (2007) Biochemistry 46(1 ):322-30). Other such mixtures of disparate split intein partners have been shown to be unreactive one with another (see, Brenzel, et al. , 2006 Biochemistry 45(6): 1571 -8). However, it is within the ability of a person skilled in the relevant art to determine whether a particular pair of polypeptides is able to associate with each other to provide a functional intein, using routine methods and without the exercise of inventive skill.
In another aspect the PHI-4 polypeptide is a circular permuted variant. In certain embodiments the PHI-4 polypeptide is a circular permuted variant of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526. The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and Erfle, (1990) J. Bacteriol. 172:3837-3841 ; Schimming, et al. , (1992) Eur. J. Biochem. 204:13-19; Yamiuchi and Minamikawa, (1991 ) FEBS Lett. 260:127-130; MacGregor, et al. , (1996) FEBS Lett. 378:263-266). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). In creating a circular permuted variant a new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C- terminus of the chain. The length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. 20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157:105-132; solvent exposed surface area, Lee and Richards, (1971 ) J. Mol. Biol. 55:379-400) and the ability to adopt the necessary conformation without deranging the configuration of the pesticidal polypeptide (conformationally flexible; Karplus and Schulz, (1985) Naturwissenschaften 72:212-213. Assuming an average of translation of 2.0 to 3.8 A per residue, this would mean the length to test would be between 0 to 30 residues, with 0 to 15 residues being the preferred range. Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated n times, where n is 1 , 2, 3 or 4. Those skilled in the art will recognize that there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short (cf., Sandhu, (1992) Critical Rev. Biotech. 12:437-462); if they are too long, entropy effects will likely destabilize the three- dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain. Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used or at least to limit the number of possibilities that must be tested in an empirical selection of linkers. They will also recognize that it is sometimes the case that the positions of the ends of the polypeptide chain are ill-defined in structural models derived from x-ray diffraction or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their c-alpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8 A per residue) are then selected. These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly- Gly-Ser cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used. Sequences of pesticidal polypeptides capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above. Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, those skilled in the art will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to either amino acid deletions or additions to the amino or carboxyl portions of the new sequence. It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch and Sander, (1983) Biopolymers 22:2577-2637; the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol. 154:51 1 -533). In some cases additional information is known about solvent exposure of residues; one example is a site of post-translational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available or is not feasible to obtain, methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops. Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile and Salvatore, (1993) Eur. J. Biochem. 218:603- 621 ). Thus using either the experimentally derived structural information or predictive methods (e.g., Srinivisan and Rose, (1995) Proteins: Struct, Funct. & Genetics 22:81-99) the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occurrence of sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. In contrast, those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region. Polynucleotides encoding circular permuted PHI-4 polypeptides with new N-terminus/C-terminus which contain a linker region separating the original C-terminus and N-terminus can be made essentially following the method described in Mullins, et al., (1994) J. Am. Chem. Soc. 1 16:5529-5533. Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. Polynucleotides encoding circular permuted PHI-4 polypeptides with new N- terminus/C-terminus which contain a linker region separating the original C-terminus and N-terminus can be made based on the tandem-duplication method described in Horlick, et al. , (1992) Protein Eng. 5:427-431. Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA.
In another aspect fusion proteins are provided that include within its amino acid sequence an amino acid sequence comprising a PHI-4 polypeptide including but not limited to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162, and SEQ ID NOs: 1518-1526 and active fragments thereof.
In another aspect fusion proteins are provided comprising a PHI-4 polypeptide and a second pesticidal polypeptide such a Cry protein. Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. Polynucleotides encoding a PHI-4 polypeptide may be fused to signal sequences which will direct the localization of the PHI-4 polypeptide to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of the PHI-4 polypeptide of the embodiments from a prokaryotic or eukaryotic cell. For example, in E. coli, one may wish to direct the expression of the protein to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the PHI-4 polypeptide may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase. Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs. In a specific embodiment, the PHI-4 polypeptide may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria (see, US Patent Numbers 5,576,195 and 5,846,818). Plant plastid transit peptide/polypeptide fusions are well known in the art (see, US Patent Number 7,193,133). Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also well known in the art. The plastid transit peptide is generally fused N-terminal to the polypeptide to be targeted (e.g., the fusion partner). In one embodiment, the fusion protein consists essentially of the peptide transit plastid and the PHI-4 polypeptide to be targeted. In another embodiment, the fusion protein comprises the peptide transit plastid and the polypeptide to be targeted. In such embodiments, the plastid transit peptide is preferably at the N-terminus of the fusion protein. However, additional amino acid residues may be N-terminal to the plastid transit peptide providing that the fusion protein is at least partially targeted to a plastid. In a specific embodiment, the plastid transit peptide is in the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein. Most or all of the plastid transit peptide is generally cleaved from the fusion protein upon insertion into the plastid. The position of cleavage may vary slightly between plant species, at different plant developmental stages, as a result of specific intercellular conditions or the particular combination of transit peptide/fusion partner used. In one embodiment, the plastid transit peptide cleavage is homogenous such that the cleavage site is identical in a population of fusion proteins. In another embodiment, the plastid transit peptide is not homogenous, such that the cleavage site varies by 1-10 amino acids in a population of fusion proteins. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at a position corresponding to its C-terminal end and the same or a compatible site can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminal end. Care must be taken in designing these sites to ensure that the coding sequences of the transit peptide and the second protein are kept "in frame" to allow the synthesis of the desired fusion protein. In some cases, it may be preferable to remove the initiator methionine codon of the second protein when the new restriction site is introduced. The introduction of restriction endonuclease recognition sites on both parent molecules and their subsequent joining through recombinant DNA techniques may result in the addition of one or more extra amino acids between the transit peptide and the second protein. This generally does not affect targeting activity as long as the transit peptide cleavage site remains accessible and the function of the second protein is not altered by the addition of these extra amino acids at its N-terminus. Alternatively, one skilled in the art can create a precise cleavage site between the transit peptide and the second protein (with or without its initiator methionine) using gene synthesis (Stemmer, et a/. , (1995) Gene 164:49-53) or similar methods. In addition, the transit peptide fusion can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of the mature protein can affect the ability of the transit peptide to target proteins to plastids and/or the efficiency of cleavage following protein import. This may be dependent on the protein to be targeted. See, e.g., Comai, et al. , (1988) J. Biol. Chem. 263(29):15104-9.
In some embodiments fusion proteins are provide comprising a PHI-4 polypeptide, a pesticidal protein such as a cry protein, and an amino acid linker.
In some embodiments fusion proteins are provided represented by a formula selected from the group consisting of
R1-L-R2, R2-L- R1, R1- R2 or R2- R1
where R1 is a PHI-4 polypeptide, R2 is a pesticidal protein with a different but complementary activity to the PHI-4 polypeptide, including but not limited to cry proteins; a polypeptide that increases the solubility and/or stability of the PHI-4 polypeptide; or a transit peptide or leader sequence. The R1 polypeptide is fused either directly or through a linker segment to the R2 polypeptide. The term "directly" defines fusions in which the polypeptides are joined without a peptide linker. Thus L represents a chemical bound or polypeptide segment to which both R1 and R2 are fused in frame, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and carboxy terminus of L to the amino terminus of R2. By "fused in frame" is meant that there is no translation termination or disruption between the reading frames of R1 and R2. The linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers joining the two molecules are preferably designed to (1 ) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, (3) have minimal hydrophobic or charged characteristic which could interact with the functional protein domains and (4) provide steric separation of R1 and R2 such that R1 and R2 could interact simultaneously with their corresponding receptors on a single cell. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions.
In some embodiments the linkers comprise sequences selected from the group of formulas: (Gly3Ser)n, (Gly4Ser)n, (Gly5Ser)n, (GlynSer)n or (AlaGlySer)n where n is an integer. One example of a highly-flexible linker is the (GlySer)-rich spacer region present within the pill protein of the filamentous bacteriophages, e.g., bacteriophages M13 or fd (Schaller, et al., 1975). This region provides a long, flexible spacer region between two domains of the pill surface protein. Also included are linkers in which an endopeptidase recognition sequence is included. Such a cleavage site may be valuable to separate the individual components of the fusion to determine if they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue Plasminogen activator, clostripain, Chymosin, Collagenase, Russell's Viper Venom Protease, Postproline cleavage enzyme, V8 protease, Thrombin and factor Xa. In some embodiments the linker comprises the amino acids EEKKN from the multi-gene expression vehicle (MGEV), which is cleaved by vacuolar proteases as disclosed in US 2007/0277263. In other embodiments, peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. Preferred linkers of the present disclosure include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere adversely with the folding and function of the individual molecules of the fusion. In another embodiment chimeric PHI-4 polypeptide are provided that are created through joining two or more portions of genes, which originally encoded separate insecticidal proteins from different species, to create a chimeric gene. The translation of the chimeric gene results in a single chimeric pesticidal polypeptide with regions, motifs or domains derived from each of the original polypeptides. In certain embodiments the chimeric protein comprises portions, motifs or domains of PHI-4 polypeptides in any combination. In certain embodiments the chimeric insecticidal polypeptide includes but not limited to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526.
It is recognized that DNA sequences may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by the wild-type (or native) pesticidal protein. These proteins may be altered in various ways including amino acid substitutions, amino acid deletions, amino acid truncations, and insertions of one or more amino acids, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155 or more amino acid substitutions, amino acid deletions and/or insertions or combinations thereof compared to any one of SEQ ID NO: 35, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526. In some embodiments a PHI-4 polypeptide comprises the deletion of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or more amino acids from the C-terminus of the PHI-4 polypeptide relative to the amino acid position of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a PHI-4 polypeptide can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess the desired pesticidal activity. However, it is understood that the ability of a PHI-4 polypeptide to confer pesticidal activity may be improved by the use of such techniques upon the compositions of this disclosure.
For example, conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a PHI-4 polypeptide without altering the biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., Alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference.
In making such changes, the hydropathic index of amino acids may be considered.
The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1 ): 105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and the like. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, ibid). These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1 .9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1 .3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred and those within +0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. US Patent Number 4,554,101 , states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in US Patent Number 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1 ); glutamate (+3.0.+0.1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+0.1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1 .3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include amino acid insertions, amino acid deletions or amino acid alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1 ) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity or epitope to facilitate either protein purification, protein detection or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, mitochondria or chloroplasts of plants or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein. In specific embodiments, the substitution is an alanine for the native amino acid at the recited position(s). Also encompassed are the nucleic acid sequence(s) encoding the variant protein or polypeptide.
Variant nucleotide and amino acid sequences of the disclosure also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different PHI-4 polypeptide coding regions can be used to create a new PHI-4 polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91 :10747-10751 ; Stemmer, (1994) Nature 370:389-391 ; Crameri, et al. , (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et al. , (1998) Nature 391 :288-291 and US Patent Numbers 5,605,793 and 5,837,458.
Domain swapping or shuffling is another mechanism for generating altered PHI-4 polypeptides. Domains may be swapped between PHI-4 polypeptides, resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov, et al., (2001 ) Appl. Environ. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al. , (1991 ) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ. Microbiol. 65:2918-2925).
Antibodies
Antibodies to a PHI-4 polypeptide of the embodiments or to variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; US Patent Number 4,196,265).
A kit for detecting the presence of a PHI-4 polypeptide or detecting the presence of a nucleotide sequence encoding a PHI-4 polypeptide, in a sample is provided. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a PHI-4 polypeptide in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding PHI-4 polypeptide(s). The kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit."
Receptor identification and isolation
Receptors to the PHI-4 polypeptide of the embodiments or to variants or fragments thereof, are also encompassed. Methods for identifying receptors are well known in the art (see, Hofmann, et. al., (1988) Eur. J. Biochem. 173:85-91 ; Gill, et al., (1995) J. Biol. Chem. 27277-27282) can be employed to identify and isolate the receptor that recognizes the PHI-4 polypeptides using the brush-border membrane vesicles from susceptible insects. In addition to the radioactive labeling method listed in the cited literatures, PHI-4 polypeptide can be labeled with fluorescent dye and other common labels such as streptavidin. Brush-border membrane vesicles (BBMV) of susceptible insects such as soybean looper and stink bugs can be prepared according to the protocols listed in the references and separated on SDS-PAGE gel and blotted on suitable membrane. Labeled PHI-4 polypeptides can be incubated with blotted membrane of BBMV and labeled the PHI-4 polypeptides can be identified with the labeled reporters. Identification of protein band(s) that interact with the PHI-4 polypeptides can be detected by N-terminal amino acid gas phase sequencing or mass spectrometry based protein identification method (Patterson, (1998) 10(22): 1 -24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Once the protein is identified, the corresponding gene can be cloned from genomic DNA or cDNA library of the susceptible insects and binding affinity can be measured directly with the PHI-4 polypeptides. Receptor function for insecticidal activity by the PHI-4 polypeptides can be verified by accomplished by RNAi type of gene knock out method (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849-46851 ).
Nucleotide Constructs, Expression Cassettes and Vectors
The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences. Further, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
A further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.
The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments. The term "operably linked" as used herein refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and where necessary to join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.
Such a DNA construct is provided with a plurality of restriction sites for insertion of the PHI-4 polypeptide gene sequence to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.
The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign or heterologous to the host organism and/or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked sequence of the embodiments. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.
In some embodiments the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an "enhancer" refers to a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al. , (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96) and the enhancers of US Patent Number 7,803,992 may also be used, each of which is incorporated by reference. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).
Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al. , (1991 ) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991 ) Cell 64:671-674; Sanfacon, et al., (1991 ) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261 -1272; Munroe, et al., (1990) Gene 91 : 151 -158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891 -7903 and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.
Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-1 1 for a discussion of host- preferred codon usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant- preferred genes. See, for example, US Patent Numbers 5,380,831 , and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-1 1 for a discussion of host- preferred codon usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant- preferred genes. See, for example, US Patent Numbers 5,380,831 , and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep 37:677-684, 2010, herein incorporated by reference. A Zea maize codon usage table can be also found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can be accessed using the www prefix. Table 2 shows a maize optimal codon analysis (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).
Table 2
Figure imgf000099_0001
CUU 223 0.11 2,396 1.43 Gin CAA 99 0.05 2, 312 1.04 cue* 5, 979 3.08 1,109 0.66 CAG* 3, 557 1.95 2, 130 0.96
CUA 106 0.05 1,280 0.76 Arg CGU 153 0.12 751 0.74
CUG* 5, 161 2.66 1, 646 0.98 CGC* 4,278 3.25 466 0.46
Pro ecu 427 0.22 1, 900 1.47 CGA 92 0.07 659 0.65
CCC* 3, 035 1.59 601 0.47 CGG* 1, 793 1.36 631 0.62
CCA 311 0.16 2,140 1.66 AGA 83 0.06 1, 948 1.91
CCG* 3, 846 2.02 513 0.4 AGG* 1, 493 1.14 1, 652 1.62
He AUU 138 0.09 2, 388 1.3 Asn AAU 131 0.07 3, 074 1.26
AUC* 4, 380 2.85 1, 353 0.74 AAC* 3, 814 1.93 1, 807 0.74
AUA 88 0.06 1,756 0.96 Lys AAA 130 0.05 3,215 0.98
Thr ACU 136 0.09 1, 990 1.43 AAG* 5, 047 1.95 3, 340 1.02
ACC* 3, 398 2.25 991 0.71 Asp GAU 312 0.09 4,217 1.38
ACA 133 0.09 2,075 1.5 GAC* 6, 729 1.91 1, 891 0.62
ACG* 2,378 1.57 495 0.36 Gly GGU 363 0.13 2, 301 1.35
Val GUU 182 0.07 2, 595 1.51 GGC* 7, 842 2.91 1,282 0.75
GUC* 4, 584 1.82 1,096 0.64 GGA 397 0.15 2,044 1.19
GUA 74 0.03 1, 325 0.77 GGG* 2, 186 0.81 1,215 0.71
GUG* 5,257 2.08 1, 842 1.07 Glu GAA 193 0.06 4, 080 1.1
GAG* 6, 010 1.94 3, 307 0.9
Codon usage was compared using Chi squared contingency test to identify optimal codons. Codons that occur significantly more often (P\0.01 ) are indicated with an asterisk.
A Glycine max codon usage table is shown in Table 3 and can also be found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1 &style=N, which can be accessed using the www prefix.
Table 3
Figure imgf000100_0001
TAG * 0.5 (263) TGG W 13.0 (6412)
CAT H 14.0 (6930) CGT R 6.6 (3291)
CAC H 11.6 (5759) CGC R 6.2 (3093)
CAA Q 20.5 (10162) CGA R 4.1 (2018)
CAG Q 16.2 (8038) CGG R 3.1 (1510)
AAT N 22.4 (11088) AGT S 12.6 (6237)
AAC N 22.8 (11284) AGC S 11.3 (5594)
AAA K 26.9 (13334) AGA R 14.8 (7337)
AAG K 35.9 (17797) AGG R 13.3 (6574)
GAT D 32.4 (16040) GGT G 20.9 (10353)
GAC D 20.4 (10097) GGC G 13.4 (6650)
GAA E 33.2 (16438) GGA G 22.3 (11022)
GAG E 33.2 (16426) GGG G 13.0 (6431)
In some embodiments the recombinant nucleic acid molecule encoding a PHI-4 polypeptide has maize optimized codons.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well- characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term "host cell" as used herein refers to a cell which contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991 ) Nature 353:90- 94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al. , (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991 ) Virology 81 :382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Such constructs may also contain a "signal sequence" or "leader sequence" to facilitate co-translational or post- translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus.
By "signal sequence" is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are protolytically activated in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments. By "leader sequence" is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria and the like. Nuclear-encoded proteins targeted to the chloroplast thylakoid lumen compartment have a characteristic bipartite transit peptide, composed of a stromal targeting signal peptide and a lumen targeting signal peptide. The stromal targeting information is in the amino-proximal portion of the transit peptide. The lumen targeting signal peptide is in the carboxyl-proximal portion of the transit peptide, and contains all the information for targeting to the lumen. Recent research in proteomics of the higher plant chloroplast has achieved in the identification of numerous nuclear- encoded lumen proteins (Kieselbach et al. FEBS LETT 480:271 -276, 2000; Peltier et al. Plant Cell 12:319-341 , 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001 ), the lumen targeting signal peptide of which can potentially be used in accordance with the present disclosure. About 80 proteins from Arabidopsis, as well as homologous proteins from spinach and garden pea, are reported by Kieselbach et al., Photosynthesis Research, 78:249-264, 2003. In particular, table 2 of this publication, which is incorporated into the description herewith by reference, discloses 85 proteins from the chloroplast lumen, identified by their accession number (see also US Patent Application Publication 2009/09044298). In addition, the recently published draft version of the rice genome (Goff et al, Science 296:92-100, 2002) is a suitable source for lumen targeting signal peptide which may be used in accordance with the present disclosure.
Suitable chloroplast transit peptides (CTP) are well known to one skilled in the art also include chimeric CTPs comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza sativa 1-deoxy-D xyulose-5- Phosphate Synthase, Oryza saf/Va-Superoxide dismutase, Oryza sai/Va-soluble starch synthase, Oryza sai/Va-NADP-dependent Malic acid enzyme, Oryza sai/Va-Phospho-2- dehydro-3-deoxyheptonate Aldolase 2, Oryza sai/Va-L-Ascorbate peroxidase 5, Oryza sai/Va-Phosphoglucan water dikinase, Zea Mays ssRUBISCO, Zea Mays-beta- glucosidase, Zea ΛZays-Malate dehydrogenase, Zea Mays Thioredoxin M-type US Patent Application Publication 2012/0304336).
The PHI-4 polypeptide gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, US Patent Number 5,380,831 , herein incorporated by reference.
In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999/43838 and US Patent Number 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al. , (1990) Plant Cell 2: 163-171 ); ubiquitin (Christensen, et al. , (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991 ) Theor. Appl. Genet. 81 :581 -588); MAS (Velten, et al. , (1984) EMBO J. 3:2723-2730); ALS promoter (US Patent Number 5,659,026) and the like. Other constitutive promoters include, for example, those discussed in US Patent Numbers 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,61 1.
Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. 28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wunl and wun2, US Patent Number 5,428,148; winl and win2 (Stanford, et al., (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al. , (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73- 76); MPI gene (Corderok, et al., (1994) Plant J. 6(2):141 -150) and the like, herein incorporated by reference.
Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1 ,3-glucanase, chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4:645-656 and Van Loon, (1985) Plant Mol. Virol. 4:1 1 1 -1 16. See also, WO 1999/43819, herein incorporated by reference.
Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331 ; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen, et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :2507- 251 1 ; Warner, et al., (1993) Plant J. 3:191-201 ; Siebertz, et al., (1989) Plant Cell 1 :961 - 968; US Patent Number 5,750,386 (nematode-inducible) and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41 :189-200).
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991 ) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991 ) Mol. Gen. Genet. 227:229-237 and US Patent Numbers 5,814,618 and 5,789,156), herein incorporated by reference.
Tissue-preferred promoters can be utilized to target enhanced PHI-4 polypeptide expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7)792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al. , (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 1 12(3):1331-1341 ; Van Camp, et al., (1996) Plant Physiol. 1 12(2):525-535; Canevascini, et al., (1996) Plant Physiol. 1 12(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1 129-1 138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al. , (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1 129-1 138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al. , (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991 ) Plant Cell 3(10): 1051 -1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., (1991 ) Plant Cell 3(1 ):1 1 -22 (full- length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633- 641 , where two root-specific promoters isolated from hemoglobin genes from the nitrogen- fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β- glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi, (1991 ) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(1 ):69-76). They concluded that enhancer and tissue- preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1 ' gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD- GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4)759-772) and rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691. See also, US Patent Numbers 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401 ,836; 5,1 10,732 and 5,023,179.
"Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed-germinating" promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin- induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1 -phosphate synthase) (see, US Patent Number 6,225,529, herein incorporated by reference). Gamma-zein and Glb-1 are endosperm-specific promoters. For dicots, seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku, K.D. and Goldberg, R.B. Plant Cell 1 :1079-1093, 1989), bean β-phaseolin, napin, β- conglycinin, glycinin 1 , soybean lectin, cruciferin, and the like. For monocots, seed- specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1 , shrunken 2, globulin 1 , etc. See also, WO 2000/12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference. In dicots, seed specific promoters include but are not limited to seed coat promoter from Arabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr (US Patent Numbers 7,294,760 and 7,847,153). A promoter that has "preferred" expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue.
Where low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts is intended. Alternatively, it is recognized that the term "weak promoters" also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.
Such weak constitutive promoters include, for example the core promoter of the
Rsyn7 promoter (WO 1999/43838 and US Patent Number 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in US Patent Numbers 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,61 1 , herein incorporated by reference.
The above list of promoters is not meant to be limiting. Any appropriate promoter can be used in the embodiments.
Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213 and Meijer, et al., (1991 ) Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91 ); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5: 131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. 7:171 -176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423); glyphosate (Shaw, et al., (1986) Science 233:478-481 and US Patent Application Serial Numbers 10/004,357 and 10/427,692); phosphinothricin (DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-51 1 ; Christopherson, et al, (1992) Proc. Natl. Acad. Sci. USA 89:6314- 6318; Yao, et al, (1992) Cell 71 :63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, et al, (1980) in The Operon, pp. 177-220; Hu, et al, (1987) Cell 48:555-566; Brown, et al, (1987) Cell 49:603-612; Figge, et al, (1988) Cell 52:713-722; Deuschle, et al, (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst, et al, (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al, (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al, (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921 ; Labow, et al, (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al, (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn, et al, (1991 ) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991 ) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991 ) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1 104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 ; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer- Verlag, Berlin) and Gill, et al., (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments.
Plant Transformation
The methods of the embodiments involve introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods and virus-mediated methods.
"Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. By "plant" is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),
Figure imgf000108_0001
transformation (US Patent Numbers 5,563,055 and 5,981 ,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, US Patent Numbers 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926) and Led transformation (WO 2000/28058). For potato transformation see, Tu, et al., (1998) Plant Molecular Biology 37:829-838 and Chong, et al., (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991 ) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988) Plant Physiol. 91 :440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 31 1 :763-764; US Patent Number 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the PHI-4 polypeptide or variants and fragments thereof directly into the plant or the introduction of the PHI-4 polypeptide transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al. , (1986) Mol Gen. Genet. 202:179-185; Nomura, et al. , (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91 :2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775- 784, all of which are herein incorporated by reference. Alternatively, the PHI-4 polypeptide polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 1999/25821 , WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the embodiments can be contained in transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
Plant transformation vectors may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as "binary vectors". Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium- mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "gene of interest" (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5:446-451 ). Several types of Agrobacterium strains (e.g. LBA4404, GV3101 , EHA101 , EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.
Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park, (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non- transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81- 84. These plants may then be grown, and either pollinated with the same transformed strain or different strains and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.
The nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired PHI-4 polypeptide. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a PHI-4 polypeptide of the embodiments, may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules are known in the art. See, for example, US Patent Numbers 5,889,191 ; 5,889,190; 5,866,785; 5,589,367 and 5,316,931 , herein incorporated by reference.
Methods for transformation of chloroplasts are known in the art. See, for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid- directed RNA polymerase. Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :7301 -7305.
The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots.
The embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa {Medicago sativa), rice {Oryza sativa), rye {Secale cereale), sorghum {Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet {Pennisetum glaucum), proso millet {Panicum miliaceum), foxtail millet {Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton {Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees {Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana {Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (O/ea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the embodiments include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta) and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.
Turf grasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewing's fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass {Agropyron smithii); Bermuda grass {Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass {Zoysia spp.); Bahia grass {Paspalum notatum); carpet grass {Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue gramma {Bouteloua gracilis); buffalo grass {Buchloe dactyloids); sideoats gramma {Bouteloua curtipendula).
Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
Evaluation of Plant Transformation
Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell, (2001 ) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
Plant transformation may be confirmed by Southern blot analysis of genomic DNA
(Sambrook and Russell, (2001 ) supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled 32P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, (2001 ) supra).
In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, (2001 ) supra). Expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, (2001 ) supra).
Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001 , supra) using antibodies that bind to one or more epitopes present on the PHI-4 polypeptide.
Stacking of traits in transgenic plant
Transgenic plants may comprise a stack of one or more insecticidal polynucleotides disclosed herein with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene, and co- transformation of genes into a single plant cell. As used herein, the term "stacked" includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid). In one non-limiting example, "stacked traits" comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 1999/25821 , WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are herein incorporated by reference.
In some embodiments the polynucleotides encoding the PHI-4 polypeptides disclosed herein, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance or stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the polynucleotide embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests. Transgenes useful for stacking include but are not limited to:
1. Transgenes that Confer Resistance to Insects or Disease and that Encode:
(A) Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example, Jones, et al., (1994) Science 266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos, et al. , (1994) Cell 78:1089 {Arabidopsis RSP2 gene for resistance to Pseudomonas syringae), McDowell and Woffenden, (2003) Trends Biotechnol. 21 (4):178-83 and Toyoda, et al., (2002) Transgenic Res. 1 1 (6):567-82. A plant resistant to a disease is one that is more resistant to a pathogen as compared to the wild type plant.
(B) Genes encoding a Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser, et al. , (1986) Gene
48:109, who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Numbers 40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications and hereby are incorporated by reference for this purpose: US Patent Numbers 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594, 6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826, 7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521 ,235, 7,544,862, 7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846, 7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731 ; WO 1999/24581 and WO 1997/40162.
Genes encoding pesticidal proteins may also be stacked including but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (201 1 ) PLoS Pathogens, 7:1-13), from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et al, (2010) J. Agric. Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al. , (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal 3:101-1 18 and Morgan, et al., (2001 ) Applied and Envir. Micro. 67:2062-2069), US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US Serial Number 13792861 ; an AflP-1A and/or AflP-1 B polypeptide of US Serial Number 13/800233; a PHI-4 polypeptide of US Serial Number 13/839702; and δ-endotoxins including, but not limited to, the Cry1 , Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry1 1 , Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21 , Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31 , Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41 , Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51 , Cry 52, Cry 53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61 , Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71 , Cry72, And Cry73 classes of δ-endotoxin genes and the B. thuringiensis cytolytic Cyt1 and Cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins include, but are not limited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8 (Accession # 126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa1 1 (Accession # CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession # AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession # HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession # HQ439790); Cry1Aa19 (Accession # HQ685121 ); Cry1Aa20 (Accession # JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession # KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession # AAA22613); Cry1Ab3 (Accession # AAA22561 ); Cry1Ab4 (Accession # BAA00071 ); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession # AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession # AAA22551 ); Cry1Ab9 (Accession # CAA38701 ); Cry1Ab10 (Accession # A29125); Cry1Ab1 1 (Accession # 112419); Cry1Ab12 (Accession # AAC64003); Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877); Cry1Ab15 (Accession # AAO13302); Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259); Cry1Ab19 (Accession # AAW31761 ); Cry1Ab20 (Accession # ABB72460); Cry1Ab21 (Accession # ABS18384); Cry1Ab22 (Accession # ABW87320); Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778); Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729); Cry1Ab27 (Accession # JN 135249); Cry1Ab28 (Accession # JN 135250); Cry1Ab29 (Accession # JN 135251 ); Cry1Ab30 (Accession # JN 135252); Cry1Ab31 (Accession # JN 135253); Cry1Ab32 (Accession # JN 135254); Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668); Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337); Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858); Cry1Ac1 (Accession # AAA22331 ); Cry1Ac2 (Accession # AAA22338); Cry1Ac3 (Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5 (Accession # AAA22339); Cry1Ac6 (Accession # AAA86266); Cry1Ac7 (Accession # AAB46989); Cry1Ac8 (Accession # AAC44841 ); Cry1Ac9 (Accession # AAB49768); CrylAd O (Accession # CAA05505 ); Cry1Ac1 1 (Accession # CAA10270); Cry1Ac12 (Accession # 112418); Cry1Ac13 (Accession # AAD38701 ); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15 (Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17 (Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19 (Accession # ABD37053); Cry1Ac20 (Accession # ABB89046 ); Cry1Ac21 (Accession # AAY66992 ); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23 (Accession # CAQ30431 ); Cry1Ac24 (Accession # ABL01535); Cry1Ac25 (Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27 (Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29 (Accession # DQ438941 ); Cry1Ac30 (Accession # GQ227507); Cry1Ac31 (Accession # GU446674); Cry1Ac32 (Accession # HM061081 ); Cry1Ac33 (Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35 (Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37 (Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2 (Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1 (Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1 (Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3 (Accession # HQ439779); Cry 1 AM (Accession # AA039719); Cry1Ai2 (Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1 (Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3 (Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5 (Accession # ABO20894); Cry1Ba6 (Accession # ABL60921); Cry1Ba7 (Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2 (Accession # HQ439782); CrylBd (Accession # CAA86568); Cry1Bd1 (Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1 (Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3 (Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1 (Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1 (Accession # AAO39720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1 (Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2 (Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4 (Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6 [1] (Accession # AAF37224 ); Cry1Ca7 (Accession # AAG50438); Cry1Ca8 (Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10 (Accession # AAN 16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12 (Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14 (Accession # JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession # AAG35409); Cry1Cb3 (Accession # ACD50894 ); Cry1Cb-like (Accession # AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession # 176415); Cry1Da3 (Accession # HQ439784); Cry1Db1 (Accession # CAA80234 ); Cry1 Db2 (Accession # AAK48937 ); Cry1 Del (Accession # ABK35074); Cry1Ea1 (Accession # CAA37933); Cry1Ea2 (Accession # CAA39609); Cry1Ea3 (Accession # AAA22345); Cry1Ea4 (Accession # AAD04732); Cry1Ea5 (Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession # AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession # HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession # JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession # AAA22348); Cry1Fa2 (Accession # AAA22347); Cry1Fa3 (Accession # HM070028); Cry1Fa4 (Accession # HM439638); Cry1Fb1 (Accession # CAA80235); Cry1Fb2 (Accession # BAA25298); Cry1Fb3 (Accession # AAF21767); Cry1Fb4 (Accession # AAC10641); Cry1Fb5 (Accession # AA013295); Cry1Fb6 (Accession # ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1 (Accession # CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1 (Accession # AAD10291); Cry1Gb2 (Accession # AA013756); Cry1Gc1 (Accession # AAQ52381); Cry1Ha1 (Accession # CAA80236); Cry1Hb1 (Accession # AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like (Accession # AAF01213); Cry1la1 (Accession # CAA44633); Cry1la2 (Accession # AAA22354); Cry1la3 (Accession # AAC36999); Cry1la4 (Accession # AAB00958); Cry1la5 (Accession # CAA70124); Cry1la6 (Accession
# AAC26910); Cry1la7 (Accession # AAM73516); Cry1la8 (Accession # AAK66742); Cry1la9 (Accession # AAQ08616); Cry1la10 (Accession # AAP86782); Cry1 Ia11 (Accession # CAC85964 ); Cry1la12 (Accession # AAV53390); Cry1la13 (Accession # ABF83202); Cry1la14 (Accession # ACG63871); Cry1la15 (Accession # FJ617445); Cry1la16 (Accession # FJ617448); Cry1la17 (Accession # GU989199); Cry1la18 (Accession # ADK23801); Cry1la19 (Accession # HQ439787); Cry1la20 (Accession # JQ228426); Cry1la21 (Accession # JQ228424); Cry1la22 (Accession # JQ228427); Cry1la23 (Accession # JQ228428); Cry1la24 (Accession # JQ228429); Cry1la25 (Accession # JQ228430); Cry1la26 (Accession # JQ228431); Cry1la27 (Accession # JQ228432); Cry1la28 (Accession # JQ228433); Cry1la29 (Accession # JQ228434); Cry1la30 (Accession # JQ317686); Cry1la31 (Accession # JX944038); Cry1la32 (Accession # JX944039); Cry1la33 (Accession # JX944040); Cry1lb1 (Accession # AAA82114); Cry1lb2 (Accession # ABW88019); Cry1lb3 (Accession # ACD75515); Cry1lb4 (Accession # HM051227); Cry1lb5 (Accession # HM070028); Cry1lb6 (Accession
# ADK38579); Cry1lb7 (Accession # JN571740); Cry1lb8 (Accession # JN675714); Cry1lb9 (Accession # JN675715); Cry1lb10 (Accession # JN675716); Cry1lb11 (Accession # JQ228423); Crylld (Accession # AAC62933); Cry1lc2 (Accession # AAE71691); Cry1ld1 (Accession # AAD44366); Cry1ld2 (Accession # JQ228422); Cry1 Ie1 (Accession # AAG43526); Cry1 Ie2 (Accession # HM439636); Cry1 Ie3 (Accession
# KC156647); Cry1le4 (Accession # KC156681); Cry1 If 1 (Accession # AAQ52382); Cry1lg1 (Accession # KC156701); Cry1 l-like (Accession # AAC31094); Cry1 l-like (Accession # ABG88859); CryUal (Accession # AAA22341); Cry1Ja2 (Accession # HM070030); Cry1Ja3 (Accession # JQ228425); CryUbl (Accession # AAA98959); CryUd (Accession # AAC31092); Cry1Jc2 (Accession # AAQ52372); CryUdl (Accession # CAC50779); Cry1Ka1 (Accession # AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1 (Accession # AAS60191); Cry1La2 (Accession # HM070031); Cry1Ma1 (Accession # FJ884067); Cry1Ma2 (Accession # KC156659); Cry1Na1 (Accession # KC156648); Cry1Nb1 (Accession # KC156678); Cry1-like (Accession # AAC31091); Cry2Aa1 (Accession # AAA22335); Cry2Aa2 (Accession # AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession # AAC04867); Cry2Aa5 (Accession
# CAA10671); Cry2Aa6 (Accession # CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8 (Accession # AA013734); Cry2Aa9 (Accession # AA013750 ); Cry2Aa10 (Accession # AAQ04263); Cry2Aa11 (Accession # AAQ52384); Cry2Aa12 (Accession # ABI83671); Cry2Aa13 (Accession # ABL01536); Cry2Aa14 (Accession # ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1 (Accession # AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3 (Accession # AAG36762); Cry2Ab4 (Accession # AA013296 ); Cry2Ab5 (Accession # AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7 (Accession # AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9 (Accession # ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab1 1 (Accession # CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13 (Accession # ACG76120); Cry2Ab14 (Accession # ACG76121 ); Cry2Ab15 (Accession # HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17 (Accession # HQ439789); Cry2Ab18 (Accession # JN 135255); Cry2Ab19 (Accession # JN 135256); Cry2Ab20 (Accession # JN 135257); Cry2Ab21 (Accession # JN 135258); Cry2Ab22 (Accession # JN 135259); Cry2Ab23 (Accession # JN 135260); Cry2Ab24 (Accession # JN 135261 ); Cry2Ab25 (Accession # JN415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27 (Accession # JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1 (Accession # CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3 (Accession # AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5 (Accession # ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7 (Accession # CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9 (Accession # CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac1 1 (Accession # CAM83895); Cry2Ac12 (Accession # CAM83896); Cry2Ad1 (Accession # AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3 (Accession # CAK29504); Cry2Ad4 (Accession # CAM32331 ); Cry2Ad5 (Accession # CA078739 ); Cry2Ae1 (Accession # AAQ52362); Cry2Af1 (Accession # ABO30519); Cry2Af2 (Accession # GQ866915); Cry2Ag1 (Accession # ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2 (Accession # ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4 (Accession # KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj (Accession # ); Cry2Ak1 (Accession # KC156660); Cry2Ba1 (Accession # KC156658); Cry3Aa1 (Accession # AAA22336); Cry3Aa2 (Accession # AAA22541 ); Cry3Aa3 (Accession # CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5 (Accession # AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7 (Accession # CAB4141 1 ); Cry3Aa8 (Accession # AAS79487); Cry3Aa9 (Accession # AAW05659); Cry3Aa10 (Accession # AAU2941 1 ); Cry3Aa1 1 (Accession # AAW82872); Cry3Aa12 (Accession # ABY49136 ); Cry3Ba1 (Accession # CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3 (Accession # JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2 (Accession # AAA74198); Cry3Bb3 (Accession # 115475); Cry3Ca1 (Accession # CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession # BAA00179); Cry4Aa3 (Accession # CAD30148); Cry4Aa4 (Accession # AFB18317); Cry4A-like (Accession # AAY96321 ); Cry4Ba1 (Accession # CAA30312); Cry4Ba2 (Accession # CAA301 14); Cry4Ba3 (Accession # AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5 (Accession # CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1 (Accession # EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2 (Accession # FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1 (Accession # AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1 (Accession # I34543); Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2 (Accession # ABW88931 ); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1 (Accession # HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1 (Accession # HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1 (Accession # HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1 (Accession # AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3 (Accession # ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7Aa1 (Accession # AAA22351 ); Cry7Ab1 (Accession # AAA21 120); Cry7Ab2 (Accession # AAA21 121 ); Cry7Ab3 (Accession # ABX24522); Cry7Ab4 (Accession # EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6 (Accession # ACI44005); Cry7Ab7 (Accession # ADB89216); Cry7Ab8 (Accession # GU145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession # ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession # ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession # ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession # KC156679); Cry7Ea1 (Accession # HM035086); Cry7Ea2 (Accession # HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession # HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession # HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession # HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession # KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession # KC156697); Cry7Ha1 (Accession # KC156651 ); Cry7la1 (Accession # KC156665); Cry7Ja1 (Accession # KC156671 ); Cry7Ka1 (Accession # KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession # BAM99307); Cry8Aa1 (Accession # AAA21 1 17); Cry8Ab1 (Accession # EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession # KC156684); Cry8Ba1 (Accession # AAA21 1 18); Cry8Bb1 (Accession # CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession # AAA21 1 19); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession # EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession # BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession # BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession # AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession # KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession # HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession # AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession # FJ198072); Cry8Ha1 (Accession # AAW81032); Cry8la1 (Accession # EU381044); Cry8la2 (Accession # GU073381 ); Cry8la3 (Accession # HM044664); Cry8la4 (Accession # KC156674); Cry8lb1 (Accession # GU325772); Cry8lb2 (Accession # KC156677); Cry8Ja1 (Accession # EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession # ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession # KC156675); Cry8La1 (Accession # GU325771 ); Cry8Ma1 (Accession # HM044665); Cry8Ma2 (Accession # EEM86551 ); Cry8Ma3 (Accession # HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession # HQ388415); Cry8Qa1 (Accession # HQ441 166); Cry8Qa2 (Accession # KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession # JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession # FJ770571 ); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession # CAA41 122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession # GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession # JX1741 10); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession # CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession # AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession # AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession # AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession # GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession # KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession # AAO12908); Cry9Ea3 (Accession # ABM21765); Cry9Ea4 (Accession # ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession # ACG63872 ); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession # GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession # CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession # KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession # AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession # KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession # KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession # AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession # CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession # DQ167578); Cry1 1Aa1 (Accession # AAA22352); Cry1 1Aa2 (Accession # AAA2261 1 ); Cry1 1Aa3 (Accession # CAD30081 ); Cry1 1Aa4 (Accession # AFB18319); Cry1 1Aa-like (Accession # DQ166531 ); Cry1 1 Ba1 (Accession # CAA60504); Cry1 1 Bb1 (Accession # AAC97162); Cry1 1 Bb2 (Accession # HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession # AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession # KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession # CAA63860); Cry17Aa1 (Accession # CAA67841 ); Cry18Aa1 (Accession # CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession # AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession # BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession # AAB93476); Cry20Ba1 (Accession # ACS93601 ); Cry20Ba2 (Accession # KC156694); Cry20-like (Accession # GQ144333); Cry21Aa1 (Accession # I32932); Cry21Aa2 (Accession # I66477); Cry21 Ba1 (Accession # BAC06484); Cry21 Ca1 (Accession # JF521577); Cry21 Ca2 (Accession # KC156687); Cry21 Da1 (Accession # JF521578); Cry22Aa1 (Accession # I34547); Cry22Aa2 (Accession # CAD43579); Cry22Aa3 (Accession # ACD9321 1 ); Cry22Ab1 (Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1 (Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1 (Accession # AAF76375); Cry24Aa1 (Accession # AAC61891 ); Cry24Ba1 (Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1 (Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1 (Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2 (Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1 (Accession # CAC80986); Cry30Ba1 (Accession # BAD00052); Cry30Ca1 (Accession # BAD67157); Cry30Ca2 (Accession # ACU24781 ); Cry30Da1 (Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1 (Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1 (Accession # ACI22625 ); Cry30Ga1 (Accession # ACG60020); Cry30Ga2 (Accession # HQ638217); Cry31Aa1 (Accession # BAB1 1757); Cry31Aa2 (Accession # AAL87458); Cry31Aa3 (Accession # BAE79808); Cry31Aa4 (Accession # BAF32571 ); Cry31Aa5 (Accession # BAF32572); Cry31Aa6 (Accession # BAI44026); Cry31Ab1 (Accession # BAE79809); Cry31Ab2 (Accession # BAF32570); Cry31Ac1 (Accession # BAF34368); Cry31Ac2 (Accession # AB731600); Cry31Ad1 (Accession # BAI44022); Cry32Aa1 (Accession # AAG3671 1 ); Cry32Aa2 (Accession # GU063849); Cry32Ab1 (Accession # GU063850); Cry32Ba1 (Accession # BAB78601 ); Cry32Ca1 (Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32Da1 (Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2 (Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1 (Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1 (Accession # KC156661 ); Cry32Hb1 (Accession # KC156666); Cry32la1 (Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1 (Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1 (Accession # KC156690); Cry32Mb1 (Accession # KC156704); Cry32Na1 (Accession # KC156691 ); Cry320a1 (Accession # KC156703); Cry32Pa1 (Accession # KC156705); Cry32Qa1 (Accession # KC156706); Cry32Ra1 (Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1 (Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1 (Accession # AAL26871 ); Cry34Aa1 (Accession # AAG50341 ); Cry34Aa2 (Accession # AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4 (Accession # AAT29030); Cry34Ab1 (Accession # AAG41671 ); Cry34Ac1 (Accession # AAG501 18); Cry34Ac2 (Accession # AAK64562); Cry34Ac3 (Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2 (Accession # AAT29033); Cry34Ba3 (Accession # AAT29031 ); Cry35Aa1 (Accession # AAG50342); Cry35Aa2 (Accession # AAK64561 ); Cry35Aa3 (Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1 (Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3 (Accession # AY536891 ); Cry35Ac1 (Accession # AAG501 17); Cry35Ba1 (Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3 (Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1 (Accession # AAF76376 ); Cry38Aa1 (Accession # AAK64559); Cry39Aa1 (Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1 (Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1 (Accession # ACF15199); Cry41Aa1 (Accession # BAD35157); Cry41Ab1 (Accession # BAD35163); Cry41 Ba1 (Accession # HM461871 ); Cry41 Ba2 (Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1 (Accession # BAD15301 ); Cry43Aa2 (Accession # BAD95474 ); Cry43Ba1 (Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1 (Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like (Accession # BAD15305); Cry44Aa (Accession # BAD08532); Cry45Aa (Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2 (Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47Aa (Accession # AAY24695); Cry48Aa (Accession # CAJ 18351 ); Cry48Aa2 (Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546 ); Cry48Ab (Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa (Accession # CAH56541 ); Cry49Aa2 (Accession # CAJ86541 ); Cry49Aa3 (Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1 (Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1 (Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1 (Accession # ABI 14444); Cry51Aa2 (Accession # GU570697); Cry52Aa1 (Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1 (Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1 (Accession # ACA52194); Cry54Aa2 (Accession # GQ140349); Cry54Ba1 (Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1 (Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1 (Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3 (Accession # JX025567); Cry57Aa1 (Accession # ANC87261 ); Cry58Aa1 (Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1 (Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2 (Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1 (Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3 (Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2 (Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1 (Accession # HM054509); Cry63Aa1 (Accession # BAI44028); Cry64Aa1 (Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2 (Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581 ); Cry66Aa2 (Accession # ZP_04099945); Cry67Aa1 (Accession # HM485582); Cry67Aa2 (Accession # ZP_04148882); Cry68Aa1 (Accession # HQ1 131 14); Cry69Aa1 (Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1 (Accession # JN209957); Cry70Aa1 (Accession # JN646781 ); Cry70Ba1 (Accession # ADO51070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1 (Accession # JX025568); Cry72Aa1 (Accession # JX025569); and Cry73Aa (Accession # AEH76822).
Examples of δ-endotoxins also include but are not limited to CrylA proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-1 1 toxin (N-terminal deletion of a-helix 1 and/or a-helix 2 variants of Cry proteins such as CrylA) of US Patent Numbers 8,304,604 and 8.304,605, Cryl B of US Patent Application Serial Number 10/525,318; Cryl C of US Patent Number 6,033,874; Cryl F of US Patent Numbers 5,188,960, 6,218,188; Cry1A/F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of US Patent Numbers 7,329,736, 7,449,552, 7,803,943, 7,476,781 , 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology 74:7145-7151 ; a Cry22, a Cry34Ab1 protein of US Patent Numbers 6,127,180, 6,624,145 and 6,340,593; a CryET33 and CryET34 protein of US Patent Numbers 6,248,535, 6,326,351 , 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006/0191034, 2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1 protein of US Patent Numbers 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI-027, AXMI-036, and AXMI-038 of US Patent Number 8,236,757; AXMI-031 , AXMI-039, AXMI- 040, AXMI-049 of US7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891 ; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US 2004/025031 1 ; AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965; AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/1 19457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462; AXMI-150 of US Patent Number 8,084,416; AXMI-205 of US20110023184; AXMI-011, AXMI-012, AXMI- 013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI- R1 and related proteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224Z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 of W011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of US Patent Number 8,334,431; AXMI- 001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211; AXMI-066 and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of US Patent Number 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543; Axmi115 variants of US2013097728Cry proteins such as CrylA and Cry3A having modified proteolytic sites of US Patent Number 8,319,019; and a CrylAc, Cry2Aa and CryICa toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011/0064710. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the "www" prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to CrylAc, Cry1Ac+Cry2Ab, CrylAb, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database which can be accessed on the world-wide web using the "www" prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cryl Fa (US2012/0317682), Cryl BE & Cryl F (US2012/031 1746), CryI CA & CrylAB (US2012/031 1745), Cryl F & CryCa (US2012/0317681 ), Cryl DA & Cryl BE (US2012/0331590), Cryl DA & Cryl Fa (US2012/0331589), CrylAB & Cryl BE (US2012/0324606), and Cryl Fa & Cry2Aa, Cry1 l or Cryl E (US2012/0324605). Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491 ,869, and cholesterol oxidases such as from Streptomyces (Purcell et al.
(1993) Bioc em Biop ys Res Commun 15:1406-1413). Pesticidal proteins also include VI P (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279,
6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can be accessed on the world-wide web using the "www" prefix). Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491 ,698 and 8,084,418). Some TC proteins have "stand alone" insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a "stand-alone" TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein "potentiators" derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins ("Protein A") are stand-alone toxins. Class B proteins ("Protein B") and Class C proteins ("Protein C") enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptBIXb and XptCIWi. Examples of Class C proteins are TccC, XptCIXb and XptBIWi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but are not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366).
(C) A polynucleotide encoding an insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon or an antagonist or agonist thereof. See, for example, the disclosure by Hammock, et al., (1990) Nature 344:458, of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
(D) A polynucleotide encoding an insect-specific peptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of, Regan,
(1994) J. Biol. Chem. 269:9 (expression cloning yields DNA coding for insect diuretic hormone receptor); Pratt, et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin is identified in Diploptera puntata); Chattopadhyay, et al., (2004) Critical Reviews in Microbiology 30(1 ):33-54; Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon 40(1 1 ):1515-1539; Ussuf, et al., (2001 ) Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, US Patent Number 5,266,317 to Tomalski, et al., who disclose genes encoding insect- specific toxins.
(E) A polynucleotide encoding an enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
(F) A polynucleotide encoding an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See, PCT Application WO 1993/02197 in the name of Scott, et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Numbers 39637 and 67152. See also, Kramer, et al., (1993) Insect Biochem. Molec. Biol. 23:691 , who teach the nucleotide sequence of a cDNA encoding tobacco hookworm chitinase and Kawalleck, et al., (1993) Plant Molec. Biol. 21 :673, who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and US Patent Numbers 6,563,020; 7,145,060 and 7,087,810.
(G) A polynucleotide encoding a molecule that stimulates signal transduction. For example, see the disclosure by Botella, et al., (1994) Plant Molec. Biol. 24:757, of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol. 104:1467, who provide the nucleotide sequence of a maize calmodulin cDNA clone.
(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCT Application WO 1995/16776 and US Patent Number 5,580,852 disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT Application WO 1995/18855 and US Patent Number 5,607,914 (teaches synthetic antimicrobial peptides that confer disease resistance).
(I) A polynucleotide encoding a membrane permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43, of heterologous expression of a cecropin-beta lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.
(J) A gene encoding a viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See, Beachy, et al. , (1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
(K) A gene encoding an insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).
(L) A gene encoding a virus-specific antibody. See, for example, Tavladoraki, et al. , (1993) Nature 366:469, who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.
(M) A polynucleotide encoding a developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo alpha-1 ,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo- alpha-1 ,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology 10:1436. The cloning and characterization of a gene which encodes a bean endopolygalacturonase- inhibiting protein is described by Toubart, et al., (1992) Plant J. 2:367.
(N) A polynucleotide encoding a developmental-arrestive protein produced in nature by a plant. For example, Logemann, et al., (1992) Bio/Technology 10:305, have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.
(O) Genes involved in the Systemic Acquired Resistance (SAR) Response and/or the pathogenesis related genes. Briggs, (1995) Current Biology 5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64 and Somssich, (2003) Cell 1 13(7):815-6.
(P) Antifungal genes (Cornelissen and Melchers, (1993) PI. Physiol. 101 :709-712 and Parijs, et al., (1991 ) Planta 183:258-264 and Bushnell, et al., (1998) Can. J. of Plant Path. 20(2): 137-149. Also see, US Patent Application Serial Numbers 09/950,933; 1 1/619,645; 1 1/657,710; 1 1/748,994; 1 1/774,121 and US Patent Numbers 6,891 ,085 and 7,306,946. LysM Receptor-like kinases for the perception of chitin fragments as a first step in plant defense response against fungal pathogens (US 2012/01 10696).
(Q) Detoxification genes, such as for fumonisin, beauvericin, moniliformin and zearalenone and their structurally related derivatives. For example, see, US Patent Numbers 5,716,820; 5,792,931 ; 5,798,255; 5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.
(R) A polynucleotide encoding a Cystatin and cysteine proteinase inhibitors. See, US Patent Number 7,205,453.
(S) Defensin genes. See, WO 2003/000863 and US Patent Numbers 6,91 1 ,577; 6,855,865; 6,777,592 and 7,238,781.
(T) Genes conferring resistance to nematodes. See, e.g., PCT Application WO 1996/30517; PCT Application WO 1993/19181 , WO 2003/033651 and Urwin, et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin Plant Bio. 2(4):327-31 ; US Patent Numbers 6,284,948 and 7,301 ,069 and miR164 genes (WO 2012/058266).
(U) Genes that confer resistance to Phytophthora Root Rot, such as the Rps 1 ,
Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes. See, for example, Shoemaker, et al., Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif. (1995).
(V) Genes that confer resistance to Brown Stem Rot, such as described in US
Patent Number 5,689,035 and incorporated by reference for this purpose.
(W) Genes that confer resistance to Colletotrichum, such as described in US Patent Application Publication US 2009/0035765 and incorporated by reference for this purpose. This includes the Reg locus that may be utilized as a single locus conversion.
2. Transgenes that Confer Resistance to a Herbicide, for Example:
(A) A polynucleotide encoding resistance to a herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively. See also, US Patent Numbers 5,605,01 1 ; 5,013,659; 5,141 ,870; 5,767,361 ; 5,731 ,180; 5,304,732; 4,761 ,373; 5,331 ,107; 5,928,937 and 5,378,824; US Patent Application Serial Number 1 1/683,737 and International Publication WO 1996/33270.
(B) A polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, US Patent Number 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. US Patent Number 5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes. See also, US Patent Numbers 6,566,587; 6,338,961 ; 6,248,876 B1 ; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971 ,908; 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; 6,225,1 14 B1 ; 6,130,366; 5,310,667; 4,535,060; 4,769,061 ; 5,633,448; 5,510,471 ; Re. 36,449; RE 37,287 E and 5,491 ,288 and International Publications EP 1 173580; WO 2001/66704; EP 1 173581 and EP 1 173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido- reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase. See, for example, US Patent Numbers 7,462,481 ; 7,405,074 and US Patent Application Publication Number US 2008/0234130. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Patent Number 4,769,061 to Comai. EP Application Number 0 333 033 to Kumada, et al., and US Patent Number 4,975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L- phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans, et al.,; De Greef, et al., (1989) Bio/Technology 7:61 , describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. See also, US Patent Numbers 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561 ,236; 5,648,477; 5,646,024; 6,177,616 B1 and 5,879,903, which are incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1 , Acc1 -S2 and Acc1 -S3 genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.
(C) A polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991 ) Plant Cell 3:169, describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in US Patent Number 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Bioc em. J. 285:173.
(D) A polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet. 246:419). Other genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).
(E) A polynucleotide encoding resistance to a herbicide targeting Protoporphyrinogen oxidase (protox) which is necessary for the production of chlorophyll.
The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in US Patent Numbers 6,288,306 B1 ; 6,282,837 B1 and 5,767,373 and International Publication WO 2001/12825.
(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-1 ) protein. The trait confers tolerance to 2,4- dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as "fop" herbicides such as quizalofop) herbicides. The aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005/107437 (see also, US 2009/0093366). The aad- 12 gene, derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).
(G) A polynucleotide encoding a herbicide resistant dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 for imparting dicamba tolerance;
(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in US Patent Number 4,810,648 for imparting bromoxynil tolerance; (I) A polynucleotide molecule encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al. , (1994) Plant J. 6:481-489 for norflurazon tolerance. 3. Transgenes that Confer or Contribute to an Altered Grain Characteristic
Such as:
(A) Altered fatty acids, for example, by
(1 ) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).
(2) Elevating oleic acid via FAD-2 gene modification and/or decreasing linolenic acid via FAD-3 gene modification (see, US Patent Numbers 6,063,947; 6,323,392; 6,372,965 and WO 1993/1 1245).
(3) Altering conjugated linolenic or linoleic acid content, such as in WO 2001/12800.
(4) Altering LEC1 , AGP, Dek1 , SuperaH , mil ps, various Ipa genes such as Ipa1 , Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO 1998/22604, WO 2003/01 1015, WO 2002/057439, WO 2003/01 1015, US Patent Numbers 6,423,886, 6,197,561 , 6,825,397 and US Patent Application Publication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid, et al. , (1995) Proc. Natl. Acad. Sci. 92:5620-5624.
(5) Genes encoding delta-8 desaturase for making long-chain polyunsaturated fatty acids (US Patent Numbers 8,058,571 and 8,338,152), delta-9 desaturase for lowering saturated fats (US Patent Number 8,063,269), Primula A6-desaturase for improving omega-3 fatty acid profiles.
(6) Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation, in particular, lipid metabolism protein (LMP) used in methods of producing transgenic plants and modulating levels of seed storage compounds including lipids, fatty acids, starches or seed storage proteins and use in methods of modulating the seed size, seed number, seed weights, root length and leaf size of plants (EP 2404499).
(7) Altering expression of a High-Level Expression of Sugar-lnducible 2 (HSI2) protein in the plant to increase or decrease expression of HSI2 in the plant. Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and/or increases drought resistance (US Patent Application Publication Number 2012/0066794).
(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particular to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 201 1/0191904).
(9) Nucleic acid molecules encoding wrinkled1 -like polypeptides for modulating sugar metabolism (US Patent Number 8,217,223).
(B) Altered phosphorus content, for example, by the
(1 ) Introduction of a phytase-encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for a disclosure of the nucleotide sequence of an Aspergillus n/'ger phytase gene.
(2) Modulating a gene that reduces phytate content. In maize, this, for example, could be accomplished, by cloning and then re-introducing DNA associated with one or more of the alleles, such as the LPA alleles, identified in maize mutants characterized by low levels of phytic acid, such as in WO 2005/1 13778 and/or by altering inositol kinase activity as in WO 2002/059324, US Patent Application Publication Number 2003/000901 1 , WO 2003/027243, US Patent Application Publication Number 2003/0079247, WO 1999/05298, US Patent Number 6,197,561 , US Patent Number 6,291 ,224, US Patent Number 6,391 ,348, WO 2002/059324, US Patent Application Publication Number 2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.
(C) Altered carbohydrates affected, for example, by altering a gene for an enzyme that affects the branching pattern of starch or, a gene altering thioredoxin such as NTR and/or TRX (see, US Patent Number 6,531 ,648. which is incorporated by reference for this purpose) and/or a gamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, US Patent Number 6,858,778 and US Patent Application Publication Number 2005/0160488, US Patent Application Publication Number 2005/0204418, which are incorporated by reference for this purpose). See, Shiroza, et al., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcus mutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrase gene), Pen, et al., (1992) Bio/Technology 10:292 (production of transgenic plants that express Bacillus licheniformis alpha-amylase), Elliot, et al., (1993) Plant Molec. Biol. 21 :515 (nucleotide sequences of tomato invertase genes), S0gaard, et al., (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of barley alpha-amylase gene) and Fisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starch branching enzyme II), WO 1999/10498 (improved digestibility and/or starch extraction through modification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1 , HCHL, C4H), US Patent Number 6,232,529 (method of producing high oil seed by modification of starch levels (AGP)). The fatty acid modification genes mentioned herein may also be used to affect starch content and/or composition through the interrelationship of the starch and oil pathways.
(D) Altered antioxidant content or composition, such as alteration of tocopherol or tocotrienols. For example, see, US Patent Number 6,787,683, US Patent Application Publication Number 2004/0034886 and WO 2000/68393 involving the manipulation of antioxidant levels and WO 2003/082899 through alteration of a homogentisate geranyl geranyl transferase (hggt).
(E) Altered essential seed amino acids. For example, see, US Patent Number 6,127,600 (method of increasing accumulation of essential amino acids in seeds), US Patent Number 6,080,913 (binary methods of increasing accumulation of essential amino acids in seeds), US Patent Number 5,990,389 (high lysine), WO 1999/40209 (alteration of amino acid compositions in seeds), WO 1999/29882 (methods for altering amino acid content of proteins), US Patent Number 5,850,016 (alteration of amino acid compositions in seeds), WO 1998/20133 (proteins with enhanced levels of essential amino acids), US Patent Number 5,885,802 (high methionine), US Patent Number 5,885,801 (high threonine), US Patent Number 6,664,445 (plant amino acid biosynthetic enzymes), US Patent Number 6,459,019 (increased lysine and threonine), US Patent Number 6,441 ,274 (plant tryptophan synthase beta subunit), US Patent Number 6,346,403 (methionine metabolic enzymes), US Patent Number 5,939,599 (high sulfur), US Patent Number 5,912,414 (increased methionine), WO 1998/56935 (plant amino acid biosynthetic enzymes), WO 1998/45458 (engineered seed protein having higher percentage of essential amino acids), WO 1998/42831 (increased lysine), US Patent Number 5,633,436 (increasing sulfur amino acid content), US Patent Number 5,559,223 (synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants), WO 1996/01905 (increased threonine), WO 1995/15392 (increased lysine), US Patent Application Publication Number 2003/0163838, US Patent Application Publication Number 2003/0150014, US Patent Application Publication Number 2004/0068767, US Patent Number 6,803,498, WO 2001/79516.
4. Genes that Control Male-Sterility:
There are several methods of conferring genetic male sterility available, such as multiple mutant genes at separate locations within the genome that confer male sterility, as disclosed in US Patent Numbers 4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocations as described by Patterson in US Patent Numbers 3,861 ,709 and 3,710,51 1. In addition to these methods, Albertsen, et al., US Patent Number 5,432,068, describe a system of nuclear male sterility which includes: identifying a gene which is critical to male fertility; silencing this native gene which is critical to male fertility; removing the native promoter from the essential male fertility gene and replacing it with an inducible promoter; inserting this genetically engineered gene back into the plant; and thus creating a plant that is male sterile because the inducible promoter is not "on" resulting in the male fertility gene not being transcribed. Fertility is restored by inducing or turning "on", the promoter, which in turn allows the gene that confers male fertility to be transcribed.
(A) Introduction of a deacetylase gene under the control of a tapetum-specific promoter and with the application of the chemical N-Ac-PPT (WO 2001/29237).
(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO 1992/13957).
(C) Introduction of the barnase and the barstar gene (Paul, et al., (1992) Plant Mol. Biol. 19:61 1-622).
For additional examples of nuclear male and female sterility systems and genes, see also, US Patent Numbers 5,859,341 ; 6,297,426; 5,478,369; 5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporated by reference.
5. Genes that create a site for site specific DNA integration.
This includes the introduction of FRT sites that may be used in the FLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system. For example, see, Lyznik, et al., (2003) Plant Cell Rep 21 :925-932 and WO 1999/25821 , which are hereby incorporated by reference. Other systems that may be used include the Gin recombinase of phage Mu (Maeser, et al., (1991 ) Vicki Chandler, The Maize Handbook ch. 1 18 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and the R/RS system of the pSRi plasmid (Araki, et al., 1992).
6. Genes that affect abiotic stress resistance
Including but not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance and salt resistance or tolerance and increased yield under stress.
(A) For example, see: WO 2000/73475 where water use efficiency is altered through alteration of malate; US Patent Numbers 5,892,009, 5,965,705, 5,929,305, 5,891 ,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034, 6,801 ,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO 2004/076638, WO 199809521 .
(B) WO 199938977 describing genes, including CBF genes and transcription factors effective in mitigating the negative effects of freezing, high salinity and drought on plants, as well as conferring other positive effects on plant phenotype.
(C) US Patent Application Publication Number 2004/0148654 and WO 2001/36596 where abscisic acid is altered in plants resulting in improved plant phenotype such as increased yield and/or increased tolerance to abiotic stress.
(D) WO 2000/006341 , WO 2004/090143, US Patent Numbers 7,531 ,723 and
6,992,237 where cytokinin expression is modified resulting in plants with increased stress tolerance, such as drought tolerance, and/or increased yield. Also see, WO 2002/02776, WO 2003/052063, JP 2002/281975, US Patent Number 6,084,153, WO 2001/64898, US Patent Number 6,177,275 and US Patent Number 6,107,547 (enhancement of nitrogen utilization and altered nitrogen responsiveness).
(E) For ethylene alteration, see, US Patent Application Publication Number 2004/0128719, US Patent Application Publication Number 2003/0166197 and WO 2000/32761.
(F) For plant transcription factors or transcriptional regulators of abiotic stress, see, e.g., US Patent Application Publication Number 2004/0098764 or US Patent Application
Publication Number 2004/0078852.
(G) Genes that increase expression of vacuolar pyrophosphatase such as AVP1 (US Patent Number 8,058,515) for increased yield; nucleic acid encoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5) polypeptides, an oligopeptide transporter protein (OPT4-like) polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschel related homeobox 1 -like (WOX1 -like) polypeptide (U. Patent Application Publication Number US 201 1/0283420).
(H) Down regulation of polynucleotides encoding poly (ADP-ribose) polymerase (PARP) proteins to modulate programmed cell death (US Patent Number 8,058,510) for increased vigor.
(I) Polynucleotide encoding DTP21 polypeptides for conferring drought resistance (US Patent Application Publication Number US 201 1/0277181 ).
(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins for modulating development, modulating response to stress, and modulating stress tolerance (US Patent Application Publication Number US 2010/0287669). (K) Polynucleotides that encode proteins that confer a drought tolerance phenotype (DTP) for conferring drought resistance (WO 2012/058528).
(L) Tocopherol cyclase (TC) genes for conferring drought and salt tolerance (US Patent Application Publication Number 2012/0272352).
(M) CAAX amino terminal family proteins for stress tolerance (US Patent Number
8,338,661 ).
(N) Mutations in the SAL1 encoding gene have increased stress tolerance, including increased drought resistant (US Patent Application Publication Number 2010/0257633).
(O) Expression of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide increasing yield-related traits (US Patent Application Publication Number 201 1/0061 133).
(P) Modulating expression in a plant of a nucleic acid encoding a Class III Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancing yield-related traits in plants, particularly increasing seed yield (US Patent Application Publication Number 2010/0024067).
Other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth and/or plant structure, can be introduced or introgressed into plants, see e.g., WO 1997/4981 1 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and US Patent Number 6,573,430 (TFL), US Patent Number 6,713,663 (FT), WO 1996/14414 (CON), WO 1996/38560, WO 2001/21822 (VRN1 ), WO 2000/44918 (VRN2), WO 1999/49064 (Gl), WO 2000/46358 (FR1 ), WO 1997/29123, US Patent Number 6,794,560, US Patent Number 6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO 2004/031349 (transcription factors).
7. Genes that confer increased yield
(A) A transgenic crop plant transformed by a 1 -AminoCyclopropane-1 - Carboxylate Deaminase-like Polypeptide (ACCDP) coding nucleic acid, wherein expression of the nucleic acid sequence in the crop plant results in the plant's increased root growth, and/or increased yield, and/or increased tolerance to environmental stress as compared to a wild type variety of the plant (US Patent Number 8,097,769).
(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1 ) using a seed preferred promoter has been shown to enhance plant growth, increase kernel number and total kernel weight per plant (US Patent Application Publication Number 2012/0079623). (C) Constitutive over-expression of maize lateral organ boundaries (LOB) domain protein (Zm-LOBDP1 ) has been shown to increase kernel number and total kernel weight per plant (US Patent Application Publication Number 2012/0079622).
(D) Enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a VIM1 (Variant in Methylation 1 )-like polypeptide or a VTC2- like (GDP-L-galactose phosphorylase) polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin Responsive Factor) polypeptide (WO 2012/038893).
(E) Modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof gives plants having increased yield relative to control plants (EP 2431472).
(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptides and homologs thereof for modifying the plant's root architecture (US Patent Application Publication Number 2009/0064373).
8. Genes that confer plant digestibility.
(A) Altering the level of xylan present in the cell wall of a plant by modulating expression of xylan synthase (US 8,173,866).
In some embodiment the stacked trait may be a trait or event that has received regulatory approval including but not limited to the events in Table 2A -1 F. Table 2A Heliant us annuus Sunflower
Event Company Description
X81359 BASF Inc. Tolerance to imidazolinone herbicides by
selection of a naturally occurring mutant.
Table 2B Medicago sativa Alfalfa
Event Company Description
J101 , J163 Monsanto Company Glyphosate herbicide tolerant alfalfa (lucerne) and Forage Genetics produced by inserting a gene encoding the International enzyme 5-enolypyruvylshikimate-3-phosphate synthase (EPSPS) from the CP4 strain of Agrobacterium tumefaciens.
Table 2C Oryza sativa Rice
Event Company Description
CL121 , CL141 , CFX51 BASF Inc. Tolerance to the imidazolinone herbicide,
imazethapyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).
IMINTA-1 , IMINTA-4 BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using sodium azide. Event Company Description
LLRICE06, LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerant rice produced by inserting a modified
phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium
Streptomyces hygroscopicus).
LLRICE601 Bayer CropScience Glufosinate ammonium herbicide tolerant rice
(Aventis produced by inserting a modified
CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT)
encoding gene from the soil bacterium
Streptomyces hygroscopicus).
PWC16 BASF Inc. Tolerance to the imidazolinone herbicide,
imazethapyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).
Table 2D Glycine max L. Soybean
Event Company Description
A5547-127 Bayer CropScience Glufosinate ammonium herbicide tolerant
(Aventis CropScience soybean produced by inserting a modified (AgrEvo)) phosphinothricin acetyltransferase (PAT)
encoding gene from the soil bacterium
Streptomyces viridochromogenes .
BPS-CV127-9 BASF Inc. The introduced csr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacid synthase protein that confers tolerance to imidazolinone herbicides due to a point mutation that results in a single amino acid substitution in which the serine residue at position 653 is replaced by asparagine (S653N).
DP-305423 Pioneer Hi-Bred High oleic acid soybean produced by inserting
International Inc. additional copies of a portion of the omega-6 desaturase encoding gene, gm-fad2-1 resulting in silencing of the endogenous omega-6 desaturase gene (FAD2-1).
DP356043 Pioneer Hi-Bred Soybean event with two herbicide tolerance
International Inc. genes: glyphosate N-acetlytransferase, which detoxifies glyphosate, and a modified acetolactate synthase (ALS) gene which is tolerant to ALS-inhibiting herbicides.
G94-1 , G94-19, G168 DuPont Canada High oleic acid soybean produced by inserting a
Agricultural Products second copy of the fatty acid desaturase
(GmFad2-1 ) encoding gene from soybean, which resulted in "silencing" of the endogenous host gene.
GTS 40-3-2 Monsanto Company Glyphosate tolerant soybean variety produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.
GU262 Bayer CropScience Glufosinate ammonium herbicide tolerant
(Aventis soybean produced by inserting a modified
CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT)
encoding gene from the soil bacterium
Streptomyces viridochromogenes .
MON87701 Monsanto Company Resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens). Event Company Description
MON87701 x Monsanto Company Glyphosate herbicide tolerance through
MON89788 expression of the EPSPS encoding gene from A.
tumefaciens strain CP4, and resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens) via expression of the Cry1 Ac encoding gene from B. thuringiensis.
MON89788 Monsanto Company Glyphosate-tolerant soybean produced by
inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding aroA (epsps) gene from Agrobacterium tumefaciens CP4.
OT96-15 Agriculture & Agri-Food Low linolenic acid soybean produced through
Canada traditional cross-breeding to incorporate the novel trait from a naturally occurring fanl gene mutant that was selected for low linolenic acid.
W62, W98 Bayer CropScience Glufosinate ammonium herbicide tolerant
(Aventis soybean produced by inserting a modified
CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT)
encoding gene from the soil bacterium
Streptomyces hygroscopicus.
Table 2E Zea mays L. Maize
Figure imgf000143_0001
Event Company Description
BT11 x MIR162 x Syngenta Seeds, Inc. Resistance to coleopteran pests, particularly MIR604 x GA21 corn rootworm pests (Diabrotica spp.) and
several lepidopteran pests of corn, including European corn borer (ECB, Ostrinia nubilalis), corn earworm (CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), and black cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate and glufosinate-ammonium containing herbicides.
BT11 X MIR604 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines BT1 1 (OECD unique identifier: SYN-BT01 1-1 ) and MIR604 (OECD unique identifier: SYN-IR605-5). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT1 1 , which contains the crylAb gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.
viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacillus thuringiensis.
BT11 x MIR604 x GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines BT1 1 (OECD unique identifier: SYN-BT01 1-1 ), MIR604 (OECD unique identifier: SYN-IR605-5) and GA21 (OECD unique identifier: MON-00021-9). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT1 1 , which contains the crylAb gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.
viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacillus thuringiensis. Tolerance to glyphosate herbicide is derived from GA21 which contains a modified EPSPS gene from maize.
CBH-351 Aventis CropScience Insect-resistant and glufosinate ammonium herbicide tolerant maize developed by inserting genes encoding Cry9C protein from Bacillus thuringiensis subsp tolworthi and
phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus.
DAS-06275-8 DOW AgroSciences Lepidopteran insect resistant and glufosinate
LLC ammonium herbicide-tolerant maize variety produced by inserting the crylF gene from Bacillus thuringiensis var aizawai and the phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus.
DAS-59122-7 DOW AgroSciences Corn rootworm-resistant maize produced by
LLC and Pioneer Hi- inserting the cry34Ab1 and cry35Ab1 genes from Bred International Inc. Bacillus thuringiensis strain PS149B1. The PAT encoding gene from Streptomyces
viridochromogenes was introduced as a selectable marker. Event Company Description
DAS-59122-7 X NK603 DOW AgroSciences Stacked insect resistant and herbicide tolerant
LLC and Pioneer Hi- maize produced by conventional cross breeding Bred International Inc. of parental lines DAS-59122-7 (OECD unique identifier: DAS-59122-7) with NK603 (OECD unique identifier: MON-00603-6). Corn rootworm-resistance is derived from DAS-59122- 7 which contains the cry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis strain
PS149B1. Tolerance to glyphosate herbicide is derived from NK603.
DAS-59122-7 x TC 1507 DOW AgroSciences Stacked insect resistant and herbicide tolerant x NK603 LLC and Pioneer Hi- maize produced by conventional cross breeding
Bred International Inc. of parental lines DAS-59122-7 (OECD unique identifier: DAS-59122-7) and TC1507 (OECD unique identifier: DAS-01507-1 ) with NK603 (OECD unique identifier: MON-00603-6). Corn rootworm-resistance is derived from DAS-59122- 7 which contains the cry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis strain
PS149B1. Lepidopteran resistance and tolerance to glufosinate ammonium herbicide is derived from TC1507. Tolerance to glyphosate herbicide is derived from NK603.
DBT418 Dekalb Genetics Insect-resistant and glufosinate ammonium
Corporation herbicide tolerant maize developed by inserting genes encoding CrylAC protein from Bacillus thuringiensis subsp kurstaki and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus
DK404SR BASF Inc. Somaclonal variants with a modified acetyl-CoA- carboxylase (ACCase) were selected by culture of embryos on sethoxydim enriched medium.
Event 3272 Syngenta Seeds, Inc. Maize line expressing a heat stable alpha- amylase gene amy797E for use in the dry-grind ethanol process. The phosphomannose isomerase gene from E.coli was used as a selectable marker.
Event 98140 Pioneer Hi-Bred Maize event expressing tolerance to glyphosate
International Inc. herbicide, via expression of a modified bacterial glyphosate N-acetlytransferase, and ALS- inhibiting herbicides, vial expression of a modified form of the maize acetolactate synthase enzyme.
EXP1910IT Syngenta Seeds, Inc. Tolerance to the imidazolinone herbicide,
(formerly Zeneca imazethapyr, induced by chemical mutagenesis Seeds) of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).
GA21 Syngenta Seeds, Inc. Introduction, by particle bombardment, of a
(formerly Zeneca modified 5-enolpyruvyl shikimate-3-phosphate Seeds) synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids.
GA21 x MON810 Monsanto Company Stacked insect resistant and herbicide tolerant corn hybrid derived from conventional crossbreeding of the parental lines GA21 (OECD identifier: MON-00021-9) and MON810 (OECD identifier: MON-00810-6).
Figure imgf000146_0001
Company gene from Bacillus thuringiensis subsp. kumamotoensis. MON863 x Monsanto Company Stacked insect resistant corn hybrid derived from MON810 conventional cross-breeding of the parental lines
MON863 (OECD identifier: MON-00863-5) and MON810 (OECD identifier: MON-00810-6)
MON863 x Monsanto Company Stacked insect resistant and herbicide tolerant corn MON810 X hybrid derived from conventional cross-breeding of the NK603 stacked hybrid MON-00863-5 x MON-00810-6 and
NK603 (OECD identifier:MON-00603-6).
MON863 x Monsanto Company Stacked insect resistant and herbicide tolerant corn NK603 hybrid derived from conventional cross-breeding of the parental lines MON863 (OECD identifier:MON-00863- 5) and NK603 (OECD identifier: MON-00603-6).
MON87460 Monsanto Company MON 87460 was developed to provide reduced yield loss under water-limited conditions compared to conventional maize. Efficacy in MON 87460 is derived by expression of the inserted Bacillus subtilis cold shock protein B (CspB).
MON88017 Monsanto Company Corn rootworm-resistant maize produced by inserting the cry3Bb1 gene from Bacillus thuringiensis subspecies kumamotoensis strain EG4691.
Glyphosate tolerance derived by inserting a 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4.
MON89034 Monsanto Company Maize event expressing two different insecticidal proteins from Bacillus thuringiensis providing resistance to number of lepidopteran pests.
MON89034 x Monsanto Company Stacked insect resistant and glyphosate tolerant maize MON88017 derived from conventional cross-breeding of the
parental lines MON89034 (OECD identifier: MON- 89034-3) and MON88017 (OECD identifier:MON- 88017-3). Resistance to Lepidopteran insects is derived from two cry genes present in MON89043. Corn rootworm resistance is derived from a single cry genes and glyphosate tolerance is derived from the 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens present in MON88017.
MON89034 x Monsanto Company Stacked insect resistant and herbicide tolerant maize NK603 produced by conventional cross breeding of parental lines MON89034 (OECD identifier: MON-89034-3) with NK603 (OECD unique identifier: MON-00603-6). Resistance to Lepidopteran insects is derived from two cry genes present in MON89043. Tolerance to glyphosate herbicide is derived from NK603.
MON89034 x Monsanto Company and Stacked insect resistant and herbicide tolerant maize TC1507 X Mycogen Seeds c/o Dow produced by conventional cross breeding of parental MON88017 X AgroSciences LLC lines: MON89034, TC1507, MON88017, and DAS- DAS-59122-7 59122. Resistance to the above-ground and below- ground insect pests and tolerance to glyphosate and glufosinate-ammonium containing herbicides.
MS3 Bayer CropScience Male sterility caused by expression of the barnase
(Aventis ribonuclease gene from Bacillus amyloliquefaciens;
CropScience(AgrEvo)) PPT resistance was via PPT-acetyltransferase (PAT).
MS6 Bayer CropScience Male sterility caused by expression of the barnase
(Aventis ribonuclease gene from Bacillus amyloliquefaciens;
CropScience(AgrEvo)) PPT resistance was via PPT-acetyltransferase (PAT). NK603 Monsanto Company Introduction, by particle bombardment, of a
modified 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids.
NK603 x Monsanto Company Stacked insect resistant and herbicide tolerant MON810 corn hybrid derived from conventional crossbreeding of the parental lines NK603 (OECD identifier: MON-00603-6) and MON810 (OECD identifier: MON-00810-6).
NK603 x Monsanto Company Stacked glufosinate ammonium and glyphosate T25 herbicide tolerant maize hybrid derived from
conventional cross-breeding of the parental lines NK603 (OECD identifier: MON-00603-6) and T25 (OECD identifier: ACS-ZM003-2).
T14, T25 Bayer CropScience (Aventis Glufosinate herbicide tolerant maize produced by
CropScience(AgrEvo)) inserting the phosphinothricin N-acetyltransferase
(PAT) encoding gene from the aerobic actinomycete Streptomyces viridochromogenes.
T25 x Bayer CropScience (Aventis Stacked insect resistant and herbicide tolerant MON810 CropScience(AgrEvo)) corn hybrid derived from conventional crossbreeding of the parental lines T25 (OECD identifier: ACS-ZM003-2) and MON810 (OECD identifier:MON-00810-6).
TC1507 Mycogen (c/o Dow AgroSciences); Insect-resistant and glufosinate ammonium
Pioneer (c/o DuPont) herbicide tolerant maize produced by inserting the cry IF gene from Bacillus thuringiensis var.
aizawai and the phosphinothricin N- acetyltransferase encoding gene from
Streptomyces viridochromogenes.
TC1507 X DOW AgroSciences LLC and Stacked insect resistant and herbicide tolerant DAS- Pioneer Hi-Bred International Inc. maize produced by conventional cross breeding 59122-7 of parental lines TC1507 (OECD unique identifier:
DAS-01507-1 ) with DAS-59122-7 (OECD unique identifier: DAS-59122-7). Resistance to lepidopteran insects is derived from TC1507 due the presence of the crylF gene from Bacillus thuringiensis var. aizawai. Corn rootworm- resistance is derived from DAS-59122-7 which contains the cry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis strain PS149B1. Tolerance to glufosinate ammonium herbicide is derived from TC1507 from the phosphinothricin N- acetyltransferase encoding gene from
Streptomyces viridochromogenes.
TC1507 X DOW AgroSciences LLC Stacked insect resistant and herbicide tolerant NK603 corn hybrid derived from conventional crossbreeding of the parental lines 1507 (OECD identifier: DAS-01507-1 ) and NK603 (OECD identifier: MON-00603-6). Table 2F Triticum aestivum Wheat
Figure imgf000149_0001
Other events with regulatory approval are well known to one skilled in the art and can be found at the Center for Environmental Risk Assessment (cera- gmc.org/?action=gm_crop_database, which can be accessed using the www prefix) and at the International Service for the Acquisition of Agri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp, which can be accessed using the www prefix).
Gene silencing
In some embodiments the stacked trait may be in the form of silencing of one or more polynucleotides of interest resulting in suppression of one or more target pest polypeptides. In some embodiments the silencing is achieved through the use of a suppression DNA construct.
In some embodiments one or more of the PHI-4 polypeptides or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussed infra.
"Suppression DNA construct" is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in "silencing" of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. "Silencing," as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The term "suppression" includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. "Silencing" or "gene silencing" does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi- based approaches and small RNA-based approaches.
A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50% or any integer between 51 % and 100% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.
Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
"Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
"Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (US Patent Number 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non- coding sequence, 3' non-coding sequence, introns or the coding sequence.
"Cosuppression" refers to the production of sense RNA transcripts capable of suppressing the expression of the target protein. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see, Vaucheret, et al. (1998) Plant J. 16:651-659 and Gura, (2000) Nature 404:804-808).
Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication WO 1998/36083).
Recent work has described the use of "hairpin" structures that incorporate all or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication Number WO 1999/53050). In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti-sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct. This increases the frequency of cosuppression or silencing in the recovered transgenic plants. For review of hairpin suppression see, Wesley, et al., (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286.
A construct where the stem is formed by at least 30 nucleotides from a gene to be suppressed and the loop is formed by a random nucleotide sequence has also effectively been used for suppression (WO 1999/61632).
The use of poly-T and poly-A sequences to generate the stem in the stem-loop structure has also been described (WO 2002/00894).
Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. Transgenic organisms prepared with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment forming the loop as described in PCT Publication Number WO 2002/00904.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391 :806). The corresponding process in plants is commonly referred to as post- transcriptional gene silencing (PTGS) or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire, et al., (1999) Trends Genet. 15:358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein, et al., (2001 ) Nature 409:363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir, et al. , (2001 ) Genes Dev. 15:188). Dicer has also been implicated in the excision of 21 - and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner, et al., (2001 ) Science 293:834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir, et al., (2001 ) Genes Dev. 15:188). In addition, RNA interference can also involve small RNA (e.g., miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819; Volpe, et al. , (2002) Science 297:1833-1837; Jenuwein, (2002) Science 297:2215-2218; and Hall, et al., (2002) Science 297:2232-2237). As such, miRNA molecules of the disclosure can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post-transcriptional level.
Methods and compositions are further provided which allow for an increase in RNAi produced from the silencing element. In such embodiments, the methods and compositions employ a first polynucleotide comprising a silencing element for a target pest sequence operably linked to a promoter active in the plant cell; and, a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof operably linked to a promoter active in the plant cell. The combined expression of the silencing element with suppressor enhancer element leads to an increased amplification of the inhibitory RNA produced from the silencing element over that achievable with only the expression of the silencing element alone. In addition to the increased amplification of the specific RNAi species itself, the methods and compositions further allow for the production of a diverse population of RNAi species that can enhance the effectiveness of disrupting target gene expression. As such, when the suppressor enhancer element is expressed in a plant cell in combination with the silencing element, the methods and composition can allow for the systemic production of RNAi throughout the plant; the production of greater amounts of RNAi than would be observed with just the silencing element construct alone; and, the improved loading of RNAi into the phloem of the plant, thus providing better control of phloem feeding insects by an RNAi approach. Thus, the various methods and compositions provide improved methods for the delivery of inhibitory RNA to the target organism. See, for example, US 2009/0188008.
As used herein, a "suppressor enhancer element" comprises a polynucleotide comprising the target sequence to be suppressed or an active fragment or variant thereof. It is recognize that the suppressor enhancer element need not be identical to the target sequence, but rather, the suppressor enhancer element can comprise a variant of the target sequence, so long as the suppressor enhancer element has sufficient sequence identity to the target sequence to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element. Similarly, the suppressor enhancer element can comprise a fragment of the target sequence, wherein the fragment is of sufficient length to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element.
It is recognized that multiple suppressor enhancer elements from the same target sequence or from different target sequences or from different regions of the same target sequence can be employed. For example, the suppressor enhancer elements employed can comprise fragments of the target sequence derived from different region of the target sequence (i.e., from the 3'UTR, coding sequence, intron, and/or 5'UTR). Further, the suppressor enhancer element can be contained in an expression cassette, as described elsewhere herein, and in specific embodiments, the suppressor enhancer element is on the same or on a different DNA vector or construct as the silencing element. The suppressor enhancer element can be operably linked to a promoter as disclosed herein. It is recognized that the suppressor enhancer element can be expressed constitutively or alternatively, it may be produced in a stage-specific manner employing the various inducible or tissue-preferred or developmental^ regulated promoters that are discussed elsewhere herein.
In specific embodiments, employing both a silencing element and the suppressor enhancer element the systemic production of RNAi occurs throughout the entire plant. In further embodiments, the plant or plant parts of the disclosure have an improved loading of RNAi into the phloem of the plant than would be observed with the expression of the silencing element construct alone and, thus provide better control of phloem feeding insects by an RNAi approach. In specific embodiments, the plants, plant parts, and plant cells of the disclosure can further be characterized as allowing for the production of a diversity of RNAi species that can enhance the effectiveness of disrupting target gene expression.
In specific embodiments, the combined expression of the silencing element and the suppressor enhancer element increases the concentration of the inhibitory RNA in the plant cell, plant, plant part, plant tissue or phloem over the level that is achieved when the silencing element is expressed alone.
As used herein, an "increased level of inhibitory RNA" comprises any statistically significant increase in the level of RNAi produced in a plant having the combined expression when compared to an appropriate control plant. For example, an increase in the level of RNAi in the plant, plant part or the plant cell can comprise at least about a 1 %, about a 1 %-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a 30%- 40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%, about a 80%-90%, about a 90%-100% or greater increase in the level of RNAi in the plant, plant part, plant cell or phloem when compared to an appropriate control. In other embodiments, the increase in the level of RNAi in the plant, plant part, plant cell or phloem can comprise at least about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold, about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40 fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70 fold, about 70 fold-80 fold, about a 80 fold- 90 fold, about a 90 fold-100 fold or greater increase in the level of RNAi in the plant, plant part, plant cell or phloem when compared to an appropriate control. Examples of combined expression of the silencing element with suppressor enhancer element for the control of Stinkbugs and Lygus can be found in US 201 1/0301223 and US 2009/01921 17.
Some embodiments relate to down-regulation of expression of target genes in insect pest species by interfering ribonucleic acid (RNA) molecules. WO 2007/074405 describes methods of inhibiting expression of target genes in invertebrate pests including Colorado potato beetle. WO 2005/1 10068 describes methods of inhibiting expression of target genes in invertebrate pests including in particular Western corn rootworm as a means to control insect infestation. Furthermore, WO 2009/091864 describes compositions and methods for the suppression of target genes from insect pest species including pests from the Lygus genus. Nucleic acid molecules including RNAi for targeting the vacuolar ATPase H subunit, useful for controlling a coleopteran pest population and infestation as described in US Patent Application Publication 2012/0198586. WO 2012/055982 describes ribonucleic acid (RNA or double stranded RNA) that inhibits or down regulates the expression of a target gene that encodes: an insect ribosomal protein such as the ribosomal protein L19, the ribosomal protein L40 or the ribosomal protein S27A; an insect proteasome subunit such as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomer of the COPI vesicle, the γ-coatomer of the COPI vesicle, the β'- coatomer protein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2 A protein which is a putative transmembrane domain protein; an insect protein belonging to the actin family such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23 protein which is a GTPase activator involved in intracellular protein transport; an insect crinkled protein which is an unconventional myosin which is involved in motor activity; an insect crooked neck protein which is involved in the regulation of nuclear alternative mRNA splicing; an insect vacuolar H+-ATPase G-subunit protein; and an insect Tbp-1 such as Tat-binding protein. US Patent Application Publications 2012/029750, US 20120297501 , and 2012/0322660 describe interfering ribonucleic acids (RNA or double stranded RNA) that functions upon uptake by an insect pest species to down-regulate expression of a target gene in said insect pest, wherein the RNA comprises at least one silencing element wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene. US Patent Application Publication 2012/0164205 describe potential targets for interfering double stranded ribonucleic acids for inhibiting invertebrate pests including: a Chd3 Homologous Sequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPase Homologous Sequence, a EF1 a Homologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, a Juvenile Hormone Epoxide Hydrolase Homologous Sequence, a Swelling Dependent Chloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate 1 -Dehydrogenase Protein Homologous Sequence, an Act42A Protein Homologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, a Transcription Factor MB Protein Homologous Sequence, a Chitinase Homologous Sequences, a Ubiquitin Conjugating Enzyme Homologous Sequence, a Glyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, an Ubiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog, and an Alpha Tubuliln Homologous Sequence. Use in Pesticidal Control
General methods for employing strains comprising a nucleic acid sequence of the embodiments or a variant thereof, in pesticide control or in engineering other organisms as pesticidal agents are known in the art. See, for example US Patent Number 5,039,523 and EP 0480762A2.
Microorganism hosts that are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the PHI-4 polypeptide, and desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas chlororaphis, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinelandii and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms. Host organisms of particular interest include yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae), Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp. (such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp., and Flavobacterium spp., and other such organisms, including Agrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereus and the like. Genes encoding the PHI-4 polypeptides of the embodiments can be introduced into microorganisms that multiply on plants (epiphytes) to deliver PHI-4 polypeptides to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria.
Root-colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain that colonizes roots can be isolated from roots of a plant (see, for example, Handelsman, et al., (1991 ) Appl. Environ. Microbiol. 56:713-718). Genes encoding the PHI-4 polypeptides of the embodiments can be introduced into a root-colonizing Bacillus cereus by standard methods known in the art.
Genes encoding PHI-4 polypeptides can be introduced, for example, into the root- colonizing Bacillus by means of electro transformation. Specifically, genes encoding the PHI-4 polypeptides can be cloned into a shuttle vector, for example, pHT3101 (Lerecius, et al., (1989) FEMS Microbiol. Letts. 60:21 1 -218. The shuttle vector pHT3101 containing the coding sequence for the particular PHI-4 polypeptide gene can, for example, be transformed into the root-colonizing Bacillus by means of electroporation (Lerecius, et al., (1989) FEMS Microbiol. Letts. 60:21 1-218).
Expression systems can be designed so that PHI-4 polypeptides are secreted outside the cytoplasm of gram-negative bacteria, such as £ coli, for example. Advantages of having PHI-4 polypeptides secreted are: (1 ) avoidance of potential cytotoxic effects of the PHI-4 polypeptide expressed; and (2) improvement in the efficiency of purification of the PHI-4 polypeptide, including, but not limited to, increased efficiency in the recovery and purification of the protein per volume cell broth and decreased time and/or costs of recovery and purification per unit protein.
PHI-4 polypeptides can be made to be secreted in £ coli, for example, by fusing an appropriate £ coli signal peptide to the amino-terminal end of the PHI-4 polypeptide. Signal peptides recognized by £. coli can be found in proteins already known to be secreted in £ coli, for example the OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437-2442). OmpA is a major protein of the £ coli outer membrane, and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (Duffaud, et al., (1987) Meth. Enzymol. 153:492).
PHI-4 polypeptides of the embodiments can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bt strains have been used as insecticidal sprays. In the case of a PHI-4 polypeptide that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the PHI-4 polypeptide into the growth medium during the fermentation process. The PHI-4 polypeptides are retained within the cell, and the cells are then processed to yield the encapsulated PHI-4 polypeptides. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bt toxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner, et al., (1993), in: Advanced Engineered Pesticides, ed. Kim).
Alternatively, the PHI-4 polypeptides are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated PHI-4 polypeptides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.
Pesticidal Compositions
In some embodiments the active ingredients can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise the formulations may be prepared into edible "baits" or fashioned into pest "traps" to permit feeding or ingestion by a target pest of the pesticidal formulation.
Methods of applying an active ingredient or an agrochemical composition that contains at least one of the PHI-4 polypeptides produced by the bacterial strains include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation, sedimentation or concentration of a culture of cells comprising the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration of from about 1 % to about 99% by weight.
Lepidopteran, dipteran, heteropteran, nematode, hemiptera or coleopteran pests may be killed or reduced in numbers in a given area by the methods of the disclosure or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest. Preferably the pest ingests or is contacted with, a pesticidally-effective amount of the polypeptide. By "pesticidally-effective amount" is intended an amount of the pesticide that is able to bring about death to at least one pest or to noticeably reduce pest growth, feeding or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop or agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.
The pesticide compositions described may be made by formulating either the bacterial cell, crystal and/or spore suspension or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material or a suspension in oil (vegetable or mineral) or water or oil/water emulsions or as a wettable powder or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally-acceptable carrier" covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in US Patent Number 6,468,523, herein incorporated by reference. The plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides or fungicides. Exemplary chemical compositions include: Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox, Methomyl, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on; Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs, Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam, Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid, Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon, Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl, Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos, Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole, Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin- Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3- yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides: Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-3- yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.
In some embodiments the herbicide is Atrazine, Bromacil, Diuron, Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-metolachlor, Hexazinne or combinations thereof.
In some embodiments the insecticide is Esfenvalerate, Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinations thereof.
Pesticidal and insecticidal activity
"Pest" includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera, and Hemiptera.
Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds of the embodiments display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery, ornamentals, food and fiber, public and animal health, domestic and commercial structure, household and stored product pests.
Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hubner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia ni Hubner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hubner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hubner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermijller (European grape vine moth); Spilonota ocellana Denis & Schiffermijller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hubner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp..
Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.
Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae, and Curculionidae (including, but not limited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug)); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae (including, but not limited to: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn rootworm); D. barberi Smith & Lawrence (northern corn rootworm,); D. undecimpunctata howardi Barber (southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle,); Phyllotreta striolata (stripped flea beetle,);; Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from the family Coccinellidae (including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle)); chafers and other beetles from the family Scarabaeidae (including, but not limited to: Popillia japonica Newman (Japanese beetle); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer); Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer (carrot beetle)); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae and beetles from the family Tenebrionidae.
Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly); and other Delia spp., Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.
Included as insects of interest are adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae, Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae, and red bugs and cotton stainers from the family Pyrrhocoridae.
Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantaginea Paaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly apple aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera Horvath (white-backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); lcerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead (persimmon psylla).
Agronomically important species of interest from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); £. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).
Furthermore, embodiments may be effective against Hemiptera such, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.; and Cimicidae spp. Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia latens Mijller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health, i.e. dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deer tick); /. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick); and scab and itch mites in the families Psoroptidae, Pyemotidae, and Sarcoptidae.
Insect pests of the order Thysanura are of interest, such as Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).
Additional arthropod pests covered include: spiders in the order Araneae such as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); and the Latrodectus mactans Fabricius (black widow spider); and centipedes in the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (house centipede).
Insect pest of interest include the superfamily of stink bugs and other related insects including but not limited to species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guild ini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria - Bean plataspid), and the family Cydnidae {Scaptocoris castanea - Root stink bug); and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker; and velvet bean caterpillar e.g., Anticarsia gemmatalis Hubner.
Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone, et al., (1985) J. of Economic Entomology 78:290-293 and US Patent Number 5,743,477, all of which are herein incorporated by reference in their entirety. Generally, the protein is mixed and used in feeding assays. See, for example Marrone, et al., (1985) J. of Economic Entomology 78:290-293. Such assays can include contacting plants with one or more pests and determining the plant's ability to survive and/or cause the death of the pests. Nematodes include parasitic nematodes such as root-knot, cyst, and lesion nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pallida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.
Seed Treatment
To protect and to enhance yield production and trait technologies, seed treatment options can provide additional crop plan flexibility and cost effective control against insects, weeds and diseases. Seed material can be treated, typically surface treated, with a composition comprising combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides, germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematocides, avicides and/or molluscicides. These compounds are typically formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. The coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Examples of the various types of compounds that may be used as seed treatments are provided in The Pesticide Manual: A World Compendium, C.D.S. Tomlin Ed., Published by the British Crop Production Council, which is hereby incorporated by reference.
Some seed treatments that may be used on crop seed include, but are not limited to, one or more of abscisic acid, acibenzolar-S-methyl, avermectin, amitrol, azaconazole, azospirillum, azadirachtin, azoxystrobin, Bacillus spp. (including one or more of cereus, firmus, megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species), bradyrhizobium spp. (including one or more of betae, canariense, elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/or yuanmingense), captan, carboxin, chitosan, clothianidin, copper, cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil, fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin, prothioconazole, pyraclostrobin, rynaxypyr, S- metolachlor, saponin, sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, triticonazole and/or zinc. PCNB seed coat refers to EPA registration number 00293500419, containing quintozen and terrazole. TCMTB refers to 2-(thiocyanomethylthio) benzothiazole. Seed varieties and seeds with specific transgenic traits may be tested to determine which seed treatment options and application rates may complement such varieties and transgenic traits in order to enhance yield. For example, a variety with good yield potential but head smut susceptibility may benefit from the use of a seed treatment that provides protection against head smut, a variety with good yield potential but cyst nematode susceptibility may benefit from the use of a seed treatment that provides protection against cyst nematode, and so on. Likewise, a variety encompassing a transgenic trait conferring insect resistance may benefit from the second mode of action conferred by the seed treatment, a variety encompassing a transgenic trait conferring herbicide resistance may benefit from a seed treatment with a safener that enhances the plants resistance to that herbicide, etc. Further, the good root establishment and early emergence that results from the proper use of a seed treatment may result in more efficient nitrogen use, a better ability to withstand drought and an overall increase in yield potential of a variety or varieties containing a certain trait when combined with a seed treatment.
Methods for inhibiting growth or killing an insect pest and controlling an insect population
In some embodiments methods are provided for inhibiting growth or killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of a recombinant PHI-4 polypeptide. In some embodiments methods are provided for inhibiting growth or killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of a recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or a variant thereof.
In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally- effective amount of a recombinant PHI-4 polypeptide. In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of a recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or a variant thereof. As used herein, by "controlling a pest population" or "controls a pest" is intended any effect on a pest that results in limiting the damage that the pest causes. Controlling a pest includes, but is not limited to, killing the pest, inhibiting development of the pest, altering fertility or growth of the pest in such a manner that the pest provides less damage to the plant, decreasing the number of offspring produced, producing less fit pests, producing pests more susceptible to predator attack or deterring the pests from eating the plant.
In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of a recombinant PHI-4 polypeptide. In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of a recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or a variant thereof.
In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a recombinant PHI-4 polypeptide. In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518- 1526 or variants thereof.
Insect Resistance Management (IRM) Strategies
Expression of B. thuringiensis δ-endotoxins in transgenic corn plants has proven to be an effective means of controlling agriculturally important insect pests (Perlak, et al., 1990; 1993). However, insects have evolved that are resistant to B. thuringiensis δ- endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing genes encoding such B. thuringiensis δ-endotoxins.
One way to increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests is to use provide non-transgenic (i.e., non-insecticidal protein) refuges (a section of non-insecticidal crops/ corn) for use with transgenic crops producing a single insecticidal protein active against target pests. The United States Environmental Protection Agency (epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which can be accessed using the www prefix) publishes the requirements for use with transgenic crops producing a single Bt protein active against target pests. In addition, the National Corn Growers Association, on their website: (ncga.com/insect-resistance-management-fact-sheet-bt- corn, which can be accessed using the www prefix) also provides similar guidance regarding refuge requirements. Due to losses to insects within the refuge area, larger refuges may reduce overall yield. Another way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests would be to have a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action.
Expression in a plant of two or more insecticidal compositions toxic to the same insect species, each insecticide being expressed at efficacious levels would be another way to achieve control of the development of resistance. This is based on the principle that evolution of resistance against two separate modes of action is far more unlikely than only one. Roush for example, outlines two-toxin strategies, also called "pyramiding" or "stacking," for management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353:777-1786). Stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. The U.S. Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non-Bt corn be planted than for single trait products (generally 20%). There are various ways of providing the IRM effects of a refuge, including various geometric planting patterns in the fields and in-bag seed mixtures, as discussed further by Roush.
In some embodiments the PHI-4 polypeptides of the disclosure are useful as an insect resistance management strategy in combination (i.e., pyramided) with other pesticidal proteins include but are not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, and the like.
Provided are methods of controlling Coleoptera insect infestation(s) in a transgenic plant that promote insect resistance management, comprising expressing in the plant at least two different insecticidal proteins having different modes of action.
In some embodiments the methods of controlling Coleoptera insect infestation in a transgenic plant and promoting insect resistance management the at least one of the insecticidal proteins comprise a PHI-4 polypeptide insecticidal to insects in the order Coleoptera.
In some embodiments the methods of controlling Coleoptera insect infestation in a transgenic plant and promoting insect resistance management the at least one of the insecticidal proteins comprises a protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51 -1 162, and SEQ ID NOs: 1518-1526 or variants thereof, insecticidal to insects in the order Coleoptera.
In some embodiments the methods of controlling Coleoptera insect infestation in a transgenic plant and promoting insect resistance management comprise expressing in the transgenic plant a PHI-4 polypeptide and a cry protein insecticidal to insects in the order Coleoptera having different modes of action.
In some embodiments the methods of controlling Coleoptera insect infestation in a transgenic plant and promoting insect resistance management comprise in the transgenic plant a protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or variants thereof and a cry protein insecticidal to insects in the order Coleoptera having different modes of action.
Also provided are methods of reducing likelihood of emergence of Coleoptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of a PHI-4 polypeptide insecticidal to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.
Also provided are methods of reducing likelihood of emergence of Coleoptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of a protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or variants thereof, insecticidal to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.
Also provided are means for effective Coleoptera insect resistance management of transgenic plants, comprising co-expressing at high levels in the plants two or more insecticidal proteins toxic to Coleoptera insects but each exhibiting a different mode of effectuating its inhibiting growth or killing activity, wherein the two or more insecticidal proteins comprise a PHI-4 polypeptide and a cry protein. Also provided are means for effective Coleoptera insect resistance management of transgenic plants, comprising co- expressing at high levels in the plants two or more insecticidal proteins toxic to Coleoptera insects but each exhibiting a different mode of effectuating its inhibiting growth or activity, wherein the two or more insecticidal proteins comprise a protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or variants thereof and a cry protein.
In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to insects in the order Coleoptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that the PHI-4 polypeptide does not compete with binding sites for cry proteins in such insects. In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to insects in the order Coleoptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that the protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-1 162, and SEQ ID NOs: 1518-1526 or variant thereof does not compete with binding sites for cry proteins in such insects. Methods for Increasing Plant Yield
Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell expressing a polynucleotide encoding the pesticidal polypeptide sequence disclosed herein and growing the plant or a seed thereof in a field infested with a pest against which the polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a lepidopteran, coleopteran, dipteran, hemipteran or nematode pest, and the field is infested with a lepidopteran, hemipteran, coleopteran, dipteran or nematode pest.
As defined herein, the "yield" of the plant refers to the quality and/or quantity of biomass produced by the plant. By "biomass" is intended any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1 % increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the pesticidal sequence.
In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing a PHI-4 polypeptide disclosed herein. Expression of the PHI-4 polypeptide results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.
These and other changes may be made to the disclosure in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the disclosure to the specific embodiments disclosed in the specification and the claims.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, manuals, books or other disclosures) in the Background, Detailed Description, and Examples is herein incorporated by reference in their entireties. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject disclosure, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees centigrade; and pressure is at or near atmospheric. EXPERI MENTALS
Example 1 : Generating PHI -4 genes
Polynucleotides having single codon substitutions compared to the PHI-4 polypeptide of SEQ ID NO: 1 were generated. As described in the examples below, the corresponding PHI-4 polypeptides were expressed, purified and assayed for WCRW insecticidal activity in order to assess the corresponding activity diversity. A reverse mutagenesis primer and a complementary forward mutagenesis primer were designed to create the desired amino acid substitution(s) at the site(s) of interest. Typically the mutagenesis primer was between 30 to 45 bases in length with two or more bases, usually 10 to 15, on both sides of the site of interest. In the case of saturation
mutagenesis, degenerated primers that cover all possible amino acid residues were used. Unless otherwise noted, the mutagenic reactions were carried out using Agilent's
QuikChange™ Lightening Site-Directed Mutagenesis kit. Materials provided in the kit are QuikChange™ Lightening Enzyme, 10X QuikChange™ Lightning Buffer, dNTP mix, QuikSolution™ reagent and Dpn1 restriction enzyme according to the manufactures directions.
PCR amplifications were typically carried out with Expand™ High Fidelity PCR system (Roche, Switzerland) in 50 ul containing 50-100 ng templates, 0.4-2 μΜ primer pair, 200 μΜ dNTPs and 2 Units of DNA polymerase. The mutagenesis reaction was initiated by pre-heating the reaction mixture to 94°C for 3 min, followed by 16 cycles of the following cycling program: 94°C for 1 min, 52°C for 1 min and 68°C for 8, 12, 16 or 24 min according to the length of template. The mutagenesis reaction was completed by incubation at 68°C for 1 h. The PCR-amplification products were evaluated by agarose gel electrophoresis. The PCR products were purified by QIAquick™ PCR purification kit (Qiagen, Germany) and further treated with the restriction enzyme Dpn1 . An aliquot of 1 μΙ of this PCR product was typically transformed into BL21 (DE3) cells and inoculated on Luria-Bertani (LB) plate containing 100 μg/ml ampicillin. About 10 colonies in the case of single amino acid mutation or 48 or more colonies for saturation mutagenesis were selected and plasmid DNA was isolated for sequencing. Two step sequencing was used, first for specific mutation site(s) with one sequencing primer followed by full length sequence confirmation with multiple sequencing primers.
Example 2: Purification of MBP::PHI-4 fusion polypeptides
Polynucleotides encoding PHI-4 polypeptides were expressed in a modified pMAL vector (New England Bio Lab) as a fusion (i.e. MBP::PHI-4; SEQ ID NO: 6) with MBP (maltose binding protein). The pMAL vector was modified to attach a 6X His tag to the N- terminus of MBP. In order to clone the polynucleotide encoding the MBP::PHI-4 fusion protein (SEQ ID NO: 6), Sph1 and BamHI sites were engineered in the vector at the cloning site. The polynucleotide (SEQ ID NO: 1 ) encoding the PHI-4 polypeptide (SEQ ID NO: 2) was amplified with a forward primer (SEQ ID NO: 32) overlapping the Sph1 site and a reverse primer (SEQ ID NO: 33) overlapping the BamHI site. This PCR product was digested with Sph1 and BamHI and cloned into pMAL that was precut with the same enzymes. The forward primer was designed such that polynucleotides encoding both MBP and PHI-4 polypeptide (SEQ ID NO: 2) within the MBP::PHI-4 gene (SEQ ID NO: 5) were ligated in frame. The plasmid containing the polynucleotide (SEQ ID NO: 5) encoding the MBP::PHI-4 polypeptide (SEQ ID NO: 6) was transformed into E. coli
BL21 (DE3) cells. The BL21 (DE3) cells were grown in MagicMedia™ (Life Technologies) in either 96 deep well plates or flasks in a shaker running at 250 rpm at 37°C for 8 hrs. followed by 16°C for 48-60 hrs. During the 16°C incubation, the MBP::PHI-4 polypeptide fusion protein accumulated in the BL21 (DE3) cells as a soluble protein.
In order to purify the MBP::PHI-4 fusion protein (SEQ ID NO: 6), the E. coli cells were harvested by centrifugation and treated in a lysozyme solution consisting of 2 mg/ml lysozyme in 50 ml sodium phosphate buffer at pH 8.0 containing 300 mM NaCI, 2 U/ml endonuclease (Epicentre) and 5 mM MgCI2 for 3 hrs. at 37°C with gentle shaking. The lysozyme treated E. coli cells were then disrupted with 1 % Triton X100 and clear lysate containing the MBP::PHI-4 proteins were prepared by centrifugation at 4000 rpm, 30 min (96 well plates) or 9000 rpm (flask produced samples). His tagged MBP-PHI-4 polypeptide fusion proteins were purified from the clear lysates by affinity chromatography using NiNTA agarose (catalog #30450; Qiagen) following the manufacturer's standard procedure. For high throughput protein purification, Pall 96 deep well filter plates (Pall Corporation; Catalogue #5051 ) were used for the affinity chromatography. The purified MPB::PHI-4 polypeptide fusion protein was eluted from NiNTA agarose and passed through Sephadex G25 to change the phosphate buffer to 25 mM HEPES-NaOH, pH 8 and used in insect bioassays for determining the insecticidal activity against Western Corn Rootworm (WCRW). Calipar GXII capillary electrophoresis with a protein chip (Agilent; catalogue #P/N760499) was used to determine the MPB::PHI-4 polypeptide
concentrations. The protein analysis was repeated at least 3 times until the final concentration was within the predetermined deviation (less than 10%). Unless otherwise noted, the PHI-4 polypeptides disclosed herein were expressed, purified and assayed for WCRW insecticidal activity as maltose binding protein fusions (i.e. MBP::PHI-4; SEQ ID NO: 6) as described above.
Example 3: Determination of WCRW insecticidal activity of MBP:: PHI-4 (SEQ ID NO: 6) and MBP::PHI-4-SFR12-004 (SEQ ID NO: 31 ) polypeptides.
The activity of MBP::PHI-4 (SEQ ID NO: 6) and MBP::PHI-4-SFR12-004 (SEQ ID NO: 31 ; Example 8) polypeptides against WCRW (western corn rootworm, Diabrotica virgifera virgifera) was determined in an artificial diet feeding assay essentially as described by Cong, R. et al. (Proceedings of the 4th Pacific Rim Conferences on
Biotechnology of Bacillus thuringiensis and its environmental impact, pp.1 18-123, ed. by R. J. Akhurst, C. E. Beard and P. Hughes, published in 2002, Canberra, Australia). The assays were conducted on an artificial diet containing dilutions of these
polypeptides. The MBP::PHI-4-SFR12-004 polypeptide fusion (SEQ ID NO: 31 ) and MBP::PHI-4 (SEQ ID NO: 6) fusions were prepared as above, and 10 μΙ_ of protein samples were mixed with 50 μΙ_ of molten (40-50°C) artificial insect diet especially prepared for Diabrotica sp. with low temperature melting agarose, whey protein and wheat germ. The diet-PHI-4 polypeptide mixture was placed in each well of a 96 well micro-titer plate. Four or more neonate WCRW larvae were placed in each well to feed for 4 days at 25°C. The response of insects towards the proteins was scored using a 0-3 numerical scoring system based on the size and mortality of the larvae in each well. If no response (or normal growth) was seen, a score of 0 was given. When the growth was slightly retarded, a score of 1 was given. A score of 2 meant that the larvae were severely retarded in growth (close to neonate size). A score of 3 meant death to all the larvae in the well. The percent response (% Response) for each treatment was calculated by dividing the total score, a sum of scores from replicated wells for each treatment, by the total highest possible scores and multiplying by 100 to yield "% Response". For example, if one treatment (one sample, one dose) had 6 replicated wells, the total highest possible score would be 3 X 6 = 18. An observed set of scores of 3, 2, 2, 3, 2, 2 for six wells at a given dose for a given variant would result in (14/18)X100 = 78% Response. Fast Activity Evaluation (FAE) analysis: The PHI-4 polypeptides at two concentrations were assayed along with 4 doses (two-fold dilutions bracketing the EC50) of the reference MBP::PHI-4 fusion protein (SEQ ID NO: 6) within one 96-well assay plate. The concentrations of the PHI-4 polypeptides were within the 4 doses of the reference protein concentrations, preferably around the middle point of the 4 dose concentrations. Each sample plate contained the reference MBP::PHI-4 protein (SEQ ID NO: 6) in a significant number of wells such as 16 wells in 4 separate doses. In each plate, up to 80 MBP::PHI-4 polypeptide variants were included and assayed for activity comparison with the reference PHI-4 polypeptide protein. From a sample plate, 10 ul of samples from each well were picked by multi-channel pipette and dispensed in one assay plate containing 50 ul molten diet in each well and mixed on a shaker. This process of producing the assay plate was repeated as many as 6 times or more to produce a desired number of replicate assay plates. After the diet was solidified and cooled to 4°C, neonate WCRW larvae were placed in each well, the plate was sealed with perforated Mylar film and incubated in a constant temperature incubator at 25°C. After 4 days, the insect responses were scored under a magnifying glass. The sigmoid dose-response values (Responses) were converted to linear probit dose-response values using SAS-JMP®, Generalized Linear Model, Binomial Response, Probit). The response for each protein in replicates was summed, this sum was compared with the probit dose-response line of the activity reference protein and the nominal fold improvement in potency was calculated. This nominal fold improvement estimated for a given dose in a given experiment is defined as the Fast Activity Evaluation Guide Number (FAEGN). For example, if a PHI-4 polypeptide showed a certain % response value at 40 ppm and comparison to the Probit curve indicated that the same response is predicted for the reference protein at 100 ppm, then the FAE Guide Number is 2.5 (100/40). According to this analysis, the PHI-4 polypeptide is nominally 2.5 times more potent than the reference PHI-4 polypeptide protein. The FAE assay was typically done with 2 different doses of PHI-4 polypeptides at a time and performed in three independent experiments, generating 6 FAEGN for each mutant in a typical FAE evaluation. The mean FAEGN is calculated to yield the Mean FAE Index (MFI). As used herein "Mean FAE Index" (MFI) refers to the mean of multiple FAEGN (typically 6 or more); unless otherwise indicated MFI is understood to be an arithmetic mean of FAEGN. For each protein, a two sided t-test was done comparing the multiple FAEGN from the clone with FAEGN values from the reference protein (typically 48-96 FAEGN of SEQ ID NO: 6). The two-sided t-test was done between these 48-96 FAEGN associated with the reference protein and the 6 or more FAEGN associated with the variant of interest. The Bonferroni correction was used to evaluate p-values (number of novel proteins/alpha) for the hypothesis that a given variant is significantly different in insecticidal activity compared to the reference protein, MBP::PHI-4 fusion protein (SEQ ID NO: 6) unless otherwise noted. The Bonferroni correction was used to evaluate p-values (number of novel proteins/alpha) for the hypothesis that a given variant is significantly different in insecticidal activity compared to the reference protein, MBP::PHI-4 fusion protein (SEQ ID NO: 6) unless otherwise noted.
Effective Concentration50 (EC50) evaluation: Variants of particular interest were assayed at higher power in more extensive dose response curves to more accurately estimate the EC50s. The preparation of dose response, infestation, incubation and scoring were as described for the FAE assay format. EC50 determinations were typically carried out with a no insecticidal protein control plus seven two-fold dilutions bracketing the expected EC50 (typically 100 ppm for MBP::PHI-4; SEQ ID NO: 6) and 24 or more replicate wells within a given experiment. As used herein, the EC50 is defined as the predicted point with 50% response in the scoring scheme. It is a combination of growth or feeding Inhibition and lethal responses. In order to determine EC50 values, each treatment (one dose) was repeated 6 or more, usually 24, times.
Data from exemplary FAE and EC50 determinations are given in Figures 1 and 2 respectively. MBP::PHI-4-SFR12-004 (SEQ ID NO: 31 ; Example 8) is a variant with improved insecticidal activity. Figure 1 shows the primary data for a typical FAE assay. Proteins were purified and quantified as described above. The % response in a typical FAE assay is given on the y axis. The protein concentration (toxin portion of the protein only) is given on the x axis. The geometric mean FAE Index in this instance is 4.0. Figure 2 shows the data for a typical EC50 measurement. Proteins were purified and quantified as described above. The fractional response (multiply by 100 to get "% Response") is given on the y axis. The inferred EC50s are 245 ppm (MBP::PHI-1 of SEQ ID NO: 6) and 48 ppm (SFR12-004; SEQ ID NO: 31 ). The data indicate that the insecticidal activity of MBP::PHI-4-SFR12-004 (SEQ ID NO: 31 ) is increased relative to that of MBP::PHI-4 (SEQ ID NO: 6).
The MBP::PHI-4 fusion is rapidly cleaved in the presence of insect gut fluid to yield MBP and mature PHI-4 protein and it is believed that the insecticidal activity is due to the cleaved toxin molecules. The MBP::PHI-4 fusion protein (SEQ ID NO: 6) was digested with 1/100 (w/w) Factor Xa (New England Biolabs) at 25° C overnight and the PHI-4 polypeptide was purified by Superdex 200 column chromatography utilizing the size difference and a weak affinity of MBP to Superdex. The MPB::PHI-4 fusion was also cleaved with trypsin and the mature PHI-4 polypeptide (derived from SEQ ID NO: 6) was purified. The mature, purified PHI-4 polypeptide derived from Factor Xa or trypsin cleavage has a MW of -60 kDa as measured on SDS-PAGE gels and is fully reactive to polyclonal antisera that also react with the MBP::PHI-4 fusion protein. The EC50 of this "mature" PHI-4 polypeptide fragment is within experimental error of the EC50 of the MBP::PHI-4 parental protein (SEQ ID NO: 6, as calculated on the basis of ppm associated with the toxin fragment and excluding ppm associated with the MBP domain of SEQ ID NO: 6.
Example 4: Single Amino Acid PHI-4 Polypeptide Variants (S1 -S4)
A set of 209 PHI-4 polypeptide single amino acid substitution variants spread across both the N and C terminal portions of the protein were made and characterized (Table 8; MUT ID: 1-209). The mutagenesis template was the polynucleotide of SEQ ID NO: 5 encoding PHI-4 (SEQ ID NO: 6) as an MBP fusion. The mutations were made using the QuickChange kit (Agilent; Catalogue #200524) essentially as described in Example 1. The particular amino acid substitutions relative to PHI-4 (SEQ ID NO: 2) are as indicated in Table 8. For example in Table 8, the PHI-4 polypeptide identified as MUT ID: 1 has a valine substituted for the native amino acid alanine at position 202 of PHI-4 (SEQ ID NO: 2) and referred to "A202V". The polypeptide variant for which activity is reported was prepared as an MBP fusion that is identical to SEQ ID NO: 6 except for this single amino acid substitution. In a similar manner, MUT ID: 1 -872 (Table 8) are all made in the context of SEQ ID NO: 6; MUT ID: 873-910 (Table 8) are made in the context of SEQ ID NO: 8; MUT ID: 91 1 -1 135 (Table 8) are made in the context of SEQ ID NO: 10. All polypeptides of Table 8 were expressed and purified as MBP fusions as described in Example 2. The PHI-4 polypeptides were expressed as MBP fusions and purified as described in Example 2. The assay protocol for WCRW insecticidal activity of the PHI-4 polypeptides was essentially as described for FAE assays in Example 3 using SEQ ID NO: 6 as the reference protein. For the analysis of the data a "Mean Deviation Score" was calculated rather than a mean FAE Index. This is a related metric that is calculated as follows. Data from four two-fold dilutions of MBP::PHI-4 (typically about 25, 50, 100 and 200 ppm final concentration of PHI-4 fragment in artificial insect diet) is plotted on a Probit plot. The difference between response expected for MBP::PHI-4 (SEQ ID NO: 6) at a given dose, based on the Probit curve, and that observed for the mean score for a PHI- 4 polypeptide variant at that given dose is calculated and this difference is defined as the Deviation Score. A negative Deviation Score indicates that the response is lower than that which is expected for the relevant parental backbone (typically MBP::PHI-4; SEQ ID NO: 6) at the same concentration and indicates that the variant is nominally less active than the parental backbone. A positive Deviation Score indicates that the variant is nominally more potent than the parental backbone. As used herein, the Mean Deviation Score refers to the arithmetic mean of multiple Deviation Scores which are typically derived from independent experiments. The Mean Deviation Score is used to estimate rank order of activities associated with a set of variants within a given experiment. The Mean Deviation Scores for the 209 variants of this example are given in Table 8 and is typically an average of at least three independent Deviation Score measurements.
Example 5: Single amino acid substitution mutants #2 (SFR)
A BLAST search revealed that AXMI-205 is a bacterial perforin-like
protein. Perforin proteins from Clavibactor michiganensis (GenBank Accession number: YP_001223127; SEQ ID NO: 49), Laccaria bicolor (GenBank Accession
number:XP_001885969; SEQ ID NO: 48), Marinomonas sp. (GenBank Accession number: ZP_01077945; SEQ ID NO: 38), Nematostella vectensis (GenBank Accession number: XP_001617993; SEQ ID NO: 50), Photorhabdus luminescens (GenBank Accession number: NP_928713; SEQ ID NO: 1507), and Serratia proteamaculans (GenBank Accession number: YP_00147861 ; SEQ ID NO: 1506) were found to be homologous to AXMI-205. The N-terminal region of up to 31 1 amino acid of AXMI-205 (SEQ ID NO: 35) is highly homologous to those perforin proteins. Among those perforin proteins, the 3D X-ray structure of Photorhabdus perforin-like protein has been published (Science 317, 1548-1551 , 2007; PDB ID: 2QP2; SEQ ID NO: 1508). According to the leading theory of perforin mode of action, the protein inserts 5 alpha helices of its N- terminal region into the target host membrane to form a large size pore (Proc. Natl. Acad. Sci. USA 102, 600-605, 2005; Immunology Today, 16, 194-201 , 1995). In the
Photorhabdus 2QP2 structure, there are two loops between Alpha C and D and Alpha I and J are considered to be the site for initiating the membrane insertion of those 5 alpha helices. These loops are called membrane insertion initiation loops. The primary sequences of those perforin proteins listed below were aligned using Vector NTI Align X function in order to identify the membrane insertion loop sequences of AXMI-205 (SEQ ID NO: 35). Amino acid sequences of V92-A103 and G21 1 -E220 of SEQ ID NO: 6 were aligned with the membrane insertion initiation loops identified in the 2QP2 structure. A number of acidic, basic and other hydrophilic amino acids were found in the AXMI-205 (SEQ ID NO: 35) membrane insertion loops indicating these loops are exposed to the solvent.
Based on this homology model and prior mutation-activity relationship data, another 664 single amino acid substitution PHI-4 polypeptide variants were made in order to assess the sequence-insecticidal activity-relationships at the selected positions. This set of 664 point mutations (Table 8; MUT ID: 210-872) were made with MBP::PHI-1 polypeptide of SEQ ID NO: 5 as the DNA template. Mutations were made according to the method of Example 1. The mutagenesis oligonucleotides used to create an exemplary mutant (PHI-4-R97D; SEQ ID NO: 7) are SEQ ID NO: 13 and SEQ ID NO: 14. The other PHI-4 polypeptides (Table 8; MUT ID: 210-872) were made in the same manner using mutagenesis oligonucleotides designed to create the selected substitutions at the desired residues of the protein. The resulting PHI-4 polypeptides were purified as MBP fusions and the activity measured in the FAE assay format or EC50 assay format as described in Examples 2 & 3. The Mean FAE Indices associated with these 664 PHI-4 polypeptides are given in lines 210-872 of Table 8.
Example 6: SFR16 point mutants
Another set of 38 single amino acid substitution PHI-4 polypeptide variants (Table 8; MUT ID: 873-910) were made with MBP::PHI-4-R97D (SEQ ID NO: 7; Example 5) as the backbone. The substitutions were selected based on the sequence-activity relationships inferred from the PHI-4 polypeptide variants of the preceding Examples. Mutations were made according to the method of Example 1 . Variants were purified as MBP fusions and activity measured in FAE assay format as described in Examples 2 & 3. The Mean FAE Indices associated with these mutants are given in lines 873-910 of Table 8. The reference for the Mean FAE Index in this example is SEQ ID NO: 8.
Example 7: PSR3 mutants
The C-terminal domain of AXMI-205 shares significant homology with proteins that have β-prism 3D structural folding patterns. In the typical β-prism, the oligosaccharide binding site is almost always in a cavity formed between the apex and belt-loops from the same Greek-key motif. The primary amino-acid sequence motif has been identified as G- X3-D (SEQ ID NO: 39) in banana lectin, which has two binding sites (Meagher JL et al Glycobiology 15:1033-42; 2005). The primary sequence of the PHI-4 polypeptide of SEQ ID NO: 2 contains 3 such motifs. The canonical structure of these motifs is indicated in Figure 3. In addition, the G-X3-D motif (SEQ ID NO: 39) can be extended toward the N- terminal direction into the D-X-G-[S/T]-G-X3-D motif (SEQ ID NO: 40), which are present 1 , 2 or 3 times in 24 proteins that are orthologous to the C-terminal portion of AXMI-205 (GenBank accession numbers: gi|136474758; gi|136444345; gi|136141087;
gi|143658948; gi|142085802; gi|135275135; gi|138446054; gi|294814724; gi|170109524; gi|156316804; gi|156377786; gi|170109526; gi|77456557; gi|1209377; gi|302823768; gi|302532087; gi|256764986; gi|302787479; gi|302823738; gi|169762636; gi|302766657; gi|270056485; gi|302792467; gi|238488445;). Each of the 3 loops in the PHI-4
polypeptide of SEQ ID NO: 2 has potential to bind an oligosaccharide, a putative binding receptor present in WCRW mid-gut cell membrane surface.
As indicated in Table 8, 225 PHI-4 polypeptide variants (MUT IDs: 91 1-1 135) were made to introduce an additional amino acid substitution into the PHI-4 polypeptide of PHI- 4-D09 (SEQ ID NO: 10) using MBP::PHI-4-D09 (SEQ ID NO: 9) as the DNA template. The PHI-4-D09 backbone contains the following substitutions relative to SEQ ID NO: 2: L40I, Y98F, L145V, L163V, I 172L, V355I, and P412A (numbers are relative to the PHI-4 polypeptide backbone of SEQ ID NO: 2. Mutagenesis was done by a modification of the method of Dominy et al (Methods in Molecular Biology, Vol. 235:209-223; 2003). Briefly, "NNK" mutagenesis at position 396 was done as follows. A pMAL vector encoding SEQ ID NO: 9 was amplified by inverse PCR for 20 cycles using SEQ ID NO: 15 & SEQ ID NO: 16. The PCR product was diluted 10-fold, subjected to one additional round of amplification using SEQ ID NO: 16 & SEQ ID NO: 17. The PCR product was purified on QuiaQuick™ column, phosphorylated with T4 polynucleotide kinase, circularized with T4 DNA ligase and transformed into E. coli BL21 (DE3) cells. Candidate colonies were amplified by colony PCR and the PCR product was sequenced first with a single primer to confirm the presence of the desired mutation and subsequently sequenced fully with multiple primers to identify clones with no additional mutations. All other PHI-4
polypeptides of this example (Table 8, lines 91 1-1 135) were made by a similar manner using mutagenesis oligonucleotides designed to create the selected substitutions at the desired residues of the protein. Positions with multiple desired mutations were made with degenerate forward primers whereas positions with only one desired mutation were made with non-degenerate primers. Clones with the desired sequences were used to express protein essentially as described in Example 2. Protein purification, activity measurements and statistical analysis was done essentially as described in example 3. The Mean FAE Index reflects the fold difference relative to PHI-4 polypeptide of SEQ ID NO: 6. The Mean EC50 of PHI-4-D09 (SEQ ID NO: 10) was measured at high statistical power and is 1.3-fold improved relative to MBP::PHI-4 (SEQ ID NO: 6). PHI-4 polypeptides of this example with Mean FAE Index > 1.3x are deemed nominally improved relative to the parental backbone (MBP::PHI-4-D09; SEQ ID NO: 10) and diversity meeting this criterion was used for production of subsequent combinatorial mutants.
Example 8: Identification of combinatorial mutants of PHI-4 polypeptides with improved insecticidal activity as measured in an artificial insect diet feeding assay Activity diversity identified in Examples 4-7 was used to create 192 combinatorial PHI-4 polypeptide variants (SEQ ID NO: 51-242; Table 9). The PHI-4 polypeptide variants were made by sequential point mutagenesis by the method of Example 1 . In all cases, the indicated PHI-4 polypeptide variants were made as MBP fusions with the same linker as is indicated in SEQ ID NO: 6. The MBP::PHI-4 polypeptides were expressed and purified as indicated in Example 2 & 3. Purified MBP::PHI-4 polypeptides were assayed in FAE assays to derive a Mean FAE Index or in EC50 assays as indicated in Example 3. Thirty-seven exemplary active PHI-4 polypeptide variants with increased Mean FAE Indices are given in Table 3, along with the sequence variation relative to SEQ ID NO: 6. The substitutions relative to PHI-4 polypeptide of SEQ ID NO: 2 are given in the right-most column. All proteins were expressed and purified as MBP fusions proteins. The reference protein for the Mean FAE Index is MPB::PHI-4 (SEQ ID NO: 6). The functional data on all of the PHI-4 polypeptides of this example is given in lines 51-242 of Table 9.
Table 3
Figure imgf000184_0001
D042N, E046N, R097D, K099L,
SFR17- E220D, K289L, R293Q, S333K, 016 12.9 5.18E-12 G336A, S401H, K402H, P412A
SFR11- 005 12.8 2.12E-10 R097D, K099L, E220D, K289L
SFR10- 042 12.0 7.73E-11 R097D, S401H
R097D, K099L, E220D, K289L,
SFR17- R293Q, S333K, G336A, S401H, 012 11.7 1.82E-07 K402H
D042N, E046N, R097D, K099L,
SFR17- E220D, K289L, S401H, K402H, 004 11.3 7.41E-06 P412A
D042N, E046N, R097D, K099L,
SFR17- E220D, K289L, S333K, G336A, 017 10.5 2.1E-15 V355I, S401H, K402H, P412A
SFR11- 014 9.9 2.08E-09 R097D, K289L
D042N, E046N, R097D, K099L,
SFR17- E220D, K289L, R293Q, S401H, 003 9.7 1E-04 K402H
SFR13- R097D, K099L, E220D, K289L, 035 9.3 1.5E-11 V355I, A396T, P412A, K520E
SFR10- 72 8.9 0.003487 R097D, S333V, K520E, Q527K
SFR17- D042N, E046N, R097D, K099L, 001 8.7 0.010038 E220D, K289L, S401H, K402H
SFR10- 056 8.4 2.98E-05 R097D, G462A, R464A, K465M
SFR10- 036 8.2 1.37E-05 R097D, S333K, G336A, E339N
R097D, K099L, E220D, K289L,
SFR17- R293Q, S333K, G336A, S401H, 015 8.2 4.75E-21 K402H, P412A
SFR11- 015 8.2 1.76E-07 R097D, R293Q
SFR11- 010 8.1 1.7E-06 R097D, E220D, K289L
SFR17- R097D, K099L, E220D, K289L, 002 7.6 5.14E-09 R293Q, S401H, K402H
SFR10- 039 7.2 9.2E-12 R097D, S333V, G336A, S338V
SFR10- R097D, S401H, K402H, K520E, 82 6.7 8.17E-06 Q527K
SFR10- 045 6.6 2.09E-14 R097D, S401H, K402H
SFR10- R097D, S401G, K402H, K520E, 87 6.4 2.15E-06 Q527K
SFR10- 060 6.3 2.89E-08 R097D, G462A, R464K, K465M
SFR5- 014 6.2 1.65E-05 R097D, R293Q, R416E, K520E
SFR13- R097D, K099L, E220D, K289L, 018 6.1 1.84E-13 V355I, S401G, P412A, K520E
SFR11- 013 5.5 1.54E-09 R097D, E220D
D042N, E046N, R097D, K099L,
SFR17- E220D, K289L, V355I, S401H, 007 5.5 5.11E-22 K402H, P412A Example 9: Identification of combinatorial mutants of PHI -4 polypeptides with improved insecticidal activity as measured in an artificial WCRW insect diet feeding assay.
Activity data from 315 PHI-4 polypeptide combinatorial variants (SEQ ID NOs: 243-558) is provided in Table 9. Libraries were initially prepared by incorporation of diversity into SEQ ID NO: 5. The diversity was largely derived from that described in Example 4. Oligonucleotides encoding diversity at forty positions were incorporated into SEQ ID NO: 5 in a DNA shuffling reaction essentially as described (Ness et al., Nature Biotec nol. 20, 1251 ; 2002). Briefly, oligonucleotides typically of 30-45 bases in length encoding the diversity elements of interest were mixed at 0.02 - 2 micromolar each in the presence of an appropriate concentration of fragments of SEQ ID NO: 5. This reaction was assembled, rescued and cloned essentially as described for synthetic genes in Example 10 and as described (Ness et al., Nature Biotechnol. 20, 1251 ; 2002). Improved variants from initial libraries were subjected to family DNA shuffling essentially as described (A. Crameri, et al Nature 391 , 288; 1998). This family shuffled library was screened by methods similar to those described in Example 3 (FAE) and to those described in Example 4 (Mean Deviation Score). Selected, improved PHI-4 polypeptide variants from the second round of DNA shuffling were further diversified by recombining N terminal and C terminal domains of elite clones using the method of splicing by overlap extension (R. Horton, et al., Gene 77:61-68; 1989) to yield novel variants. All variants were purified by the method of Example 2 and assayed by the method of Mean
Deviations of Example 4. PHI-4 polypeptides variants identified are given in Table 9 (SEQ ID NO: 243-558). Example 10: Identification of combinatorial mutants of PHI-4 polypeptides with improved insecticidal activity as measured in an artificial WCRW insect diet feeding assay
A set of 158 PHI-4 polypeptide combinatorial variants (SEQ ID NO: 559-716) were prepared by total gene synthesis, essentially as described by Stemmer et al (Gene 164:49-53; 1995). An additional treatment was implemented as described (Saaem, I. et al, Nucleic Acids Research, Published Nov. 29, 201 1 , 1 -8). Briefly, in a typical gene synthesis reaction a set of oligonucleotides of 120 bases each encoding both top and bottom strands of the target gene were designed. Complementary oligos typically overlap by 54-65 nucleotides. Oligos to make synthetic genes are combined such that a final concentration of each oligo is approximately 0.05 - 1 uM. Gene assembly is typically done with Herculase II (Agilent) using the following cycling program: 98°C 3 min followed by (96°C x 30 sec, 40°C x 30 sec, 72°C x 30 sec) x 24 cycles. The initial PCR is then used as template for a second PCR in which a second pair of primers is used to amplify the fully synthetic gene product. Typical PCR conditions for the second synthetic gene reaction were 98°C 3 min followed by (96°C 30 sec, 50°C 30 sec, 72°C 35 sec), x 24 cycles. Reactions were analyzed by 1 % E-gels (Invitrogen). A subsequent treatment consisting of a re-annealing step, treatment with Cell (Transgenomics; Catalogue #706020) and subsequent amplification (25 cycles) was done essentially as described (Saaem, I. et al, Nucleic Acids Research, Published Nov. 29, 201 1 , 1 -8). A third and final amplification of the synthetic gene was done with similar PCR conditions in a single cycle. The product of this reaction was purified and ligated by Gibson ligation method (New
England Biolabs; Catalogue ## E261 1 L) to an appropriate vector transformed to chemical competent BL21 (DE3) cells. Sequence verified clones (Lines 559-716 of Table 9) comprising the PHI-4 polypeptide were expressed as MBP fusions, purified and assayed essentially as described in Examples 2 and 3. Table 4 shows the SEQ ID NOs: and substitutions relative to the PHI-4 polypeptide of SEQ ID NO: 2 for twenty active variants. The mean FAE Index is calculated relative to the MBP::PHI-4 backbone (SEQ ID NO: 6).
Table 4
Figure imgf000187_0001
F043E, Y098F, L145V, Y171F,
PSR1- 8.39E- I172L, Y206F, E278N, V355I,
10 568 2-149 7.0 08 A417S, V455I, W457N
D042N, F043E, Y098F, L145V,
PSR1- 3.93E- Y171F, I172L, E278N, V355I,
10 569 2-087 6.9 10 Q442E, V455I, W457N
D042N, R097N, Y098F, L145V,
PSR1- 1.01E- Y171F, I172L, V355I, I410V,
10 570 2-158 6.9 07 V455I
E046N, Y098F, L145V, Y171F,
PSR1- 2.02E- I172L, D182Q, E278N, V355I,
10 571 2-086 6.7 05 Q442E, V455I, W457N
D042N, I052V, Y098F, L145V, I172L, Y206F, I283V, V355I,
PSR1- 0.01072 H370R, I410V, P412A, A417S,
10 572 1-053 6.5 6 T426S, T461S
D042N, F043E, Y098F, L145V,
PSR1- 5.28E- Y171F, I172L, V210I, I283V,
10 573 2-096 6.2 07 M354L, V355I, V455I, W457N
D042N, R097N, Y098F, L145V,
PSR1- 2.92E- Y171F, I172L, V355I, H370R,
10 574 2-135 6.1 11 Q442E, V455I
D042N, R097N, Y098F, L145V,
PSR1- 0.01827 I172L, I283V, V355I, I410V,
10 575 1-014 5.9 6 Q442E, V455I
E046N, R097N, Y098F, L145V,
PSR1- 5.02E- Y171F, I172L, V355I, Q442E,
10 576 2-141 5.8 09 V455I
E046N, Y098F, L145V, L163V,
PSR1- 0.01725 I172L, Y206F, V210I, E339Q,
10 577 1-006 5.6 2 V355I, A417S
F043E, Y098F, L145V, Y171F,
PSR1- 7.35E- I172L, Y206F, E278N, M354L,
10 578 2-095 5.3 05 V355I, V455I, W457N
Example 11 : Identification of combinatorial mutants of PHI-4 polypeptides with improved insecticidal activity as measured in an artificial insect diet feeding assay
Sixty-six PHI-4 polypeptide variants (SEQ ID NO: 717-783), containing
permutations of a number of substitutions, were made by total gene synthesis, essentially as described in Example 10. The substitutions were done in the context of a backbone (PSR1 -2-105; SEQ ID NO: 584 from Table 9) containing the following substitutions relative to PHI-4 polypeptide of SEQ ID NO: 2: E46N, R97N, Y98F, L145V, Y171 F, I 172L, V355I, 1410V, V455I, and W457N. The resulting MBP::PHI-4 polypeptide fusion proteins were expressed, purified, assayed for insecticidal activity on WCRW larvae and analyzed for insecticidal relative to MBP::PHI-4 (SEQ ID NO: 6) using the Mean FAE Index metric as described in Examples 2 & 3. Table 5 shows the Mean FAE Indices, SEQ ID NOs: and substitutions relative to PHI-4 polypeptide of SEQ ID NO: 2 for twenty active PHI-4 polypeptide variants of this example. The Mean FAE Index was calculated relative to the MBP::PHI-4 backbone (SEQ ID NO: 6). The insecticidal activities of the PHI-4 polypeptides in Table 5 (Mean FAE Index) reflect the arithmetic means of three independent experiments and are expressed as fold WCRW insecticidal activity improvement of the PHI-4 polypeptide variants relative to MBP::PHI-4 (SEQ ID NO: 6). As indicated, the mean FAE Indices range from 0.26x to >8x (fold improvement relative to MBP::PHI-4). The majority of the PHI-4 polypeptides have increased insecticidal activity relative to MBP::PHI-4 (FAE>1 ). The p values indicate that the measured differences relative to MBP::PHI-4 (SEQ ID NO: 6) are highly significant.
Table 5
Figure imgf000189_0001
E046N, R097N, Y098F, L145V, Y171F,
PSR7- I172L, V355I, S401H, K402G, D403Y,
11 730 155 3.7 I410V, V455I, W457N
E046N, R097N, Y098F, L145V, Y171F,
PSR7- I172L, V355I, S401K, K402W, D403Y,
11 731 116 3.6 I410V, V455I, W457N
E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, S401G, K402H, D403Y,
11 732 PSR7-95 3.4 I410V, V455I, W457N
E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, A396L, K402G, I410V,
11 733 PSR7-90 3.3 V455I, W457N
E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, A396G, K402H, I410V,
11 734 PSR7-97 3.3 V455I, W457N
E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, A396G, S401H, K402W,
11 735 PSR7-64 2.8 D403Y, I410V, V455I, W457N
E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, S401H, K402W, D403Y,
11 736 PSR7-93 2.8 I410V, V455I, W457N
Example 12: Identification of combinatorial mutants of PHI -4 polypeptides with improved insecticidal activity as measured in an artificial WCRW insect diet feeding assay
Thirty six PHI-4 polypeptide variants (Table 9; SEQ ID NO: 784-819), containing permutations of eleven substitutions (E220D, G336A, K099L, K289L, K402H, K520E, P412A, R097D, S333K, S401 H, V355I; numbering scheme as per SEQ ID NO: 2; all variants made as MBP fusions as indicated in Table 9), were made using site directed mutagenesis as described in Example 1 and PHI-4 polypeptide variants were expressed, purified, assayed for insecticidal activity on WCRW larvae and analyzed for insecticidal activity relative to PHI-4 polypeptide of SEQ ID NO: 2 as described in Examples 2 & 3. Table 6 shows the Mean FAE Indices, SEQ ID NOs: and substitutions relative to the PHI- 4 polypeptide of SEQ ID NO: 2 for twenty active PHI-4 polypeptide variants of this example. The mean FAE Index is calculated relative to the MBP::PHI-4 backbone (SEQ ID NO: 6). The mean FAE indices reflect the arithmetic means of three independent experiments. As indicated, the mean FAE indices range from 4.5 to >8. The p values indicate that the measured differences relative to MBP::PHI-4 (SEQ ID NO: 6) are highly significant. Table 6
Figure imgf000191_0001
Example 13: Accordance between FAE and EC50 assays.
Experimental data on Mean FAE Index and mean EC50 for twenty-five PHI-4 polypeptide variants is given in Figure 4. PHI-4 polypeptide variants were first tested in the FAE assay and then selected ones were retested in a multiple EC50 assays. In general, the fold improvement in mean FAE Index is modestly larger than the fold improvement that was subsequently measured in Mean EC50 measurements. This overall trend is as expected from the phenomenon of regression toward the mean (International Journal of Epidemiology 2005; 34:215-220). All 25 PHI-4 polypeptides elected for retesting in EC50 assays repeated as being significantly improved. Figure 4 shows the EC50 measurements for representative variants from Table 9 (SEQ ID NO: 610, SEQ ID NO: 595, SEQ ID NO: 584, SEQ ID NO: 591 , SEQ ID NO: 576, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 150, SEQ ID NO: 150, SEQ ID NO: 149, SEQ I D NO: 167, SEQ ID NO: 167, SEQ ID NO: 164, SEQ ID NO: 164, SEQ ID NO: 170, SEQ I D NO: 170, SEQ ID NO: 795, SEQ ID NO: 794, SEQ ID NO: 784, SEQ ID NO: 799, SEQ I D NO: 785, SEQ ID NO: 788, SEQ ID NO: 786, SEQ ID NO: 796, SEQ ID NO: 787). Example 14: Combinatorial substitutions
Example 1 1 yielded numerous PHI-4 polypeptide variants with improved insecticidal activity based on combinatorial substitutions of the three lectin-like motifs described in Example 7. A combinatorial library was prepared of 120 genes based on this diversity as follows. SEQ ID NOs: 760 & 761 each contains unique substitutions in loop 1. SEQ ID NOs: 717-726; 728-732; 734-737 & 760 contain unique substitutions in loop 2. SEQ ID NOs: 761 and 758 contain unique substitutions in loop 3. Gene synthesis was used to create a combinatorial library of these loop sequences essentially as described in Example 10. These genes can be expressed and assayed for activity on WCRW larvae by the methods described above.
Example 15: Mutagenesis of putative protease sensitive sites of PHI-4 polypeptides
Trypsin was used to identify the site(s) where proteases possibly attack (Protease Accessible Sites) the PHI-4 polypeptide of SEQ ID NO: 2. The PHI-4 polypeptide of SEQ ID NO: 2 in 50mM Tris-HCI, pH8 was mixed with 1/50 (weight/weight) trypsin and incubated for 1 hr. at 37°C. It was found that protein was relatively resistant to trypsin, with no immediate digestion down to the small fragments, but produced a 55 kDa major band and 24 kDa minor band by SDS-PAGE analysis after the incubation. These two bands were excised from the gel and analyzed by mass spectrometry and N-terminal sequencing. The N-terminal sequencing revealed SAANAGQLGN (amino acids 3 - 12 of SEQ ID NO: 2) for the 55 kDa protein indicating that only two amino acid residues, Methionine and Alanine were missing from the N-terminal sequence. The mass- spectrometry, however, showed a loss of C-terminal sequence from Ser at 521 to Leu at 536 of SEQ ID NO: 2. This indicates that trypsin digested the PHI-4 polypeptide protein at the C-terminal side of Lys at 520 of SEQ ID NO: 2. The N-terminal sequence of the 24 kDa band was VDKVLLMD (amino acids 314 to 321 of SEQ ID NO: 2). The mass- spectrometry analysis on the 24 kDa fragment confirmed the C-terminal region of PHI-4 polypeptide of SEQ ID NO: 2 starting with Val at 314 relative to SEQ ID NO: 2 as shown by N-terminal sequencing and ending at Lys at 520 relative to SEQ ID NO: 2. This indicated that trypsin digested the PHI-4 polypeptide of SEQ ID NO: 2 at the C-terminal side of Lys at 313 of SEQ ID NO: 2.
From this experiment, it was found that there are at least two protease accessible sites, Lys at 313 and Lys at 520 SEQ ID NO: 6. These two sites were mutated to other amino acid residues by saturation mutagenesis and it was found that mutations at Lys at 313 and Lys at 520 of SEQ ID NO: 2 increase the insecticidal activity significantly. For example, the activity of the PHI-4 polypeptide variant, K313Q (MUT ID: 889), was enhanced 2.3 fold over the activity of PHI-4 polypeptide of SEQ ID NO: 2 as measured in an FAE assay (Table 8). The activity of the PHI-4 polypeptide K520Q (MUT ID: 881 ) was increased 3.1 fold. Activity increases were also found in combinations with other mutations. For example, the activity of the PHI-4 polypeptide having the R097D and K520E substitutions (SEQ ID NO: 52) is 3.5 fold higher than that of PHI-4-R097D (MUT ID: 8) alone by FAE assay.
Example 16: Saturation mutagenesis of amino acid residues selected by site directed single amino acid mutagenesis
Certain amino acid residues showed activity changes when mutated by site directed single amino acid mutagenesis. Those residues were "Selected" for saturation mutagenesis. For the purposes of this example, "Selected" can refer to single amino acid mutations that affect the activity, positively or negatively, relative to the parental backbone in which they were made. More specifically substitutions of Table 8 with Mean FAE Indices of <0.7 or of >1.3 are deemed "Selected". For example, Selected amino acid residues were found by performing site directed mutagenesis at certain residues such as Arg and Lys. These basic amino acid residues were mutated to either acidic (Asp, Glu) or neutral, polar (e.g.: Asn and Gin) residues, and the activity of those mutants was determined by the FAE insect assay individually. Acidic amino acid residues such as Asp and Glu were changed to basic (e.g.: Arg, Lys) or neutral, polar (e.g.: Asn and Gin) residues and the mutant activity was determined. Neutral, polar amino acid residues such as Gin and Asn were mutated to either acidic (Asp, Glu) or basic (e.g.: Arg, Lys) amino acid residues to see if the activity of those mutants were changed positively (for example mean FAE Index >1.3 relative to the reference protein) or negatively (for example mean FAE Index <0.7 relative to the reference sequence). Another example of finding Selected amino acid residues is based on the sequence-function relationship. Since AXMI-205 is a member of the perforin family it is possible to identify amino acid residues of PHI-4 polypeptide of SEQ ID NO: 2 which are involved in the mode of action elements such as membrane insertion initiation and receptor binding loops. Amino acid residues found in those regions were considered Selected for saturation mutagenesis in this Example. One can use the alanine scanning to empirically define Selected residues. This technique was used to find additional Selected amino acid residues in the putative receptor binding loops.
After any amino acid residues were determined Selected, those residues were subjected to saturation or near saturation mutagenesis to produce a set of up to 19 mutants for each site (20 all possible amino acids minus the amino acid found in the wild type). The insecticidal activity of all these mutants was determined by the FAE insect assay. Saturation mutagenesis of the Selected amino acid residues, was useful for identifying substitutions with Mean FAE Indices of >1 , in many cases >1 .3. When the activity of one single amino acid mutation was found to be positive by showing increased activity over the PHI-4 polypeptide of SEQ ID NO: 2, the saturation mutagenesis enabled us to find other mutation(s) that showed further increased activity. For example, while the FAE Index of E082Q (MUT ID: 370) was positive (1.37), the saturation mutagenesis at this site revealed other mutations showing much higher FAE Indices. For example, the index of E082I (MUT ID: 219) was 7.80 and that of E082L (MUT ID: 259) was 2.71 indicating that PHI-4 polypeptide of SEQ ID NO: 2 hydrophobic residues are beneficial at this site as far as its insecticidal activity is concerned.
Other Selected amino acid substitutions resulted in decreased activity. When these sites were examined further by saturation mutagenesis, substitutions with Mean FAE Indices of >1 were observed. For example, the FAE Indices of K099Q (MUT ID: 677), K099E (MUT ID: 715) were 0.34 and 0.26, respectively. This shows that Lysine at this site is functionally involved in activity and that alternative substitutions may result in improved activity. In this example, the saturation mutagenesis revealed substitutions with Mean FAE Index > 1. For example, the substitution K099L (MUT ID: 299) has a Mean FAE Index of 5.72 (Table 8). Similar instances were found across the entire PHI-4 polypeptide of SEQ ID NO: 2, for example those indicated in Table 7. Table 7 shows the Mean FAE Indices for nine pairs of substitutions. All data is from Table 8. Table 7
Figure imgf000195_0001
The serine at position 98 of SEQ ID NO: 2 was selected by alanine scanning amino acid residues found in a region of the protein that is suspected overlap with the receptor binding loops. Point mutants with improved potency may then be used to prepare and screen combinatorial libraries based on that diversity.
Taking mean FAE Index <0.7 as a definition of Selected, the following positions are deemed Selected: P14, D24, Q38, E53, R55, R61 , Q75, D76, E83, E1 18, E126, D152, R166, K188, K191 , D193, K242, P243, R248, D254, L266, D268, A270, D274, D298, K313, D315, K316, D321 , V343, S349, Q360, R361 , D368, I373, D376, F378, D379, D394, Y404, Q413, N430, Q449, D497, R500, S504. Saturation or near saturation mutagenesis at these positions can be performed by the method of Example 7 or equivalent methods and purified and screened the variant proteins for activity by the methods of Example 2 & 3 or equivalent methods.
Example 17: Transgenic expression and activity evaluation
The polynucleotides encoding PHI-4 polypeptides of SEQ ID NOs: 22-25 were cloned under control of the maize ubiquitin promoter (Christensen and Quail, (1996) Transgenic Research 5:213-218) into a standard vector suitable for transformation of maize by Agrobacterium. Transgenic maize plants were produced by the method of Example 20. Selected TO plants were tested for susceptibility to WCRW feeding by challenging TO plants with WCRW larvae. After 19-21 days of challenge, the roots were visually examined and root nodal injury scores were recorded as described (Oleson J. et al J. Economic Entomology 98:1-8; 2005). Root nodal injury scores are indicated in Figure 5. The data support the conclusion that the three PHI-4 polypeptide variants provide measurable in planta efficacy for protection of maize transgenic plants against WCRW. Figure 5 shows the TO seedlings in the V3 - V4 growth stage were challenged as described (Oleson J. et al J. Economic Entomology 98: 1 -8; 2005) and root nodal injury scores were recorded.
Example 18: In planta expression of fusion proteins
Localization of the protein can also play an important role in in planta
accumulation. One can direct proteins such as PHI-4 polypeptides to the chloroplast using a chloroplast targeting peptide (CTP). Additionally, one can direct expression to the apoplastic space using fusions to peptides such as the barley alpha amylase-derived peptide (BAA; SEQ ID NO: 1513). One may also direct transgenically expressed proteins for retention in the endoplasmic reticulum by fusing to both BAA and the sequence "KDEL" (SEQ ID NO: 1514). Proteins can also be directed to the vacuolar space by fusion with the C terminal peptide from plant defensins such as the maize defensin 20 C- terminal propeptide (SEQ ID NO: 51 1 ). Other functionally equivalent gene elements may be combined in a similar manner. One may also direct expression specifically to the roots with root-specific promoters. Each of these modifications may be made separately or in combination and any given combination of elements to improve accumulation of protein in plant tissue or in functionally improved efficacy of the expressed protein.
Example 19: Transformation of Maize by Particle Bombardment and Regeneration of Transgenic Plants
Immature maize embryos from greenhouse donor plants are bombarded with a DNA molecule containing the PHI-4 polypeptide of nucleotide sequence (e.g., SEQ ID NO: 1 ) operably linked to an ubiquitin promoter and the selectable marker gene PAT (Wohlleben, et al., (1988) Gene 70: 25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate DNA molecule. Transformation is performed as follows. Media recipes follow below. Preparation of Target Tissue
The ears are husked and surface sterilized in 30% CLOROX™ bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment. Preparation of DNA
A plasmid vector comprising a nucleotide sequence (e.g., SEQ ID NO: 1 ) operably linked to an ubiquitin promoter is made. For example, a suitable transformation vector comprises a UBI 1 promoter from Zea mays, a 5' UTR from UBI 1 and a UBI 1 intron, in combination with a Pinll terminator. The vector additionally contains a PAT selectable marker gene driven by a CAMV35S promoter and includes a CAMV35S terminator.
Optionally, the selectable marker can reside on a separate plasmid. A DNA molecule comprising a toxin nucleotide sequence as well as a PAT selectable marker is precipitated onto 1.1 μηη (average diameter) tungsten pellets using a CaCI2 precipitation procedure as follows:
100 μΙ_ prepared tungsten particles in water
10 μΙ_ (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)
100 μΙ_ 2.5 M CaC12
10 μΙ_ 0.1 M spermidine
Each reagent is added sequentially to a tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 mL 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μΙ_ 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the
tungsten/DNA particles are briefly sonicated and 10 μΙ_ spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
Particle Gun Treatment
The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA. Subsequent Treatment
Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1 .6 gallon) and grown to maturity. Plants are monitored and scored for expression of the toxin by assays known in the art or as described above.
Bombardment and Culture Media
Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000x SIGMA-151 1 ), 0.5 mg/L thiamine HCI, 120.0 g/L sucrose, 1.0 mg/L 2,4-D and 2.88 g/L L-proline (brought to volume with deionized H20 following adjustment to pH 5.8 with KOH); 2.0 g/L Gelrite™ (added after bringing to volume with dl H20); and 8.5 mg/L silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000x SIGMA-151 1 ), 0.5 mg/L thiamine HCI, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D (brought to volume with dl H20 following adjustment to pH 5.8 with KOH); 3.0 g/L Gelrite™ (added after bringing to volume with dl H20); and 0.85 mg/L silver nitrate and 3.0 mg/L Bialaphos (both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO 1 1 1 17-
074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCI, 0.10 g/L pyridoxine HCI, and 0.40 g/L Glycine brought to volume with polished D-l H20) (Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/L sucrose, and 1 .0 mL/L of 0.1 mM abscisic acid (brought to volume with polished dl H20 after adjusting to pH 5.6); 3.0 g/L Gelrite™ (added after bringing to volume with dl H20); and 1 .0 mg/L indoleacetic acid and 3.0 mg/L Bialaphos (added after sterilizing the medium and cooling to 60 C).
Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 1 1 1 17-074), 5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCI, 0.10 g/L pyridoxine HCI, and 0.40 g/L Glycine brought to volume with polished dl H20), 0.1 g/L myo-inositol, and 40.0 g/L sucrose (brought to volume with polished dl H20 after adjusting pH to 5.6); and 6 g/L Bacto-agar (added after bringing to volume with polished dl H20), sterilized and cooled to 60°C. Example 20: Agrobacterium-Med ated Transformation of Maize and
Regeneration of Transgenic Plants For
Figure imgf000199_0001
transformation of maize with a toxin nucleotide sequence (e.g., SEQ ID NO: 1 ), the method of Zhao can be used (US Patent Number 5,981 ,840 and PCT Patent Publication Number WO 1998/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium under conditions whereby the bacteria are capable of transferring the nucleotide sequence (e.g. SEQ ID NO: 1 ) to at least one cell of at least one of the immature embryos (step 1 : the infection step). In this step the immature embryos can be immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos can be cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos can be cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium can be cultured on solid medium to regenerate the plants.
Table Legends Table 8. The definitions of the column headings are as follows: "MUT ID", a unique identifier for each substitutions; "Backbone", the SEQ ID corresponding to the polypeptide backbone in which the substitution was made; "Position", amino acid position according to the numbering convention of SEQ ID NO: 2, "Ref. A.A.", the standard single letter code for the amino acid present in the backbone sequence at the indicated position; "Substitution", the standard single letter code for the amino acid present in the mutant sequence at the indicated position; "FAE", the arithmetic Mean FAE Index as further defined in Example 3; "p-value" the calculated p value associated with the hypothesis that the variant polypeptide is significantly different than the reference protein used in that particular FAE assay, as defined further in Example 3; "EC50 (ppm)", EC50 as defined in example 3 with the EC50 dose given in ppm for the toxin portion of the sample; "Deviation", Mean Deviation Score as defined in Example 4; "Example #", the example number corresponding to the creation of the variant. The reference protein against which the variant protein is compared is: (MUT IDs: 1-872 and 91 1 -1 135) used SEQ ID NO: 6 as the reference protein; (MUT IDs: 873-910) used SEQ ID NO: 8 as the reference protein.
Table 8
Figure imgf000200_0001
MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
41 PHI-4 371 A G 0.02 4
42 PHI-4 371 A T 0.24 4
43 PHI-4 385 A G 0.08 4
44 PHI-4 385 A P -0.23 4
45 PHI-4 396 A E -0.56 4
46 PHI-4 405 A S 0.09 4
47 PHI-4 405 A W -0.38 4
48 PHI-4 409 A P 0.25 4
49 PHI-4 417 A C 0.11 4
50 PHI-4 445 C L 0.16 4
51 PHI-4 445 C T 0.06 4
52 PHI-4 331 E N 0.24 4
53 PHI-4 339 E N 0.04 4
54 PHI-4 339 E Q 0.34 4
55 PHI-4 344 F W 0.16 4
56 PHI-4 374 F 1 0.1 4
57 PHI-4 378 F 1 -0.78 4
58 PHI-4 351 G V 0.1 4
59 PHI-4 397 G R 0.29 4
60 PHI-4 428 G S -0.17 4
61 PHI-4 373 1 V -0.52 4
62 PHI-4 375 K R 0.19 4
63 PHI-4 384 K A 0.05 4
64 PHI-4 341 L V 0.19 4
65 PHI-4 380 L G 0.17 4
66 PHI-4 383 L 1 0.07 4
67 PHI-4 383 L V 0.17 4
68 PHI-4 354 M L 0.29 4
69 PHI-4 422 M V 0.05 4
70 PHI-4 345 N H 0.08 4
71 PHI-4 362 N S 0.2 4
72 PHI-4 430 N D -0.7 4
73 PHI-4 453 N D 0.02 4
74 PHI-4 372 P L -0.13 4
75 PHI-4 390 Q D -0.36 4
76 PHI-4 452 Q G -0.1 4
77 PHI-4 391 R L 0.16 4
78 PHI-4 333 S E -0.34 4
79 PHI-4 333 S R 0.14 4
80 PHI-4 349 S F -0.53 4
81 PHI-4 349 S P -0.9 4
82 PHI-4 398 S G -0.71 4
83 PHI-4 427 S N -0.08 4
84 PHI-4 427 S T -0.22 4
85 PHI-4 343 V F -0.81 4
86 PHI-4 355 V L 0.09 4
87 PHI-4 382 V D 0.23 4 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
88 PHI-4 382 V L 0.16 4
89 PHI-4 387 V T -0.33 4
90 PHI-4 392 V L -0.26 4
91 PHI-4 438 V R 0.28 4
92 PHI-4 455 V 1 0.81 4
93 PHI-4 389 W F -0.06 4
94 PHI-4 389 W Y 0.19 4
95 PHI-4 457 W N 0.34 4
96 PHI-4 352 Y C -0.34 4
97 PHI-4 352 Y F -0.2 4
98 PHI-4 404 Y F -0.04 4
99 PHI-4 404 Y G -0.7 4
100 PHI-4 429 Y E -0.36 4
101 PHI-4 437 Y 1 -0.01 4
102 PHI-4 437 Y V 0.05 4
103 PHI-4 30 A C -0.08 4
104 PHI-4 103 A G 0.03 4
105 PHI-4 127 A T 0.1 4
106 PHI-4 185 A s 0.28 4
107 PHI-4 238 A T -0.24 4
108 PHI-4 263 A s -0.01 4
109 PHI-4 270 A P -0.6 4
110 PHI-4 287 A c 0.37 4
111 PHI-4 182 D Q 0.5 4
112 PHI-4 193 D N 0.32 4
113 PHI-4 268 D N 0.08 4
114 PHI-4 46 E D 0.47 4
115 PHI-4 46 E N 0.67 4
116 PHI-4 80 E S 0.27 4
117 PHI-4 83 E S -0.62 4
118 PHI-4 162 E D 0.28 4
119 PHI-4 278 E N 0.61 4
120 PHI-4 43 F E 0.53 4
121 PHI-4 73 F Y 0.01 4
122 PHI-4 149 F A -0.05 4
123 PHI-4 149 F V 0.25 4
124 PHI-4 303 F Y 0.08 4
125 PHI-4 22 G S 0.07 4
126 PHI-4 50 1 V 0.31 4
127 PHI-4 119 1 N -0.22 4
128 PHI-4 213 1 L -0.05 4
129 PHI-4 207 K A -0.4 4
130 PHI-4 214 K S 0.11 4
131 PHI-4 36 L M 0.12 4
132 PHI-4 100 L F -0.31 4
133 PHI-4 105 L 1 0.22 4
134 PHI-4 141 L H -0.34 4 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
135 PHI-4 181 L A -0.33 4
136 PHI-4 266 L 1 -0.73 4
137 PHI-4 266 L V -0.47 4
138 PHI-4 19 M 1 -0.04 4
139 PHI-4 19 M L 0.31 4
140 PHI-4 88 M 1 0.2 4
141 PHI-4 88 M L 0 4
142 PHI-4 204 M A 0.1 4
143 PHI-4 245 M L 0.34 4
144 PHI-4 155 N K 0.11 4
145 PHI-4 231 N S 0.32 4
146 PHI-4 14 P A 0.13 4
147 PHI-4 159 P D 0.2 4
148 PHI-4 243 P L -0.54 4
149 PHI-4 282 P G 0.26 4
150 PHI-4 55 R K 0.3 4
151 PHI-4 61 R K 0.24 4
152 PHI-4 97 R N 0.59 4
153 PHI-4 292 R Q 0.27 4
154 PHI-4 56 S T 0.36 4
155 PHI-4 173 S A 0.29 4
156 PHI-4 184 S T 0.27 4
157 PHI-4 219 S N -0.4 4
158 PHI-4 230 S E 0.11 4
159 PHI-4 276 S A 0.36 4
160 PHI-4 279 S P -0.35 4
161 PHI-4 58 T S 0.09 4
162 PHI-4 112 T S 0.04 4
163 PHI-4 117 T S 0.18 4
164 PHI-4 189 T K 0.08 4
165 PHI-4 94 V 1 0.18 4
166 PHI-4 210 V 1 0.41 4
167 PHI-4 57 Y F 0.26 4
168 PHI-4 167 Y W 0.08 4
169 PHI-4 170 Y H 0.23 4
170 PHI-4 171 Y F 1.29 4
171 PHI-4 183 Y V 0.05 4
172 PHI-4 186 A V -0.34 4
173 PHI-4 342 A V 0.24 4
174 PHI-4 445 C S -0.24 4
175 PHI-4 321 D E -0.27 4
176 PHI-4 46 E G 0.02 4
177 PHI-4 222 E G -0.34 4
178 PHI-4 297 E G -0.42 4
179 PHI-4 344 F Y -0.07 4
180 PHI-4 483 F S -0.19 4
181 PHI-4 66 H R 0 4 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
182 PHI-4 441 H R -0.13 4
183 PHI-4 172 1 V 0.07 4
184 PHI-4 384 K G 0.19 4
185 PHI-4 465 K E -0.19 4
186 PHI-4 209 L P 0.4 4
187 PHI-4 236 L P 0.44 4
188 PHI-4 144 M 1 -0.03 4
189 PHI-4 158 M R -0.4 4
190 PHI-4 12 N D -0.28 4
191 PHI-4 155 N D -0.02 4
192 PHI-4 350 N S 0.08 4
193 PHI-4 14 P L -1.16 4
194 PHI-4 115 Q L -0.14 4
195 PHI-4 306 Q L -0.39 4
196 PHI-4 309 Q R -0.3 4
197 PHI-4 134 S G -0.21 4
198 PHI-4 195 S N -0.02 4
199 PHI-4 504 S C -0.59 4
200 PHI-4 189 T 1 0.26 4
201 PHI-4 233 T A -0.33 4
202 PHI-4 16 V D 0.03 4
203 PHI-4 294 V A 0.03 4
204 PHI-4 355 V G -0.31 4
205 PHI-4 438 V A -0.15 4
206 PHI-4 448 V A 0.05 4
207 PHI-4 284 w R 0.11 4
208 PHI-4 84 Y F 0.35 4
209 PHI-4 167 Y C 0.1 4
210 PHI-4 97 R D 125.8 0.01 5
211 PHI-4 447 D K 31.7 0.598 5
212 PHI-4 334 G R 25.2 0 5
213 PHI-4 527 Q K 25 0 5
214 PHI-4 109 F K 14.3 0 5
215 PHI-4 74 K E 12.4 0.152 5
216 PHI-4 402 K F 10.2 0 5
217 PHI-4 336 G A 8 0 5
218 PHI-4 527 Q P 7.8 0 5
219 PHI-4 82 E 1 7.8 0.086 5
220 PHI-4 109 F G 6.8 0.151 5
221 PHI-4 97 R E 6.7 0 5
222 PHI-4 220 E H 6.6 0 5
223 PHI-4 165 K E 6.6 0 5
224 PHI-4 289 K L 6.6 0 5
225 PHI-4 454 R Y 6.4 0 5
226 PHI-4 109 F M 6.4 0 5
227 PHI-4 247 D Y 6.1 0 5
228 PHI-4 454 R M 5.8 0 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
229 PHI-4 99 K L 5.7 0 5
230 PHI-4 289 K V 5.1 0 5
231 PHI-4 109 F S 5 0.374 5
232 PHI-4 289 K P 5 0 5
233 PHI-4 454 R S 4.6 0 5
234 PHI-4 220 E D 4.4 0 5
235 PHI-4 334 G K 4.3 0.395 5
236 PHI-4 459 K M 4.2 0 5
237 PHI-4 97 R Q 4 0 5
238 PHI-4 454 R V 4 0.001 5
239 PHI-4 517 Q 1 4 0.006 5
240 PHI-4 99 K Y 4 0 5
241 PHI-4 256 Q K 4 0.192 5
242 PHI-4 109 F D 3.9 0.596 5
243 PHI-4 220 E T 3.8 0 5
244 PHI-4 196 Q K 3.7 0.08 5
245 PHI-4 517 Q F 3.7 0.001 5
246 PHI-4 79 K E 3.6 0.006 5
247 PHI-4 454 R 1 3.5 0.002 5
248 PHI-4 454 R K 3.5 0.001 5
249 PHI-4 289 K E 3.3 0.994 5
250 PHI-4 74 K G 3.3 0 5
251 PHI-4 403 D Y 3.3 0 5
252 PHI-4 166 R Q 3.3 0.992 5
253 PHI-4 517 Q K 3.3 0.162 5
254 PHI-4 447 D Y 3.2 0 5
255 PHI-4 289 K Q 2.8 0.028 5
256 PHI-4 454 R F 2.8 0.011 5
257 PHI-4 220 E Y 2.7 0.003 5
258 PHI-4 447 D S 2.7 0 5
259 PHI-4 82 E L 2.7 0.881 5
260 PHI-4 196 Q N 2.7 0 5
261 PHI-4 216 E Q 2.7 0.017 5
262 PHI-4 334 G 1 2.6 0.893 5
263 PHI-4 151 D S 2.6 0.001 5
264 PHI-4 454 R W 2.6 0.005 5
265 PHI-4 165 K Q 2.6 0.878 5
266 PHI-4 459 K V 2.6 0.024 5
267 PHI-4 148 D F 2.6 0.001 5
268 PHI-4 220 E V 2.5 0.016 5
269 PHI-4 454 R Q 2.5 0.068 0.26 5
270 PHI-4 447 D E 2.5 0 5
271 PHI-4 527 Q C 2.5 0.001 5
272 PHI-4 196 Q D 2.5 0.004 5
273 PHI-4 82 E Y 2.5 0.938 5
274 PHI-4 527 Q E 2.4 0.423 5
275 PHI-4 402 K H 2.4 0.016 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
276 PHI-4 459 K W 2.4 0.043 5
277 PHI-4 459 K Q 2.3 0.116 5
278 PHI-4 289 K Y 2.3 0.027 5
279 PHI-4 99 K 1 2.3 0.024 5
280 PHI-4 289 K T 2.3 0.031 5
281 PHI-4 220 E s 2.3 0.037 5
282 PHI-4 459 K 1 2.3 0.049 5
283 PHI-4 462 G A 2.3 0.017 5
284 PHI-4 99 K M 2.2 0.025 5
285 PHI-4 289 K D 2.2 0.032 5
286 PHI-4 109 F N 2.2 0.061 5
287 PHI-4 220 E Q 2.2 0.166 5
288 PHI-4 203 E T 2.2 0.973 5
289 PHI-4 257 Q 1 2.1 0.013 5
290 PHI-4 203 E H 2.1 0.948 5
291 PHI-4 151 D A 2.1 0.017 5
292 PHI-4 447 D 1 2.1 0.017 5
293 PHI-4 97 R G 2.1 0.068 5
294 PHI-4 151 D N 2 0.026 5
295 PHI-4 148 D P 2 0.001 5
296 PHI-4 97 R S 2 0.106 0.45 5
297 PHI-4 151 D W 2 0.042 5
298 PHI-4 257 Q E 2 0.543 5
299 PHI-4 109 F E 2 0.926 5
300 PHI-4 527 Q S 2 0.282 5
301 PHI-4 403 D W 2 0.001 5
302 PHI-4 518 E Q 1.9 0.377 5
303 PHI-4 460 G A 1.9 0.124 5
304 PHI-4 499 E Q 1.9 0.395 5
305 PHI-4 148 D V 1.9 0.003 5
306 PHI-4 148 D E 1.9 0.004 5
307 PHI-4 459 K T 1.9 0.232 5
308 PHI-4 289 K F 1.9 0.158 5
309 PHI-4 289 K S 1.9 0.149 5
310 PHI-4 151 D V 1.9 0.079 5
311 PHI-4 402 K R 1.9 0.247 0.08 5
312 PHI-4 196 Q E 1.8 0.482 5
313 PHI-4 525 Q K 1.8 0.647 5
314 PHI-4 289 K M 1.8 0.189 5
315 PHI-4 302 E Q 1.8 0.505 5
316 PHI-4 403 D F 1.8 0.009 5
317 PHI-4 148 D H 1.8 0.011 5
318 PHI-4 165 K P 1.8 0.231 5
319 PHI-4 459 K S 1.8 0.323 5
320 PHI-4 24 D Q 1.8 0.766 5
321 PHI-4 151 D Q 1.7 0.766 5
322 PHI-4 289 K R 1.7 0.256 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
323 PHI-4 196 Q A 1.7 0.142 5
324 PHI-4 459 K H 1.7 0.408 5
325 PHI-4 454 R G 1.7 0.407 5
326 PHI-4 395 D C 1.7 0.807 5
327 PHI-4 220 E R 1.7 0.414 5
328 PHI-4 99 K F 1.6 0.363 5
329 PHI-4 289 K W 1.6 0.457 5
330 PHI-4 527 Q H 1.6 0.251 5
331 PHI-4 220 E W 1.6 0.508 5
332 PHI-4 9 Q K 1.6 0.756 5
333 PHI-4 99 K C 1.6 0.454 5
334 PHI-4 309 Q K 1.6 0.737 5
335 PHI-4 148 D W 1.6 0.054 5
336 PHI-4 216 E F 1.6 0.451 5
337 PHI-4 99 K V 1.6 0.487 5
338 PHI-4 454 R H 1.6 0.577 5
339 PHI-4 309 Q E 1.5 0.704 5
340 PHI-4 165 K T 1.5 0.519 5
341 PHI-4 165 K A 1.5 0.529 5
342 PHI-4 398 S Q 1.5 0.523 5
343 PHI-4 454 R D 1.5 0.628 5
344 PHI-4 142 R E 1.5 0.934 5
345 PHI-4 449 Q E 1.5 0.713 5
346 PHI-4 196 Q 1 1.5 0.425 5
347 PHI-4 278 E Q 1.5 0.065 5
348 PHI-4 502 R E 1.5 0.962 5
349 PHI-4 165 K L 1.5 0.602 5
350 PHI-4 90 Q E 1.5 0.721 5
351 PHI-4 399 G A 1.5 0.587 5
352 PHI-4 447 D G 1.5 0.157 0.12 5
353 PHI-4 151 D C 1.5 0.485 5
354 PHI-4 289 K 1 1.5 0.636 5
355 PHI-4 459 K C 1.5 0.73 5
356 PHI-4 220 E M 1.4 0.611 5
357 PHI-4 454 R L 1.4 0.721 5
358 PHI-4 459 K Y 1.4 0.736 5
359 PHI-4 442 Q K 1.4 0.751 5
360 PHI-4 99 K N 1.4 0.687 5
361 PHI-4 402 K W 1.4 0.758 5
362 PHI-4 216 E V 1.4 0.658 5
363 PHI-4 165 K G 1.4 0.731 5
364 PHI-4 214 K Q 1.4 0.701 5
365 PHI-4 165 K D 1.4 0.741 5
366 PHI-4 165 K V 1.4 0.744 5
367 PHI-4 220 E A 1.4 0.694 5
368 PHI-4 289 K H 1.4 0.774 5
369 PHI-4 165 K H 1.4 0.785 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
370 PHI-4 82 E Q 1.4 0.875 -0.77 5
371 PHI-4 109 F C 1.4 0.764 5
372 PHI-4 220 E F 1.4 0.726 5
373 PHI-4 51 E Q 1.4 0.853 5
374 PHI-4 459 K P 1.3 0.888 5
375 PHI-4 447 D P 1.3 0.328 5
376 PHI-4 165 K 1 1.3 0.831 5
377 PHI-4 220 E 1 1.3 0.764 5
378 PHI-4 459 K N 1.3 0.905 5
379 PHI-4 454 R T 1.3 0.926 5
380 PHI-4 87 K Q 1.3 0.688 5
381 PHI-4 402 K G 1.3 0.96 5
382 PHI-4 289 K N 1.3 0.91 5
383 PHI-4 148 D A 1.3 0.428 5
384 PHI-4 216 E Y 1.3 0.857 5
385 PHI-4 306 Q K 1.3 0.669 5
386 PHI-4 148 D R 1.3 0.572 5
387 PHI-4 151 D M 1.3 0.852 5
388 PHI-4 257 Q C 1.3 0.848 5
389 PHI-4 9 Q E 1.3 0.687 5
390 PHI-4 402 K N 1.2 0.927 5
391 PHI-4 148 D L 1.2 0.614 5
392 PHI-4 148 D S 1.2 0.628 5
393 PHI-4 436 D K 1.2 0.755 5
394 PHI-4 459 K A 1.2 0.886 5
395 PHI-4 447 D L 1.2 0.673 5
396 PHI-4 454 R P 1.2 0.981 5
397 PHI-4 398 S C 1.2 0.984 5
398 PHI-4 76 D Q 1.2 0.755 5
399 PHI-4 220 E L 1.2 0.994 5
400 PHI-4 165 K M 1.2 0.903 5
401 PHI-4 196 Q R 1.2 0.819 5
402 PHI-4 402 K L 1.2 0.848 5
403 PHI-4 220 E C 1.2 0.994 5
404 PHI-4 403 D E 1.2 0.986 5
405 PHI-4 220 E N 1.2 0.836 5
406 PHI-4 42 D Q 1.2 0.754 5
407 PHI-4 165 K W 1.2 0.873 5
408 PHI-4 466 D R 1.2 0.795 5
409 PHI-4 517 Q C 1.2 0.789 5
410 PHI-4 256 Q E 1.2 0.618 5
411 PHI-4 517 Q N 1.2 0.785 5
412 PHI-4 148 D Q 1.2 0.754 5
413 PHI-4 517 Q V 1.2 0.777 5
414 PHI-4 83 E Q 1.2 0.668 5
415 PHI-4 165 K F 1.2 0.836 5
416 PHI-4 447 D F 1.2 0.912 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
417 PHI-4 109 F A 1.2 0.71 5
418 PHI-4 86 E Q 1.2 0.565 5
419 PHI-4 61 R E 1.2 0.567 5
420 PHI-4 151 D L 1.2 0.891 5
421 PHI-4 305 K Q 1.1 0.553 5
422 PHI-4 257 Q S 1.1 0.729 5
423 PHI-4 74 K R 1.1 0.782 5
424 PHI-4 257 Q H 1.1 0.731 5
425 PHI-4 447 D W 1.1 0.933 5
426 PHI-4 402 K Y 1.1 0.74 5
427 PHI-4 257 Q W 1.1 0.72 5
428 PHI-4 517 Q P 1.1 0.831 5
429 PHI-4 331 E Q 1.1 0.661 5
430 PHI-4 334 G W 1.1 0.635 5
431 PHI-4 335 S A 1.1 0.754 5
432 PHI-4 297 E Q 1.1 0.497 5
433 PHI-4 264 E Q 1.1 0.658 5
434 PHI-4 447 D T 1.1 0.767 5
435 PHI-4 229 R E 1.1 0.494 5
436 PHI-4 298 D Q 1.1 0.752 5
437 PHI-4 527 Q F 1.1 0.763 5
438 PHI-4 74 K M 1.1 0.695 5
439 PHI-4 289 K C 1.1 0.677 5
440 PHI-4 113 D E 1.1 0.877 5
441 PHI-4 257 Q M 1.1 0.651 5
442 PHI-4 207 K Q 1.1 0.463 5
443 PHI-4 454 R N 1.1 0.624 5
444 PHI-4 502 R Q 1.1 0.454 5
445 PHI-4 402 K T 1.1 0.62 5
446 PHI-4 151 D R 1.1 0.692 5
447 PHI-4 527 Q W 1.1 0.616 5
448 PHI-4 109 F R 1 0.682 5
449 PHI-4 151 D E 1 0.655 5
450 PHI-4 289 K G 1 0.617 5
451 PHI-4 74 K L 1 0.611 5
452 PHI-4 146 R Q 1 0.424 5
453 PHI-4 113 D M 1 0.736 5
454 PHI-4 74 K H 1 0.636 5
455 PHI-4 191 K Q 1 0.649 5
456 PHI-4 291 E Q 1 0.424 5
457 PHI-4 148 D G 1 0.724 5
458 PHI-4 165 K Y 1 0.595 5
459 PHI-4 74 K D 1 0.582 5
460 PHI-4 402 K V 1 0.556 5
461 PHI-4 165 K R 1 0.575 5
462 PHI-4 497 D Q 1 0.75 5
463 PHI-4 397 G A 1 0.568 5 MUT BackPositef Substi0 DeviaExample
m ΕΔ F P- EC5
t
ID NO: bone ion A.A. tution value (ppm) tion #
464 PHI-4 289 K A 1 0.558 5
465 PHI-4 517 Q E 1 0.509 5
466 PHI-4 360 Q E 1 0.523 5
467 PHI-4 254 D K 1 0.75 5
468 PHI-4 402 K S 1 0.511 5
469 PHI-4 288 D Q 1 0.75 5
470 PHI-4 454 R C 1 0.51 5
471 PHI-4 306 Q E 1 0.547 5
472 PHI-4 82 E V 1 0.626 5
473 PHI-4 113 D N 1 0.538 5
474 PHI-4 447 D M 1 0.498 5
475 PHI-4 447 D H 1 0.497 5
476 PHI-4 151 D G 1 0.494 5
477 PHI-4 214 K E 1 0.339 5
478 PHI-4 148 D C 1 0.506 5
479 PHI-4 216 E T 1 0.601 5
480 PHI-4 87 K E 0.9 0.329 5
481 PHI-4 165 K N 0.9 0.467 5
482 PHI-4 151 D K 0.9 0.749 5
483 PHI-4 113 D S 0.9 0.441 5
484 PHI-4 402 K D 0.9 0.415 5
485 PHI-4 459 K R 0.9 0.42 0.24 5
486 PHI-4 74 K F 0.9 0.427 5
487 PHI-4 361 R E 0.9 0.304 5
488 PHI-4 165 K C 0.9 0.421 5
489 PHI-4 72 D Q 0.9 0.748 5
490 PHI-4 257 Q V 0.9 0.441 5
491 PHI-4 220 E K 0.9 0.535 5
492 PHI-4 334 G H 0.9 0.575 5
493 PHI-4 113 D H 0.9 0.394 5
494 PHI-4 517 Q R 0.9 0.421 5
495 PHI-4 436 D Q 0.9 0.748 5
496 PHI-4 235 R P 0.9 0.396 5
497 PHI-4 235 R K 0.9 0.748 -0.01 5
498 PHI-4 79 K Q 0.9 0.277 5
499 PHI-4 527 Q Y 0.9 0.414 5
500 PHI-4 113 D A 0.9 0.326 5
501 PHI-4 216 E S 0.9 0.501 5
502 PHI-4 257 Q L 0.9 0.409 5
503 PHI-4 46 E Q 0.9 0.627 5
504 PHI-4 447 D N 0.9 0.369 5
505 PHI-4 247 D E 0.9 0.321 5
506 PHI-4 72 D K 0.9 0.748 5
507 PHI-4 517 Q H 0.9 0.404 5
508 PHI-4 74 K W 0.9 0.361 5
509 PHI-4 442 Q E 0.9 0.48 0.3 5
510 PHI-4 229 R Q 0.9 0.258 5 MUT BackPositef SubstiΕΔ F P- EC50 DeviaExample mt
ID NO: bone ion A.A. tution value (ppm) tion #
511 PHI-4 527 Q R 0.9 0.388 5
512 PHI-4 74 K V 0.9 0.367 5
513 PHI-4 288 D K 0.9 0.747 5
514 PHI-4 48 D Q 0.9 0.747 5
515 PHI-4 517 Q S 0.9 0.378 5
516 PHI-4 402 K A 0.9 0.333 5
517 PHI-4 216 E A 0.8 0.452 5
518 PHI-4 216 E G 0.8 0.45 5
519 PHI-4 113 D Y 0.8 0.302 5
520 PHI-4 402 K M 0.8 0.313 5
521 PHI-4 82 E P 0.8 0.63 5
522 PHI-4 525 Q E 0.8 0.469 5
523 PHI-4 99 K R 0.8 0.312 5
524 PHI-4 257 Q G 0.8 0.35 5
525 PHI-4 48 D K 0.8 0.746 5
526 PHI-4 305 K E 0.8 0.226 5
527 PHI-4 216 E W 0.8 0.417 5
528 PHI-4 207 K E 0.8 0.22 5
529 PHI-4 447 D V 0.8 0.266 5
530 PHI-4 196 Q F 0.8 0.337 5
531 PHI-4 216 E K 0.8 0.406 5
532 PHI-4 151 D H 0.8 0.263 5
533 PHI-4 74 K S 0.8 0.285 5
534 PHI-4 90 Q K 0.8 0.466 5
535 PHI-4 459 K G 0.8 0.282 5
536 PHI-4 454 R E 0.8 0.215 5
537 PHI-4 216 E R 0.8 0.391 0.04 5
538 PHI-4 74 K Y 0.8 0.276 5
539 PHI-4 99 K S 0.8 0.277 5
540 PHI-4 248 R Q 0.8 0.201 5
541 PHI-4 142 R Q 0.8 0.193 5
542 PHI-4 82 E F 0.8 0.615 5
543 PHI-4 461 T A 0.8 0.245 5
544 PHI-4 33 E Q 0.8 0.185 5
545 PHI-4 193 D Q 0.8 0.745 5
546 PHI-4 332 D N 0.8 0.226 5
547 PHI-4 42 D K 0.8 0.745 5
548 PHI-4 74 K A 0.8 0.238 5
549 PHI-4 257 Q K 0.7 0.461 5
550 PHI-4 148 D M 0.7 0.117 5
551 PHI-4 527 Q G 0.7 0.272 5
552 PHI-4 527 Q V 0.7 0.276 5
553 PHI-4 379 D Q 0.7 0.745 5
554 PHI-4 146 R E 0.7 0.607 5
555 PHI-4 527 Q T 0.7 0.266 5
556 PHI-4 454 R A 0.7 0.213 5
557 PHI-4 216 E D 0.7 0.306 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
558 PHI-4 402 K C 0.7 0.203 5
559 PHI-4 517 Q T 0.7 0.256 5
560 PHI-4 257 Q P 0.7 0.256 5
561 PHI-4 220 E G 0.7 0.3 5
562 PHI-4 99 K H 0.7 0.2 5
563 PHI-4 55 R E 0.7 0.153 5
564 PHI-4 340 D N 0.7 0.179 5
565 PHI-4 466 D N 0.7 0.185 5
566 PHI-4 257 Q A 0.7 0.238 5
567 PHI-4 99 K T 0.7 0.191 5
568 PHI-4 74 K T 0.7 0.189 5
569 PHI-4 113 D L 0.7 0.081 5
570 PHI-4 74 K Q 0.7 0.602 5
571 PHI-4 527 Q 1 0.7 0.238 5
572 PHI-4 99 K A 0.7 0.186 5
573 PHI-4 517 Q W 0.7 0.235 5
574 PHI-4 126 E Q 0.7 0.459 -0.15 5
575 PHI-4 196 Q G 0.7 0.231 5
576 PHI-4 274 D K 0.7 0.744 5
577 PHI-4 517 Q G 0.7 0.229 5
578 PHI-4 99 K W 0.7 0.175 5
579 PHI-4 394 D Q 0.7 0.743 5
580 PHI-4 216 E 1 0.7 0.257 5
581 PHI-4 517 Q M 0.7 0.214 5
582 PHI-4 334 G S 0.7 0.507 5
583 PHI-4 517 Q D 0.7 0.207 5
584 PHI-4 196 Q L 0.7 0.21 5
585 PHI-4 298 D K 0.6 0.742 5
586 PHI-4 74 K C 0.6 0.141 5
587 PHI-4 395 D S 0.6 0.538 5
588 PHI-4 74 K 1 0.6 0.139 5
589 PHI-4 527 Q M 0.6 0.189 5
590 PHI-4 113 D Q 0.6 0.742 5
591 PHI-4 340 D A 0.6 0.142 5
592 PHI-4 216 E H 0.6 0.216 5
593 PHI-4 402 K 1 0.6 0.13 5
594 PHI-4 235 R S 0.6 0.123 5
595 PHI-4 75 Q K 0.6 0.422 5
596 PHI-4 203 E Q 0.6 0.588 -0.37 5
597 PHI-4 527 Q A 0.6 0.167 5
598 PHI-4 517 Q A 0.6 0.163 5
599 PHI-4 361 R Q 0.6 0.094 5
600 PHI-4 466 D P 0.6 0.075 5
601 PHI-4 193 D K 0.6 0.741 5
602 PHI-4 458 D V 0.6 0.075 5
603 PHI-4 268 D Q 0.6 0.741 5
604 PHI-4 61 R Q 0.6 0.086 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
605 PHI-4 151 D Y 0.6 0.068 5
606 PHI-4 196 Q Y 0.6 0.146 5
607 PHI-4 196 Q V 0.5 0.145 5
608 PHI-4 527 Q D 0.5 0.144 5
609 PHI-4 166 R E 0.5 0.084 5
610 PHI-4 334 G A 0.5 0.477 5
611 PHI-4 447 D Q 0.5 0.74 5
612 PHI-4 196 Q C 0.5 0.137 5
613 PHI-4 466 D K 0.5 0.74 5
614 PHI-4 74 K P 0.5 0.083 5
615 PHI-4 196 Q M 0.5 0.127 5
616 PHI-4 99 K D 0.5 0.083 5
617 PHI-4 82 E D 0.5 0.141 5
618 PHI-4 216 E M 0.5 0.136 5
619 PHI-4 235 R H 0.5 0.074 5
620 PHI-4 340 D P 0.5 0.078 5
621 PHI-4 216 E L 0.5 0.134 5
622 PHI-4 458 D G 0.5 0.119 5
623 PHI-4 403 D S 0.5 0.074 5
624 PHI-4 360 Q K 0.5 0.335 5
625 PHI-4 216 E P 0.5 0.127 5
626 PHI-4 257 Q Y 0.5 0.11 5
627 PHI-4 118 E Q 0.5 0.061 5
628 PHI-4 379 D K 0.5 0.739 5
629 PHI-4 402 K Q 0.5 0.061 5
630 PHI-4 254 D Q 0.5 0.739 5
631 PHI-4 257 Q T 0.5 0.107 5
632 PHI-4 458 D P 0.5 0.04 5
633 PHI-4 466 D w 0.5 0.036 5
634 PHI-4 403 D L 0.5 0.037 5
635 PHI-4 196 Q H 0.5 0.101 5
636 PHI-4 517 Q L 0.4 0.099 5
637 PHI-4 196 Q S 0.4 0.099 5
638 PHI-4 257 Q F 0.4 0.096 5
639 PHI-4 196 Q T 0.4 0.096 5
640 PHI-4 235 R G 0.4 0.055 5
641 PHI-4 332 D S 0.4 0.005 5
642 PHI-4 152 D Q 0.4 0.738 5
643 PHI-4 368 D Q 0.4 0.738 5
644 PHI-4 257 Q R 0.4 0.092 5
645 PHI-4 315 D K 0.4 0.738 5
646 PHI-4 99 K P 0.4 0.052 5
647 PHI-4 109 F P 0.4 0.103 5
648 PHI-4 113 D T 0.4 0.004 5
649 PHI-4 334 G M 0.4 0.447 5
650 PHI-4 75 Q E 0.4 0.315 5
651 PHI-4 274 D Q 0.4 0.738 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
652 PHI-4 402 K E 0.4 0.048 5
653 PHI-4 191 K E 0.4 0.048 5
654 PHI-4 332 D E 0.4 0.004 5
655 PHI-4 517 Q Y 0.4 0.085 5
656 PHI-4 340 D 1 0.4 0.025 5
657 PHI-4 459 K D 0.4 0.05 5
658 PHI-4 76 D K 0.4 0.738 5
659 PHI-4 216 E C 0.4 0.093 5
660 PHI-4 340 D R 0.4 0.048 5
661 PHI-4 466 D Y 0.4 0.025 5
662 PHI-4 340 D G 0.4 0.046 5
663 PHI-4 376 D Q 0.4 0.737 5
664 PHI-4 203 E C 0.4 0.088 5
665 PHI-4 235 R N 0.4 0.042 5
666 PHI-4 247 D S 0.4 0.076 5
667 PHI-4 332 D F 0.4 0.021 5
668 PHI-4 216 E N 0.4 0.083 5
669 PHI-4 527 Q N 0.4 0.076 5
670 PHI-4 340 D C 0.4 0.043 5
671 PHI-4 38 Q E 0.4 0.297 5
672 PHI-4 152 D K 0.4 0.737 5
673 PHI-4 413 Q E 0.4 0.316 5
674 PHI-4 247 D T 0.4 0.002 5
675 PHI-4 235 R V 0.3 0.034 5
676 PHI-4 268 D K 0.3 0.736 5
677 PHI-4 99 K Q 0.3 0.034 5
678 PHI-4 235 R D 0.3 0.032 5
679 PHI-4 394 D K 0.3 0.736 5
680 PHI-4 458 D C 0.3 0.001 5
681 PHI-4 340 D Y 0.3 0.015 5
682 PHI-4 24 D K 0.3 0.736 5
683 PHI-4 466 D G 0.3 0.001 5
684 PHI-4 466 D C 0.3 0.014 5
685 PHI-4 402 K P 0.3 0.013 5
686 PHI-4 340 D L 0.3 0.031 5
687 PHI-4 334 G Y 0.3 0.421 5
688 PHI-4 334 G Q 0.3 0.421 5
689 PHI-4 334 G L 0.3 0.42 5
690 PHI-4 398 S V 0.3 0.013 5
691 PHI-4 334 G N 0.3 0.42 5
692 PHI-4 398 S H 0.3 0.012 5
693 PHI-4 113 D R 0.3 0.001 5
694 PHI-4 235 R T 0.3 0.026 5
695 PHI-4 334 G c 0.3 0.417 5
696 PHI-4 248 R E 0.3 0.028 5
697 PHI-4 235 R 1 0.3 0.025 5
698 PHI-4 398 S R 0.3 0.011 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
699 PHI-4 334 G F 0.3 0.455 5
700 PHI-4 113 D P 0.3 0.001 5
701 PHI-4 109 F L 0.3 0.052 5
702 PHI-4 203 E S 0.3 0.412 5
703 PHI-4 109 F Y 0.3 0.412 5
704 PHI-4 466 D E 0.3 0.001 5
705 PHI-4 55 R Q 0.3 0.025 5
706 PHI-4 203 E V 0.3 0.411 5
707 PHI-4 398 S L 0.3 0.01 5
708 PHI-4 332 D G 0.3 0.001 5
709 PHI-4 203 E G 0.3 0.41 5
710 PHI-4 466 D 1 0.3 0.009 5
711 PHI-4 466 D A 0.3 0 5
712 PHI-4 235 R A 0.3 0.021 5
713 PHI-4 340 D V 0.3 0.022 5
714 PHI-4 466 D F 0.3 0.009 5
715 PHI-4 99 K E 0.3 0.023 5
716 PHI-4 188 K E 0.3 0.022 5
717 PHI-4 235 R C 0.2 0.019 5
718 PHI-4 235 R F 0.2 0.018 5
719 PHI-4 340 D W 0.2 0.02 5
720 PHI-4 235 R L 0.2 0.018 5
721 PHI-4 113 D W 0.2 0 5
722 PHI-4 459 K E 0.2 0.541 5
723 PHI-4 334 G V 0.2 0.4 5
724 PHI-4 113 D V 0.2 0 5
725 PHI-4 113 D G 0.2 0 5
726 PHI-4 235 R M 0.2 0.015 5
727 PHI-4 403 D N 0.2 0.012 5
728 PHI-4 203 E N 0.2 0.396 5
729 PHI-4 340 D F 0.2 0.006 5
730 PHI-4 340 D S 0.2 0.005 5
731 PHI-4 247 D P 0.2 0 5
732 PHI-4 203 E 1 0.2 0.035 5
733 PHI-4 148 D K 0.2 0.733 5
734 PHI-4 466 D T 0.2 0 5
735 PHI-4 332 D H 0.2 0.005 5
736 PHI-4 458 D A 0.2 0 5
737 PHI-4 235 R E 0.2 0.016 5
738 PHI-4 340 D K 0.2 0.733 5
739 PHI-4 466 D V 0.2 0 5
740 PHI-4 109 F V 0.2 0.032 5
741 PHI-4 398 S T 0.2 0.389 5
742 PHI-4 99 K G 0.2 0.012 5
743 PHI-4 466 D L 0.2 0 5
744 PHI-4 340 D T 0.2 0.013 5
745 PHI-4 332 D A 0.2 0 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
746 PHI-4 403 D R 0.2 0.013 5
747 PHI-4 113 D K 0.2 0.733 5
748 PHI-4 403 D A 0.2 0.013 5
749 PHI-4 109 F Q 0.2 0.03 5
750 PHI-4 332 D C 0.2 0 5
751 PHI-4 340 D M 0.2 0.004 5
752 PHI-4 203 E R 0.2 0.029 5
753 PHI-4 235 R Y 0.2 0.011 5
754 PHI-4 196 Q P 0.2 0.029 5
755 PHI-4 247 D W 0.2 0 5
756 PHI-4 203 E A 0.2 0.384 5
757 PHI-4 466 D H 0.2 0 5
758 PHI-4 203 E F 0.1 0.027 5
759 PHI-4 449 Q K 0.1 0.206 5
760 PHI-4 334 G T 0.1 0.382 5
761 PHI-4 413 Q K 0.1 0.223 5
762 PHI-4 38 Q K 0.1 0.242 5
763 PHI-4 82 E R 0.1 0.381 5
764 PHI-4 340 D H 0.1 0.003 5
765 PHI-4 235 R W 0.1 0.009 5
766 PHI-4 398 S P 0.1 0.026 5
767 PHI-4 458 D S 0.1 0 -0.49 5
768 PHI-4 247 D N 0.1 0 5
769 PHI-4 466 D S 0.1 0 5
770 PHI-4 395 D 1 0.1 0.379 5
771 PHI-4 466 D Q 0.1 0.732 5
772 PHI-4 220 E P 0.1 0.01 5
773 PHI-4 340 D Q 0.1 0.732 5
774 PHI-4 247 D G 0.1 0 5
775 PHI-4 203 E Y 0.1 0.025 5
776 PHI-4 334 G E 0.1 0.378 5
777 PHI-4 247 D A 0.1 0.025 5
778 PHI-4 458 D F 0.1 0.003 5
779 PHI-4 403 D C 0.1 0 5
780 PHI-4 403 D 1 0.1 0.003 5
781 PHI-4 403 D V 0.1 0.009 5
782 PHI-4 403 D G 0.1 0.009 5
783 PHI-4 203 E K 0.1 0.373 5
784 PHI-4 398 S A 0.1 0.372 5
785 PHI-4 340 D E 0.1 0.008 5
786 PHI-4 82 E H 0.1 0.371 5
787 PHI-4 203 E W 0.1 0.37 5
788 PHI-4 447 D R 0.1 0 5
789 PHI-4 109 F W 0.1 0.37 5
790 PHI-4 247 D V 0.1 0 5
791 PHI-4 203 E L 0.1 0.369 5
792 PHI-4 332 D T 0.1 0 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
793 PHI-4 332 D 1 0.1 0.002 5
794 PHI-4 82 E G 0.1 0.019 5
795 PHI-4 247 D L 0.1 0 5
796 PHI-4 368 D K 0.1 0.731 5
797 PHI-4 82 E M 0.1 0.018 5
798 PHI-4 247 D H 0.1 0 5
799 PHI-4 458 D N 0.1 0.019 5
800 PHI-4 235 R Q 0.1 0.008 5
801 PHI-4 398 S D 0.1 0.017 5
802 PHI-4 332 D Y 0.1 0 5
803 PHI-4 247 D F 0.1 0.018 5
804 PHI-4 332 D V 0.1 0 5
805 PHI-4 395 D A 0.1 0.017 5
806 PHI-4 334 G P 0.1 0.362 5
807 PHI-4 53 E Q 0.1 0.008 5
808 PHI-4 97 R T 0.1 0.016 5
809 PHI-4 458 D 1 0.1 0 5
810 PHI-4 398 S N 0.1 0.016 5
811 PHI-4 109 F H 0.1 0.361 5
812 PHI-4 403 D P 0 0 5
813 PHI-4 497 D K 0 0.73 5
814 PHI-4 458 D M 0 0.017 5
815 PHI-4 458 D T 0 0.017 5
816 PHI-4 447 D A 0 0 5
817 PHI-4 395 D N 0 0.404 180 -0.91 5
818 PHI-4 247 D R 0 0 5
819 PHI-4 82 E T 0 0.36 5
820 PHI-4 395 D Y 0 0.36 5
821 PHI-4 398 S E 0 0.36 5
822 PHI-4 332 D Q 0 0.73 5
823 PHI-4 403 D M 0 0 5
824 PHI-4 247 D Q 0 0.73 5
825 PHI-4 398 S 1 0 0.015 5
826 PHI-4 458 D Y 0 0.016 5
827 PHI-4 398 S M 0 0.015 5
828 PHI-4 403 D H 0 0 5
829 PHI-4 82 E N 0 0.358 5
830 PHI-4 403 D T 0 0 5
831 PHI-4 247 D M 0 0.016 5
832 PHI-4 395 D R 0 0.015 5
833 PHI-4 398 S Y 0 0.401 5
834 PHI-4 395 D F 0 0.357 5
835 PHI-4 395 D M 0 0.357 5
836 PHI-4 395 D P 0 0.357 5
837 PHI-4 97 R A 0 0.014 5
838 PHI-4 395 D Q 0 0.73 -0.82 5
839 PHI-4 332 D L 0 0 5 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
840 PHI-4 395 D W 0 0.357 5
841 PHI-4 332 D P 0 0 5
842 PHI-4 403 D Q 0 0.73 5
843 PHI-4 458 D L 0 0 5
844 PHI-4 398 S K 0 0.356 5
845 PHI-4 458 D E 0 0.015 5
846 PHI-4 203 E P 0 0.014 5
847 PHI-4 395 D T 0 0.356 5
848 PHI-4 82 E c 0 0.356 5
849 PHI-4 395 D G 0 0.355 5
850 PHI-4 97 R V 0 0.014 5
851 PHI-4 332 D W 0 0 5
852 PHI-4 395 D L 0 0.355 5
853 PHI-4 332 D M 0 0 5
854 PHI-4 398 S W 0 0.014 5
855 PHI-4 395 D E 0 0.355 5
856 PHI-4 458 D R 0 0.015 5
857 PHI-4 321 D K 0 0.729 5
858 PHI-4 82 E K 0 0.013 5
859 PHI-4 82 E A 0 0.353 5
860 PHI-4 332 D R 0 0 5
861 PHI-4 458 D H 0 0 5
862 PHI-4 398 S F 0 0.353 5
863 PHI-4 395 D K 0 0.729 5
864 PHI-4 395 D H 0 0.353 5
865 PHI-4 203 E M 0 0.353 5
866 PHI-4 458 D W 0 0.014 5
867 PHI-4 403 D K 0 0.729 5
868 PHI-4 247 D K 0 0.729 5
869 PHI-4 376 D K 0 0.729 5
870 PHI-4 321 D Q 0 0.729 5
871 PHI-4 332 D K 0 0.729 5
872 PHI-4 315 D Q 5
873 SFR16 293 R E 19.5 0.045 6
874 SFR16 416 R E 18 0.039 6
875 SFR16 328 K E 17.8 0.06 6
876 SFR16 500 R Q 17.1 0.061 6
877 SFR16 452 Q K 8.8 0.014 6
878 SFR16 293 R Q 6.9 0 6
879 SFR16 150 R Q 4.2 0 6
880 SFR16 471 Q K 4 0.14 6
881 SFR16 261 Q E 3.1 0.011 6
882 SFR16 520 K Q 3.1 0.006 6
883 SFR16 471 Q E 3.1 0.01 6
884 SFR16 147 R E 2.7 0.038 6
885 SFR16 520 K E 2.6 0.888 6
886 SFR16 509 K Q 2.5 0.053 6 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
887 SFR16 162 E Q 2.5 0.058 6
888 SFR16 281 Q K 2.4 0.415 6
889 SFR16 452 Q E 2.4 0.13 6
890 SFR16 313 K Q 2.3 0.204 6
891 SFR16 328 K Q 2.2 0.194 6
892 SFR16 391 R E 2.1 0.812 6
893 SFR16 281 Q E 2.1 0.535 6
894 SFR16 391 R Q 2.1 0.253 6
895 SFR16 174 E Q 2 0.138 6
896 SFR16 391 R D 1.8 0.768 6
897 SFR16 147 R Q 1.5 0.712 6
898 SFR16 261 Q K 1.4 0.739 6
899 SFR16 313 K R 1.4 0.758 6
900 SFR16 316 K Q 1.3 0.685 6
901 SFR16 150 R E 1.2 0.677 6
902 SFR16 500 R K 1.2 0.754 6
903 SFR16 509 K E 1.1 0.537 6
904 SFR16 416 R Q 1 0.416 6
905 SFR16 316 K E 0.5 0.061 6
906 SFR16 242 K R 0.4 0.738 6
907 SFR16 313 K E 0.1 0.013 6
908 SFR16 242 K E 0.1 0.011 6
909 SFR16 242 K Q 0.1 0.011 6
910 SFR16 500 R E 0.1 0.01 6
911 PSR3 339 E N 343.6 0 7
912 PSR3 401 S H 32.8 0 7
913 PSR3 401 S P 21.4 0 7
914 PSR3 339 E 1 14.8 0 7
915 PSR3 465 K N 14.7 0 7
916 PSR3 402 K R 11.7 0 7
917 PSR3 402 K G 8.3 0 7
918 PSR3 333 S G 7.4 0 7
919 PSR3 464 R D 7.1 0 7
920 PSR3 402 K W 6.8 0 7
921 PSR3 401 S G 6.2 0 7
922 PSR3 465 K V 5 0.002 7
923 PSR3 402 K H 4.7 0 7
924 PSR3 396 A L 4.6 0 7
925 PSR3 338 S H 4.6 0.001 7
926 PSR3 464 R K 4.3 0 7
927 PSR3 465 K M 4.3 0 7
928 PSR3 333 S K 4.3 0 7
929 PSR3 464 R A 4 0 7
930 PSR3 396 A K 3.9 0 7
931 PSR3 338 S V 3.2 0 7
932 PSR3 333 S V 3.1 0.005 7
933 PSR3 401 S K 3.1 0 7 MUT BackPositRef Substi50 DeviaExample
m ΕΔ F P- EC
t
ID NO: bone ion A.A. tution value (ppm) tion #
934 PS 3 339 E P 3 0.026 7
935 PSR3 465 K P 2.9 0.071 7
936 PSR3 396 A N 2.8 0.042 7
937 PSR3 402 K T 2.8 0.06 7
938 PSR3 464 R s 2.7 0.031 7
939 PSR3 463 A s 2.7 0.083 7
940 PSR3 459 K Y 2.6 0.051 7
941 PSR3 333 S N 2.6 0.051 7
942 PSR3 396 A G 2.5 0.057 7
943 PSR3 464 R G 2.5 0.079 7
944 PSR3 338 S K 2.5 0.008 7
945 PSR3 402 K N 2.3 0.127 7
946 PSR3 465 K G 2.3 0.268 7
947 PSR3 402 K Y 2.2 0.158 7
948 PSR3 401 S V 2.2 0.024 7
949 PSR3 464 R H 2.2 0.175 7
950 PSR3 465 K R 2.1 0.246 7
951 PSR3 401 S R 2.1 0.206 7
952 PSR3 402 K M 2.1 0.242 7
953 PSR3 338 S A 2 0.376 7
954 PSR3 464 R N 2 0.293 7
955 PSR3 459 K E 2 0.298 7
956 PSR3 465 K T 2 0.401 7
957 PSR3 333 S A 2 0.082 7
958 PSR3 338 S G 1.9 0.456 7
959 PSR3 339 E M 1.9 0.368 7
960 PSR3 396 A 1 1.9 0.479 7
961 PSR3 338 S T 1.8 0.182 7
962 PSR3 402 K P 1.8 0.587 7
963 PSR3 459 K P 1.8 0.466 7
964 PSR3 333 S H 1.8 0.561 7
965 PSR3 396 A M 1.8 0.213 7
966 PSR3 333 S Q 1.7 0.254 7
967 PSR3 339 E S 1.6 0.359 7
968 PSR3 401 S 1 1.6 0.753 7
969 PSR3 338 S 1 1.6 0.756 7
970 PSR3 401 S N 1.6 0.38 7
971 PSR3 333 S T 1.5 0.833 7
972 PSR3 339 E A 1.5 0.837 7
973 PSR3 339 E C 1.5 0.485 7
974 PSR3 401 S F 1.5 0.846 7
975 PSR3 463 A G 1.5 0.761 7
976 PSR3 464 R Q 1.5 0.931 7
977 PSR3 396 A R 1.5 0.538 7
978 PSR3 339 E F 1.5 0.805 7
979 PSR3 338 S E 1.5 0.907 7
980 PSR3 339 E V 1.4 0.674 7 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
981 PSR3 465 K H 1.4 0.933 7
982 PSR3 465 K C 1.4 0.935 7
983 PSR3 339 E L 1.4 0.741 7
984 PSR3 401 S T 1.4 0.753 7
985 PSR3 401 S A 1.4 0.962 7
986 PSR3 459 K R 1.4 0.962 7
987 PSR3 333 S D 1.4 0.972 7
988 PSR3 339 E Q 1.3 0.842 7
989 PSR3 464 R T 1.3 0.975 7
990 PSR3 338 S M 1.3 0.857 7
991 PSR3 338 S R 1.3 0.89 7
992 PSR3 333 S 1 1.3 0.904 7
993 PSR3 465 K W 1.3 0.864 7
994 PSR3 338 S P 1.3 0.921 7
995 PSR3 333 S L 1.3 0.847 7
996 PSR3 459 K H 1.3 0.824 7
997 PSR3 338 S D 1.3 0.979 7
998 PSR3 465 K F 1.2 0.881 7
999 PSR3 402 K F 1.2 0.793 7
1000 PSR3 339 E D 1.2 0.795 7
1001 PSR3 337 A V 1.2 0.899 7
1002 PSR3 338 S N 1.2 0.883 7
1003 PSR3 465 K A 1.2 0.796 7
1004 PSR3 396 A Y 1.1 0.747 7
1005 PSR3 401 S D 1.1 0.701 7
1006 PSR3 333 S C 1.1 0.741 7
1007 PSR3 339 E W 1.1 0.681 7
1008 PSR3 333 S E 1.1 0.658 7
1009 PSR3 337 A G 1.1 0.656 7
1010 PSR3 459 K W 1.1 0.7 7
1011 PSR3 401 S M 1.1 0.694 7
1012 PSR3 401 S Q 1.1 0.635 7
1013 PSR3 465 K L 1.1 0.634 7
1014 PSR3 396 A Q 1.1 0.697 7
1015 PSR3 402 K A 1.1 0.629 7
1016 PSR3 401 S E 1.1 0.674 7
1017 PSR3 333 S R 1.1 0.649 7
1018 PSR3 339 E H 1.1 0.642 7
1019 PSR3 338 S L 1.1 0.636 7
1020 PSR3 396 A H 1 0.562 7
1021 PSR3 464 R F 1 0.587 7
1022 PSR3 402 K L 1 0.549 7
1023 PSR3 339 E R 1 0.549 7
1024 PSR3 465 K 1 1 0.575 7
1025 PSR3 464 R 1 1 0.553 7
1026 PSR3 402 K D 1 0.54 7
1027 PSR3 333 S F 0.9 0.509 7 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
1028 PSR3 465 K Q 0.9 0.634 7
1029 PSR3 465 K D 0.9 0.488 7
1030 PSR3 396 A T 0.9 0.409 7
1031 PSR3 465 K Y 0.9 0.479 7
1032 PSR3 464 R V 0.9 0.467 7
1033 PSR3 338 S Q 0.9 0.382 7
1034 PSR3 401 S L 0.9 0.434 7
1035 PSR3 459 K V 0.9 0.457 7
1036 PSR3 464 R L 0.9 0.447 7
1037 PSR3 465 K S 0.9 0.409 7
1038 PSR3 339 E Y 0.9 0.407 7
1039 PSR3 459 K Q 0.9 0.422 7
1040 PSR3 333 S M 0.8 0.395 7
1041 PSR3 463 A 1 0.8 0.411 7
1042 PSR3 401 S Y 0.8 0.318 7
1043 PSR3 396 A V 0.8 0.287 7
1044 PSR3 463 A C 0.8 0.382 7
1045 PSR3 339 E G 0.8 0.27 7
1046 PSR3 400 K D 0.8 0.27 7
1047 PSR3 400 K Y 0.8 0.267 7
1048 PSR3 338 S F 0.8 0.259 7
1049 PSR3 459 K C 0.8 0.361 7
1050 PSR3 396 A S 0.8 0.249 7
1051 PSR3 400 K H 0.8 0.23 7
1052 PSR3 400 K R 0.8 0.228 7
1053 PSR3 459 K T 0.7 0.328 7
1054 PSR3 402 K 1 0.7 0.308 7
1055 PSR3 464 R Y 0.7 0.314 7
1056 PSR3 464 R P 0.7 0.298 7
1057 PSR3 339 E K 0.7 0.188 7
1058 PSR3 402 K Q 0.7 0.273 7
1059 PSR3 333 S P 0.7 0.169 7
1060 PSR3 400 K N 0.7 0.167 7
1061 PSR3 333 S Y 0.7 0.256 7
1062 PSR3 400 K L 0.6 0.242 7
1063 PSR3 459 K S 0.6 0.24 7
1064 PSR3 333 S W 0.6 0.246 7
1065 PSR3 339 E T 0.6 0.237 7
1066 PSR3 400 K M 0.6 0.13 7
1067 PSR3 396 A F 0.6 0.235 7
1068 PSR3 401 S W 0.6 0.233 7
1069 PSR3 459 K G 0.6 0.233 7
1070 PSR3 402 K C 0.6 0.226 7
1071 PSR3 465 K E 0.6 0.099 7
1072 PSR3 400 K T 0.6 0.108 7
1073 PSR3 396 A P 0.6 0.208 7
1074 PSR3 338 S Y 0.6 0.206 7 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
1075 PSR3 459 K 1 0.6 0.203 7
1076 PSR3 459 K A 0.6 0.201 7
1077 PSR3 396 A E 0.6 0.198 7
1078 PSR3 459 K F 0.6 0.198 7
1079 PSR3 400 K S 0.5 0.195 7
1080 PSR3 338 S W 0.5 0.188 7
1081 PSR3 463 A V 0.5 0.189 7
1082 PSR3 459 K L 0.5 0.175 7
1083 PSR3 400 K A 0.5 0.064 7
1084 PSR3 400 K Q 0.5 0.056 7
1085 PSR3 402 K V 0.4 0.146 7
1086 PSR3 459 K N 0.4 0.14 7
1087 PSR3 400 K 1 0.4 0.047 7
1088 PSR3 400 K C 0.4 0.13 7
1089 PSR3 464 R W 0.4 0.129 7
1090 PSR3 402 K S 0.4 0.124 7
1091 PSR3 459 K M 0.4 0.117 7
1092 PSR3 396 A C 0.4 0.112 7
1093 PSR3 396 A D 0.4 0.032 7
1094 PSR3 402 K E 0.4 0.114 7
1095 PSR3 400 K V 0.3 0.085 7
1096 PSR3 459 K D 0.3 0.079 7
1097 PSR3 400 K E 0.2 0.02 7
1098 PSR3 463 A F 0.2 0.066 7
1099 PSR3 463 A L 0.2 0.065 7
1100 PSR3 337 A Q 0.2 0.01 7
1101 PSR3 463 A M 0.2 0.059 7
1102 PSR3 337 A F 0.2 0.009 7
1103 PSR3 464 R E 0.2 0.014 7
1104 PSR3 400 K G 0.2 0.059 7
1105 PSR3 338 S C 0.2 0.054 7
1106 PSR3 337 A S 0.2 0.008 7
1107 PSR3 337 A T 0.2 0.008 7
1108 PSR3 463 A T 0.2 0.057 7
1109 PSR3 463 A N 0.1 0.052 7
1110 PSR3 401 S C 0.1 0.008 7
1111 PSR3 400 K F 0.1 0.049 7
1112 PSR3 400 K W 0.1 0.049 7
1113 PSR3 337 A C 0.1 0.052 7
1114 PSR3 337 A W 0.1 0.047 7
1115 PSR3 396 A W 0.1 0.051 7
1116 PSR3 463 A D 0.1 0.046 7
1117 PSR3 337 A Y 0.1 0.006 7
1118 PSR3 463 A Q 0.1 0.043 7
1119 PSR3 463 A W 0.1 0.042 7
1120 PSR3 337 A P 0.1 0.005 7
1121 PSR3 337 A H 0.1 0.004 7 MUT BackPositRef SubstiP- EC50 DeviaExample
FAE ID NO: bone ion A.A. tution value (ppm) tion #
1122 PSR3 337 A K 0.1 0.043 7
1123 PSR3 337 A L 0.1 0.004 7
1124 PSR3 463 A H 0.1 0.038 7
1125 PSR3 337 A 1 0.1 0.004 7
1126 PSR3 337 A D 0.1 0.004 7
1127 PSR3 337 A N 0.1 0.004 7
1128 PSR3 463 A K 0.1 0.037 7
1129 PSR3 337 A M 0 0.003 7
1130 PSR3 400 K P 0 0.037 7
1131 PSR3 463 A R 0 0.032 7
1132 PSR3 463 A Y 0 0.031 7
1133 PSR3 337 A R 0 0.035 7
1134 PSR3 463 A P 0 0.031 7
1135 PSR3 337 A E 0 0.035 7
Table 9. The definitions of the column headings are as follows: "SEQ ID NO:", a unique identifier for each DNA or amino acid sequence; "Trivial Name", a trivial but unique name for each DNA or protein sequence; "Reference Protein", the SEQ I D NO corresponding to the reference protein used in the FAE assays for each variant; "FAE", the arithmetic Mean FAE Index as further defined in Example 3; "p-value" the calculated p value associated with the hypothesis that the variant polypeptide is significantly different than the reference protein used in that particular FAE assay, as defined further in Example 3; "EC50 Fold", the fold increase in potency as defined by ratio of reference EC50 to variant EC50 is calculated in each of multiple experiments and then the mean of these independently measured numbers is reported as "EC50 Fold"; "Deviation", Mean Deviation Score as defined in Example 4; "Example #", the example number corresponding to the creation of the variant. The reference protein against which the variant protein is compared is: (MUT IDs: 1-872 and 91 1-1 135) used SEQ ID NO: 6 as the reference protein; (MUT IDs: 873- 910) used SEQ ID NO: 8 as the reference protein.
Table 9
SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
51 SFR5-014 6.2 0 8
52 SFR5-001 3.8 0 8
53 SFR5-007 3.6 0 8
54 SFR5-010 3.3 0 8
55 SFR5-009 3.3 0 8
56 SFR5-003 3.2 0 8
57 SFR5-017 3.1 0 8 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
58 SF 5-006 2.4 0.015 8
59 SFR5-013 2.2 0.046 8
60 SFR5-016 2.1 0.057 8
61 SFR5-004 1.9 0.139 8
62 SFR5-005 1.9 0.146 8
63 SFR5-020 1.7 0.328 8
64 SFR5-002 1.5 0.487 8
65 SFR5-008 1.4 0.927 8
66 SFR5-015 1.3 0.753 8
67 SFR09-007 3.3 8
68 SFR09-005 2.4 8
69 SFR09-002 2.2 8
70 SFR09-004 2.1 8
71 SFR09-006 2 8
72 SFR09-003 2 8
73 SFR10-032 17.1 0 8
74 SFR10-042 12 0 8
75 SFR10-72 8.9 0.003 8
76 SFR10-056 8.4 0 8
77 SFR10-036 8.2 0 8
78 SFR10-039 7.2 0 8
79 SFR10-82 6.7 0 8
80 SFR10-045 6.6 0 8
81 SFR10-87 6.4 0 8
82 SFR10-060 6.3 0 8
83 SFR10-052 5.3 0 8
84 SFR10-059 4.9 0 8
85 SFR10-84 4.6 0.025 8
86 SFR10-031 4.4 0 8
87 SFR10-054 4.2 0 8
88 SFR10-064 3.9 0 8
89 SFR10-76 3.8 0 8
90 SFR10-008 3.7 0 8
91 SFR10-035 3.7 0 8
92 SFR10-015 3.5 0 8
93 SFR10-71 3.5 0 8
94 SFR10-74 3.4 0 8
95 SFR10-047 3.4 0 8
96 SFR10-043 3.4 0.006 8
97 SFR10-055 2.9 0 8
98 SFR10-065 2.8 0 8
99 SFR10-041 2.8 0 8
100 SFR10-83 2.8 0.006 8
101 SFR10-002 2.7 0.001 8
102 SFR10-046 2.5 0.002 8
103 SFR10-037 2.5 0.002 8
104 SFR10-048 2.5 0.002 8 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
105 SF 10-78 2.4 0 8
106 SFR10-050 2.4 0 8
107 SFR10-058 2.4 0.017 8
108 SFR10-020 2.4 0.009 8
109 SFR10-057 2.3 0.018 8
110 SFR10-040 2.3 0.008 8
111 SFR10-75 2.3 0 8
112 SFR10-73 2.3 0 8
113 SFR10-049 2.2 0.044 8
114 SFR10-003 2.2 0.022 8
115 SFR10-038 2.2 0.015 8
116 SFR10-024 2.2 0.022 8
117 SFR10-79 2.1 0.006 8
118 SFR10-81 2 0.001 8
119 SFR10-77 2 0.006 8
120 SFR10-88 1.9 0.205 8
121 SFR10-051 1.9 0.109 8
122 SFR10-016 1.9 0.12 8
123 SFR10-025 1.8 0.152 8
124 SFR10-017 1.6 0.364 8
125 SFR10-018 1.6 0.393 8
126 SFR10-004 1.6 0.39 8
127 SFR10-029 1.6 0.395 8
128 SFR10-053 1.6 0.434 8
129 SFR10-006 1.5 0.637 8
130 SFR10-80 1.4 0.113 8
131 SFR10-021 1.4 0.697 8
132 SFR10-009 1.4 0.726 8
133 SFR10-007 1.4 0.728 8
134 SFR10-030 1.3 0.965 8
135 SFR10-014 1.2 0.79 8
136 SFR10-044 1.2 0.8 8
137 SFR10-89 1.1 0.673 8
138 SFR10-013 1.1 0.596 8
139 SFRlO-011 1.1 0.588 8
140 SFRlO-010 1 0.457 8
141 SFR10-023 0.9 0.356 8
142 SFR10-022 0.9 0.288 8
143 SFR10-027 0.9 0.264 8
144 SFR10-005 0.8 0.233 8
145 SFR10-019 0.7 0.148 8
146 SFR10-028 0.7 0.093 8
147 SFR10-026 0.6 0.079 8
148 SFRll-001 27.4 0 8
149 SFR11-012 14.8 0 8
150 SFR11-005 12.8 0 8
151 SFR11-014 9.9 0 8 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
152 SF 11-015 8.2 0 8
153 SFRll-010 8.1 0 8
154 SFR11-013 5.5 0 8
155 SFR11-004 4.3 0 8
156 SFR11-009 4 0 8
157 SFR11-002 4 0 8
158 SFRll-011 3.9 0 8
159 SFR11-007 3.8 0.007 8
160 SFR11-008 3.1 0 8
161 SFR11-006 2.3 0.012 8
162 SFR12-028 7.6 8
163 SFR12-022 5.7 8
164 SFR12-004 5 8
165 SFR12-006 4.9 8
166 SFR12-015 4.6 8
167 SFR12-001 4.6 8
168 SFR12-014 4.6 8
169 SFR12-002 4.5 8
170 SFR12-005 4.4 8
171 SFR12-017 4.2 8
172 SFR12-018 3.9 8
173 SFR12-003 3.9 8
174 SFR12-032 3.5 8
175 SFR12-016 3.2 8
176 SFR12-029 3.2 8
177 SFR12-011 3.2 8
178 SFR12-007 3 8
179 SFR12-010 3 8
180 SFR12-012 2.8 8
181 SFR12-009 2.8 8
182 SFR12-031 2.3 8
183 SFR13-035 9.3 0 8
184 SFR13-018 6.1 0 8
185 SFR13-039 5.4 0 8
186 SFR13-008 5.3 0 8
187 SFR13-012 5.3 0 8
188 SFR13-036 5.2 0 8
189 SFR13-009 4.8 0 8
190 SFR13-025 4.8 0 8
191 SFR13-033 4.7 0 8
192 SFR13-038 4.5 0 8
193 SFR13-003 4.5 0 8
194 SFR13-021 4.3 0 8
195 SFR13-030 4.3 0 8
196 SFR13-017 4.2 0 8
197 SFR13-004 4.2 0 8
198 SFR13-006 4.1 0 8 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
199 SF 13-026 4.1 0 8
200 SFR13-016 4.1 0 8
201 SFR13-031 4.1 0 8
202 SFR13-034 3.7 0 8
203 SFR13-007 3.7 0 8
204 SFR13-029 3.7 0 8
205 SFR13-024 3.6 0 8
206 SFR13-020 3.4 0 8
207 SFR13-001 3 0 8
208 SFR13-005 3 0 8
209 SFR13-037 2.8 0 8
210 SFR13-032 2.8 0 8
211 SFR13-027 2.5 0 8
212 SFR13-028 2.4 0 8
213 SFR13-010 2.4 0 8
214 SFR13-011 2.2 0 8
215 SFR13-019 2.2 0 8
216 SFR13-023 2 0 8
217 SFR13-022 1.3 0.322 8
218 SFR14-004 3.3 8
219 SFR14-007 3.2 8
220 SFR14-008 3.2 8
221 SFR14-005 2.8 8
222 SFR14-001 2.8 8
223 SFR14-002 2 8
224 SFR14-003 1.3 8
225 SFR17-013 20.9 0.002 8
226 SFR17-019 20.1 0 8
227 SFR17-014 19.5 0 8
228 SFR17-011 19 0 8
229 SFR17-005 17.1 0 8
230 SFR17-018 16.5 0 8
231 SFR17-009 13.5 0 8
232 SFR17-006 13.1 0 8
233 SFR17-016 12.9 0 8
234 SFR17-012 11.7 0 8
235 SFR17-004 11.3 0 8
236 SFR17-017 10.5 0 8
237 SFR17-003 9.7 0 8
238 SFR17-001 8.7 0.01 8
239 SFR17-015 8.2 0 8
240 SFR17-002 7.6 0 8
241 SFR17-007 5.5 0 8
242 SFR17-008 4.7 0 8
243 P053168-D-01_S03721995 0.39 9
244 P053168-A-10_S03723031 0.34 9
245 P053168-D-06_S03722432 0.26 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
246 P053168-F-07_S03722451 0.25 9
247 P053168-F-10_S03723024 0.24 9
248 P053169-E-10_S03723571 0.23 9
249 P053168-B-03_S03722281 0.19 9
250 P053168-E-10_S03722994 0.19 9
251 P053168-G-09_S03723000 0.17 9
252 P053168-H-05_S03722286 0.16 9
253 P053168-F-04_S03722292 0.15 9
254 P053168-A-03_S03722326 0.14 9
255 P053168-C-04_S03722283 0.14 9
256 P053168-D-07_S03722516 0.14 9
257 P053168-A-12_S03722983 0.14 9
258 P053169-H-01_S03723182 0.13 9
259 P053168-H-11_S03723019 0.13 9
260 P053168-A-02_S03723761 0.12 9
261 P053168-C-10_S03722993 0.12 9
262 P053168-A-07_S03722449 0.12 9
263 P053168-B-08_S03722529 0.1 9
264 P053168-B-12_S03722998 0.09 9
265 P053169-E-12_S03723656 0.08 9
266 P053168-A-08_S03722523 0.07 9
267 P053168-B-04_S03722320 0.07 9
268 P053168-B-10_S03722972 0.07 9
269 P053169-G-11_S03723594 0.07 9
270 P053168-D-11_S03722996 0.06 9
271 P053169-G-01_S03723175 0.06 9
272 P053168-E-05_S03722315 0.06 9
273 P053168-F-08_S03722955 0.05 9
274 P053168-D-05_S03722300 0.04 9
275 P053169-H-02_S03723149 0.03 9
276 P053168-C-06_S03722354 0.02 9
277 P053169-D-06_S03723260 0.02 9
278 P053169-B-08_S03723469 0.02 9
279 P053169-G-08_S03723462 0.01 9
280 P053168-G-04_S03722314 0.01 9
281 P053168-H-08_S03722875 0.01 9
282 P053169-B-12_S03723595 0.01 9
283 P053169-D-01_S03723219 -0.01 9
284 P053168-G-05_S03722346 -0.01 9
285 P053168-A-09_S03722919 -0.01 9
286 P053168-A-04_S03722290 -0.01 9
287 P053169-F-11_S03723652 -0.02 9
288 P053169-H-12_S03723599 -0.03 9
289 P053168-F-01_S03723758 -0.03 9
290 P053169-H-04_S03723234 -0.04 9
291 P053168-A-06_S03722294 -0.05 9
292 P053169-G-04_S03723174 -0.05 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
293 P053169-A-11_S03723493 -0.06 9
294 P053169-B-09_S03723434 -0.06 9
295 P053169-G-12_S03723613 -0.06 9
296 P053168-B-02_S03723770 -0.06 9
297 P053168-G-02_S03722325 -0.06 9
298 P053168-H-06_S03722508 -0.07 9
299 P053168-C-09_S03723029 -0.08 9
300 P053169-E-11_S03723504 -0.08 9
301 P053168-F-06_S03722352 -0.08 9
302 P053168-D-09_S03722970 -0.08 9
303 P053169-D-08_S03723410 -0.08 9
304 P053169-F-03_S03723179 -0.08 9
305 P053169-G-10_S03723505 -0.09 9
306 P053168-D-12_S03723072 -0.09 9
307 P053169-H-09_S03723489 -0.09 9
308 P053169-A-02_S03723190 -0.09 9
309 P053169-B-02_S03723176 -0.1 9
310 P053169-B-05_S03723255 -0.1 9
311 P053169-H-06_S03723276 -0.11 9
312 P053169-A-03_S03723185 -0.11 9
313 P053168-D-10_S03723046 -0.11 9
314 P053168-E-11_S03722989 -0.12 9
315 P053169-F-10_S03723498 -0.12 9
316 P053169-G-05_S03723273 -0.12 9
317 P053168-H-09_S03722964 -0.12 9
318 P053168-E-07_S03722450 -0.12 9
319 P053168-G-01_S03723771 -0.12 9
320 P053169-F-09_S03723457 -0.13 9
321 P053169-B-01_S03723205 -0.13 9
322 P053168-F-09_S03723030 -0.14 9
323 P053169-C-10_S03723416 -0.14 9
324 P053169-A-09_S03723486 -0.14 9
325 P053168-C-11_S03723040 -0.15 9
326 P053169-D-09_S03723487 -0.15 9
327 P053169-A-06_S03723289 -0.16 9
328 P053169-C-04_S03723203 -0.17 9
329 P053169-C-12_S03723581 -0.17 9
330 P053169-C-03_S03723201 -0.17 9
331 P053169-C-01_S03723212 -0.17 9
332 P053169-F-05_S03723296 -0.18 9
333 P053169-B-04_S03723195 -0.18 9
334 P053169-C-09_S03723456 -0.18 9
335 P053169-E-05_S03723236 -0.18 9
336 P053169-D-02_S03723169 -0.19 9
337 P053169-D-11_S03723568 -0.19 9
338 P053169-E-04_S03723145 -0.19 9
339 P053169-F-01_S03723167 -0.19 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
340 P053169-B-07_S03723314 -0.19 9
341 P053168-G-12_S03723112 -0.21 9
342 P053168-H-03_S03722342 -0.21 9
343 P053169-E-01_S03723146 -0.22 9
344 P053169-B-03_S03723193 -0.22 9
345 P053168-C-02_S03723834 -0.22 9
346 P053169-G-06_S03723253 -0.22 9
347 P053169-C-02_S03723148 -0.23 9
348 P053169-B-06_S03723231 -0.23 9
349 P053169-G-09_S03723466 -0.24 9
350 P053169-H-11_S03723608 -0.24 9
351 P053168-H-02_S03722266 -0.24 9
352 P053169-H-05_S03723280 -0.25 9
353 P053169-E-02_S03723214 -0.27 9
354 P053168-H-12_S03723098 -0.27 9
355 P053169-B-10_S03723467 -0.27 9
356 P053168-E-12_S03723094 -0.28 9
357 P053168-F-11_S03722997 -0.29 9
358 P053169-H-07_S03723341 -0.29 9
359 P053169-F-06_S03723313 -0.3 9
360 P053168-E-09_S03722977 -0.3 9
361 P053168-G-11_S03723004 -0.31 9
362 P053169-E-09_S03723450 -0.31 9
363 P053168-A-11_S03722967 -0.31 9
364 P053169-D-05_S03723295 -0.33 9
365 P053169-F-04_S03723166 -0.34 9
366 P053169-F-08_S03723446 -0.4 9
367 P053169-A-04_S03723180 -0.4 9
368 P053169-E-08_S03723424 -0.43 9
369 P053168-G-08_S03722933 -0.45 9
370 P053169-C-05_S03723285 -0.46 9
371 P053168-B-01_S03722061 -0.47 9
372 P053168-E-02_S03722184 -0.54 9
373 P053168-G-10_S03722966 -0.55 9
374 P053168-D-08_S03722699 -0.55 9
375 P053168-G-06_S03722434 -0.55 9
376 P053169-D-07_S03723336 -0.55 9
377 P053168-C-01_S03722075 -0.55 9
378 P053168-C-07_S03722502 -0.57 9
379 P053169-C-07_S03723247 -0.63 9
380 P053168-E-08_S03722830 -0.75 9
381 P053168-B-09_S03722914 -0.79 9
382 P053168-F-12_S03723066 -0.96 9
383 P053168-D-02_S03722190 -0.97 9
384 P053169-G-07_S03723338 -1.19 9
385 P053169-F-12_S03723612 -1.33 9
386 P053169-H-08_S03723485 -1.33 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
387 P053168-G-07_S03722459 -1.36 9
388 P053169-E-03_S03723164 -1.41 9
389 P053364-A-09_P053168-B-071 P053169-H-01 0.55 9
390 P053364-G-01_P053168-D-03 1 P053169-D-12 0.51 9
391 P053364-H-01_P053168-B-071 P053169-E-12 0.5 9
392 P053364-A-01_P053168-B-071 P053169-D-12 0.46 9
393 P053364-H-08_P053168-B-061 P053169-H-03 0.42 9
394 P053364-G-05_P053168-D-03 1 P053169-H-03 0.41 9
395 P053364-A-08_P053168-B-071 P053169-A-05 0.41 9
396 P053364-C-01_P053168-H-01 1 P053169-D-12 0.39 9
397 P053364-F-01_P053168-E-031 P053169-D-12 0.37 9
398 P053364-C-05_P053168-H-01 1 P053169-H-03 0.36 9
399 P053364-B-ll_P053168-B-06 | Axmi205 0.36 9
400 P053364-A-05_P053168-B-071 P053169-H-03 0.34 9
401 P053364-F-10_P053168-B-05 | Axmi205 0.33 9
402 P053364-C-09_P053168-H-01 1 P053169-H-01 0.31 9
403 P053364-B-01_P053168-B-051 P053169-D-12 0.3 9
404 P053364-B-09_P053168-B-051 P053169-H-01 0.29 9
405 P053364-A-03_P053168-B-071 P053169-E-10 0.29 9
406 P053364-A-02_P053168-B-071 P053169-A-10 0.28 9
407 P053364-B-04_P053168-B-051 P053169-A-12 0.28 9
408 P053364-G-10_P053168-H-011 Axmi205 0.27 9
409 P053364-D-01_P053168-H-041 P053169-D-12 0.27 9
410 P053364-G-03_P053168-D-03 1 P053169-E-10 0.25 9
411 P053364-B-10_P053168-B-051 P053169-A-07 0.24 9
412 P053364-A-10_P053168-B-071 P053169-A-07 0.24 9
413 P053364-E-01_P053168-F-051 P053169-D-12 0.23 9
414 P053364-H-03_P053168-H-01 1 P053169-E-12 0.23 9
415 P053364-H-10_P053168-H-041 Axmi205 0.23 9
416 P053364-B-05_P053168-B-051 P053169-H-03 0.21 9
417 P053364-F-02_P053168-E-031 P053169-A-10 0.2 9
418 P053364-E-ll_P053168-D-03 | Axmi205 0.19 9
419 P053364-C-03_P053168-H-01 1 P053169-E-10 0.19 9
420 P053364-C-02_P053168-H-01 1 P053169-A-10 0.18 9
421 P053364-B-03_P053168-B-051 P053169-E-10 0.18 9
422 P053364-D-09_P053168-H-041 P053169-H-01 0.16 9
423 P053364-H-02_P053168-B-051 P053169-E-12 0.15 9
424 P053364-G-08_P053168-B-061 P053169-A-10 0.15 9
425 P053364-F-04_P053168-E-031 P053169-A-12 0.15 9
426 P053364-C-08_P053168-H-01 1 P053169-A-05 0.15 9
427 P053364-F-11_P053168-E-011 Axmi205 0.14 9
428 P053364-E-10_P053168-B-071 Axmi205 0.14 9
429 P053364-H-09_P053168-A-101 P053169-H-03 0.13 9
430 P053364-G-02_P053168-D-03 1 P053169-A-10 0.13 9
431 P053364-E-12_Axmi2051 P053169-A-05 0.13 9
432 P053364-D-03_P053168-H-041 P053169-E-10 0.12 9
433 P053364-F-07_P053168-B-061 P053169-D-12 0.12 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
434 P053364-B-02_P053168-B-051 P053169-A-10 0.1 9
435 P053364-F-08_P053168-B-061 P053169-E-10 0.1 9
436 P053364-A-04_P053168-B-071 P053169-A-12 0.1 9
437 P053364-E-02_P053168-F-051 P053169-A-10 0.1 9
438 P053364-D-ll_P053168-F-051 Axmi205 0.1 9
439 P053364-G-04_P053168-D-03 1 P053169-A-12 0.09 9
440 P053364-C-10_P053168-H-01 1 P053169-A-07 0.09 9
441 P053364-C-ll_P053168-E-031 Axmi205 0.09 9
442 P053364-G-12_Axmi2051 P053169-A-07 0.07 9
443 P053364-G-07_P053168-A-101 P053169-D-12 0.06 9
444 P053364-A-07_P053168-B-07 1 P053169-G-02 0.05 9
445 P053364-C-12_Axmi2051 P053169-H-03 0.03 9
446 P053364-H-ll_Axmi2051 P053169-A-10 0.02 9
447 P053364-D-02_P053168-H-041 P053169-A-10 0.01 9
448 P053364-B-07_P053168-B-051 P053169-G-02 0.01 9
449 P053364-E-05_P053168-F-051 P053169-H-03 0 9
450 P053364-F-09_P053168-B-061 P053169-A-12 -0.01 9
451 P053364-H-07_P053168-E-01 1 P053169-H-03 -0.01 9
452 P053364-B-08_P053168-B-051 P053169-A-05 -0.02 9
453 P053364-G-09_P053168-A-101 P053169-E-10 -0.02 9
454 P053364-C-04_P053168-H-01 1 P053169-A-12 -0.03 9
455 P053364-G-ll_Axmi205 1 P053169-D-12 -0.04 9
456 P053364-F-05_P053168-E-031 P053169-H-03 -0.05 9
457 P053364-D-12_Axmi2051 P053169-G-02 -0.05 9
458 P053364-H-05_P053168-A-101 P053169-A-12 -0.08 9
459 P053364-E-03_P053168-F-051 P053169-E-10 -0.09 9
460 P053364-C-07_P053168-H-01 1 P053169-G-02 -0.09 9
461 P053364-D-08_P053168-H-041 P053169-A-05 -0.1 9
462 P053364-H-04_P053168-H-041 P053169-E-12 -0.11 9
463 P053364-D-05_P053168-H-041 P053169-H-03 -0.12 9
464 P053364-D-10_P053168-H-041 P053169-A-07 -0.2 9
465 P053364-E-07_P053168-E-01 1 P053169-D-12 -0.22 9
466 P053364-D-07_P053168-H-041 P053169-G-02 -0.28 9
467 P053364-B-12_Axmi2051 P053169-A-12 -0.3 9
468 P053364-F-03_P053168-E-031 P053169-E-10 -0.32 9
469 P053364-D-04_P053168-H-041 P053169-A-12 -0.45 9
470 P053364-E-08_P053168-E-01 1 P053169-A-10 -0.47 9
471 P053364-E-09_P053168-E-01 1 P053169-A-12 -0.49 9
472 P052569-H-01_S03656474 0.8 9
473 P052569-H-12_S03657014 0.79 9
474 P052569-A-08_S03656726 0.76 9
475 P052569-G-12_S03657008 0.76 9
476 P052569-G-11_S03656954 0.59 9
477 P052569-F-06_S03656660 0.46 9
478 P052569-H-04_S03656592 0.46 9
479 P052569-E-12_S03656987 0.43 9
480 P052569-A-07_S03656670 0.42 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
481 P052569-H-11_S03656955 0.4 9
482 P052569-F-12_S03657007 0.38 9
483 P052569-C-09_S03656798 0.37 9
484 P052570-E-01_S03657050 0.36 9
485 P052569-E-09_S03656809 0.35 9
486 P052569-D-08_S03656755 0.34 9
487 P052570-H-01_S03657072 0.32 9
488 P052570-D-06_S03657721 0.32 9
489 P052569-D-10_S03656861 0.3 9
490 P052569-F-10_S03656870 0.29 9
491 P052569-E-11_S03656903 0.28 9
492 P052569-B-06_S03656630 0.24 9
493 P052570-C-09_S03657822 0.2 9
494 P052569-C-11_S03656884 0.2 9
495 P052569-C-12_S03656973 0.2 9
496 P052570-H-04_S03657568 0.19 9
497 P052569-A-11_S03656874 0.16 9
498 P052569-A-09_S03656786 0.12 9
499 P052569-A-12_S03656968 0.08 9
500 P052570-B-03_S03657111 0.08 9
501 P052570-A-03_S03657109 0.08 9
502 P052570-E-09_S03657840 0.05 9
503 P052569-C-03_S03656567 0 9
504 P052569-A-10_S03656822 -0.02 9
505 P052569-B-05_S03656597 -0.03 9
506 P052569-G-07_S03656711 -0.04 9
507 P052569-D-11_S03656887 -0.05 9
508 P052569-C-01_S03656390 -0.08 9
509 P052569-F-08_S03656761 -0.1 9
510 P052569-E-03_S03656569 -0.1 9
511 P052569-E-01_S03656453 -0.11 9
512 P052569-D-06_S03656641 -0.12 9
513 P052569-E-04_S03656588 -0.13 9
514 P052570-H-09_S03657859 -0.13 9
515 P052569-G-09_S03656819 -0.14 9
516 P052570-B-01_S03657015 -0.15 9
517 P052569-C-02_S03656518 -0.15 9
518 P052569-G-05_S03656616 -0.18 9
519 P052569-B-02_S03656510 -0.21 9
520 P052569-G-02_S03656544 -0.25 9
521 P052569-F-05_S03656612 -0.25 9
522 P052569-B-03_S03656560 -0.26 9
523 P052570-E-04_S03657538 -0.27 9
524 P052570-G-03_S03657389 -0.27 9
525 P052569-A-06_S03656628 -0.27 9
526 P052570-E-05_S03657649 -0.27 9
527 P052569-F-07_S03656686 -0.28 9 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
528 P052569-B-10_S03656825 -0.29 9
529 P052569-C-05_S03656602 -0.32 9
530 P052569-E-05_S03656609 -0.36 9
531 P052570-G-01_S03657062 -0.39 9
532 P052570-F-03_S03657348 -0.41 9
533 P052570-F-09_S03657844 -0.41 9
534 P052569-C-04_S03656585 -0.44 9
535 P052569-F-01_S03656460 -0.44 9
536 P052570-F-04_S03657542 -0.45 9
537 P052569-A-04_S03656581 -0.47 9
538 P052569-H-06_S03656667 -0.48 9
539 P052570-H-08_S03657813 -0.49 9
540 P052569-F-04_S03656589 -0.49 9
541 P052569-D-01_S03656438 -0.5 9
542 P052569-H-02_S03656556 -0.56 9
543 P052569-C-10_S03656837 -0.57 9
544 P052570-A-09_S03657814 -0.62 9
545 P052570-A-07_S03657735 -0.64 9
546 P052569-H-08_S03656779 -0.66 9
547 P052569-D-02_S03656527 -0.69 9
548 P052570-C-07_S03657741 -0.8 9
549 P052569-D-03_S03656568 -0.86 9
550 P052569-B-04_S03656583 -0.91 9
551 P052569-G-08_S03656766 -0.92 9
552 P052570-E-10_S03657894 -1.04 9
553 P052569-G-01_S03656472 -1.11 9
554 P052569-D-07_S03656678 -1.18 9
555 P052570-A-10_S03657861 -1.23 9
556 P052569-B-07_S03656671 -1.38 9
557 P052570-B-06_S03657713 -1.47 9
558 P052570-A-06_S03657712 -1.83 9
559 PS l-1-076 25.6 0 10
560 PSRl-1-074 22.7 0 10
561 PSR1-2-145 15.9 0 10
562 PSR1-2-082 11.6 0 10
563 PSR1-2-088 11 0 10
564 PSR1-2-094 10.1 0 10
565 PSR1-2-110 9.7 0 10
566 PSRl-1-073 8.5 0.003 10
567 PSR1-2-091 7.2 0 10
568 PSR1-2-149 7 0 10
569 PSR1-2-087 6.9 0 10
570 PSR1-2-158 6.9 0 10
571 PSR1-2-086 6.7 0 10
572 PSRl-1-053 6.5 0.011 10
573 PSR1-2-096 6.2 0 10
574 PSR1-2-135 6.1 0 10 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
575 PS l-1-014 5.9 0.018 10
576 PSR1-2-141 5.8 0 2.5 10
577 PSRl-1-006 5.6 0.017 10
578 PSR1-2-095 5.3 0 10
579 PSR1-2-097 5.2 0 10
580 PSRl-1-039 5 0.046 10
581 PSRl-1-049 4.8 0.07 10
582 PSR1-2-127 4.6 0 10
583 PSR1-2-109 4.5 0 10
584 PSR1-2-105 4.5 0 1.9 10
585 PSR1-2-150 4.4 0 10
586 PSRl-1-061 4.3 0.118 10
587 PSRl-1-028 4.1 0.164 10
588 PSR1-2-128 4 0 10
589 PSR1-2-151 4 0 10
590 PSRl-1-045 3.9 0.185 10
591 PSR1-2-113 3.9 0 1.8 10
592 PSRl-1-005 3.9 0.001 10
593 PSRl-1-036 3.8 0.229 10
594 PSRl-1-003 3.7 0.241 10
595 PSR1-2-098 3.7 0 2.1 10
596 PSR1-2-138 3.7 0 10
597 PSR1-2-107 3.7 0 10
598 PSR1-2-143 3.6 0 10
599 PSRl-1-001 3.5 0.282 10
600 PSR1-2-102 3.5 0 10
601 PSR1-2-093 3.5 0 10
602 PSRl-1-051 3.5 0.302 10
603 PSR1-2-101 3.5 0 10
604 PSRl-1-052 3.4 0.317 10
605 PSRl-1-033 3.4 0.324 10
606 PSRl-1-072 3.3 0.341 10
607 PSR1-2-081 3.3 0.001 10
608 PSRl-1-065 3.3 0.383 10
609 PSRl-1-012 3.2 0.371 10
610 PSR1-2-089 3.2 0 2.7 10
611 PSRl-1-063 3.2 0.399 10
612 PSRl-1-004 3.2 0.388 10
613 PSRl-1-048 3.1 0.415 10
614 PSRl-1-058 3.1 0.425 10
615 PSRl-1-057 3.1 0.446 10
616 PSRl-1-060 3.1 0.442 10
617 PSR1-2-142 3 0.001 10
618 PSR1-2-136 3 0 10
619 PSR1-2-083 2.8 0.001 10
620 PSRl-1-066 2.7 0.575 10
621 PSR1-2-111 2.7 0.002 10 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
622 PS 1-2-130 2.7 0.002 10
623 PSR1-2-112 2.7 0.022 10
624 PSRl-1-059 2.6 0.634 10
625 PS l-1-041 2.6 0.655 10
626 PSRl-1-030 2.6 0.656 10
627 PSRl-1-043 2.5 0.665 10
628 PSRl-1-011 2.5 0.677 10
629 PSRl-1-021 2.5 0.684 10
630 PSRl-1-077 2.5 0.699 10
631 PSR1-2-121 2.5 0.006 10
632 PSR1-2-114 2.4 0.01 10
633 PSR1-2-154 2.3 0.032 10
634 PSRl-1-068 2.3 0.785 10
635 PSR1-2-134 2.3 0.017 10
636 PSRl-1-019 2.3 0.787 10
637 PSR1-2-084 2.3 0.019 10
638 PSRl-1-018 2.2 0.833 10
639 PSRl-1-013 2.2 0.837 10
640 PSRl-1-015 2.2 0.837 10
641 PSRl-1-042 2.2 0.838 10
642 PSR1-2-090 2.2 0.035 10
643 PSRl-1-020 2.1 0.861 10
644 PSRl-1-056 2.1 0.865 10
645 PSR1-2-157 2.1 0.055 10
646 PSRl-1-055 2.1 0.902 10
647 PSRl-1-008 2.1 0.906 10
648 PSR1-2-147 2 0.075 10
649 PSR1-2-117 2 0.097 10
650 PSRl-1-034 2 0.911 10
651 PSR1-2-137 2 0.092 10
652 PSRl-1-009 2 0.922 10
653 PSRl-1-046 2 0.956 10
654 PSR1-2-099 1.9 0.153 10
655 PSRl-1-031 1.9 0.979 10
656 PSRl-1-067 1.9 0.996 10
657 PSR1-2-106 1.9 0.17 10
658 PSRl-1-075 1.9 0.99 10
659 PSR1-2-085 1.8 0.216 10
660 PSRl-1-029 1.7 0.933 10
661 PSR1-2-120 1.7 0.334 10
662 PSRl-1-002 1.7 0.899 10
663 PSRl-1-024 1.6 0.879 10
664 PSRl-1-079 1.6 0.878 10
665 PSR1-2-144 1.6 0.4 10
666 PSR1-2-122 1.6 0.454 10
667 PSR1-2-156 1.6 0.46 10
668 PSR1-2-104 1.6 0.447 10 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
669 PS l-1-026 1.6 0.854 10
670 PS l-1-071 1.5 0.832 10
671 PSRl-1-054 1.5 0.833 10
672 PSRl-1-064 1.5 0.83 10
673 PSR1-2-153 1.5 0.536 10
674 PSR1-2-146 1.5 0.59 10
675 PSRl-1-032 1.5 0.814 10
676 PSRl-1-050 1.5 0.81 10
677 PSR1-2-140 1.5 0.597 10
678 PSR1-2-125 1.5 0.59 10
679 PSR1-2-092 1.5 0.607 10
680 PSRl-1-044 1.5 0.798 10
681 PSRl-1-010 1.5 0.791 10
682 PSRl-1-070 1.4 0.766 10
683 PSR1-2-116 1.4 0.773 10
684 PSRl-1-035 1.4 0.745 10
685 PSRl-1-016 1.4 0.74 10
686 PSRl-1-022 1.3 0.729 10
687 PSR1-2-152 1.3 0.881 10
688 PSR1-2-129 1.2 0.962 10
689 PSR1-2-115 1.2 0.91 10
690 PSR1-2-100 1.2 0.845 10
691 PSRl-1-037 1.1 0.606 10
692 PSR1-2-123 1.1 0.649 10
693 PSRl-1-038 1 0.57 10
694 PSR1-2-118 1 0.544 10
695 PSRl-1-027 1 0.567 10
696 PSR1-2-124 1 0.541 10
697 PSRl-1-069 1 0.564 10
698 PSR1-2-148 1 0.523 10
699 PSR1-2-103 0.9 0.35 10
700 PSR1-2-126 0.9 0.336 10
701 PSRl-1-017 0.8 0.501 10
702 PSRl-1-023 0.8 0.499 10
703 PSR1-2-131 0.8 0.294 10
704 PSR1-2-155 0.8 0.256 10
705 PSR1-2-132 0.7 0.17 10
706 PSR1-2-139 0.6 0.144 10
707 PSR1-2-108 0.6 0.111 10
708 PSR1-2-133 0.6 0.11 10
709 PSR1-2-080 0.4 0.051 10
710 PSRl-1-062 0.3 0.315 10
711 PSRl-1-078 0.3 0.303 10
712 PSR1-2-119 0.3 0.017 10
713 PSRl-1-025 0.2 0.277 10
714 PSRl-1-040 0.2 0.263 10
715 PSRl-1-047 0.1 0.251 10 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
716 PS l-1-007 0.204 10
717 PSR7-141 10 11
718 PSR7-63 8.4 11
719 PSR7-89 8.1 11
720 PSR7-94 6.6 11
721 PSR7-106 6.1 11
722 PSR7-96 6.1 11
723 PSR7-100 5 11
724 PSR7-148 4.7 11
725 PSR7-98 4.7 11
726 PSR7-113 4.5 11
727 PSR7-121 4.5 11
728 PSR7-7 4.5 11
729 PSR7-86 4.2 11
730 PSR7-155 3.7 11
731 PSR7-116 3.6 11
732 PSR7-95 3.4 11
733 PSR7-90 3.3 11
734 PSR7-97 3.3 11
735 PSR7-64 2.8 11
736 PSR7-93 2.8 11
737 PSR7-101 2.7 11
738 PSR7-112 2.5 11
739 PSR7-133 2.4 11
740 PSR7-110 2.3 11
741 PSR7-111 2.1 11
742 PSR7-103 2.1 11
743 PSR7-32 2.1 11
744 PSR7-91 1.9 11
745 PSR7-124 1.9 11
746 PSR7-146 1.7 11
747 PSR7-136 1.6 11
748 PSR7-41 1.5 11
749 PSR7-159 1.5 11
750 PSR7-138 1.3 11
751 PSR7-156 1.1 11
752 PSR7-135 1 11
753 PSR7-122 1 11
754 PSR7-134 0.8 11
755 PSR7-92 0.8 11
756 PSR7-154 0.6 11
757 PSR7-115 0.5 11
758 PSR8-64 15 11
759 PSR8-11 7.9 11
760 PSR8-40 7.5 11
761 PSR8-58 6.6 11
762 PSR8-70 3.3 11 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
763 PS 8-69 3.1 11
764 PSR8-49 2.9 11
765 PSR8-55 2.4 11
766 PSR8-43 2.1 11
767 PSR8-28 2 11
768 PSR8-47 2 11
769 PSR8-31 1.6 11
770 PSR8-33 1.3 11
771 PSR8-37 1.2 11
772 PSR8-23 1.1 11
773 PSR8-4 1.1 11
774 PSR8-50 1 11
775 PSR8-39 0.9 11
776 PSR8-42 0.9 11
777 PSR8-9 0.9 11
778 PSR8-63 0.7 11
779 PSR8-8 0.6 11
780 PSR8-17 0.6 11
781 PSR8-34 0.5 11
782 PSR8-53 0.3 11
783 PSR8-44 0.3 11
784 SFR15-009 18.8 0 6.3 12
785 SFR15-019 8 0 6.8 12
786 SFR15-021 7.9 0 6.9 12
787 SFR15-033 7.9 0 4.8 12
788 SFR15-020 7.3 0 8.5 12
789 SFR15-027 7 0 12
790 SFR15-036 6.6 0 12
791 SFR15-007 6.6 0 12
792 SFR15-017 5.5 0 12
793 SFR15-015 5.5 0 12
794 SFR15-005 5.4 0 5.4 12
795 SFR15-001 5.3 0 4.7 12
796 SFR15-030 5.2 0 3.2 12
797 SFR15-025 5.1 0 12
798 SFR15-011 5 0 12
799 SFR15-012 4.9 0 6.6 12
800 SFR15-029 4.7 0 12
801 SFR15-016 4.7 0 12
802 SFR15-010 4.5 0 12
803 SFR15-003 4.5 0 12
804 SFR15-023 4.4 0 12
805 SFR15-006 4.3 0 12
806 SFR15-028 4 0 12
807 SFR15-026 4 0 12
808 SFR15-032 3.9 0 12
809 SFR15-013 3.7 0 12 SEQ ID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
810 SF 15-004 3.7 0 12
811 SFR15-018 3.5 0 12
812 SFR15-034 3.5 0 12
813 SFR15-008 3.5 0 12
814 SFR15-022 3.5 0 12
815 SFR15-035 3.4 0 12
816 SFR15-031 3.3 0 12
817 SFR15-002 3.1 0 12
818 SFR15-014 2.4 0 12
819 SFR15-024 2.1 0 12
820 SFR12-035 12.37
821 SFR12-037 9.61
822 SFR12-038 12.63
823 SFR12-040 9.05
824 SFR17-020 27.97
825 SFR17-021 5.86
826 SFR17-022 7.98
827 SFR18-001 7.54
828 SFR18-002 7.25
829 SFR18-003 5.94
830 SFR18-004 7.2
831 SFR18-005 8.63
832 SFR18-006 6.94
833 SFR18-007 11.53
834 SFR18-009 7.31
835 SFR19-001 21.43
836 SFR19-002 11.63
837 SFR19-003 10
838 SFR19-004 18.62
839 SFR19-005 9.79
840 SFR19-007 15.94
841 SFR19-008 11.06
842 SFR19-009 27.29
843 SFR19-010 12.85
844 SFR19-011 18.24
845 SFR19-012 10.92
846 SFR19-013 9.13
847 SFR19-014 14.13
848 SFR19-015 11.32
849 SFR19-016 20.96
850 SFR19-017 12.41
851 SFR19-018 6.18
852 SFR19-019 8.49
853 SFR19-020 11.78
854 SFR19-021 23.45
855 SFR19-022 13.51
856 SFR19-023 9.31 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
857 SF 19-024 12.15
858 SFR19-025 15.25
859 SFR19-026 10.59
860 SFR19-028 6.97
861 SFR19-031 6.39
862 SFR19-032 6.54
863 SFR19-034 6.71
864 SFR19-035 20.38
865 SFR19-037 7.3
866 SFR19-038 9.24
867 SFR19-039 6.83
868 SFR19-041 14.94
869 SFR19-044 13.24
870 SFR19-045 9.01
871 SFR19-046 7.13
872 SFR19-047 16.25
873 SFR19-048 19.11
874 SFR19-049 16.73
875 SFR19-051 8.65
876 SFR19-052 9.84
877 SFR19-053 9.75
878 SFR19-054 10.39
879 SFR19-056 10.21
880 SFR19-057 15.88
881 SFR19-063 10.27
882 SFR19-065 8.49
883 SFR19-071 9.78
884 SFR19-072 9.81
885 SFR19-075 17.4
886 SFR19-077 37.92
887 SFR19-079 37.38
888 SFR19-080 18.2
889 SFR20-001 7.45
890 SFR20-007 6.03
891 SFR20-010 8.12
892 SFR20-011 7.15
893 SFR20-015 6.65
894 SFR20-016 6.07
895 SFR20-018 7.13
896 SFR20-019 9.3
897 SFR20-020 16.62
898 SFR20-021 7.98
899 SFR20-022 6.66
900 SFR20-024 7.44
901 SFR20-026 7.96
902 SFR20-028 11.67
903 SFR20-029 7.72 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
904 SF 20-030 6.57
905 SFR20-031 5.97
906 SFR20-033 6.64
907 SFR20-036 7.39
908 SFR20-044 8.65
909 SFR20-045 14.21
910 SFR20-046 15.77
911 SFR20-047 17.88
912 SFR20-048 6.82
913 SFR20-049 13.4
914 SFR20-051 6.06
915 SFR20-053 11.18
916 SFR20-054 12.84
917 SFR20-056 10.69
918 SFR20-057 7.2
919 SFR20-064 9.57
920 SFR20-066 5.93
921 SFR20-074 27.75
922 SFR20-075 14.4
923 SFR20-076 12.11
924 SFR21-001 18.77
925 SFR21-004 9.04
926 SFR21-007 10.77
927 SFR21-008 16.87
928 SFR21-010 11.23
929 SFR21-011 12.73
930 SFR21-012 13.63
931 SFR21-013 8.61
932 SFR21-014 9.57
933 SFR21-015 9.74
934 SFR21-018 9.47
935 SFR21-024 13.82
936 SFR22-031 21.7 21.71
937 SFR22-032 17.97 17.98
938 SFR23-001 31.56 31.58
939 SFR23-002 20.38 20.39
940 SFR23-003 14.13 14.14
941 SFR23-004 20.27 20.28
942 SFR23-005 12.83 12.83
943 SFR23-006 13.49 13.49
944 SFR23-007 24.97 24.99
945 SFR23-008 12.02 12.03
946 SFR23-009 11.6 11.61
947 SFR23-010 22.49 22.5
948 SFR23-012 27.88 27.89
949 SFR23-013 8.05 8.06
950 SFR24-006 22.58 22.59 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
951 SFR24-007 9.44 9.44
952 SFR24-008 20.56 20.57
953 SFR24-015 5.87 5.87
954 SFR24-016 18.63 18.64
955 SFR24-017 6.73 6.74
956 PSR10_SB-003 34.27 6.12
957 PSR10_SA-009 31.62 6.17
958 PSR10_SB-041 29.57 7.42
959 PSR10_SA-077 27.77 6.79
960 PSR10_SA-080 26.44 12.65
961 PSR10_SA-021 25.6 10.8
962 PSR10_SA-061 24.99 11.01
963 PSR10_SB-001 23.63 9.38
964 PSR10_SA-002 23.63
965 PSR10_SB-011 22.63
966 PSR10_SA-067 22.46
967 PSR10_SA-041 22.01
968 PSR10_SA-065 22 8.01
969 PSR10_SB-044 21.57
970 PSR10_SA-089 21.47
971 PSR10_SB-033 21.1
972 PSR10_SA-085 20.89
973 PSR10_SB-015 20.68
974 PSR10_SB-121 19.58
975 PSR10_SA-001 19.11
976 PSR10_SA-040 19.1
977 PSR10_SB-028 19.09
978 PSR10_SB-045 18.89 6.99
979 PSR10_SB-037 18.68
980 PSR10_SB-013 18.68
981 PSR10_SA-070 18.58
982 PSR10_SB-022 18.32
983 PSR10_SA-020 18.1
984 PSR10_SA-100 18.07
985 PSR10_SB-052 17.8
986 PSR10_SB-118 17.75
987 PSR10_SA-019 17.41
988 PSR10_SB-089 17.36
989 PSR10_SB-043 17.29
990 PSR10_SB-110 17.07
991 PSR10_SB-039 16.79
992 PSR10_SA-101 16.66
993 PSR10_SA-062 16.53
994 PSR10_SA-010 16.52
995 PSR10_SA-022 16.46
996 PSR10_SA-047 16.28
997 PSR10_SB-070 16 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
998 PSR10_SA-017 15.89
999 PSR10_SB-020 15.72
1000 PSR10_SA-003 15.25
1001 PSR10_SB-064 15.25
1002 PSR10_SB-069 15.12
1003 PSR10_SA-081 15.02
1004 PSR10_SB-106 14.91
1005 PSR10_SB-012 14.88
1006 PSR10_SB-073 14.87
1007 PSR10_SA-006 14.86
1008 PSR10_SB-068 14.82
1009 PSR10_SB-063 14.65
1010 PSR10_SB-055 14.62
1011 PSR10_SA-050 14.62
1012 PSR10_SB-024 14.59
1013 PSR10_SB-014 14.58
1014 PSR10_SB-081 14.57
1015 PSR10_SA-069 14.49
1016 PSR10_SA-084 14.44
1017 PSR10_SB-108 14.42
1018 PSR10_SB-062 14.31
1019 PSR10_SB-077 14.31
1020 PSR10_SB-102 14.31
1021 PSR10_SB-046 14.28
1022 PSR10_SB-059 14.28
1023 PSR10_SB-016 14.21
1024 PSR10_SB-101 14.16
1025 PSR10_SA-005 14.07 7.58
1026 PSR10_SB-067 14.06
1027 PSR10_SB-002 14.06
1028 PSR10_SA-024 14.02
1029 PSR10_SB-082 13.96
1030 PSR10_SB-095 13.93
1031 PSR10_SB-065 13.82
1032 PSR10_SB-104 13.63
1033 PSR10_SB-117 13.57
1034 PSR10_SB-057 13.47
1035 PSR10_SA-090 13.3
1036 PSR10_SB-019 13.23
1037 PSR10_SB-038 13.21
1038 PSR10_SA-042 13.09
1039 PSR10_SB-010 12.93
1040 PSR10_SB-103 12.85
1041 PSR10_SA-096 12.77
1042 PSR10_SA-068 12.74
1043 PSR10_SB-026 12.68
1044 PSR10_SB-097 12.68 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
1045 PSR10_SB-009 12.59
1046 PS 10_SA-004 12.43
1047 PSR10_SA-064 12.33
1048 PSR10_SB-025 12.3
1049 PSR10_SB-088 12.19
1050 PSR10_SB-090 12.18
1051 PSR10_SB-036 12.15
1052 PSR10_SA-045 12.14
1053 PSR10_SB-049 12.14
1054 PSR10_SB-021 12.13
1055 PSR10_SB-030 12.07
1056 PSR10_SA-029 12.04
1057 PSR10_SB-066 11.97
1058 PSR10_SA-025 11.84
1059 PSR10_SB-119 11.81
1060 PSR10_SB-076 11.79
1061 PSR10_SA-058 11.68
1062 PSR10_SB-120 11.63
1063 PSR10_SB-004 11.61
1064 PSR10_SB-114 11.57
1065 PSR10_SA-082 11.57
1066 PSR10_SB-053 11.55
1067 PSR10_SB-051 11.46
1068 PSR10_SB-098 11.35
1069 PSR10_SA-118 11.3
1070 PSR10_SA-097 11.26
1071 PSR10_SB-099 11.21
1072 PSR10_SA-099 11.17
1073 PSR10_SB-083 11.16
1074 PSR10_SA-060 11.16
1075 PSR10_SA-095 11.13
1076 PSR10_SB-112 11.09
1077 PSR10_SA-102 11.08
1078 PSR10_SB-007 11.02
1079 PSR10_SB-113 10.99
1080 PSR10_SB-084 10.98
1081 PSR10_SB-115 10.98
1082 PSR10_SA-007 10.95
1083 PSR10_SA-087 10.94
1084 PSR10_SB-111 10.93
1085 PSR10_SA-030 10.84
1086 PSR10_SB-006 10.82
1087 PSR10_SB-105 10.8
1088 PSR10_SB-075 10.74
1089 PSR10_SA-086 10.65
1090 PSR10_SB-040 10.59
1091 PSR10_SB-060 10.5 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
1092 PS 10_SA-098 10.45
1093 PSR10_SA-011 10.42
1094 PSR10_SA-023 10.41
1095 PSR10_SB-017 10.41 6.94
1096 PSR10_SB-116 10.36
1097 PSR10_SB-029 10.35
1098 PSR10_SA-038 10.3
1099 PSR10_SA-033 10.25
1100 PSR10_SA-093 10.18
1101 PSR10_SA-057 10.16
1102 PSR10_SB-092 10.09
1103 PSR10_SB-078 10.05
1104 PSR10_SB-023 10.02
1105 PSR10_SB-027 9.95
1106 PSR10_SA-037 9.93
1107 PSR10_SB-042 9.92
1108 PSR10_SB-080 9.9
1109 PSR10_SB-031 9.88
1110 PSR10_SB-107 9.87
1111 PSR10_SB-050 9.84
1112 PSR10_SB-005 9.81
1113 PSR10_SA-107 9.79
1114 PSR10_SB-074 9.74
1115 PSR10_SA-018 9.58
1116 PSR10_SB-054 9.53
1117 PSR10_SB-035 9.48
1118 PSR10_SA-106 9.21
1119 PSR10_SA-120 9.13
1120 PSR10_SB-091 8.84
1121 PSR10_SB-058 8.76
1122 PSR10_SA-026 8.7
1123 PSR10_SA-052 8.69
1124 PSR10_SA-078 8.66
1125 PSR10_SB-061 8.63
1126 PSR10_SA-110 8.59
1127 PSR10_SB-048 8.54
1128 PSR10_SB-032 8.49
1129 PSR10_SB-008 8.45
1130 PSR11-2-016 32.82
1131 PSR11-2-077 30.06
1132 PSR11-2-061 29.88
1133 PSR11-2-072 29.67
1134 PSR11-2-038 29.12
1135 PSR11-2-008 27.23
1136 PSR11-2-059 27.22
1137 PSR11-2-063 25.89
1138 PSR11-2-009 25 SEQID P-
Trivial Name FAE EC50 Fold Deviation Example # NO: value
1139 PSR11-2-065 21.97
1140 PSR11-2-022 20.39
1141 PSR11_188 18.95
1142 PSR11-2-070 18.79
1143 PSR11-2-024 18.6
1144 PSR11-2-051 17.84
1145 PSR11_004 16.81
1146 PSR11_065 16.55
1147 PSR11_071 15.61
1148 PSR11-2-068 15.48
1149 PSR11_120 14.6
1150 PSR11_002 14.22
1151 PSRll-2-020 14.15
1152 PSR11-2-053 13.8
1153 PSR11-2-067 13.16
1154 PSR11-2-021 13.1
1155 PSR11-2-047 11.41
1156 PSR11_069 11.3
1157 PSR11-2-033 10.94
1158 PSR11_010 10.22
1159 PSR11-2-044 10.2
1160 PSR11-2-004 9.91
1161 PSR11-2-025 8.62
1162 PSR11-2-054 8.45
1518 SFR12-020 5.66
1519 SFR12-033 9.74
1520 SFR12-034 8.87
1521 SFR12-036 9.11
1522 SFR18-008 5.36
1523 SFR18-010 5.67
1524 SFR18-011 5.81
1525 SFR21-016 6.61
1526 SFR21-017 7.89

Claims

THAT WHICH IS CLAIMED:
1. A PHI-4 polypeptide having improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35).
2. The PHI-4 polypeptide of claim 1 , wherein the PHI-4 polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 40, 42, 43, 46, 52, 97, 98, 99, 145, 150, 151 , 153, 163, 171 , 172, 182, 196, 206, 210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342, 346, 354, 355, 370, 389, 393, 396, 401 , 402, 403, 410, 412, 416, 417, 426, 442, 447, 452, 454, 455, 457, 461 , 462, 500, 509, 520 or 527 of SEQ ID NO: 35.
3. The PHI-4 polypeptide of claim 2, wherein the amino acid at position 40 is Leu or lie; the amino acid at position 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu; the amino acid at position 46 is Glu or Asn; the amino acid at position 52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acid at position 99 is Lys or Leu; the amino acid at position 145 is Leu or Val; the amino acid at position 150 is Arg or Gin; the amino acid at position 151 is Asp or Ser; the amino acid at position 153 is Leu or lie; the amino acid at position 163 is Leu or Val; the amino acid at position 171 is Tyr or Phe; the amino acid at position 172 is lie or Leu; the amino acid at position 182 is Asp or Gin; the amino acid at position 196 is Gin or Asn; the amino acid at position 206 is Tyr or Phe; the amino acid at position 210 is Val or lie; the amino acid at position 216 is Glu or Gin; the amino acid at position 220 is Glu, Gin, His or Asp; the amino acid at position 278 is Glu or Asn; the amino acid at position 283 is lie or Val; the amino acid at position 289 is Lys, Gin or Leu; the amino acid at position 293 is Arg, Gin or Glu; the amino acid at position 328 is Lys or Glu; the amino acid at position 333 is Ser, Lys or Val; the amino acid at position 334 is Gly, Lys or Arg; the amino acid at position 336 is Gly or Ala; the amino acid at position 338 is Ser or Val; the amino acid at position 339 is Glu, Asn or Gin; the amino acid at position 342 is Ala or Ser; the amino acid at position 346 is Pro or Ala; the amino acid at position 354 is Met or Leu; the amino acid at position 355 is Val or lie; the amino acid at position 370 is His or Arg; the amino acid at position 389 is Trp or Leu; the amino acid at position 393 is Trp or Leu; the amino acid at position 396 is Ala, Leu, Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly, Lys or Pro; the amino acid at position 402 is Lys, His, Gly or Trp; the amino acid at position 403 is Asp or Tyr; the amino acid at position 410 is lie or Val; the amino acid at position 412 is Pro or Ala; the amino acid at position 416 is Arg or Glu; the amino acid at position 417 is Ala or Ser; the amino acid at position 426 is Thr or Ser; the amino acid at position 442 is Gin or Glu; the amino acid at position 447 is Asp or Lys; the amino acid at position 452 is Gin or Lys; the amino acid at position 454 is Arg or Gin; the amino acid at position 455 is Val or lie; the amino acid at position 457 is Trp or Asn; the amino acid at position 461 is Thr or Ser; the amino acid at position 462 is Gly or Ala; the amino acid at position 500 is Arg or Gin; the amino acid at position 509 is Lys or Gin; the amino acid at position 520 is Lys, Glu or Gin; and the amino acid at position 527 is Gin or Lys.
4. The PHI-4 polypeptide of claim 1 , wherein the PHI-4 polypeptide comprises an amino acid sequence of the formula,
5 10 15
Met Xaa Ser Ala Ala Asn Ala Gly Xaa Leu Gly Asn Leu Xaa Gly
20 25 30
Xaa Thr Ser Xaa Gly Met Xaa Tyr Xaa Val Asn Gly Leu Tyr Ala
35 40 45
Ser Pro Glu Ser Leu Xaa Gly Gin Pro Leu Phe Xaa Xaa Gly Gly
50 55 60 Xaa Leu Asp Ser Xaa Xaa lie Glu Gly Xaa Xaa Xaa Xaa Phe Pro
65 70 75
Xaa Ser Met His Val His Thr Tyr Phe His Ser Asp Xaa Xaa Gin
80 85 90
Xaa Val Ser Xaa Xaa lie Xaa Xaa Xaa Arg Xaa Xaa Xaa Ser Xaa
95 100 105
His Val Gly Xaa Ser Gly Xaa Xaa Xaa Leu Phe Ser Xaa Ser Xaa
110 115 120
Ser Val Asp Xaa Thr Thr Xaa Xaa Gin Gin Leu Xaa Glu lie Thr
125 130 135 Xaa Ser Ser Thr Arg Glu Xaa His Val Leu Trp Tyr lie Ser Leu
140 145 150
Pro Gly Ala Ala Thr Leu Xaa Ser Met Leu Xaa Xaa Xaa Xaa Xaa
155 160 165
Xaa Asp Xaa Xaa Xaa Pro Asn Met Xaa Ala Met Xaa Leu Phe Xaa
170 175 180
Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa Ala Ala Val Gly Gly Arg
185 190 195
Leu Xaa Xaa Xaa Xaa Ala Ser Lys Xaa Leu Xaa Met Xaa Ser Ser
200 205 210 Xaa Ser Leu Ser Thr Thr Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Xaa 215 220 225
Gly Glu He Xaa He Xaa His Gly Ser Xaa Met Glu Lys Gin Val
230 235 240 Asn Ser Phe Xaa Xaa Xaa Ser Thr He Arg Xaa Thr Ala Thr Gly
245 250 255
Gly Lys Pro Gly Xaa Thr Xaa Arg He Leu His Gly Pro Asp Ser
260 265 270
Xaa Xaa Ala Phe Ser Xaa Trp Ala Xaa Ser Leu Leu Xaa Tyr Ala
275 280 285
Thr Leu Met Asp Phe Xaa Thr Xaa Ser Leu Xaa Xaa He Xaa Ala
290 295 300
Leu Xaa Asp Xaa Pro Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Ala Xaa
305 310 315 Pro Xaa Xaa Met Xaa Xaa Ser Gin Xaa Ser He Pro Xaa Val Asp
320 325 330
Xaa Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly
335 340 345
Xaa Asp Xaa XcL cL XcL cL XcL cL XcL cL Xaa Xaa Asp Xaa XcL cL XcL cL XcL cL XcL cL
350 355 360
Xaa Ser Thr Ser Xaa Xaa Tyr Lys Xaa Xaa Gly Gin Phe Xaa Gin
365 370 375
Arg Xaa His Xaa Ser Val Ala Asp Gly His Xaa Pro He Xaa Xaa
380 385 390 Asp Leu Phe Asp Xaa Gly Xaa Xaa Xaa Xaa Pro Val Gly Xaa Gin
395 400 405
Xaa Val Trp Asp Xaa Xaa Xaa Xaa Gly Lys Xaa Xaa Xaa Tyr Xaa
410 415 420
Cys Trp Arg XcL cL XcL cL XcL cL XcL cL Gin Gly Tyr Xaa Xaa Xaa Gly Asp
425 430 435
Val Xaa Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro
440 445 450
XcL cL XcL cL XcL cL Cys Xaa His Xaa Ser Leu Xaa Ala Xaa Xaa Xaa Thr
455 460 465 Leu Xaa Xaa Xaa Xaa Trp Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa
470 475 480
Xaa Val Ser Leu Trp Xaa Xaa Gly Ala Ala Gly Ala Val Ala Ser
485 490 495
Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser
500 505 510
Gly Xaa lie Xaa Xaa Leu Xaa Gly Ser lie Ala Cys Val Xaa Thr
515 520 525
Ser Ala lie Ala Ser Met Xaa Xaa Met Xaa Ser Met Leu Ser Xaa
530 535
His Xaa Gly Met Glu [ Ala Met Met Ser Lys Leu
wherein
Xaa at position 2 is . Ala or Arg;
Xaa at position 9 is Gin, Lys or Glu;
Xaa at position 14 is Pro or Ala;
Xaa at position 16 is Val or Asp;
Xaa at position 19 is Met or Leu;
Xaa at position 22 is Gly or Ser;
Xaa at position 24 is Asp, Asn or Gin;
Xaa at position 36 is Leu or Met;
Xaa at position 42 is Asp, Asn or Gin;
Xaa at position 43 is Phe or Glu;
Xaa at position 46 is Glu, Asp, Asn or Gly;
Xaa at position 50 is lie or Val;
Xaa at position 51 is Glu or Gin;
Xaa at position 55 is Arg or Lys;
Xaa at position 56 is Ser or Thr;
Xaa at position 57 is Tyr or Phe;
Xaa at position 58 is Thr or Ser;
Xaa at position 61 is Arg, Lys or Glu;
Xaa at position 73 is Phe or Tyr;
Xaa at position 74 is Lys, Glu, Gly, Arg,
Asp;
Xaa at position 76 is Asp or Gin; Xaa at position 79 is Lys or Glu;
Xaa at position 80 is Glu or Ser;
Xaa at position 82 is Glu, He, Leu, Tyr or Gin;
Xaa at position 83 is Glu or Gin;
Xaa at position 84 is Tyr or Phe;
Xaa at position 86 is Glu or Gin;
Xaa at position 87 is Lys or Gin;
Xaa at position 88 is Met, He or Leu;
Xaa at position 90 is Gin or Glu;
Xaa at position 94 is Val or lie;
Xaa at position 97 is Arg, Asn, Asp, Glu, Gin,
He, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys,
Val or Leu;
Xaa at position 98 is Tyr or Phe;
Xaa at position 99 is Lys, Leu, Tyr, He, Met, Phe,
or Asn;
Xaa at position 103 is Ala or Gly;
Xaa at position 105 is Leu or lie;
Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn, Glu, Cys, Ala or Arg;
Xaa at position 112 is Thr or Ser;
Xaa at position 113 is Asp, Glu or Met;
Xaa at position 117 is Thr or Ser;
Xaa at position 121 is Tyr or Phe;
Xaa at position 127 is Ala or Thr;
Xaa at position 142 is Arg or Glu;
Xaa at position 146 is Arg or Gin;
Xaa at position 147 is Arg, Glu or Gin;
Xaa at position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly;
Xaa at position 149 is Phe or Val;
Xaa at position 150 is Arg, Gin, Glu or Asn;
Xaa at position 151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaa at position 153 is Leu or lie;
Xaa at position 154 is Asn or Asp;
Xaa at position 155 is Asn or Lys;
Xaa at position 159 is Pro or Asp;
Xaa at position 162 is Glu, Asp, Gin, Asn or Leu;
Xaa at position 165 is Lys, Glu, Gin, Pro, Thr, Ala, Leu,
Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg;
Xaa at position 166 is Arg or Gin;
Xaa at position 167 is Tyr, Trp or Cys;
Xaa at position 170 is Tyr or His;
Xaa at position 171 is Tyr or Phe;
Xaa at position 172 is He, Leu or Val;
Xaa at position 173 is Ser or Ala;
Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser;
Xaa at position 182 is Asp or Gin;
Xaa at position 183 is Tyr or Val;
Xaa at position 184 is Ser or Thr;
Xaa at position 185 is Ala or Ser;
Xaa at position 189 is Thr, Lys or lie;
Xaa at position 191 is Lys or Gin;
Xaa at position 193 is Asp or Asn;
Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala,
Arg;
Xaa at position 202 is Ala or Val;
Xaa at position 203 is Glu, Thr or His;
Xaa at position 204 is Met or Ala;
Xaa at position 206 is Tyr or Phe;
Xaa at position 207 is Lys or Gin;
Xaa at position 209 is Leu or Pro;
Xaa at position 210 is Val or lie;
Xaa at position 214 is Lys, Ser or Gin;
Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg;
Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met, Ala, Phe, He, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu;
Xaa at position 230 is Ser or Glu;
Xaa at position 231 is Asn or Ser;
Xaa at position 236 is Leu or Pro;
Xaa at position 245 is Met or Leu;
Xaa at position 247 is Asp or Tyr;
Xaa at position 256 is Gin, Lys or Glu;
Xaa at position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or
Met;
Xaa at position 261 is Gin, Glu, Lys or Ala;
Xaa at position 264 is Glu or Gin;
Xaa at position 268 is Asp or Asn;
Xaa at position 276 is Ser or Ala;
Xaa at position 278 is Glu, Asn or Gin;
Xaa at position 281 is Gin, Lys or Glu;
Xaa at position 282 is Pro or Gly;
Xaa at position 284 is Trp or Arg;
Xaa at position 287 is Ala or Cys;
Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr,
Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala;
Xaa at position 291 is Glu or Gin;
Xaa at position 292 is Arg or Gin;
Xaa at position 293 is Arg, Glu or Gin;
Xaa at position 294 is Val or Ala;
Xaa at position 296 is Leu or lie;
Xaa at position 297 is Glu or Gin;
Xaa at position 298 is Asp or Gin;
Xaa at position 300 is Phe or Tyr;
Xaa at position 302 is Glu or Gin;
Xaa at position 303 is Phe or Tyr;
Xaa at position 305 is Lys, Gin, Ala, lie, Met, Asn, Thr or
Val;
Xaa at position 306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu;
Xaa at position 313 is Lys, Gin or Arg;
Xaa at position 316 is Lys or Gin;
Xaa at position 328 is Lys, Glu or Gin;
Xaa at position 331 is Glu, Asn or Gin;
Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala,
His, Gin, Thr, Asp, lie, Leu, Cys or Glu;
Xaa at position 334 is Gly, Arg, Lys, lie or Trp;
Xaa at position 335 is Ser or Ala;
Xaa at position 336 is Gly or Ala;
Xaa at position 337 is Ala, Val or Gly;
Xaa at position 338 is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asn or Leu;
Xaa at position 339 is Glu, Asn, Gin, He,
Ala, Cys, Phe, Val, Leu, Asp, rp, His or Arg;
Xaa at position 341 is Leu or Val;
Xaa at position 342 is Ala, Ser or Val;
Xaa at position 343 is Val or He;
Xaa at position 344 is Phe or Trp;
Xaa at position 345 is Asn or His;
Xaa at position 346 is Pro or Ala;
Xaa at position 350 is Asn or Ser;
Xaa at position 351 is Gly or Val;
Xaa at position 354 is Met or Leu;
Xaa at position 355 is Val, lie or Leu;
Xaa at position 359 is Gly or Ala;
Xaa at position 362 is Asn or Ser;
Xaa at position 364 is Ala or Sen¬
Xaa at position 371 is Ala, Gly or Thr;
Xaa at position 374 is Phe or ile;
Xaa at position 375 is Lys or Arg;
Xaa at position 380 is Leu or Gly;
Xaa at position 382 is Val, Asp or Leu;
Xaa at position 383 is Leu, lie or Val; Xaa at position 384 is Lys, Ala or Gly;
Xaa at position 385 is Ala or Gly;
Xaa at position 389 is Trp or Tyr;
Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His;
Xaa at position 395 is Asp or Cys;
Xaa at position 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin, His or Thr;
Xaa at position 397 is Gly, Arg or Ala;
Xaa at position 398 is Ser, Gin or Cys;
Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie, Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu;
Xaa at position 402 is Lys, Phe, His, Arg, Trp, Gly, Asn,
Leu, Tyr, Thr, Val, Met, Pro or Ala;
Xaa at position 403 is Asp, Tyr, Trp, Phe
Xaa at position 405 is Ala or Ser;
Xaa at position 409 is Ala or Pro ;
Xaa at position 410 is He or Val;
Xaa at position 411 is Pro or Ala;
Xaa at position 412 is Pro or Ala;
Xaa at position 416 is Arg, Glu or Gin;
Xaa at position 417 is Ala, Ser or Cys;
Xaa at position 418 is Leu or Met;
Xaa at position 422 is Met or Val;
Xaa at position 426 is Thr or Ser;
Xaa at position 436 is Asp or Lys;
Xaa at position 437 is Tyr or Val;
Xaa at position 438 is Val or Arg;
Xaa at position 440 is Val or Leu;
Xaa at position 442 is Gin, Lys or Glu;
Xaa at position 445 is Cys, Leu or Thr;
Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu, He, Gly,
Pro, Leu, Phe, Trp or Thr;
Xaa at position 448 is Val or Ala;
Xaa at position 449 is Gin or Glu; Xaa at position 452 is Gin, Lys or Glu, Ala;
Xaa at position 453 is Asn or Asp;
Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp, Gin, Gly, His, Asp, Leu, Thr, Pro or Asn;
Xaa at position 455 is Val or lie;
Xaa at position 457 is Trp or Asn;
Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie, Thr,
Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu;
Xaa at position 460 is Gly or Ala;
Xaa at position 461 is Thr or Ser;
Xaa at position 462 is Gly or Ala;
Xaa at position 463 is Ala, Ser or Gly;
Xaa at position 464 is Arg, Gly, His, Gin, Thr, Phe, Ala, Asp, Ser or Lys;
Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu;
Xaa at position 466 is Asp or Arg;
Xaa at position 471 is Gin, Lys, Glu or Met;
Xaa at position 472 is Pro or Ser;
Xaa at position 497 is Asp or Gin;
Xaa at position 499 is Glu or Gin;
Xaa at position 500 is Arg, Gin or Lys;
Xaa at position 502 is Arg, Glu or Gin;
Xaa at position 509 is Lys, Gin, Glu or Ala;
Xaa at position 517 is Gin, Cys, Asn, Val or Pro ;
Xaa at position 518 is Glu or Gin;
Xaa at position 520 is Lys, Gin, Glu, His or Ala;
Xaa at position 525 is Gin or Lys; and
Xaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp; wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 3 is an amino acid different from the corresponding amino acid of SEQ ID NO: 35 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
5. A PHI-4 polypeptide, comprising an amino acid sequence of the formula
5 10 15
Met Xaa Ser Ala Ala Asn Ala Gly Gin Leu Gly Asn Leu Pro Gly
20 25 30 Val Thr Ser Met Gly Met Gly Tyr Xaa Val Asn Gly Leu Tyr Ala
35 40 45
Ser Pro Glu Ser Leu Leu Gly Gin Pro Leu Phe Xaa Xaa Gly Gly
50 55 60
Xaa Leu Asp Ser lie Glu lie Glu Gly Arg Ser Tyr Thr Phe Pro
65 70 75
Arg Ser Met His Val His Thr Tyr Phe His Ser Asp Phe Xaa Gin
80 85 90
Asp Val Ser Xaa Glu lie Xaa Glu Tyr Arg Glu Lys Met Ser Gin
95 100 105 His Val Gly Val Ser Gly Xaa Xaa Xaa Leu Phe Ser Ala Ser Leu
110 115 120
Ser Val Asp Xaa Thr Thr Thr Asp Gin Gin Leu Thr Glu lie Thr
125 130 135
Tyr Ser Ser Thr Arg Glu Ala His Val Leu Trp Tyr lie Ser Leu
140 145 150
Pro Gly Ala Ala Thr Leu Arg Ser Met Leu Arg Xaa Xaa Phe Xaa
155 160 165
Xaa Asp Xaa Asn Asn Pro Asn Met Pro Ala Met Xaa Leu Phe Xaa
170 175 180 Xaa Tyr Gly Pro Tyr Xaa lie Ser Xaa Ala Ala Val Gly Gly Arg
185 190 195
Leu Xaa Tyr Ser Ala Ala Ser Lys Thr Leu Lys Met Asp Ser Ser
200 205 210
Xaa Ser Leu Ser Thr Thr Ala Xaa Met Ser Xaa Lys Ala Leu Val
215 220 225
Gly Glu lie Lys lie Xaa His Gly Ser Xaa Met Glu Lys Gin Val
230 235 240
Asn Ser Phe Arg Ser Asn Ser Thr lie Arg Leu Thr Ala Thr Gly
245 250 255 Gly Lys Pro Gly Met Thr Xaa Arg lie Leu His Gly Pro Asp Ser 260 265 270
Xaa Xaa Ala Phe Ser Xaa Trp Ala Glu Ser Leu Leu Asp Tyr Ala
275 280 285 Thr Leu Met Asp Phe Ser Thr Xaa Ser Leu Xaa Pro lie Trp Ala
290 295 300
Leu Ala Asp Xaa Pro Glu Arg Xaa Val Glu Leu Glu Asp Ala Phe
305 310 315
Pro Glu Phe Met Lys Gin Ser Gin Gin Ser lie Pro Xaa Val Asp
320 325 330
Lys Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly
335 340 345
Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Leu Ala Xaa Phe Asn
350 355 360 Xaa Ser Thr Ser Asn Gly Tyr Lys Met Xaa Gly Gin Phe Xaa Gin
365 370 375
Arg Asn His Ala Ser Val Ala Asp Gly His Ala Pro lie Phe Lys
380 385 390
Asp Leu Phe Asp Leu Gly Val Leu Lys Ala Pro Val Gly Trp Gin
395 400 405
Xaa Val Trp Asp Asp Xaa Gly Ser Gly Lys Xaa Xaa Xaa Tyr Ala
410 415 420
Cys Trp Arg Ala lie Xaa Xaa Gin Gly Tyr Xaa Xaa Xaa Gly Asp
425 430 435 Val Met Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro
440 445 450
Asp Tyr Val Cys Xaa His Gin Ser Leu Cys Ala Xaa Val Gin Thr
455 460 465
Leu Xaa Asn Xaa Xaa Trp Trp Asp Xaa Gly Xaa XcL cL XcL cL XcL cL XcL cL
470 475 480
Asp Val Ser Leu Trp Xaa Xaa Gly Ala Ala Gly Ala Val Ala Ser
485 490 495
Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser
500 505 510 Gly Asp He Glu Xaa Leu Arg Gly Ser He Ala Cys Val Xaa Thr
515 520 525
Ser Ala He Ala Ser Met Gin Glu Met Xaa Ser Met Leu Ser Gin
530 535
His Xaa Gly Met Glu Ala Met Met Ser Lys Leu (SEQ ID NO: 4), wherein
Xaa at position 2 is Ala or Arg;
Xaa at position 24 is Asp or Asn;
Xaa at position 42 is Asp or Asn;
Xaa at position 43 is Phe or Glu;
Xaa at position 46 is Glu or Asn;
Xaa at position 74 is Lys, Glu or Gly;
Xaa at position 79 is Lys or Glu;
Xaa at position 82 is Glu, He, Leu or Tyr;
Xaa at position 97 is Arg, Asn, Asp, Glu, Gin, Gly, Ser,
He, Phe, His, Lys, Thr, Asn, Tyr, Trp, Pro, Cys, Ala, Met,
Val or Leu;
Xaa at position 98 is Tyr or Phe;
Xaa at position 99 is Lys, Leu, Tyr, He or Met;
Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn;
Xaa at position 147 is Arg or Glu;
Xaa at position 148 is Asp, Phe or Pro;
Xaa at position 150 is Arg, Gin, Glu or Asn;
Xaa at position 151 is Asp, Ser, Ala or Asn;
Xaa at position 153 is Leu or lie;
Xaa at position 162 is Glu, Asp, Gin, Asn or Leu;
Xaa at position 165 is Lys, Glu or Gin;
Xaa at position 166 is Arg or Gin;
Xaa at position 171 is Tyr or Phe;
Xaa at position 174 is Glu, Gin, Asn, Lys, Val or Ser;
Xaa at position 182 is Asp or Gin;
Xaa at position 196 is Gin, Lys, Asn or Asp;
Xaa at position 203 is Glu, Thr or His;
Xaa at position 206 is Tyr or Phe; Xaa at position 216 is Glu or Gin;
Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser
Gin;
Xaa at position 247 is Asp or Tyr;
Xaa at position 256 is Gin or Lys;
Xaa at position 257 is Gin or lie;
Xaa at position 261 is Gin, Glu, Lys or Ala;
Xaa at position 278 is Glu or Asn;
Xaa at position 281 is Gin, Lys or Glu;
Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Ty
Thr or Asp;
Xaa at position 293 is Arg, Glu or Gin;
Xaa at position 313 is Lys or Gin;
Xaa at position 328 is Lys, Glu or Gin;
Xaa at position 333 is Ser, Gly, Lys, Val or Asn;
Xaa at position 334 is Gly, Arg, Lys or lie;
Xaa at position 336 is Gly or Ala;
Xaa at position 338 is Ser, His, Val, Lys or Ala;
Xaa at position 339 is Glu, Asn, lie or Pro;
Xaa at position 343 is Val or lie;
Xaa at position 346 is Pro or Ala;
Xaa at position 355 is Val or lie;
Xaa at position 359 is Gly or Ala;
Xaa at position 391 is Arg, Leu, Glu, Gin, Asp or His;
Xaa at position 396 is Ala, Leu, Lys, Asn, Gly or Thr;
Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val or Arg;
Xaa at position 402 is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn, Tyr or Met;
Xaa at position 403 is Asp or Tyr;
Xaa at position 411 is Pro or Ala;
Xaa at position 412 is Pro or Ala;
Xaa at position 416 is Arg or Glu;
Xaa at position 417 is Ala or Ser;
Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr or Ser;
Xaa at position 440 is Val or Leu;
Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu or lie;
Xaa at position 452 is Gin, Lys or Glu;
Xaa at position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys,
Phe, Trp or Gin;
Xaa at position 455 is Val or lie;
Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie or Tyr;
Xaa at position 461 is Thr or Ser;
Xaa at position 462 is Gly or Ala;
Xaa at position 463 is Ala or Ser;
Xaa at position 464 is Arg, Gly, His, Ala, Asp, Ser or Lys;
Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly or Arg;
Xaa at position 471 is Gin, Lys, Glu or Met;
Xaa at position 472 is Pro or Ser;
Xaa at position 500 is Arg or Gin;
Xaa at position 509 is Lys, Gin or Ala;
Xaa at position 520 is Lys, Gin, Glu, His or Ala; and
Xaa at position 527 is Gin, Lys, Pro, Cys or Glu; wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 4 is an amino acid different from the corresponding amino acid of SEQ ID NO: 35 and wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
6. The PHI-4 polypeptide of any one of claims 2 to 5, further comprising one or more amino acid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468, 499 or 517 of
SEQ ID NO: 35.
7. The PHI-4 polypeptide of claim 6, wherein the amino acid at position 86 is Glu or Thr; the amino acid at position 359 is Gly or Ala; the amino acid at position 399 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; the amino acid at position 465 is Lys or Met, the amino acid at position 467 is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu; the amino acid at position 499 is Glu or Ala or the amino acid at position 517 is Glu or Arg.
8. The PHI-4 polypeptide of claim 1 , wherein the PHI-polypeptide comprises at least one amino acid substitution at a residue relative to SEQ ID NO: 35 in a structural domain selected from:
a hydrophilic residue;
a residue in a membrane insertion initiation loop;
a residue in a receptor binding loop; and
a residue in a protease sensitive region,
wherein the PHI-4 polypeptide has increased insecticidal activity compared to the polypeptide of SEQ ID NO: 35.
9. The PHI-4 polypeptide of claim 8, wherein the hydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin, and Cys.
10. The PHI-4 polypeptide of claim 8 or 9, wherein the membrane insertion loops are between about amino acid at position 92 (Val) and 101 (Ala) and at position 21 1 (Gly) and 220 (Glu) relative to SEQ ID NO: 35.
1 1 . The PHI-4 polypeptide of any one of claims 8 to 10, wherein the receptor binding loops are between about amino acid 332 (Asp) and 340 (Asp), 395 (Asp) and 403 (Asp) , 458 (Asp) and 466 (Asp) relative to SEQ ID NO: 35.
12. The PHI-4 polypeptide of claim 1 1 , wherein the receptor binding loop residue is selected from positions 332, 333, 334, 335, 336, 337, 338, 339, 340, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 458, 459, 460, 461 , 462, 463, 464, 465, 466 of SEQ ID NO: 35.
13. The PHI-4 polypeptide of any one of claims 8 to 12, wherein the protease sensitive region residue is selected from about amino acid residues between 305 (Lys) and 316 (Lys) and 500 (Arg) and 535 (Lys) relative to SEQ ID NO: 35.
14. The PHI-4 polypeptide of any one of claims 1 to 13, wherein the PHI-4 polypeptide comprises 1 to 54 amino acid substitutions compared to SEQ ID NO: 35.
15. The PHI-4 polypeptide of any one of claims 1 to 13, wherein the PHI-4 polypeptide comprises 1 to 15 amino acid substitutions compared to SEQ ID NO: 35.
16. The PHI-4 polypeptide of any one of claims 1 to 13, wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO: 35.
17. The PHI-4 polypeptide of any one of claims 1 to 16, wherein the insecticidal activity is increased about 1.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).
18. The PHI-4 polypeptide of any one of claims 1 to 17, wherein the improved insecticidal activity compared to AXMI-205 (SEQ ID NO: 35) is against Western Corn Root Worm (WCRW) larvae.
19. The PHI-4 polypeptide of any one of claims 1-18, wherein 1-25 amino acids are deleted from the N-terminus of the PHI-4 polypeptide and/or C-terminus of the PHI-4 polypeptide.
20. A polynucleotide encoding a PHI-4 polypeptide of any one of claims 1 to 19.
21 . A composition, comprising an insecticidally-effective amount of the PHI-4 polypeptide of any one of claims 1-19.
22. A method of inhibiting growth or killing an insect pest, comprising contacting the insect pest with the composition of claim 21.
23. A method for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the resistant insect pest population with the composition of claim
21 .
24. The method of controlling an insect pest population resistant to an pesticidal protein of claim 23, wherein the pesticidal protein is selected from CrylAc, CrylAb, Cry1A.105, CrylAc, Cryl F, Cry1 Fa2, Cry1 F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1 , Cry34Ab1 , Cry35Ab1 , Vip3A, Cry9c, eCry3.1Ab and CBI-Bt.
25. A transgenic plant or progeny thereof, comprising the polynucleotide of claim 20.
26. The transgenic plant or progeny thereof of claim 25, wherein the transgenic plant is a monocotyledon.
27. The transgenic plant or progeny thereof of claim 25, wherein the plant is selected from barley, corn, oat, rice, rye, sorghum, turf grass, sugarcane, wheat, alfalfa, banana, broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower, safflower, soybean, strawberry, sugar beet, sweet potato, tobacco, tomato ornamental, shrub, nut, chickpea, pigeon pea, millets, hops and pasture grasses.
28. The transgenic plant or progeny thereof of claim 25, further comprising one or more additional transgenic traits.
29. The transgenic plant of claim 28, wherein the one or more additional transgenic trait is selected from insect resistance, herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stalk strength, increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, salt resistance or tolerance and increased yield under stress.
30. Seed, grain or processed product thereof of the transgenic plant of any one of claims 25 to 29, wherein the seed, grain or processed product thereof comprises the polynucleotide.
31 . An expression cassette, comprising the polynucleotide of of claim 20 operably linked to one or more regulatory sequences directing expression of the PHI-4 polypeptide.
32. The seed of claim 30, wherein one or more seed treatment has been applied to the seed.
33. A method for expressing in a plant a polynucleotide encoding an insecticidal protein, comprising
(a) inserting into a plant cell the polynucleotide of claim 20 encoding the PHI-4 polypeptide;
(b) obtaining a transformed plant cell comprising the nucleic acid sequence of step (a); and
(c) generating from the transformed plant cell a plant capable of expressing the polynucleotide encoding the PHI-4 polypeptide.
34. A method for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof, an insecticidally-effective amount of the PHI-4 polypeptide of any one of claims 1 to 19.
35. A method for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of the PHI-4 polypeptide of any one of claims 1 to 19.
36. A method of inhibiting growth or killing an insect pest, comprising contacting the insect pest with a composition comprising an insecticidally-effective amount of the the PHI-4 polypeptide of any one of claims 1 to 19.
37. A method for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of the PHI-4 polypeptide of any one of claims 1 to 19.
38. A fusion protein comprising the PHI-4 polypeptide of any one of claims 1 to 19.
PCT/US2014/024524 2013-03-15 2014-03-12 Phi-4 polypeptides and methods for their use WO2014150914A2 (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
MX2015013269A MX360160B (en) 2013-03-15 2014-03-12 PHI-4 POLYPEPTIDES and METHODS FOR THEIR USE.
RU2015143825A RU2015143825A (en) 2013-03-15 2014-03-12 PHI-4 POLYPEPTIDES AND WAYS OF THEIR APPLICATION
CA2901316A CA2901316A1 (en) 2013-03-15 2014-03-12 Phi-4 polypeptides and methods for their use
US14/776,212 US10023877B2 (en) 2013-03-15 2014-03-12 PHI-4 polypeptides and methods for their use
EP14768504.4A EP2971000A4 (en) 2013-03-15 2014-03-12 Phi-4 polypeptides and methods for their use
CN201480027131.7A CN105473605A (en) 2013-03-15 2014-03-12 Phi-4 polypeptides and methods for their use
BR112015023709A BR112015023709A2 (en) 2013-03-15 2014-03-12 phi-4 polypeptide, polynucleotide, composition, method for inhibiting growth, method for controlling a population, plant, seed, expression cassette, method for expressing a polynucleotide in a plant, method for protecting a plant, fusion protein
ZA2015/05705A ZA201505705B (en) 2013-03-15 2015-08-07 Phi-4 polypeptides and methods for their use
US16/013,580 US10577626B2 (en) 2013-03-15 2018-06-20 PHI-4 polypeptides and methods for their use
US16/741,271 US11180775B2 (en) 2013-03-15 2020-01-13 PHI-4 polypeptides and methods for their use
US17/451,031 US11697821B2 (en) 2013-03-15 2021-10-15 PHI-4 polypeptides and methods for their use

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361793439P 2013-03-15 2013-03-15
US61/793,439 2013-03-15
US201361859883P 2013-07-30 2013-07-30
US61/859,883 2013-07-30

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14/776,212 A-371-Of-International US10023877B2 (en) 2013-03-15 2014-03-12 PHI-4 polypeptides and methods for their use
US16/013,580 Division US10577626B2 (en) 2013-03-15 2018-06-20 PHI-4 polypeptides and methods for their use

Publications (3)

Publication Number Publication Date
WO2014150914A2 true WO2014150914A2 (en) 2014-09-25
WO2014150914A3 WO2014150914A3 (en) 2014-11-13
WO2014150914A8 WO2014150914A8 (en) 2015-11-26

Family

ID=51581621

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/024524 WO2014150914A2 (en) 2013-03-15 2014-03-12 Phi-4 polypeptides and methods for their use

Country Status (9)

Country Link
US (4) US10023877B2 (en)
EP (1) EP2971000A4 (en)
CN (1) CN105473605A (en)
BR (1) BR112015023709A2 (en)
CA (1) CA2901316A1 (en)
MX (1) MX360160B (en)
RU (1) RU2015143825A (en)
WO (1) WO2014150914A2 (en)
ZA (1) ZA201505705B (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104365250A (en) * 2014-10-31 2015-02-25 山东省农业科学院作物研究所 Soil basic fertility-based efficient application method of nitrogenous fertilizer for wheat in Huang-Huai-Hai wheat growing region
WO2015120276A1 (en) 2014-02-07 2015-08-13 Pioneer Hi Bred International Inc Insecticidal proteins and methods for their use
WO2015130893A1 (en) 2014-02-28 2015-09-03 E. I. Du Pont De Nemours And Company Synthetic oligoglucosamines for improvement of plant growth and yield
WO2015130890A1 (en) 2014-02-28 2015-09-03 E. I. Du Pont De Nemours And Company Synthetic salt complexes for improvement of plant growth and yield
WO2015134256A1 (en) 2014-02-28 2015-09-11 E. I. Du Pont De Nemours And Company Combinatorial libraries
WO2018084936A1 (en) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2018140214A1 (en) 2017-01-24 2018-08-02 Pioneer Hi-Bred International, Inc. Nematicidal protein from pseudomonas
US20190330652A1 (en) * 2016-09-09 2019-10-31 Syngenta Pariticpations AG Insecticidal proteins
US10472642B2 (en) 2015-02-05 2019-11-12 British American Tobacco (Investments) Limited Method for the reduction of tobacco-specific nitrosamines or their precursors in tobacco plants
EP3691453A4 (en) * 2017-10-02 2021-07-07 Syngenta Participations AG Engineered pesticidal proteins and methods of controlling plant pests
CN113677809A (en) * 2018-10-30 2021-11-19 先正达参股股份有限公司 Insecticidal proteins

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9394345B2 (en) 2013-03-15 2016-07-19 Pioneer Hi-Bred International, Inc. PHI-4 polypeptides and methods for their use
CA2985198A1 (en) 2015-05-19 2016-11-24 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US10647995B2 (en) 2015-06-16 2020-05-12 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
WO2017023486A1 (en) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Plant derived insecticidal proteins and methods for their use
EP3442994A4 (en) 2016-04-14 2019-11-27 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having improved activity spectrum and uses thereof
BR112018072417B1 (en) 2016-05-04 2023-03-14 E. I. Du Pont De Nemours And Company RECOMBINANT INSECTICIDAL POLYPEPTIDE, CHIMERIC POLYPEPTIDE, COMPOSITION, RECOMBINANT POLYNUCLEOTIDE, DNA CONSTRUCTS, METHODS FOR OBTAINING A TRANSGENIC PLANT, METHODS FOR INHIBITING THE GROWTH OR EXTERMINATION OF AN INSECT PEST OR PEST POPULATION, METHOD FOR OBTAINING A TRANSFORMED PROKARYOTIC CELL TRANSFORMED AND METHOD TO GENETICALLY MODIFY THE INSECTICIDAL POLYPEPTIDE
CN109312359A (en) 2016-06-16 2019-02-05 先锋国际良种公司 To prevent and treat the composition and method of insect pest
US11155829B2 (en) 2016-07-01 2021-10-26 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
US20210292778A1 (en) 2016-07-12 2021-09-23 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
EP3555118B1 (en) 2016-12-14 2021-08-18 Pioneer Hi-Bred International Inc. Insecticidal proteins and methods for their use
MX2019007491A (en) 2016-12-22 2019-09-06 Pioneer Hi Bred Int Insecticidal proteins and methods for their use.
MX2019007962A (en) * 2017-01-12 2019-09-04 Syngenta Participations Ag Insecticidal proteins.
CN110621780B (en) 2017-05-11 2024-03-19 先锋国际良种公司 Insecticidal proteins and methods of use thereof
BR112019024827A2 (en) 2017-05-26 2020-06-16 Pioneer Hi-Bred International, Inc. DNA CONSTRUCTION, TRANSGENIC PLANT OR PROGENY OF THE SAME, COMPOSITION AND METHOD TO CONTROL A POPULATION OF INSECT PEST
WO2019074598A1 (en) 2017-10-13 2019-04-18 Pioneer Hi-Bred International, Inc. Virus-induced gene silencing technology for insect control in maize
US20210130844A1 (en) * 2018-01-03 2021-05-06 Uwm Research Foundation, Inc. Sterile mutant and two line breeding system
WO2019169150A1 (en) 2018-03-02 2019-09-06 Pioneer Hi-Bred International, Inc. Plant health assay
US11525144B2 (en) * 2018-03-14 2022-12-13 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
CN112533625A (en) * 2018-08-27 2021-03-19 先正达参股股份有限公司 Compositions and methods for protein detection
CN109456976B (en) * 2018-12-26 2021-09-28 菏泽学院 Lymantria dispar JHAT gene, encoding protein thereof and application of dsRNA thereof in pest control
EP3935581A4 (en) 2019-03-04 2022-11-30 Iocurrents, Inc. Data compression and communication using machine learning
CN112794892B (en) * 2019-11-13 2022-09-09 中国科学院动物研究所 Antifungal peptide mutant and preparation method and application thereof

Citations (397)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710511A (en) 1971-04-21 1973-01-16 Univ Illinois Procedures for use of genic male sterility in production of commercial hybrid maize
US3861709A (en) 1973-07-12 1975-01-21 Amsted Ind Inc Shiftable fifth wheel construction
US4196265A (en) 1977-06-15 1980-04-01 The Wistar Institute Method of producing antibodies
US4535060A (en) 1983-01-05 1985-08-13 Calgene, Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase, production and use
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US4654465A (en) 1985-07-18 1987-03-31 Agracetus Genic male-sterile maize
EP0242246A1 (en) 1986-03-11 1987-10-21 Plant Genetic Systems N.V. Plant cells resistant to glutamine synthetase inhibitors, made by genetic engineering
US4727219A (en) 1986-11-28 1988-02-23 Agracetus Genic male-sterile maize using a linked marker gene
US4761373A (en) 1984-03-06 1988-08-02 Molecular Genetics, Inc. Herbicide resistance in plants
US4810648A (en) 1986-01-08 1989-03-07 Rhone Poulenc Agrochimie Haloarylnitrile degrading gene, its use, and cells containing the gene
EP0333033A1 (en) 1988-03-09 1989-09-20 Meiji Seika Kaisha Ltd. Glutamine synthesis gene and glutamine synthetase
US4940835A (en) 1985-10-29 1990-07-10 Monsanto Company Glyphosate-resistant plants
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US4971908A (en) 1987-05-26 1990-11-20 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US4975374A (en) 1986-03-18 1990-12-04 The General Hospital Corporation Expression of wild type and mutant glutamine synthetase in foreign hosts
US5013659A (en) 1987-07-27 1991-05-07 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5023179A (en) 1988-11-14 1991-06-11 Eric Lam Promoter enhancer element for gene expression in plant roots
US5039523A (en) 1988-10-27 1991-08-13 Mycogen Corporation Novel Bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin
WO1991014778A2 (en) 1990-03-20 1991-10-03 Ecogen Inc. BACILLUS THURINGIENSIS cryIIIC GENE AND PROTEIN TOXIC TO COLEOPTERAN INSECTS
US5094945A (en) 1983-01-05 1992-03-10 Calgene, Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthase, production and use
EP0480762A2 (en) 1990-10-12 1992-04-15 Mycogen Corporation Novel bacillus thuringiensis isolates active against dipteran pests
US5107065A (en) 1986-03-28 1992-04-21 Calgene, Inc. Anti-sense regulation of gene expression in plant cells
US5110732A (en) 1989-03-14 1992-05-05 The Rockefeller University Selective gene expression in plants
WO1992013957A1 (en) 1991-02-07 1992-08-20 Plant Genetic Systems, N.V. Stamen-specific promoters from corn
WO1992013956A1 (en) 1991-02-08 1992-08-20 Plant Genetic Systems, N.V. Stamen-specific promoters from rice
US5145783A (en) 1987-05-26 1992-09-08 Monsanto Company Glyphosate-tolerant 5-endolpyruvyl-3-phosphoshikimate synthase
WO1993002197A1 (en) 1991-07-23 1993-02-04 Nickerson Biocem Limited CALLASE-RELATED DNAs AND THEIR USE IN ARTIFICIAL MALE STERILITY
US5188960A (en) 1989-06-27 1993-02-23 Mycogen Corporation Bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins
US5188642A (en) 1985-08-07 1993-02-23 Monsanto Company Glyphosate-resistant plants
WO1993011245A1 (en) 1991-12-04 1993-06-10 E.I. Du Pont De Nemours And Company Fatty acid desaturase genes from plants
US5240855A (en) 1989-05-12 1993-08-31 Pioneer Hi-Bred International, Inc. Particle gun
WO1993019181A1 (en) 1992-03-25 1993-09-30 Sandoz Ltd. Biological material
US5266317A (en) 1990-10-04 1993-11-30 University Of Georgia Research Foundation, Inc. Insect-specific paralytic neurotoxin genes for use in biological insect control: methods and compositions
US5268463A (en) 1986-11-11 1993-12-07 Jefferson Richard A Plant promoter α-glucuronidase gene construct
US5304732A (en) 1984-03-06 1994-04-19 Mgi Pharma, Inc. Herbicide resistance in plants
US5310667A (en) 1989-07-17 1994-05-10 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5312910A (en) 1987-05-26 1994-05-17 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5316931A (en) 1988-02-26 1994-05-31 Biosource Genetics Corp. Plant viral vectors having heterologous subgenomic promoters for systemic expression of foreign genes
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5324646A (en) 1992-01-06 1994-06-28 Pioneer Hi-Bred International, Inc. Methods of regeneration of Medicago sativa and expressing foreign DNA in same
US5331107A (en) 1984-03-06 1994-07-19 Mgi Pharma, Inc. Herbicide resistance in plants
US5378824A (en) 1986-08-26 1995-01-03 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5399680A (en) 1991-05-22 1995-03-21 The Salk Institute For Biological Studies Rice chitinase promoter
US5401836A (en) 1992-07-16 1995-03-28 Pioneer Hi-Bre International, Inc. Brassica regulatory sequence for root-specific or root-abundant gene expression
WO1995015392A1 (en) 1993-11-30 1995-06-08 E.I. Du Pont De Nemours And Company Chimeric geens and methods for increasing the lysine content of the seeds of corn, soybean and rapeseed plants
WO1995016776A1 (en) 1993-12-17 1995-06-22 Pioneer Hi-Bred International, Inc. Derivatives of tachyplesin having inhibitory activity towards plant pathogenic fungi
US5428148A (en) 1992-04-24 1995-06-27 Beckman Instruments, Inc. N4 - acylated cytidinyl compounds useful in oligonucleotide synthesis
US5432068A (en) 1990-06-12 1995-07-11 Pioneer Hi-Bred International, Inc. Control of male fertility using externally inducible promoter sequences
WO1995018855A2 (en) 1994-01-07 1995-07-13 Pioneer Hi-Bred International, Inc. Synthetic antimicrobial peptides
US5436391A (en) 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
WO1995022625A1 (en) 1994-02-17 1995-08-24 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
US5459252A (en) 1991-01-31 1995-10-17 North Carolina State University Root specific gene promoter
US5463175A (en) 1990-06-25 1995-10-31 Monsanto Company Glyphosate tolerant plants
US5466785A (en) 1990-04-12 1995-11-14 Ciba-Geigy Corporation Tissue-preferential promoters
US5478369A (en) 1990-06-12 1995-12-26 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating male fertility and method of using same
WO1996001905A1 (en) 1994-07-08 1996-01-25 E.I. Du Pont De Nemours And Company Chimeric genes and method for increasing the threonine content of the seeds of plants
US5489520A (en) 1990-04-17 1996-02-06 Dekalb Genetics Corporation Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
US5491288A (en) 1991-03-05 1996-02-13 Rhone Poulenc Agrochimie Chimeric gene comprising the arabidopsis histone H4 promoter for the transformation of plants
US5496714A (en) 1992-12-09 1996-03-05 New England Biolabs, Inc. Modification of protein by use of a controllable interveining protein sequence
US5500365A (en) 1989-02-24 1996-03-19 Monsanto Company Synthetic plant genes
US5510471A (en) 1991-03-05 1996-04-23 Rhone-Poulenc Agrochimie Chimeric gene for the transformation of plants
WO1996014414A1 (en) 1994-11-02 1996-05-17 John Innes Centre Innovations Limited Genetic control of flowering
US5559223A (en) 1991-08-09 1996-09-24 E. I. Dupont De Nemours And Company Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants
WO1996030517A1 (en) 1995-03-31 1996-10-03 Commonwealth Scientific And Industrial Research Organisation Genetic sequences conferring nematode resistance in plants and uses therefor
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
WO1996033270A1 (en) 1995-04-20 1996-10-24 American Cyanamid Company Structure-based designed herbicide resistant products
WO1996033207A1 (en) 1995-04-18 1996-10-24 Glaxo Group Limited End-complementary polymerase reaction
US5569597A (en) 1985-05-13 1996-10-29 Ciba Geigy Corp. Methods of inserting viral DNA into plant material
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
WO1996038560A2 (en) 1995-06-02 1996-12-05 John Innes Centre Innovations Limited Genetic control of flowering
US5604121A (en) 1991-08-27 1997-02-18 Agricultural Genetics Company Limited Proteins with insecticidal properties against homopteran insects and their use in plant protection
US5605011A (en) 1986-08-26 1997-02-25 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5608144A (en) 1994-08-12 1997-03-04 Dna Plant Technology Corp. Plant group 2 promoters and uses thereof
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US5608142A (en) 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
WO1997010339A1 (en) 1995-09-13 1997-03-20 John Innes Centre Innovations Limited Flowering genes
US5627061A (en) 1990-08-31 1997-05-06 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5633436A (en) 1993-03-02 1997-05-27 E. I. Du Pont De Nemours And Company Feedcrops enriched in sulfur amino acids and methods for improvements
US5633363A (en) 1994-06-03 1997-05-27 Iowa State University, Research Foundation In Root preferential promoter
WO1997020078A1 (en) 1995-11-30 1997-06-05 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
WO1997029123A2 (en) 1996-02-12 1997-08-14 Plant Bioscience Limited Nucleic acid encoding gai gene of arabidopsis thaliana
US5659026A (en) 1995-03-24 1997-08-19 Pioneer Hi-Bred International ALS3 promoter
WO1997035966A1 (en) 1996-03-25 1997-10-02 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
WO1997040162A2 (en) 1996-04-19 1997-10-30 Mycogen Corporation Pesticidal toxins
US5689035A (en) 1995-09-26 1997-11-18 Pioneer Hi-Bred International, Inc. Brown stem rot resistance in soybeans
US5689052A (en) 1993-12-22 1997-11-18 Monsanto Company Synthetic DNA sequences having enhanced expression in monocotyledonous plants and method for preparation thereof
WO1997049811A1 (en) 1996-06-21 1997-12-31 Plant Bioscience Limited Genetic control of flowering
US5716820A (en) 1994-08-12 1998-02-10 Pioneer Hi-Bred International, Inc. Fumonisin detoxification enzymes
US5723323A (en) 1985-03-30 1998-03-03 Kauffman; Stuart Alan Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof
WO1998009521A1 (en) 1996-09-04 1998-03-12 Michigan State University Dna and encoded protein which regulates cold and dehydration regulated genes
US5731180A (en) 1991-07-31 1998-03-24 American Cyanamid Company Imidazolinone resistant AHAS mutants
US5736369A (en) 1994-07-29 1998-04-07 Pioneer Hi-Bred International, Inc. Method for producing transgenic cereal plants
US5743477A (en) 1992-08-27 1998-04-28 Dowelanco Insecticidal proteins and method for plant protection
US5750386A (en) 1991-10-04 1998-05-12 North Carolina State University Pathogen-resistant transgenic plants
WO1998020133A2 (en) 1996-11-01 1998-05-14 Pioneer Hi-Bred International, Inc. Proteins with enhanced levels of essential amino acids
WO1998022604A1 (en) 1996-11-20 1998-05-28 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US5767373A (en) 1994-06-16 1998-06-16 Novartis Finance Corporation Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms
WO1998027230A1 (en) 1996-12-18 1998-06-25 Maxygen, Inc. Methods and compositions for polypeptide engineering
WO1998031837A1 (en) 1997-01-17 1998-07-23 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
WO1998032326A2 (en) 1997-01-24 1998-07-30 Pioneer Hi-Bred International, Inc. Methods for $i(agrobacterium)-mediated transformation
US5789156A (en) 1993-06-14 1998-08-04 Basf Ag Tetracycline-regulated transcriptional inhibitors
WO1998036083A1 (en) 1997-02-14 1998-08-20 Plant Bioscience Limited Methods and means for gene silencing in transgenic plants
US5798255A (en) 1996-11-22 1998-08-25 Pioneer Hi-Bred International, Inc. Beauvericin detoxification compositions and methods
WO1998041653A1 (en) 1997-03-18 1998-09-24 Novo Nordisk A/S An in vitro method for construction of a dna library
WO1998041622A1 (en) 1997-03-18 1998-09-24 Novo Nordisk A/S Method for constructing a library using dna shuffling
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
WO1998042831A2 (en) 1997-03-27 1998-10-01 E.I. Du Pont De Nemours And Company Chimeric genes and methods for increasing the lysine content of the seeds of plants
WO1998042832A1 (en) 1997-03-25 1998-10-01 California Institute Of Technology Recombination of polynucleotide sequences using random or defined primers
WO1998042727A1 (en) 1997-03-21 1998-10-01 Sri International Sequence alterations using homologous recombination
WO1998045448A1 (en) 1997-04-08 1998-10-15 E.I. Du Pont De Nemours And Company Soybean plant producing seeds with reduced levels of raffinose saccharides and phytic acid
WO1998045458A1 (en) 1997-04-08 1998-10-15 E.I. Du Pont De Nemours And Company An engineered seed protein having a higher percentage of essential amino acids
US5824524A (en) 1990-06-12 1998-10-20 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating fertility and method of using same
US5834247A (en) 1992-12-09 1998-11-10 New England Biolabs, Inc. Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein
US5837876A (en) 1995-07-28 1998-11-17 North Carolina State University Root cortex specific gene promoter
US5846812A (en) 1996-11-22 1998-12-08 Pioneer Hi-Bred International, Inc. Zearalenone detoxification compositions and methods
US5850016A (en) 1996-03-20 1998-12-15 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
WO1998056935A2 (en) 1997-06-12 1998-12-17 E.I. Du Pont De Nemours And Company Plant amino acid biosynthetic enzymes
WO1998056918A1 (en) 1997-06-13 1998-12-17 Plant Bioscience Limited Methods and means for control of flowering in plants
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
WO1999005298A1 (en) 1997-07-22 1999-02-04 Pioneer Hi-Bred International, Inc. Genes controlling phytate metabolism in plants and uses thereof
WO1999009174A1 (en) 1997-08-13 1999-02-25 Plant Bioscience Limited Genetic control of plant growth and development
US5877012A (en) 1993-03-25 1999-03-02 Novartis Finance Corporation Class of proteins for the control of plant pests
WO1999010498A2 (en) 1997-08-27 1999-03-04 Pioneer Hi-Bred International, Inc. Genes encoding enzymes for lignin biosynthesis and uses thereof
US5879903A (en) 1986-08-23 1999-03-09 Hoechst Aktiengesellschaft Phosphinothricin-resistance gene, and its use
US5879918A (en) 1989-05-12 1999-03-09 Pioneer Hi-Bred International, Inc. Pretreatment of microprojectiles prior to using in a particle gun
US5885801A (en) 1995-06-02 1999-03-23 Pioneer Hi-Bred International, Inc. High threonine derivatives of α-hordothionin
US5885802A (en) 1995-06-02 1999-03-23 Pioneer Hi-Bred International, Inc. High methionine derivatives of α-hordothionin
US5886244A (en) 1988-06-10 1999-03-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
US5889191A (en) 1992-12-30 1999-03-30 Biosource Technologies, Inc. Viral amplification of recombinant messenger RNA in transgenic plants
WO1999021979A1 (en) 1997-10-28 1999-05-06 Maxygen, Inc. Human papillomavirus vectors
WO1999023107A1 (en) 1997-10-31 1999-05-14 Maxygen, Incorporated Modification of virus tropism and host range by viral genome shuffling
WO1999024581A2 (en) 1997-11-12 1999-05-20 Mycogen Corporation Plant-optimized genes encoding pesticidal toxins
WO1999025855A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from t-dna using site-specific recombination systems
WO1999025840A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. A novel method for the integration of foreign dna into eukaryoticgenomes
WO1999025821A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Compositions and methods for genetic modification of plants
WO1999025853A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Targeted manipulation of herbicide-resistance genes in plants
US5912414A (en) 1994-05-13 1999-06-15 E. I. Du Pont De Nemours And Company Nucleic acid fragments, chimeric genes and methods for increasing the methionine content of the seeds of plants
WO1999029882A2 (en) 1997-12-10 1999-06-17 Pioneer Hi-Bred International, Inc. Methods for altering the nutritional value of a plant protein, vegetative storage protein, vsp, by altering the amino acid content of proteins
WO1999029902A1 (en) 1997-12-08 1999-06-17 California Institute Of Technology Method for creating polynucleotide and polypeptide sequences
WO1999031248A1 (en) 1997-12-18 1999-06-24 Ecogen, Inc. INSECT-RESISTANT TRANSGENIC PLANTS AND METHODS FOR IMPROVING δ-ENDOTOXIN ACTIVITY AGAINST TARGET INSECTS
US5929305A (en) 1997-10-14 1999-07-27 Michigan State University Plant material containing non-naturally introduced binding protein for regulating cold and dehydration regulatory genes
US5928937A (en) 1995-04-20 1999-07-27 American Cyanamid Company Structure-based designed herbicide resistant products
US5932782A (en) 1990-11-14 1999-08-03 Pioneer Hi-Bred International, Inc. Plant transformation method using agrobacterium species adhered to microprojectiles
EP0932670A1 (en) 1996-03-25 1999-08-04 Maxygen, Inc. Evolving cellular dna uptake by recursive sequence recombination
WO1999038977A2 (en) 1998-02-03 1999-08-05 Mendel Biotechnology, Inc. Plant having altered environmental stress tolerance
WO1999040209A1 (en) 1998-02-09 1999-08-12 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
US5939599A (en) 1991-02-14 1999-08-17 E. I. Du Pont De Nemours And Company High sulfur seed protein gene and method for increasing the sulfur amino acid contact of plants
WO1999041369A2 (en) 1998-02-11 1999-08-19 Maxygen, Inc. Genetic vaccine vector engineering
WO1999041383A1 (en) 1998-02-11 1999-08-19 Maxygen, Inc. Antigen library immunization
WO1999043819A1 (en) 1998-02-26 1999-09-02 Pioneer Hi-Bred International, Inc. Family of maize pr-1 genes and promoters
WO1999043838A1 (en) 1998-02-24 1999-09-02 Pioneer Hi-Bred International, Inc. Synthetic promoters
WO1999049064A2 (en) 1998-03-20 1999-09-30 Plant Bioscience Limited Plant control genes
WO1999053050A1 (en) 1998-04-08 1999-10-21 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
WO1999055882A1 (en) 1998-04-24 1999-11-04 E.I. Du Pont De Nemours And Company Phytic acid biosynthetic enzymes
US5986177A (en) 1997-01-10 1999-11-16 Agricultural Genetic Engineering Research Institute Bacillus thuringiensis isolates with broad spectrum activity
US5990389A (en) 1993-01-13 1999-11-23 Pioneer Hi-Bred International, Inc. High lysine derivatives of α-hordothionin
WO1999061632A1 (en) 1998-05-22 1999-12-02 Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) A process for inhibiting expression of genes
WO1999064579A2 (en) 1998-06-11 1999-12-16 E.I. Du Pont De Nemours And Company Corn oleosin promoter and genes encoding corn desaturases
WO2000000632A1 (en) 1998-06-29 2000-01-06 Phylos, Inc. Methods for generating highly diverse libraries
WO2000004190A1 (en) 1998-07-15 2000-01-27 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6023013A (en) 1997-12-18 2000-02-08 Monsanto Company Insect-resistant transgenic plants
WO2000006341A1 (en) 1998-07-31 2000-02-10 Minnesota Mining And Manufacturing Company Cleaning pads formed from non-woven abrasive web material, especially for domestic use
WO2000009679A1 (en) 1998-08-12 2000-02-24 Proteus (S.A.) Method for obtaining in vitro recombined polynucleotide sequences, sequence banks and resulting sequences
US6033874A (en) 1996-11-27 2000-03-07 Ecogen, Inc. CRY1C polypeptides having improved toxicity to lepidopteran insects
WO2000012733A1 (en) 1998-08-28 2000-03-09 Pioneer Hi-Bred International, Inc. Seed-preferred promoters from end genes
US6040497A (en) 1997-04-03 2000-03-21 Dekalb Genetics Corporation Glyphosate resistant maize lines
WO2000018906A2 (en) 1998-09-29 2000-04-06 Maxygen, Inc. Shuffling of codon altered genes
US6048838A (en) 1997-05-05 2000-04-11 Dow Agrosciences Llc Insecticidal protein toxins from xenorhabdus
US6060594A (en) 1997-12-18 2000-05-09 Ecogen, Inc. Nucleic acid segments encoding modified bacillus thuringiensis coleopteran-toxic crystal proteins
US6063947A (en) 1996-07-03 2000-05-16 Cargill, Incorporated Canola oil having increased oleic acid and decreased linolenic acid content
US6063597A (en) 1997-12-18 2000-05-16 Monsanto Company Polypeptide compositions toxic to coleopteran insects
WO2000028058A2 (en) 1998-11-09 2000-05-18 Pioneer Hi-Bred International, Inc. Transcriptional activator lec1 nucleic acids, polypeptides and their uses
WO2000032761A1 (en) 1998-11-25 2000-06-08 The Trustees Of The University Of Pennsylvania Ethylene-response-factor1 (erf1) in plants
US6077824A (en) 1997-12-18 2000-06-20 Ecogen, Inc. Methods for improving the activity of δ-endotoxins against insect pests
US6080913A (en) 1996-09-25 2000-06-27 Pioneer Hi-Bred International, Inc. Binary methods of increasing accumulation of essential amino acids in seeds
US6084153A (en) 1996-02-14 2000-07-04 The Governors Of The University Of Alberta Plants having enhanced nitrogen assimilation/metabolism
US6083736A (en) 1996-11-22 2000-07-04 Pioneer Hi-Bred International, Inc. Ochrobactrum anthropi bacteria which degrade moniliformin
WO2000042561A2 (en) 1999-01-19 2000-07-20 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
WO2000042559A1 (en) 1999-01-18 2000-07-20 Maxygen, Inc. Methods of populating data structures for use in evolutionary simulations
WO2000044918A1 (en) 1999-01-28 2000-08-03 Plant Bioscience Limited Methods and means for modification of plant characteristics using the vernalization gene vrn2
WO2000046358A2 (en) 1999-02-05 2000-08-10 Plant Bioscience Limited Arabidopsis thaliana derived frigida gene conferring late flowering
US6107547A (en) 1993-10-06 2000-08-22 New York University Transgenic plants that exhibit enhanced nitrogen assimilation
US6127600A (en) 1995-05-31 2000-10-03 Pioneer Hi-Bred International, Inc. Methods of increasing accumulation of essential amino acids in seeds
US6130366A (en) 1984-12-28 2000-10-10 Plant Genetic Systems Chimaeric gene coding for a transit peptide and a heterologous polypeptide
WO2000060089A1 (en) 1999-04-07 2000-10-12 Mendel Biotechnology, Inc. Genetic trait breeding method
WO2000068393A1 (en) 1999-05-07 2000-11-16 Pioneer Hi-Bred International, Inc. Phytyl/prenyltransferase nucleic acids, polypeptides and uses thereof
WO2000073475A1 (en) 1999-05-28 2000-12-07 Pioneer Hi-Bred International, Inc. Methods for modulating water-use efficiency or productivity in a plant
WO2001004147A2 (en) 1999-07-12 2001-01-18 E.I. Du Pont De Nemours And Company Plant inositol polyphosphate phosphatase homologs
US6177616B1 (en) 1996-12-16 2001-01-23 Hoecsht Schering Agrevo Gmbh Genes coding for amino acid deacetylases with specificity for N-acetyl-L-phosphinothricin, their isolation and their use
US6177611B1 (en) 1998-02-26 2001-01-23 Pioneer Hi-Bred International, Inc. Maize promoters
US6177275B1 (en) 1996-07-24 2001-01-23 New York University Plant nitrogen regulatory P-PII genes
WO2001012731A1 (en) 1999-08-19 2001-02-22 Ppg Industries Ohio, Inc. Hydrophobic particulate inorganic oxides and polymeric compositions containing same
WO2001012825A1 (en) 1999-08-13 2001-02-22 Syngenta Participations Ag Herbicide-tolerant protoporphyrinogen oxidase
WO2001012800A2 (en) 1999-08-16 2001-02-22 E.I. Du Pont De Nemours And Company Method for the production of calendic acid, a fatty acid containing delta-8,10,12 conjugated double bonds and dimorphecolic acid, a fatty acid containing a 9-hydroxy group and delta-10,12 conjugated double bonds
US6197561B1 (en) 1997-07-22 2001-03-06 Pioneer Hi-Bred International, Inc. Genes controlling phytate metabolism in plants and uses thereof
WO2001021822A1 (en) 1999-09-17 2001-03-29 Plant Bioscience Limited Methods and means for modification of plant flowering characteristics
WO2001023401A2 (en) 1999-09-28 2001-04-05 Maxygen, Inc. Use of codon-varied oligonucleotide synthesis for synthetic sequence recombination
WO2001026459A2 (en) 1999-10-12 2001-04-19 Mendel Biotechnology, Inc. Flowering time modification
WO2001029237A2 (en) 1999-10-15 2001-04-26 Aventis Cropscience N.V. Conditional sterility in wheat
US6225529B1 (en) 1998-08-20 2001-05-01 Pioneer Hi-Bred International, Inc. Seed-preferred promoters
WO2001034726A2 (en) 1999-10-21 2001-05-17 Fluor Corporation Methods and apparatus for high propane recovery
WO2001036444A1 (en) 1999-11-17 2001-05-25 Mendel Biotechnology, Inc. Plant developmental genes
WO2001036596A2 (en) 1999-11-17 2001-05-25 Pioneer Hi-Bred International, Inc. Modulation of plant response to abscisic acid
US6248535B1 (en) 1999-12-20 2001-06-19 University Of Southern California Method for isolation of RNA from formalin-fixed paraffin-embedded tissue specimens
US6284948B1 (en) 1998-05-18 2001-09-04 Pioneer Hi-Bred International, Inc. Genes and methods for control of nematodes in plants
WO2001064898A2 (en) 2000-02-29 2001-09-07 E.I. Dupont De Nemours And Company Genes encoding denitrification enzymes
WO2001066704A2 (en) 2000-03-09 2001-09-13 Monsanto Technology Llc Methods for making plants tolerant to glyphosate and compositions thereof
US6291224B1 (en) 1998-07-17 2001-09-18 Pioneer Hi-Bred International, Inc. Genes controlling phytate metabolism in plants and uses thereof
US6297426B1 (en) 1990-06-12 2001-10-02 Pioneer Hi-Bred International, Inc. Methods of mediating female fertility in plants
WO2001079516A2 (en) 2000-04-14 2001-10-25 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US6323392B1 (en) 1999-03-01 2001-11-27 Pioneer Hi-Bred International, Inc. Formation of brassica napus F1 hybrid seeds which exhibit a highly elevated oleic acid content and a reduced linolenic acid content in the endogenously formed oil of the seeds
US6326351B1 (en) 1996-09-24 2001-12-04 Monsanto Technology Llc Bacillus thuringiensis CryET33 and CryET34 compositions and uses therefor
WO2002000904A2 (en) 2000-06-23 2002-01-03 E. I. Du Pont De Nemours And Company Recombinant constructs and their use in reducing gene expression
WO2002000894A2 (en) 2000-06-30 2002-01-03 Cropdesign N.V. Gene silencing vector
WO2002002776A1 (en) 2000-07-06 2002-01-10 Institute Of Microbiology, Chinese Academy Of Sciences A method of improving nitrogen assimilation efficiency in plants
US6338961B1 (en) 1995-07-19 2002-01-15 Rhone-Poulenc Agrochimie Isolated DNA sequence capable of serving as regulatory element in a chimeric gene which can be used for the transformation of plants
US6340593B1 (en) 1998-10-23 2002-01-22 Mycogen Corporation Plant-optimized polynucleotides encoding approximately 15 kDa and approximately 45 kDa pesticidal proteins
EP1173580A1 (en) 1999-04-29 2002-01-23 Syngenta Limited Herbicide resistant plants
EP1173582A1 (en) 1999-04-29 2002-01-23 Syngenta Limited Herbicide resistant plants
EP1173581A1 (en) 1999-04-29 2002-01-23 Syngenta Limited Herbicide resistant plants
US6346403B1 (en) 1998-09-08 2002-02-12 E.I. Du Pont De Nemours And Company Methionine metabolic enzymes
WO2002015675A1 (en) 2000-08-22 2002-02-28 Mendel Biotechnology, Inc. Genes for modifying plant traits iv
WO2002017430A1 (en) 2000-08-22 2002-02-28 Paratek Microwave, Inc. Combline filters with tunable dielectric capacitors
US6365377B1 (en) 1999-03-05 2002-04-02 Maxygen, Inc. Recombination of insertion modified nucleic acids
US6372965B1 (en) 1992-11-17 2002-04-16 E.I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and hydroxylases from plants
US6388171B1 (en) 1999-07-12 2002-05-14 Pioneer Hi-Bred International, Inc. Compositions and methods for fumonisin detoxification
US6391348B1 (en) 1998-11-20 2002-05-21 Pioneer Hi-Bred International, Inc. Method of reducing cholesterol in eggs
WO2002042424A2 (en) 2000-11-22 2002-05-30 Pioneer Hi-Bred International, Inc. Seed-preferred regulatory elements and uses thereof
US6423886B1 (en) 1999-09-02 2002-07-23 Pioneer Hi-Bred International, Inc. Starch synthase polynucleotides and their use in the production of new starches
WO2002057439A2 (en) 2000-10-24 2002-07-25 E. I. Du Pont De Nemours And Company Plant transcription factors
WO2002059324A2 (en) 2001-01-12 2002-08-01 Pioneer Hi-Bred International, Inc. Novel inositol polyphosphate kinase genes and uses thereof
US6441274B1 (en) 1999-06-16 2002-08-27 E. I. Du Pont De Nemours & Company Plant tryptophan synthase beta subunit
US6459019B1 (en) 1992-03-19 2002-10-01 E.I. Du Pont De Nemours And Company Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants
JP2002281975A (en) 2001-03-28 2002-10-02 Yamaguchi Technology Licensing Organization Ltd Gene belonging to soy nitrate transporter 1 gene family
WO2002077185A2 (en) 2001-03-27 2002-10-03 Mendel Biotechnology, Inc. Methods for modifying flowering phenotypes
WO2002079403A2 (en) 2001-03-30 2002-10-10 Mendel Biotechnology, Inc. Method for modifying plant biomass
US6468523B1 (en) 1998-11-02 2002-10-22 Monsanto Technology Llc Polypeptide compositions toxic to diabrotic insects, and methods of use
WO2003000863A2 (en) 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Defensin polynucleotides and methods of use
WO2003011015A2 (en) 2001-08-02 2003-02-13 Pioneer Hi-Bred International, Inc. Methods for improving seed and grain characteristics
WO2003013228A2 (en) 2001-08-09 2003-02-20 Mendel Biotechnology, Inc. Biochemistry-related polynucleotides and polypeptides in plants
US6531316B1 (en) 1999-03-05 2003-03-11 Maxyag, Inc. Encryption of traits using split gene sequences and engineered genetic elements
US6531648B1 (en) 1998-12-17 2003-03-11 Syngenta Participations Ag Grain processing method and transgenic plants useful therein
US6538177B1 (en) 1998-07-15 2003-03-25 Pioneer Hi-Bred International, Inc. Compositions and methods for fumonisin detoxification
WO2003027243A2 (en) 2001-09-27 2003-04-03 Pioneer Hi-Bred International, Inc. Phytate polynucleotides and methods of use
WO2003033651A2 (en) 2001-10-16 2003-04-24 Pioneer Hi-Bred International, Inc. Compositions and methods for promoting nematode resistance in plants
US6563020B1 (en) 1999-03-24 2003-05-13 Pioneer Hi-Bred International, Inc. Maize chitinases and their use in enhancing disease resistance in crop plants
US6566587B1 (en) 1995-07-19 2003-05-20 Bayer Cropscience S.A. Mutated 5-enolpyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene
WO2003052063A2 (en) 2001-12-14 2003-06-26 The Nitrate Elimination Company, Inc. Simplified eukaryotic nitrate reductase
US20030135879A1 (en) 1997-04-04 2003-07-17 Board Of Regents Of University Of Nebraska. Methods and materials for making and using transgenic dicamba-degrading organisms
US20030150014A1 (en) 1998-08-17 2003-08-07 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US20030163838A1 (en) 1998-08-17 2003-08-28 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US20030166197A1 (en) 2001-05-10 2003-09-04 Ecker Joseph R. Ethylene insensitive plants
WO2003082899A2 (en) 2002-03-22 2003-10-09 E.I. Dupont De Nemours & Company Compositions and methods for altering tocotrienol content
US20030204870A1 (en) 2001-06-29 2003-10-30 Allen Stephen M. Alteration of oil traits in plants
US6664445B1 (en) 1997-06-06 2003-12-16 E. I. Du Pont De Nemours And Company Plant amino acid biosynthetic enzymes
US6664446B2 (en) 1999-03-23 2003-12-16 Mendel Biotechnology, Inc. Transgenic plants comprising polynucleotides encoding transcription factors that confer disease tolerance
US6706866B1 (en) 1996-09-04 2004-03-16 Michigan State University Plant having altered environmental stress tolerance
US6713259B2 (en) 2000-09-13 2004-03-30 Monsanto Technology Llc Corn event MON810 and compositions and methods for detection thereof
US6713663B2 (en) 1998-04-15 2004-03-30 The Salk Institute For Biological Studies Flowering locus T (FT) and genetically modified plants having delayed flower development
US6713063B1 (en) 1996-11-20 2004-03-30 Monsanto Technology, Llc Broad-spectrum δ-endotoxins
US20040068767A1 (en) 1998-08-17 2004-04-08 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
WO2004031349A2 (en) 2002-09-18 2004-04-15 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
US20040078852A1 (en) 2002-08-02 2004-04-22 Thomashow Michael F. Transcription factors to improve plant stress tolerance
US20040098764A1 (en) 2001-03-16 2004-05-20 Heard Jacqueline E. Plant transcriptional regulators of abiotic stress
US20040128719A1 (en) 2002-06-21 2004-07-01 Klee Harry J. Materials and methods for tissue-specific targeting of ethylene insensitivity in transgenic plants
US6777592B2 (en) 2000-04-14 2004-08-17 E.I. Du Pont Denemours And Company Arthropod defensins of Scolopendra canidens, Vaejovis carolinianus, and Argiope spp.
WO2004074462A2 (en) 2003-02-20 2004-09-02 Athenix Corporation Delta-endotoxin genes and methods for their use
WO2004076638A2 (en) 2003-02-25 2004-09-10 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
US20040197917A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-014, delta-endotoxin gene and methods for its use
US20040197916A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-004, a delta-endotoxin gene and methods for its use
US6803498B2 (en) 1998-08-17 2004-10-12 Pioneer Hi-Bred Int'l. Inc. Maize cellulose synthases and uses thereof
US20040210965A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-007, a delta-endotoxin gene and methods for its use
WO2004090143A2 (en) 2003-04-04 2004-10-21 Pioneer Hi-Bred International, Inc. Modulation of cytokinin activity in plants
US20040210964A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-009, a delta-endotoxin gene and methods for its use
US20040216186A1 (en) 2003-02-20 2004-10-28 Athenix Corporation AXMI-006, a delta-endotoxin gene and methods for its use
US6812380B2 (en) 2001-03-27 2004-11-02 Pioneer Hi-Bred International, Inc. Compositions and methods of zearalenone detoxification
US6825397B1 (en) 1998-11-09 2004-11-30 Pioneer Hi-Bred International, Inc. LEC1 trancriptional activator nucleic acids and methods of use thereof
US20040250311A1 (en) 2003-02-20 2004-12-09 Athenix Corporation AXMI-008, a delta-endotoxin gene and methods for its use
US6855865B2 (en) 1999-05-07 2005-02-15 E.I. Du Pont De Nemours And Company Nucleic acids encoding plant defensins and methods of use thereof
US6858778B1 (en) 2000-11-07 2005-02-22 Pioneer Hi-Bred International, Inc. Plants transformed with a DNA construct comprising a nucleic acid molecule encoding an 18 kD α-globulin
WO2005021585A2 (en) 2003-08-28 2005-03-10 Athenix Corporation Axmi-003, a delta-endotoxin gene and methods for its use
WO2005038032A1 (en) 2003-10-14 2005-04-28 Athenix Corporation Axmi-010, a delta-endotoxin gene and methods for its use
US6891085B2 (en) 2001-04-20 2005-05-10 Pioneer Hi-Bred International, Inc. Nucleic acid encoding the FUS6 antimicrobial polypeptide of Agrotis ipsilon and its use to enhance disease resistance in a plant
US6893826B1 (en) 2000-11-17 2005-05-17 Monsanto Technology Llc Cotton event PV-GHBK04 (757) and compositions and methods for detection thereof
US20050160488A1 (en) 2000-11-07 2005-07-21 Pioneer Hi-Bred International, Inc. Grain quality through altered expression of seed proteins
US20050204418A1 (en) 2000-11-07 2005-09-15 Pioneer Hi-Bred International, Inc. Grain quality through altered expression of seed proteins
US6962705B2 (en) 1996-11-20 2005-11-08 Monsanto Technolgy Llc Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity
WO2005107437A2 (en) 2004-04-30 2005-11-17 Dow Agrosciences Llc Novel herbicide resistance genes
WO2005110068A2 (en) 2004-04-09 2005-11-24 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
WO2005113778A2 (en) 2004-05-20 2005-12-01 Pioneer Hi-Bred International, Inc. Maize multidrug resistance-associated protein polynucleotides and methods of use
US6992237B1 (en) 1999-04-16 2006-01-31 Pioneer Hi-Bred International Inc. Regulated expression of genes in plant seeds
US7064249B2 (en) 1998-11-04 2006-06-20 Monsanto Technology Llc Plants transformed to express Cry2A δ-endotoxins
US7087810B2 (en) 2001-11-07 2006-08-08 Muller Mathis L Isolated nucleic acids encoding proteins with chitinase activity and uses thereof
WO2006083891A2 (en) 2005-01-31 2006-08-10 Athenix Corporation Axmi-018, axmi-020, and axmi-021, a family of delta-endotoxin genes and methods for their use
US20060191034A1 (en) 2003-07-07 2006-08-24 Baum James A Insecticidal proteins secreted from bacillus thuringiensis and uses therefor
US7105332B2 (en) 2002-06-26 2006-09-12 E.I. Du Pont De Nemours And Company Genes encoding proteins with pesticidal activity
WO2006119457A1 (en) 2005-05-02 2006-11-09 Athenix Corporation Axmi-028 and axmi-029, family of novel delta-endotoxin genes and methods for their use
US7145060B2 (en) 2001-11-07 2006-12-05 Pioneer Hi-Bred International, Inc. Nucleic acid encoding a chitinase and methods of using it to make fungal resistant plants
US7179965B2 (en) 2004-03-26 2007-02-20 Dow Agrosciences Llc Cry1F and Cry1Ac transgenic cotton lines and event-specific identification thereof
US7193133B2 (en) 2004-06-09 2007-03-20 Michael Lassner Plastid transit peptides
US7205453B2 (en) 2003-09-25 2007-04-17 Pioneer Hi-Bred International, Inc. Crop plant cystatin proteinase inhibitors encoding nucleic acids and methods of use
US7208474B2 (en) 2004-02-25 2007-04-24 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
WO2007074405A2 (en) 2005-09-16 2007-07-05 Devgen Nv Dsrna as insect control agent
US7244820B2 (en) 2001-03-30 2007-07-17 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7288643B2 (en) 2003-05-02 2007-10-30 Pioneer Hi-Bred International, Inc. Corn event TC1507 and methods for detection thereof
US7294760B2 (en) 2003-03-28 2007-11-13 Monsanto Technology Llc Plant promoters for use in early seed development
US7301069B2 (en) 2004-06-30 2007-11-27 Pioneer Hi-Bred International, Inc. Methods of protecting plants from pathogenic fungi and nematodes
US20070277263A1 (en) 2006-05-25 2007-11-29 Hexima Ltd. Multi-gene expression vehicle
US7306946B2 (en) 2004-07-02 2007-12-11 Pioneer Hi-Bred International, Inc. Antifungal polypeptides
US7323556B2 (en) 2004-09-29 2008-01-29 Pioneer Hi-Bred International, Inc. Corn event DAS-59122-7 and methods for detection thereof
US7329736B2 (en) 2006-04-14 2008-02-12 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7405074B2 (en) 2004-04-29 2008-07-29 Pioneer Hi-Bred International, Inc. Glyphosate-N-acetyltransferase (GAT) genes
US20080234130A1 (en) 2005-08-24 2008-09-25 Pioneer Hi-Bred International, Inc. Compositions providing tolerance to multiple herbicides and methods of use thereof
US7449552B2 (en) 2006-04-14 2008-11-11 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US20080295207A1 (en) 2007-04-27 2008-11-27 Monsanto Technology Llc Hemipteran-and Coleopteran Active Toxin Proteins from Bacillus Thuringiensis
US7462760B2 (en) 2002-06-26 2008-12-09 Pioneer Hi-Bred International, Inc. Genes encoding plant protease-resistant pesticidal proteins and method of their use
US7462481B2 (en) 2000-10-30 2008-12-09 Verdia, Inc. Glyphosate N-acetyltransferase (GAT) genes
US7468278B2 (en) 2006-07-21 2008-12-23 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis gene with coleopteran activity
US20090035765A1 (en) 2005-04-04 2009-02-05 E.I. Du Pont De Nemours And Company Polynucleotides and methods for making plants resistant to fungal pathogens
US20090044298A1 (en) 2005-09-16 2009-02-12 Bayer Cropscience S.A. Transplastomic Plants Expressing Lumen-Targeted Protein
US7491698B2 (en) 2003-01-21 2009-02-17 Dow Agrosciences Llc Mixing and matching TC proteins for pest control
US7491869B2 (en) 2004-02-20 2009-02-17 Pioneer Hi-Bred International, Inc. Methods of using non-plant insecticidal lipase encoding nucleic acids in transgenic plants
US20090064373A1 (en) 2007-08-29 2009-03-05 Graziana Taramino Plants with altered root architecture, related constructs and methods involving genes encoding nucleoside diphosphatase kinase (ndk) polypeptides and homologs thereof
US7521235B2 (en) 2006-07-21 2009-04-21 Pioneer Hi-Bred International, Inc. Unique novel Bacillus thuringiensis gene with Lepidopteran activity
US7531723B2 (en) 1999-04-16 2009-05-12 Pioneer Hi-Bred International, Inc. Modulation of cytokinin activity in plants
US20090144852A1 (en) 2007-10-16 2009-06-04 Athenix Corporation Axmi-066 and axmi-076: delta-endotoxin proteins and methods for their use
US20090144863A1 (en) 2005-03-08 2009-06-04 Basf Plant Science Gmbh Expression Enhancing Intron Sequences
US20090188008A1 (en) 2008-01-17 2009-07-23 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides
WO2009091864A2 (en) 2008-01-17 2009-07-23 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides from lygus
US7605304B2 (en) 2000-10-24 2009-10-20 E.I. Du Pont De Nemours And Company Genes encoding novel bacillus thuringiensis proteins with pesticidal activity against coleopterans
US7629504B2 (en) 2003-12-22 2009-12-08 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry9 nucleic acids
US20100005543A1 (en) 2008-06-25 2010-01-07 Athenix Corporation Toxin genes and methods for their use
US20100017914A1 (en) 2007-03-28 2010-01-21 Syngenta Participations Ag Insecticidal proteins
US20100024067A1 (en) 2006-12-15 2010-01-28 Cropdesign N.V. Plants having enhanced seed yield-related traits and a method for making the same
US7705216B2 (en) 2002-07-29 2010-04-27 Monsanto Technology Llc Corn event PV-ZMIR13 (MON863) plants and compositions and methods for detection thereof
US20100197592A1 (en) 2009-02-05 2010-08-05 Athenix Corporation Variant axmi-r1 delta endotoxin genes and methods for their use
US7772465B2 (en) 2007-06-26 2010-08-10 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis gene with lepidopteran activity
US7803943B2 (en) 2005-03-17 2010-09-28 Biotium, Inc. Methods of using dyes in association with nucleic acid staining or detection and associated technology
US20100257633A1 (en) 2007-06-20 2010-10-07 The Australian National University Method for improving stress resistance in plants and materials therefor
US20100287669A1 (en) 2009-04-14 2010-11-11 Pioneer Hi-Bred International, Inc. Modulation of acc synthase improves plant yield under low nitrogen conditions
US20100298211A1 (en) 2009-03-11 2010-11-25 Athenix Corporation Axmi-001, axmi-002, axmi-030, axmi-035, and axmi-045: toxin genes and methods for their use
US7858849B2 (en) 2006-12-08 2010-12-28 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US20110023184A1 (en) 2009-07-02 2011-01-27 Nalini Manoj Desai Axmi-205 pesticidal gene and methods for its use
US20110061133A1 (en) 2007-09-14 2011-03-10 Basf Plant Science Gmbh Plants having increased yield-related traits and a method for making the same
US20110064710A1 (en) 2009-09-11 2011-03-17 Benson Terry A Novel bacillus thuringiensis isolate
US7923602B2 (en) 2006-06-14 2011-04-12 Athenix Corp. AXMI-031, AXMI-039, AXMI-040 and AXMI-049, a family of novel delta endotoxin genes and methods for their use
US20110191904A1 (en) 2009-12-17 2011-08-04 Rajesh Kumar Plant Genes Associated With Seed Oil Content And Methods Of Their Use
WO2011103247A2 (en) 2010-02-18 2011-08-25 Athenix Corp. Axmi218, axmi219, axmi220, axmi226, axmi227, axmi228, axmi229, axmi230, and axmi231 delta-endotoxin genes and methods for their use
WO2011103248A2 (en) 2010-02-18 2011-08-25 Athenix Corp. AXMI221z, AXMI222z, AXMI223z, AXMI224z, AND AXMI225z DELTA-ENDOTOXIN GENES AND METHODS FOR THEIR USE
US20110263488A1 (en) 2006-06-15 2011-10-27 Athenix Corp. Family of pesticidal proteins and methods for their use
US20110277181A1 (en) 2009-10-30 2011-11-10 Ei Du Pont De Nemours And Company Drought tolerant plants and related constructs and methods involving genes encoding dtp21 polypeptides
US8058510B2 (en) 1998-07-17 2011-11-15 Bayer Bioscience N.V. Method and means to modulate programmed cell death in eukaryotic cells
US8058571B2 (en) 2008-10-08 2011-11-15 Research In Motion Limited Dual-action single-key mechanism
US8058515B2 (en) 1999-11-10 2011-11-15 University Of Connecticut Plant cells and plants overexpressing vacuolar proton pyrophosphatases
US20110283420A1 (en) 2009-01-28 2011-11-17 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and a method for making the same
US8063269B2 (en) 2003-12-18 2011-11-22 Alberta Research Council Inc. Transgenic plants with reduced level of saturated fatty acid and methods for making them
US20110301223A1 (en) 2010-06-04 2011-12-08 E.I. Du Pont De Nemours And Company Compositions and methods for insecticidal control of stinkbugs
US8084416B2 (en) 2008-12-23 2011-12-27 Athenix Corp. AXMI-150 delta-endotoxin gene and methods for its use
EP2404499A2 (en) 2004-06-16 2012-01-11 BASF Plant Science GmbH Nucleic acid molecules encoding Lipid Metabolism Protein (LMP) and methods of use in plants
US8097769B2 (en) 2005-07-18 2012-01-17 Basf Plant Science Gmbh Yield increase in plants overexpressing the ACCDP genes
US20120029750A1 (en) 2009-03-12 2012-02-02 Ford Global Technologies, Llc Auto-seek electrical connection for a plug-in hybrid electric vehicle
US20120066794A1 (en) 2009-05-28 2012-03-15 Nirmala Sharma Increased Seed Oil and Abiotic Stress Tolerance Mediated by HSI2
EP2431472A1 (en) 2005-07-06 2012-03-21 CropDesign N.V. Plant yield improvement by STE20-like gene expression
US20120079623A1 (en) 2010-09-27 2012-03-29 Pioneer Hi-Bred International, Inc. Yield Enhancement in Plants by Modulation of a ZM-ZFP1 Protein
WO2012038893A1 (en) 2010-09-24 2012-03-29 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and method for making the same
US20120079622A1 (en) 2010-09-24 2012-03-29 Pioneer Hi-Bred International, Inc. Yield Enhancement in Plants by Modulation of a ZM-LOBDP1 Protein
WO2012055982A2 (en) 2010-10-27 2012-05-03 Devgen Nv Down-regulating gene expression in insect pests
WO2012058266A1 (en) 2010-10-29 2012-05-03 Syngenta Participations Ag Overexpression of plant mirnas for parasite control
US20120110696A1 (en) 2006-08-07 2012-05-03 Jinrong Wan LysM Receptor-Like Kinases To Improve Plant Defense Response Against Fungal Pathogens
WO2012058528A2 (en) 2010-10-28 2012-05-03 E. I. Du Pont De Nemours And Company Drought tolerant plants and related constructs and methods involving genes encoding dtp6 polypeptides
US8173866B1 (en) 2008-01-11 2012-05-08 Pioneer Hi-Bred International, Inc. Modulation of plant xylan synthases
US20120198586A1 (en) 2010-12-30 2012-08-02 Dow Agrosciences Llc Nucleic acid molecules that target the vacuolar atpase h subunit and confer resistance to coleopteran pests
US8236757B2 (en) 2005-04-01 2012-08-07 Athenix Corp AXMI-027, AXMI-036 and AXMI-038, a family of delta-endotoxin genes and methods for their use
WO2012139004A2 (en) 2011-04-07 2012-10-11 Monsanto Technology Llc Insect inhibitory toxin family active against hemipteran and/or lepidopteran insects
US20120272352A1 (en) 2009-10-30 2012-10-25 Syngenta Participations Ag Genes Conferring Drought and Salt Tolerance and Uses Thereof
US20120278954A1 (en) 2011-02-11 2012-11-01 Bowen David J Pesticidal Nucleic Acids and Proteins and Uses Thereof
US8304604B2 (en) 2009-04-17 2012-11-06 Dow Agrosciences, Llc. DIG-3 insecticidal Cry toxins
US8304605B2 (en) 2009-06-16 2012-11-06 Dow Agrosciences, Llc. DIG-11 insecticidal cry toxins
US20120297501A1 (en) 2011-04-20 2012-11-22 Devgen Nv Plants resistant to insect pests
US8318900B2 (en) 2009-02-27 2012-11-27 Athenix Corp. Pesticidal proteins and methods for their use
US8319019B2 (en) 2009-01-23 2012-11-27 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
US20120304336A1 (en) 2011-05-23 2012-11-29 E.I. Du Pont De Nemours And Company Chloroplast transit peptides and methods of their use
US20120311745A1 (en) 2009-12-16 2012-12-06 Dow Agrosciences Llc Combined use of cry1ca and cry1ab proteins for insect resistance management
US20120311746A1 (en) 2009-12-16 2012-12-06 Dow Agroscience Llc Insect resistance management with combinations of cry1be and cry1f proteins
US20120317681A1 (en) 2009-12-16 2012-12-13 Thomas Meade COMBINED USE OF CRY1Ca AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20120317682A1 (en) 2009-12-16 2012-12-13 Dow Agrosciences Llc Combined use of vip3ab and cry1fa for management of resistant insects
US8334366B1 (en) 2009-04-29 2012-12-18 The United States Of America, As Represented By The Secretary Of Agriculture Mutant lycotoxin-1 peptide sequences for insecticidal and cell membrane altering properties
US8334431B2 (en) 2008-07-02 2012-12-18 Athenix Corporation AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184: insecticidal proteins and methods for their use
US20120324605A1 (en) 2009-12-16 2012-12-20 Dow Agrosciences Llc Insectcidal protein combinations for controlling fall armyworm and european corn borer, and methods for insect resistance management
US8338661B2 (en) 2007-07-13 2012-12-25 Basf Plant Science Gmbh Transgenic plants with increased stress tolerance and yield
US8338152B2 (en) 2007-04-10 2012-12-25 E. I. Du Pont De Nemours And Company Delta-8 desaturases and their use in making polyunsaturated fatty acids
US20120331589A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc COMBINED USE OF CRY1Da AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20120331590A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc Use of cry1da in combination with cry1be for management of resistant insects
WO2013016617A1 (en) 2011-07-28 2013-01-31 Athenix Corp. Axmi205 variant proteins and methods for their use
US20130097728A1 (en) 2011-04-05 2013-04-18 Volker Heinrichs Axmi115 variant insecticidal gene and methods for its use

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7461900A (en) 1999-09-02 2001-03-26 Agresearch Limited Nucleotide sequences
US7244607B2 (en) 2003-10-01 2007-07-17 The United States Of America, As Represented By The Secretary Of Agriculture Chromobacterium subtsugae sp. nov. for control of insect pests
CA2811694A1 (en) 2010-09-22 2012-03-29 Bayer Intellectual Property Gmbh Use of biological or chemical control agents for controlling insects and nematodes in resistant crops
US9394345B2 (en) * 2013-03-15 2016-07-19 Pioneer Hi-Bred International, Inc. PHI-4 polypeptides and methods for their use

Patent Citations (500)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710511A (en) 1971-04-21 1973-01-16 Univ Illinois Procedures for use of genic male sterility in production of commercial hybrid maize
US3861709A (en) 1973-07-12 1975-01-21 Amsted Ind Inc Shiftable fifth wheel construction
US4196265A (en) 1977-06-15 1980-04-01 The Wistar Institute Method of producing antibodies
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US4535060A (en) 1983-01-05 1985-08-13 Calgene, Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase, production and use
US5094945A (en) 1983-01-05 1992-03-10 Calgene, Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthase, production and use
US4769061A (en) 1983-01-05 1988-09-06 Calgene Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthase, production and use
US5304732A (en) 1984-03-06 1994-04-19 Mgi Pharma, Inc. Herbicide resistance in plants
US4761373A (en) 1984-03-06 1988-08-02 Molecular Genetics, Inc. Herbicide resistance in plants
US5331107A (en) 1984-03-06 1994-07-19 Mgi Pharma, Inc. Herbicide resistance in plants
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US6130366A (en) 1984-12-28 2000-10-10 Plant Genetic Systems Chimaeric gene coding for a transit peptide and a heterologous polypeptide
US5723323A (en) 1985-03-30 1998-03-03 Kauffman; Stuart Alan Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof
US5817483A (en) 1985-03-30 1998-10-06 Stuart Kauffman Process for the production of stochastically-generated peptides,polypeptides or proteins having a predetermined property
US5814476A (en) 1985-03-30 1998-09-29 Stuart Kauffman Process for the production of stochastically-generated transcription or translation products
US5976862A (en) 1985-03-30 1999-11-02 Ixsys Corporation Process for obtaining DNA, RNA, peptides, polypeptides, or proteins, by recombinant DNA technique
US5824514A (en) 1985-03-30 1998-10-20 Stuart A. Kauffman Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides
US5569597A (en) 1985-05-13 1996-10-29 Ciba Geigy Corp. Methods of inserting viral DNA into plant material
US4654465A (en) 1985-07-18 1987-03-31 Agracetus Genic male-sterile maize
US5188642A (en) 1985-08-07 1993-02-23 Monsanto Company Glyphosate-resistant plants
US4940835A (en) 1985-10-29 1990-07-10 Monsanto Company Glyphosate-resistant plants
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5846818A (en) 1985-11-01 1998-12-08 Xoma Corporation Pectate lyase signal sequence
US4810648A (en) 1986-01-08 1989-03-07 Rhone Poulenc Agrochimie Haloarylnitrile degrading gene, its use, and cells containing the gene
EP0242246A1 (en) 1986-03-11 1987-10-21 Plant Genetic Systems N.V. Plant cells resistant to glutamine synthetase inhibitors, made by genetic engineering
US5648477A (en) 1986-03-11 1997-07-15 Plant Genetic Systems, N.V. Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
EP0242236A1 (en) 1986-03-11 1987-10-21 Plant Genetic Systems N.V. Plant cells resistant to glutamine synthetase inhibitors, made by genetic engineering
US5646024A (en) 1986-03-11 1997-07-08 Plant Genetic Systems, N.V. Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US5561236A (en) 1986-03-11 1996-10-01 Plant Genetic Systems Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US4975374A (en) 1986-03-18 1990-12-04 The General Hospital Corporation Expression of wild type and mutant glutamine synthetase in foreign hosts
US5107065A (en) 1986-03-28 1992-04-21 Calgene, Inc. Anti-sense regulation of gene expression in plant cells
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5879903A (en) 1986-08-23 1999-03-09 Hoechst Aktiengesellschaft Phosphinothricin-resistance gene, and its use
US5605011A (en) 1986-08-26 1997-02-25 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5378824A (en) 1986-08-26 1995-01-03 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5268463A (en) 1986-11-11 1993-12-07 Jefferson Richard A Plant promoter α-glucuronidase gene construct
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US4727219A (en) 1986-11-28 1988-02-23 Agracetus Genic male-sterile maize using a linked marker gene
US5608142A (en) 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
US4971908A (en) 1987-05-26 1990-11-20 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5145783A (en) 1987-05-26 1992-09-08 Monsanto Company Glyphosate-tolerant 5-endolpyruvyl-3-phosphoshikimate synthase
US5312910A (en) 1987-05-26 1994-05-17 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5013659A (en) 1987-07-27 1991-05-07 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5141870A (en) 1987-07-27 1992-08-25 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5316931A (en) 1988-02-26 1994-05-31 Biosource Genetics Corp. Plant viral vectors having heterologous subgenomic promoters for systemic expression of foreign genes
US5889190A (en) 1988-02-26 1999-03-30 Biosource Technologies, Inc. Recombinant plant viral nucleic acids
US5589367A (en) 1988-02-26 1996-12-31 Biosource Technologies, Inc. Recombinant plant viral nucleic acids
US5866785A (en) 1988-02-26 1999-02-02 Biosource Technologies, Inc. Recombinant plant viral nucleic acids
EP0333033A1 (en) 1988-03-09 1989-09-20 Meiji Seika Kaisha Ltd. Glutamine synthesis gene and glutamine synthetase
US5886244A (en) 1988-06-10 1999-03-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
US5039523A (en) 1988-10-27 1991-08-13 Mycogen Corporation Novel Bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin
US5023179A (en) 1988-11-14 1991-06-11 Eric Lam Promoter enhancer element for gene expression in plant roots
US5500365A (en) 1989-02-24 1996-03-19 Monsanto Company Synthetic plant genes
US5880275A (en) 1989-02-24 1999-03-09 Monsanto Company Synthetic plant genes from BT kurstaki and method for preparation
US5110732A (en) 1989-03-14 1992-05-05 The Rockefeller University Selective gene expression in plants
US5240855A (en) 1989-05-12 1993-08-31 Pioneer Hi-Bred International, Inc. Particle gun
US5879918A (en) 1989-05-12 1999-03-09 Pioneer Hi-Bred International, Inc. Pretreatment of microprojectiles prior to using in a particle gun
US5188960A (en) 1989-06-27 1993-02-23 Mycogen Corporation Bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins
US5310667A (en) 1989-07-17 1994-05-10 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
WO1991014778A2 (en) 1990-03-20 1991-10-03 Ecogen Inc. BACILLUS THURINGIENSIS cryIIIC GENE AND PROTEIN TOXIC TO COLEOPTERAN INSECTS
US5466785A (en) 1990-04-12 1995-11-14 Ciba-Geigy Corporation Tissue-preferential promoters
US5874265A (en) 1990-04-17 1999-02-23 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof
US5969213A (en) 1990-04-17 1999-10-19 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof
US5489520A (en) 1990-04-17 1996-02-06 Dekalb Genetics Corporation Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
US5919675A (en) 1990-04-17 1999-07-06 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5550318A (en) 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US6297426B1 (en) 1990-06-12 2001-10-02 Pioneer Hi-Bred International, Inc. Methods of mediating female fertility in plants
US6265640B1 (en) 1990-06-12 2001-07-24 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating fertility and method of using same
US5824524A (en) 1990-06-12 1998-10-20 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating fertility and method of using same
US5432068A (en) 1990-06-12 1995-07-11 Pioneer Hi-Bred International, Inc. Control of male fertility using externally inducible promoter sequences
US5478369A (en) 1990-06-12 1995-12-26 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating male fertility and method of using same
US5859341A (en) 1990-06-12 1999-01-12 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating fertility and method of using same
US5850014A (en) 1990-06-12 1998-12-15 Pioneer Hi-Bred International Nucleotide sequences mediating ferility and method of using same
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US5463175A (en) 1990-06-25 1995-10-31 Monsanto Company Glyphosate tolerant plants
US5776760A (en) 1990-06-25 1998-07-07 Monsanto Company Glyphosate tolerant plants
US5627061A (en) 1990-08-31 1997-05-06 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5804425A (en) 1990-08-31 1998-09-08 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US6248876B1 (en) 1990-08-31 2001-06-19 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5633435A (en) 1990-08-31 1997-05-27 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US6225114B1 (en) 1990-09-28 2001-05-01 Monsanto Company Modified gene encoding glyphosate-tolerant 5-enolpruvyl-3-phosphoshikimate synthase
US5266317A (en) 1990-10-04 1993-11-30 University Of Georgia Research Foundation, Inc. Insect-specific paralytic neurotoxin genes for use in biological insect control: methods and compositions
EP0480762A2 (en) 1990-10-12 1992-04-15 Mycogen Corporation Novel bacillus thuringiensis isolates active against dipteran pests
US5932782A (en) 1990-11-14 1999-08-03 Pioneer Hi-Bred International, Inc. Plant transformation method using agrobacterium species adhered to microprojectiles
US5459252A (en) 1991-01-31 1995-10-17 North Carolina State University Root specific gene promoter
WO1992013957A1 (en) 1991-02-07 1992-08-20 Plant Genetic Systems, N.V. Stamen-specific promoters from corn
WO1992013956A1 (en) 1991-02-08 1992-08-20 Plant Genetic Systems, N.V. Stamen-specific promoters from rice
US5939599A (en) 1991-02-14 1999-08-17 E. I. Du Pont De Nemours And Company High sulfur seed protein gene and method for increasing the sulfur amino acid contact of plants
US5633448A (en) 1991-03-05 1997-05-27 Rhone-Poulenc Agrochimie Chimeric gene for the transformation of plants
US5491288A (en) 1991-03-05 1996-02-13 Rhone Poulenc Agrochimie Chimeric gene comprising the arabidopsis histone H4 promoter for the transformation of plants
US5510471A (en) 1991-03-05 1996-04-23 Rhone-Poulenc Agrochimie Chimeric gene for the transformation of plants
US5399680A (en) 1991-05-22 1995-03-21 The Salk Institute For Biological Studies Rice chitinase promoter
WO1993002197A1 (en) 1991-07-23 1993-02-04 Nickerson Biocem Limited CALLASE-RELATED DNAs AND THEIR USE IN ARTIFICIAL MALE STERILITY
US5731180A (en) 1991-07-31 1998-03-24 American Cyanamid Company Imidazolinone resistant AHAS mutants
US5767361A (en) 1991-07-31 1998-06-16 American Cyanamid Company Imidazolinone resistant AHAS mutants
US5559223A (en) 1991-08-09 1996-09-24 E. I. Dupont De Nemours And Company Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants
US5604121A (en) 1991-08-27 1997-02-18 Agricultural Genetics Company Limited Proteins with insecticidal properties against homopteran insects and their use in plant protection
US5750386A (en) 1991-10-04 1998-05-12 North Carolina State University Pathogen-resistant transgenic plants
US5436391A (en) 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
WO1993011245A1 (en) 1991-12-04 1993-06-10 E.I. Du Pont De Nemours And Company Fatty acid desaturase genes from plants
US5324646A (en) 1992-01-06 1994-06-28 Pioneer Hi-Bred International, Inc. Methods of regeneration of Medicago sativa and expressing foreign DNA in same
US6459019B1 (en) 1992-03-19 2002-10-01 E.I. Du Pont De Nemours And Company Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants
WO1993019181A1 (en) 1992-03-25 1993-09-30 Sandoz Ltd. Biological material
US5428148A (en) 1992-04-24 1995-06-27 Beckman Instruments, Inc. N4 - acylated cytidinyl compounds useful in oligonucleotide synthesis
US5401836A (en) 1992-07-16 1995-03-28 Pioneer Hi-Bre International, Inc. Brassica regulatory sequence for root-specific or root-abundant gene expression
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5743477A (en) 1992-08-27 1998-04-28 Dowelanco Insecticidal proteins and method for plant protection
US6372965B1 (en) 1992-11-17 2002-04-16 E.I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and hydroxylases from plants
US5496714A (en) 1992-12-09 1996-03-05 New England Biolabs, Inc. Modification of protein by use of a controllable interveining protein sequence
US5834247A (en) 1992-12-09 1998-11-10 New England Biolabs, Inc. Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein
US5889191A (en) 1992-12-30 1999-03-30 Biosource Technologies, Inc. Viral amplification of recombinant messenger RNA in transgenic plants
US5607914A (en) 1993-01-13 1997-03-04 Pioneer Hi-Bred International, Inc. Synthetic antimicrobial peptides
US5990389A (en) 1993-01-13 1999-11-23 Pioneer Hi-Bred International, Inc. High lysine derivatives of α-hordothionin
US5633436A (en) 1993-03-02 1997-05-27 E. I. Du Pont De Nemours And Company Feedcrops enriched in sulfur amino acids and methods for improvements
US5633436C1 (en) 1993-03-02 2001-01-30 Du Pont Feedcrops enriched in sulfur amino acids and methods for improvements
US6137033A (en) 1993-03-25 2000-10-24 Novartis Finance Corporation Class of proteins for the control of plant pests
US5877012A (en) 1993-03-25 1999-03-02 Novartis Finance Corporation Class of proteins for the control of plant pests
US6107279A (en) 1993-03-25 2000-08-22 Novartis Finance Corporation Class of proteins for the control of plant pests
US5789156A (en) 1993-06-14 1998-08-04 Basf Ag Tetracycline-regulated transcriptional inhibitors
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
US6107547A (en) 1993-10-06 2000-08-22 New York University Transgenic plants that exhibit enhanced nitrogen assimilation
WO1995015392A1 (en) 1993-11-30 1995-06-08 E.I. Du Pont De Nemours And Company Chimeric geens and methods for increasing the lysine content of the seeds of corn, soybean and rapeseed plants
US5580852A (en) 1993-12-17 1996-12-03 Pioneer Hi-Bred International, Inc. Derivatives of tachyplesin having inhibitory activity towards plant pathogenic fungi
WO1995016776A1 (en) 1993-12-17 1995-06-22 Pioneer Hi-Bred International, Inc. Derivatives of tachyplesin having inhibitory activity towards plant pathogenic fungi
US5689052A (en) 1993-12-22 1997-11-18 Monsanto Company Synthetic DNA sequences having enhanced expression in monocotyledonous plants and method for preparation thereof
WO1995018855A2 (en) 1994-01-07 1995-07-13 Pioneer Hi-Bred International, Inc. Synthetic antimicrobial peptides
US5811238A (en) 1994-02-17 1998-09-22 Affymax Technologies N.V. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5830721A (en) 1994-02-17 1998-11-03 Affymax Technologies N.V. DNA mutagenesis by random fragmentation and reassembly
WO1995022625A1 (en) 1994-02-17 1995-08-24 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
EP0752008A1 (en) 1994-02-17 1997-01-08 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5912414A (en) 1994-05-13 1999-06-15 E. I. Du Pont De Nemours And Company Nucleic acid fragments, chimeric genes and methods for increasing the methionine content of the seeds of plants
US5633363A (en) 1994-06-03 1997-05-27 Iowa State University, Research Foundation In Root preferential promoter
US6288306B1 (en) 1994-06-16 2001-09-11 Syngenta Investment Corporation Methods of selecting plants, plant tissue or plant cells resistant to a protoporphyrinogen oxidase inhibitor
US6282837B1 (en) 1994-06-16 2001-09-04 Novartis Finance Corporation Methods of controlling the growth of undesired vegetation with herbicide tolerant plants or plant seeds having altered protoporphyrinogen oxidase activity
US5767373A (en) 1994-06-16 1998-06-16 Novartis Finance Corporation Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms
WO1996001905A1 (en) 1994-07-08 1996-01-25 E.I. Du Pont De Nemours And Company Chimeric genes and method for increasing the threonine content of the seeds of plants
US5736369A (en) 1994-07-29 1998-04-07 Pioneer Hi-Bred International, Inc. Method for producing transgenic cereal plants
US5716820A (en) 1994-08-12 1998-02-10 Pioneer Hi-Bred International, Inc. Fumonisin detoxification enzymes
US5608144A (en) 1994-08-12 1997-03-04 Dna Plant Technology Corp. Plant group 2 promoters and uses thereof
US5792931A (en) 1994-08-12 1998-08-11 Pioneer Hi-Bred International, Inc. Fumonisin detoxification compositions and methods
WO1996014414A1 (en) 1994-11-02 1996-05-17 John Innes Centre Innovations Limited Genetic control of flowering
US5659026A (en) 1995-03-24 1997-08-19 Pioneer Hi-Bred International ALS3 promoter
WO1996030517A1 (en) 1995-03-31 1996-10-03 Commonwealth Scientific And Industrial Research Organisation Genetic sequences conferring nematode resistance in plants and uses therefor
US5834252A (en) 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction
WO1996033207A1 (en) 1995-04-18 1996-10-24 Glaxo Group Limited End-complementary polymerase reaction
WO1996033270A1 (en) 1995-04-20 1996-10-24 American Cyanamid Company Structure-based designed herbicide resistant products
US5928937A (en) 1995-04-20 1999-07-27 American Cyanamid Company Structure-based designed herbicide resistant products
US6127600A (en) 1995-05-31 2000-10-03 Pioneer Hi-Bred International, Inc. Methods of increasing accumulation of essential amino acids in seeds
US5885801A (en) 1995-06-02 1999-03-23 Pioneer Hi-Bred International, Inc. High threonine derivatives of α-hordothionin
WO1996038560A2 (en) 1995-06-02 1996-12-05 John Innes Centre Innovations Limited Genetic control of flowering
US5885802A (en) 1995-06-02 1999-03-23 Pioneer Hi-Bred International, Inc. High methionine derivatives of α-hordothionin
US6566587B1 (en) 1995-07-19 2003-05-20 Bayer Cropscience S.A. Mutated 5-enolpyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene
US6338961B1 (en) 1995-07-19 2002-01-15 Rhone-Poulenc Agrochimie Isolated DNA sequence capable of serving as regulatory element in a chimeric gene which can be used for the transformation of plants
US5837876A (en) 1995-07-28 1998-11-17 North Carolina State University Root cortex specific gene promoter
WO1997010339A1 (en) 1995-09-13 1997-03-20 John Innes Centre Innovations Limited Flowering genes
US6573430B1 (en) 1995-09-13 2003-06-03 Plant Bioscience Limited Cen gene and a method for delaying flowering
US5689035A (en) 1995-09-26 1997-11-18 Pioneer Hi-Bred International, Inc. Brown stem rot resistance in soybeans
WO1997020078A1 (en) 1995-11-30 1997-06-05 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US6307126B1 (en) 1996-02-12 2001-10-23 Plant Bioscience Limited Nucleic acid encoding GAI gene of Arabidopsis thaliana
WO1997029123A2 (en) 1996-02-12 1997-08-14 Plant Bioscience Limited Nucleic acid encoding gai gene of arabidopsis thaliana
US6794560B2 (en) 1996-02-12 2004-09-21 Pioneer Hi-Bred International, Inc. Nucleic acid encoding GAI gene of Arabidopsis thaliana
US6084153A (en) 1996-02-14 2000-07-04 The Governors Of The University Of Alberta Plants having enhanced nitrogen assimilation/metabolism
US5850016A (en) 1996-03-20 1998-12-15 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
WO1997035966A1 (en) 1996-03-25 1997-10-02 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
EP0932670A1 (en) 1996-03-25 1999-08-04 Maxygen, Inc. Evolving cellular dna uptake by recursive sequence recombination
WO1997040162A2 (en) 1996-04-19 1997-10-30 Mycogen Corporation Pesticidal toxins
US6083499A (en) 1996-04-19 2000-07-04 Mycogen Corporation Pesticidal toxins
US6548291B1 (en) 1996-04-19 2003-04-15 Mycogen Corporation Pesticidal toxins
US6624145B1 (en) 1996-04-19 2003-09-23 Mycogen Corporation Pesticidal toxins
US6127180A (en) 1996-04-19 2000-10-03 Mycogen Corporation Pesticidal toxins
US6072050A (en) 1996-06-11 2000-06-06 Pioneer Hi-Bred International, Inc. Synthetic promoters
WO1997049811A1 (en) 1996-06-21 1997-12-31 Plant Bioscience Limited Genetic control of flowering
US6063947A (en) 1996-07-03 2000-05-16 Cargill, Incorporated Canola oil having increased oleic acid and decreased linolenic acid content
US6177275B1 (en) 1996-07-24 2001-01-23 New York University Plant nitrogen regulatory P-PII genes
US5892009A (en) 1996-09-04 1999-04-06 Michigan State University DNA and encoded protein which regulates cold and dehydration regulated genes
US5891859A (en) 1996-09-04 1999-04-06 Michigan State University Method for regulating cold and dehydration regulatory genes in a plant
US6417428B1 (en) 1996-09-04 2002-07-09 Michael F. Thomashow Plant having altered environmental stress tolerance
WO1998009521A1 (en) 1996-09-04 1998-03-12 Michigan State University Dna and encoded protein which regulates cold and dehydration regulated genes
US5965705A (en) 1996-09-04 1999-10-12 Board Of Trustees, Michigan State University DNA and encoded protein which regulates cold and dehydration regulated genes
US6706866B1 (en) 1996-09-04 2004-03-16 Michigan State University Plant having altered environmental stress tolerance
US6399330B1 (en) 1996-09-24 2002-06-04 Monsanto Technology Llc Bacillus thuringiensis cryet33 and cryet34 compositions and uses thereof
US6326351B1 (en) 1996-09-24 2001-12-04 Monsanto Technology Llc Bacillus thuringiensis CryET33 and CryET34 compositions and uses therefor
US7504229B2 (en) 1996-09-24 2009-03-17 Monsanto Technology Llc Methods for detecting Bacillus thuringiensis cryET33 and cryET34 polypeptides
US7385107B2 (en) 1996-09-24 2008-06-10 Monsanto Technologies Llc Insect-resistant transgenic plants transformed with CryET33 and CryET34-encoding nucleic acids
US6949626B2 (en) 1996-09-24 2005-09-27 Monsanto Technology Llc Bacillus thuringiensis cryET33 and cryET34 compositions and uses therefor
US6080913A (en) 1996-09-25 2000-06-27 Pioneer Hi-Bred International, Inc. Binary methods of increasing accumulation of essential amino acids in seeds
WO1998020133A2 (en) 1996-11-01 1998-05-14 Pioneer Hi-Bred International, Inc. Proteins with enhanced levels of essential amino acids
US7070982B2 (en) 1996-11-20 2006-07-04 Monsanto Technology Llc Polynucleotide compositions encoding broad spectrum delta-endotoxins
WO1998022604A1 (en) 1996-11-20 1998-05-28 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
US6232529B1 (en) 1996-11-20 2001-05-15 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
US6962705B2 (en) 1996-11-20 2005-11-08 Monsanto Technolgy Llc Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity
US6713063B1 (en) 1996-11-20 2004-03-30 Monsanto Technology, Llc Broad-spectrum δ-endotoxins
US5846812A (en) 1996-11-22 1998-12-08 Pioneer Hi-Bred International, Inc. Zearalenone detoxification compositions and methods
US6083736A (en) 1996-11-22 2000-07-04 Pioneer Hi-Bred International, Inc. Ochrobactrum anthropi bacteria which degrade moniliformin
US5798255A (en) 1996-11-22 1998-08-25 Pioneer Hi-Bred International, Inc. Beauvericin detoxification compositions and methods
US6033874A (en) 1996-11-27 2000-03-07 Ecogen, Inc. CRY1C polypeptides having improved toxicity to lepidopteran insects
US6177616B1 (en) 1996-12-16 2001-01-23 Hoecsht Schering Agrevo Gmbh Genes coding for amino acid deacetylases with specificity for N-acetyl-L-phosphinothricin, their isolation and their use
WO1998027230A1 (en) 1996-12-18 1998-06-25 Maxygen, Inc. Methods and compositions for polypeptide engineering
US5986177A (en) 1997-01-10 1999-11-16 Agricultural Genetic Engineering Research Institute Bacillus thuringiensis isolates with broad spectrum activity
WO1998031837A1 (en) 1997-01-17 1998-07-23 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US5981840A (en) 1997-01-24 1999-11-09 Pioneer Hi-Bred International, Inc. Methods for agrobacterium-mediated transformation
WO1998032326A2 (en) 1997-01-24 1998-07-30 Pioneer Hi-Bred International, Inc. Methods for $i(agrobacterium)-mediated transformation
WO1998036083A1 (en) 1997-02-14 1998-08-20 Plant Bioscience Limited Methods and means for gene silencing in transgenic plants
WO1998041653A1 (en) 1997-03-18 1998-09-24 Novo Nordisk A/S An in vitro method for construction of a dna library
WO1998041622A1 (en) 1997-03-18 1998-09-24 Novo Nordisk A/S Method for constructing a library using dna shuffling
WO1998042727A1 (en) 1997-03-21 1998-10-01 Sri International Sequence alterations using homologous recombination
WO1998042832A1 (en) 1997-03-25 1998-10-01 California Institute Of Technology Recombination of polynucleotide sequences using random or defined primers
WO1998042831A2 (en) 1997-03-27 1998-10-01 E.I. Du Pont De Nemours And Company Chimeric genes and methods for increasing the lysine content of the seeds of plants
US6040497A (en) 1997-04-03 2000-03-21 Dekalb Genetics Corporation Glyphosate resistant maize lines
US20030135879A1 (en) 1997-04-04 2003-07-17 Board Of Regents Of University Of Nebraska. Methods and materials for making and using transgenic dicamba-degrading organisms
WO1998045448A1 (en) 1997-04-08 1998-10-15 E.I. Du Pont De Nemours And Company Soybean plant producing seeds with reduced levels of raffinose saccharides and phytic acid
WO1998045458A1 (en) 1997-04-08 1998-10-15 E.I. Du Pont De Nemours And Company An engineered seed protein having a higher percentage of essential amino acids
US6048838A (en) 1997-05-05 2000-04-11 Dow Agrosciences Llc Insecticidal protein toxins from xenorhabdus
US6379946B1 (en) 1997-05-05 2002-04-30 Wisconsin Alumn Research Foundation Insecticidal protein toxins from Xenorhabdus
US6664445B1 (en) 1997-06-06 2003-12-16 E. I. Du Pont De Nemours And Company Plant amino acid biosynthetic enzymes
WO1998056935A2 (en) 1997-06-12 1998-12-17 E.I. Du Pont De Nemours And Company Plant amino acid biosynthetic enzymes
WO1998056918A1 (en) 1997-06-13 1998-12-17 Plant Bioscience Limited Methods and means for control of flowering in plants
WO1999005298A1 (en) 1997-07-22 1999-02-04 Pioneer Hi-Bred International, Inc. Genes controlling phytate metabolism in plants and uses thereof
US6197561B1 (en) 1997-07-22 2001-03-06 Pioneer Hi-Bred International, Inc. Genes controlling phytate metabolism in plants and uses thereof
WO1999009174A1 (en) 1997-08-13 1999-02-25 Plant Bioscience Limited Genetic control of plant growth and development
WO1999010498A2 (en) 1997-08-27 1999-03-04 Pioneer Hi-Bred International, Inc. Genes encoding enzymes for lignin biosynthesis and uses thereof
US5929305A (en) 1997-10-14 1999-07-27 Michigan State University Plant material containing non-naturally introduced binding protein for regulating cold and dehydration regulatory genes
WO1999021979A1 (en) 1997-10-28 1999-05-06 Maxygen, Inc. Human papillomavirus vectors
WO1999023107A1 (en) 1997-10-31 1999-05-14 Maxygen, Incorporated Modification of virus tropism and host range by viral genome shuffling
WO1999024581A2 (en) 1997-11-12 1999-05-20 Mycogen Corporation Plant-optimized genes encoding pesticidal toxins
US6218188B1 (en) 1997-11-12 2001-04-17 Mycogen Corporation Plant-optimized genes encoding pesticidal toxins
WO1999025821A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Compositions and methods for genetic modification of plants
WO1999025840A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. A novel method for the integration of foreign dna into eukaryoticgenomes
WO1999025854A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. A method for directional stable transformation of eukaryotic cells
WO1999025853A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Targeted manipulation of herbicide-resistance genes in plants
WO1999025855A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from t-dna using site-specific recombination systems
WO1999029902A1 (en) 1997-12-08 1999-06-17 California Institute Of Technology Method for creating polynucleotide and polypeptide sequences
WO1999029882A2 (en) 1997-12-10 1999-06-17 Pioneer Hi-Bred International, Inc. Methods for altering the nutritional value of a plant protein, vegetative storage protein, vsp, by altering the amino acid content of proteins
US7227056B2 (en) 1997-12-18 2007-06-05 Monsanto Technology Llc Coleopteran-resistant transgenic plants and methods of their production
US7544862B2 (en) 1997-12-18 2009-06-09 Monsanto Technology Llc Coleopteran-resistant transgenic plants and methods of their production
US6023013A (en) 1997-12-18 2000-02-08 Monsanto Company Insect-resistant transgenic plants
US6060594A (en) 1997-12-18 2000-05-09 Ecogen, Inc. Nucleic acid segments encoding modified bacillus thuringiensis coleopteran-toxic crystal proteins
US6063597A (en) 1997-12-18 2000-05-16 Monsanto Company Polypeptide compositions toxic to coleopteran insects
US6077824A (en) 1997-12-18 2000-06-20 Ecogen, Inc. Methods for improving the activity of δ-endotoxins against insect pests
WO1999031248A1 (en) 1997-12-18 1999-06-24 Ecogen, Inc. INSECT-RESISTANT TRANSGENIC PLANTS AND METHODS FOR IMPROVING δ-ENDOTOXIN ACTIVITY AGAINST TARGET INSECTS
US6620988B1 (en) 1997-12-18 2003-09-16 Monsanto Technology, Llc Coleopteran-resistant transgenic plants and methods of their production using modified Bacillus thuringiensis Cry3Bb nucleic acids
US6642030B1 (en) 1997-12-18 2003-11-04 Monsanto Technology, Llc Nucleic acid compositions encoding modified Bacillus thuringiensis coleopteran-toxic crystal proteins
WO1999038977A2 (en) 1998-02-03 1999-08-05 Mendel Biotechnology, Inc. Plant having altered environmental stress tolerance
WO1999040209A1 (en) 1998-02-09 1999-08-12 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
WO1999041402A2 (en) 1998-02-11 1999-08-19 Maxygen, Inc. Targeting of genetic vaccine vectors
WO1999041383A1 (en) 1998-02-11 1999-08-19 Maxygen, Inc. Antigen library immunization
WO1999041369A2 (en) 1998-02-11 1999-08-19 Maxygen, Inc. Genetic vaccine vector engineering
WO1999041368A2 (en) 1998-02-11 1999-08-19 Maxygen, Inc. Optimization of immunomodulatory properties of genetic vaccines
WO1999043838A1 (en) 1998-02-24 1999-09-02 Pioneer Hi-Bred International, Inc. Synthetic promoters
WO1999043819A1 (en) 1998-02-26 1999-09-02 Pioneer Hi-Bred International, Inc. Family of maize pr-1 genes and promoters
US6177611B1 (en) 1998-02-26 2001-01-23 Pioneer Hi-Bred International, Inc. Maize promoters
WO1999049064A2 (en) 1998-03-20 1999-09-30 Plant Bioscience Limited Plant control genes
WO1999053050A1 (en) 1998-04-08 1999-10-21 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US6713663B2 (en) 1998-04-15 2004-03-30 The Salk Institute For Biological Studies Flowering locus T (FT) and genetically modified plants having delayed flower development
WO1999055882A1 (en) 1998-04-24 1999-11-04 E.I. Du Pont De Nemours And Company Phytic acid biosynthetic enzymes
US6284948B1 (en) 1998-05-18 2001-09-04 Pioneer Hi-Bred International, Inc. Genes and methods for control of nematodes in plants
WO1999061632A1 (en) 1998-05-22 1999-12-02 Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) A process for inhibiting expression of genes
WO1999064579A2 (en) 1998-06-11 1999-12-16 E.I. Du Pont De Nemours And Company Corn oleosin promoter and genes encoding corn desaturases
WO2000000632A1 (en) 1998-06-29 2000-01-06 Phylos, Inc. Methods for generating highly diverse libraries
US6538177B1 (en) 1998-07-15 2003-03-25 Pioneer Hi-Bred International, Inc. Compositions and methods for fumonisin detoxification
WO2000004190A1 (en) 1998-07-15 2000-01-27 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6291224B1 (en) 1998-07-17 2001-09-18 Pioneer Hi-Bred International, Inc. Genes controlling phytate metabolism in plants and uses thereof
US8058510B2 (en) 1998-07-17 2011-11-15 Bayer Bioscience N.V. Method and means to modulate programmed cell death in eukaryotic cells
WO2000006341A1 (en) 1998-07-31 2000-02-10 Minnesota Mining And Manufacturing Company Cleaning pads formed from non-woven abrasive web material, especially for domestic use
WO2000009679A1 (en) 1998-08-12 2000-02-24 Proteus (S.A.) Method for obtaining in vitro recombined polynucleotide sequences, sequence banks and resulting sequences
US20040068767A1 (en) 1998-08-17 2004-04-08 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US6803498B2 (en) 1998-08-17 2004-10-12 Pioneer Hi-Bred Int'l. Inc. Maize cellulose synthases and uses thereof
US20030163838A1 (en) 1998-08-17 2003-08-28 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US20030150014A1 (en) 1998-08-17 2003-08-07 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US6225529B1 (en) 1998-08-20 2001-05-01 Pioneer Hi-Bred International, Inc. Seed-preferred promoters
WO2000012733A1 (en) 1998-08-28 2000-03-09 Pioneer Hi-Bred International, Inc. Seed-preferred promoters from end genes
US6346403B1 (en) 1998-09-08 2002-02-12 E.I. Du Pont De Nemours And Company Methionine metabolic enzymes
WO2000018906A2 (en) 1998-09-29 2000-04-06 Maxygen, Inc. Shuffling of codon altered genes
US6340593B1 (en) 1998-10-23 2002-01-22 Mycogen Corporation Plant-optimized polynucleotides encoding approximately 15 kDa and approximately 45 kDa pesticidal proteins
US6468523B1 (en) 1998-11-02 2002-10-22 Monsanto Technology Llc Polypeptide compositions toxic to diabrotic insects, and methods of use
US7064249B2 (en) 1998-11-04 2006-06-20 Monsanto Technology Llc Plants transformed to express Cry2A δ-endotoxins
US6825397B1 (en) 1998-11-09 2004-11-30 Pioneer Hi-Bred International, Inc. LEC1 trancriptional activator nucleic acids and methods of use thereof
WO2000028058A2 (en) 1998-11-09 2000-05-18 Pioneer Hi-Bred International, Inc. Transcriptional activator lec1 nucleic acids, polypeptides and their uses
US6391348B1 (en) 1998-11-20 2002-05-21 Pioneer Hi-Bred International, Inc. Method of reducing cholesterol in eggs
WO2000032761A1 (en) 1998-11-25 2000-06-08 The Trustees Of The University Of Pennsylvania Ethylene-response-factor1 (erf1) in plants
US6531648B1 (en) 1998-12-17 2003-03-11 Syngenta Participations Ag Grain processing method and transgenic plants useful therein
WO2000042559A1 (en) 1999-01-18 2000-07-20 Maxygen, Inc. Methods of populating data structures for use in evolutionary simulations
WO2000042560A2 (en) 1999-01-19 2000-07-20 Maxygen, Inc. Methods for making character strings, polynucleotides and polypeptides
WO2000042561A2 (en) 1999-01-19 2000-07-20 Maxygen, Inc. Oligonucleotide mediated nucleic acid recombination
WO2000044918A1 (en) 1999-01-28 2000-08-03 Plant Bioscience Limited Methods and means for modification of plant characteristics using the vernalization gene vrn2
WO2000046358A2 (en) 1999-02-05 2000-08-10 Plant Bioscience Limited Arabidopsis thaliana derived frigida gene conferring late flowering
US6323392B1 (en) 1999-03-01 2001-11-27 Pioneer Hi-Bred International, Inc. Formation of brassica napus F1 hybrid seeds which exhibit a highly elevated oleic acid content and a reduced linolenic acid content in the endogenously formed oil of the seeds
US6531316B1 (en) 1999-03-05 2003-03-11 Maxyag, Inc. Encryption of traits using split gene sequences and engineered genetic elements
US6365377B1 (en) 1999-03-05 2002-04-02 Maxygen, Inc. Recombination of insertion modified nucleic acids
US6664446B2 (en) 1999-03-23 2003-12-16 Mendel Biotechnology, Inc. Transgenic plants comprising polynucleotides encoding transcription factors that confer disease tolerance
US6563020B1 (en) 1999-03-24 2003-05-13 Pioneer Hi-Bred International, Inc. Maize chitinases and their use in enhancing disease resistance in crop plants
WO2000060089A1 (en) 1999-04-07 2000-10-12 Mendel Biotechnology, Inc. Genetic trait breeding method
US7531723B2 (en) 1999-04-16 2009-05-12 Pioneer Hi-Bred International, Inc. Modulation of cytokinin activity in plants
US6992237B1 (en) 1999-04-16 2006-01-31 Pioneer Hi-Bred International Inc. Regulated expression of genes in plant seeds
EP1173580A1 (en) 1999-04-29 2002-01-23 Syngenta Limited Herbicide resistant plants
EP1173581A1 (en) 1999-04-29 2002-01-23 Syngenta Limited Herbicide resistant plants
EP1173582A1 (en) 1999-04-29 2002-01-23 Syngenta Limited Herbicide resistant plants
WO2000068393A1 (en) 1999-05-07 2000-11-16 Pioneer Hi-Bred International, Inc. Phytyl/prenyltransferase nucleic acids, polypeptides and uses thereof
US6855865B2 (en) 1999-05-07 2005-02-15 E.I. Du Pont De Nemours And Company Nucleic acids encoding plant defensins and methods of use thereof
US6787683B1 (en) 1999-05-07 2004-09-07 Pioneer Hi-Bred International, Inc. Phytyl/prenyltransferase nucleic acids, polypeptides and uses thereof
US7238781B2 (en) 1999-05-07 2007-07-03 E. I. Dupont De Nemours And Company Plant defensin polynucleotides and methods of use thereof
WO2000073475A1 (en) 1999-05-28 2000-12-07 Pioneer Hi-Bred International, Inc. Methods for modulating water-use efficiency or productivity in a plant
US6441274B1 (en) 1999-06-16 2002-08-27 E. I. Du Pont De Nemours & Company Plant tryptophan synthase beta subunit
US6388171B1 (en) 1999-07-12 2002-05-14 Pioneer Hi-Bred International, Inc. Compositions and methods for fumonisin detoxification
WO2001004147A2 (en) 1999-07-12 2001-01-18 E.I. Du Pont De Nemours And Company Plant inositol polyphosphate phosphatase homologs
WO2001012825A1 (en) 1999-08-13 2001-02-22 Syngenta Participations Ag Herbicide-tolerant protoporphyrinogen oxidase
WO2001012800A2 (en) 1999-08-16 2001-02-22 E.I. Du Pont De Nemours And Company Method for the production of calendic acid, a fatty acid containing delta-8,10,12 conjugated double bonds and dimorphecolic acid, a fatty acid containing a 9-hydroxy group and delta-10,12 conjugated double bonds
WO2001012731A1 (en) 1999-08-19 2001-02-22 Ppg Industries Ohio, Inc. Hydrophobic particulate inorganic oxides and polymeric compositions containing same
US6423886B1 (en) 1999-09-02 2002-07-23 Pioneer Hi-Bred International, Inc. Starch synthase polynucleotides and their use in the production of new starches
WO2001021822A1 (en) 1999-09-17 2001-03-29 Plant Bioscience Limited Methods and means for modification of plant flowering characteristics
WO2001023401A2 (en) 1999-09-28 2001-04-05 Maxygen, Inc. Use of codon-varied oligonucleotide synthesis for synthetic sequence recombination
WO2001026459A2 (en) 1999-10-12 2001-04-19 Mendel Biotechnology, Inc. Flowering time modification
WO2001029237A2 (en) 1999-10-15 2001-04-26 Aventis Cropscience N.V. Conditional sterility in wheat
WO2001034726A2 (en) 1999-10-21 2001-05-17 Fluor Corporation Methods and apparatus for high propane recovery
US8058515B2 (en) 1999-11-10 2011-11-15 University Of Connecticut Plant cells and plants overexpressing vacuolar proton pyrophosphatases
WO2001036598A1 (en) 1999-11-17 2001-05-25 Mendel Biotechnology, Inc. Environmental stress tolerance genes
WO2001036597A1 (en) 1999-11-17 2001-05-25 Mendel Biotechnology, Inc. Plant biochemistry-related genes
WO2001036444A1 (en) 1999-11-17 2001-05-25 Mendel Biotechnology, Inc. Plant developmental genes
WO2001035727A1 (en) 1999-11-17 2001-05-25 Mendel Biotechnology, Inc. Seed trait genes
WO2001036596A2 (en) 1999-11-17 2001-05-25 Pioneer Hi-Bred International, Inc. Modulation of plant response to abscisic acid
WO2001035725A1 (en) 1999-11-17 2001-05-25 Mendel Biotechnology, Inc. Yield-related genes
US20040148654A1 (en) 1999-11-17 2004-07-29 Pioneer Hi-Bred International, Inc. Modulation of abscisic acid
US6248535B1 (en) 1999-12-20 2001-06-19 University Of Southern California Method for isolation of RNA from formalin-fixed paraffin-embedded tissue specimens
WO2001064898A2 (en) 2000-02-29 2001-09-07 E.I. Dupont De Nemours And Company Genes encoding denitrification enzymes
WO2001066704A2 (en) 2000-03-09 2001-09-13 Monsanto Technology Llc Methods for making plants tolerant to glyphosate and compositions thereof
WO2001079516A2 (en) 2000-04-14 2001-10-25 Pioneer Hi-Bred International, Inc. Maize cellulose synthases and uses thereof
US6777592B2 (en) 2000-04-14 2004-08-17 E.I. Du Pont Denemours And Company Arthropod defensins of Scolopendra canidens, Vaejovis carolinianus, and Argiope spp.
WO2002000904A2 (en) 2000-06-23 2002-01-03 E. I. Du Pont De Nemours And Company Recombinant constructs and their use in reducing gene expression
WO2002000894A2 (en) 2000-06-30 2002-01-03 Cropdesign N.V. Gene silencing vector
WO2002002776A1 (en) 2000-07-06 2002-01-10 Institute Of Microbiology, Chinese Academy Of Sciences A method of improving nitrogen assimilation efficiency in plants
WO2002017430A1 (en) 2000-08-22 2002-02-28 Paratek Microwave, Inc. Combline filters with tunable dielectric capacitors
WO2002015675A1 (en) 2000-08-22 2002-02-28 Mendel Biotechnology, Inc. Genes for modifying plant traits iv
US6801104B2 (en) 2000-08-22 2004-10-05 Paratek Microwave, Inc. Electronically tunable combline filters tuned by tunable dielectric capacitors
US6713259B2 (en) 2000-09-13 2004-03-30 Monsanto Technology Llc Corn event MON810 and compositions and methods for detection thereof
US7696412B2 (en) 2000-10-24 2010-04-13 E.I. Du Pont De Nemours And Company Genes encoding novel Bacillus thuringiensis proteins with pesticidal activity against Coleopterans
WO2002057439A2 (en) 2000-10-24 2002-07-25 E. I. Du Pont De Nemours And Company Plant transcription factors
US7605304B2 (en) 2000-10-24 2009-10-20 E.I. Du Pont De Nemours And Company Genes encoding novel bacillus thuringiensis proteins with pesticidal activity against coleopterans
US7462481B2 (en) 2000-10-30 2008-12-09 Verdia, Inc. Glyphosate N-acetyltransferase (GAT) genes
US20050204418A1 (en) 2000-11-07 2005-09-15 Pioneer Hi-Bred International, Inc. Grain quality through altered expression of seed proteins
US20050160488A1 (en) 2000-11-07 2005-07-21 Pioneer Hi-Bred International, Inc. Grain quality through altered expression of seed proteins
US6858778B1 (en) 2000-11-07 2005-02-22 Pioneer Hi-Bred International, Inc. Plants transformed with a DNA construct comprising a nucleic acid molecule encoding an 18 kD α-globulin
US6893826B1 (en) 2000-11-17 2005-05-17 Monsanto Technology Llc Cotton event PV-GHBK04 (757) and compositions and methods for detection thereof
WO2002042424A2 (en) 2000-11-22 2002-05-30 Pioneer Hi-Bred International, Inc. Seed-preferred regulatory elements and uses thereof
WO2002059324A2 (en) 2001-01-12 2002-08-01 Pioneer Hi-Bred International, Inc. Novel inositol polyphosphate kinase genes and uses thereof
US20030009011A1 (en) 2001-01-12 2003-01-09 Jinrui Shi Novel inositol polyphosphate kinase genes and uses thereof
US20040098764A1 (en) 2001-03-16 2004-05-20 Heard Jacqueline E. Plant transcriptional regulators of abiotic stress
WO2002077185A2 (en) 2001-03-27 2002-10-03 Mendel Biotechnology, Inc. Methods for modifying flowering phenotypes
US6812380B2 (en) 2001-03-27 2004-11-02 Pioneer Hi-Bred International, Inc. Compositions and methods of zearalenone detoxification
JP2002281975A (en) 2001-03-28 2002-10-02 Yamaguchi Technology Licensing Organization Ltd Gene belonging to soy nitrate transporter 1 gene family
US6717034B2 (en) 2001-03-30 2004-04-06 Mendel Biotechnology, Inc. Method for modifying plant biomass
WO2002079403A2 (en) 2001-03-30 2002-10-10 Mendel Biotechnology, Inc. Method for modifying plant biomass
US8237020B2 (en) 2001-03-30 2012-08-07 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7244820B2 (en) 2001-03-30 2007-07-17 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7615686B2 (en) 2001-03-30 2009-11-10 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US6891085B2 (en) 2001-04-20 2005-05-10 Pioneer Hi-Bred International, Inc. Nucleic acid encoding the FUS6 antimicrobial polypeptide of Agrotis ipsilon and its use to enhance disease resistance in a plant
US20030166197A1 (en) 2001-05-10 2003-09-04 Ecker Joseph R. Ethylene insensitive plants
WO2003000863A2 (en) 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Defensin polynucleotides and methods of use
US6911577B2 (en) 2001-06-22 2005-06-28 Pioneer Hi-Bred International, Inc. Defensin polynucleotides and methods of use
US20030204870A1 (en) 2001-06-29 2003-10-30 Allen Stephen M. Alteration of oil traits in plants
WO2003011015A2 (en) 2001-08-02 2003-02-13 Pioneer Hi-Bred International, Inc. Methods for improving seed and grain characteristics
WO2003013228A2 (en) 2001-08-09 2003-02-20 Mendel Biotechnology, Inc. Biochemistry-related polynucleotides and polypeptides in plants
WO2003014327A2 (en) 2001-08-09 2003-02-20 Mendel Biotechnology, Inc. Stress-related polynucleotides and polypeptides in plants
WO2003013227A2 (en) 2001-08-09 2003-02-20 Mendel Biotechnology, Inc. Yield-related polynucleotides and polypeptides in plants
WO2003027243A2 (en) 2001-09-27 2003-04-03 Pioneer Hi-Bred International, Inc. Phytate polynucleotides and methods of use
US20030079247A1 (en) 2001-09-27 2003-04-24 Pioneer Hi-Bred International, Inc. Phytate polynucleotides and methods of use
WO2003033651A2 (en) 2001-10-16 2003-04-24 Pioneer Hi-Bred International, Inc. Compositions and methods for promoting nematode resistance in plants
US7087810B2 (en) 2001-11-07 2006-08-08 Muller Mathis L Isolated nucleic acids encoding proteins with chitinase activity and uses thereof
US7145060B2 (en) 2001-11-07 2006-12-05 Pioneer Hi-Bred International, Inc. Nucleic acid encoding a chitinase and methods of using it to make fungal resistant plants
WO2003052063A2 (en) 2001-12-14 2003-06-26 The Nitrate Elimination Company, Inc. Simplified eukaryotic nitrate reductase
WO2003082899A2 (en) 2002-03-22 2003-10-09 E.I. Dupont De Nemours & Company Compositions and methods for altering tocotrienol content
US20040034886A1 (en) 2002-03-22 2004-02-19 E. I. Du Pont De Nemours And Company Compositions and methods for altering tocotrienol content
US20040128719A1 (en) 2002-06-21 2004-07-01 Klee Harry J. Materials and methods for tissue-specific targeting of ethylene insensitivity in transgenic plants
US7105332B2 (en) 2002-06-26 2006-09-12 E.I. Du Pont De Nemours And Company Genes encoding proteins with pesticidal activity
US7462760B2 (en) 2002-06-26 2008-12-09 Pioneer Hi-Bred International, Inc. Genes encoding plant protease-resistant pesticidal proteins and method of their use
US7378499B2 (en) 2002-06-26 2008-05-27 Pioneer Hi-Bred International, Inc. Genes encoding proteins with pesticidal activity
US7705216B2 (en) 2002-07-29 2010-04-27 Monsanto Technology Llc Corn event PV-ZMIR13 (MON863) plants and compositions and methods for detection thereof
US20040078852A1 (en) 2002-08-02 2004-04-22 Thomashow Michael F. Transcription factors to improve plant stress tolerance
WO2004031349A2 (en) 2002-09-18 2004-04-15 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
US8084418B2 (en) 2003-01-21 2011-12-27 Dow Agrosciences Llc Methods of inhibiting insects by treatment with a complex comprising a Photorhabdus insecticidal protein and one or two Xenorhabdus enhancer proteins
US7491698B2 (en) 2003-01-21 2009-02-17 Dow Agrosciences Llc Mixing and matching TC proteins for pest control
WO2004074462A2 (en) 2003-02-20 2004-09-02 Athenix Corporation Delta-endotoxin genes and methods for their use
US20040197916A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-004, a delta-endotoxin gene and methods for its use
US20040210965A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-007, a delta-endotoxin gene and methods for its use
US20040250311A1 (en) 2003-02-20 2004-12-09 Athenix Corporation AXMI-008, a delta-endotoxin gene and methods for its use
US20040197917A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-014, delta-endotoxin gene and methods for its use
US20040210964A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-009, a delta-endotoxin gene and methods for its use
US20040216186A1 (en) 2003-02-20 2004-10-28 Athenix Corporation AXMI-006, a delta-endotoxin gene and methods for its use
WO2004076638A2 (en) 2003-02-25 2004-09-10 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
US7294760B2 (en) 2003-03-28 2007-11-13 Monsanto Technology Llc Plant promoters for use in early seed development
US7847153B2 (en) 2003-03-28 2010-12-07 Monsanto Technology Llc Plant promoters for use in early seed development
WO2004090143A2 (en) 2003-04-04 2004-10-21 Pioneer Hi-Bred International, Inc. Modulation of cytokinin activity in plants
US7288643B2 (en) 2003-05-02 2007-10-30 Pioneer Hi-Bred International, Inc. Corn event TC1507 and methods for detection thereof
US20060191034A1 (en) 2003-07-07 2006-08-24 Baum James A Insecticidal proteins secreted from bacillus thuringiensis and uses therefor
WO2005021585A2 (en) 2003-08-28 2005-03-10 Athenix Corporation Axmi-003, a delta-endotoxin gene and methods for its use
US7205453B2 (en) 2003-09-25 2007-04-17 Pioneer Hi-Bred International, Inc. Crop plant cystatin proteinase inhibitors encoding nucleic acids and methods of use
WO2005038032A1 (en) 2003-10-14 2005-04-28 Athenix Corporation Axmi-010, a delta-endotoxin gene and methods for its use
US8063269B2 (en) 2003-12-18 2011-11-22 Alberta Research Council Inc. Transgenic plants with reduced level of saturated fatty acid and methods for making them
US7790846B2 (en) 2003-12-22 2010-09-07 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis Cry9 toxins
US7629504B2 (en) 2003-12-22 2009-12-08 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry9 nucleic acids
US7491869B2 (en) 2004-02-20 2009-02-17 Pioneer Hi-Bred International, Inc. Methods of using non-plant insecticidal lipase encoding nucleic acids in transgenic plants
US7208474B2 (en) 2004-02-25 2007-04-24 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US7179965B2 (en) 2004-03-26 2007-02-20 Dow Agrosciences Llc Cry1F and Cry1Ac transgenic cotton lines and event-specific identification thereof
WO2005110068A2 (en) 2004-04-09 2005-11-24 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US20120164205A1 (en) 2004-04-09 2012-06-28 Baum James A Compositions and methods for control of insect infestations in plants
US7405074B2 (en) 2004-04-29 2008-07-29 Pioneer Hi-Bred International, Inc. Glyphosate-N-acetyltransferase (GAT) genes
US20090093366A1 (en) 2004-04-30 2009-04-09 Dow Agrosciences Llc Novel Herbicide Resistance Genes
WO2005107437A2 (en) 2004-04-30 2005-11-17 Dow Agrosciences Llc Novel herbicide resistance genes
WO2005113778A2 (en) 2004-05-20 2005-12-01 Pioneer Hi-Bred International, Inc. Maize multidrug resistance-associated protein polynucleotides and methods of use
US7193133B2 (en) 2004-06-09 2007-03-20 Michael Lassner Plastid transit peptides
EP2404499A2 (en) 2004-06-16 2012-01-11 BASF Plant Science GmbH Nucleic acid molecules encoding Lipid Metabolism Protein (LMP) and methods of use in plants
US8217223B2 (en) 2004-06-16 2012-07-10 Basf Plant Science Gmbh Nucleic acid molecules encoding WRINKLED1-like polypeptides and methods of use in plants
US7301069B2 (en) 2004-06-30 2007-11-27 Pioneer Hi-Bred International, Inc. Methods of protecting plants from pathogenic fungi and nematodes
US7306946B2 (en) 2004-07-02 2007-12-11 Pioneer Hi-Bred International, Inc. Antifungal polypeptides
US7323556B2 (en) 2004-09-29 2008-01-29 Pioneer Hi-Bred International, Inc. Corn event DAS-59122-7 and methods for detection thereof
WO2006083891A2 (en) 2005-01-31 2006-08-10 Athenix Corporation Axmi-018, axmi-020, and axmi-021, a family of delta-endotoxin genes and methods for their use
US20090144863A1 (en) 2005-03-08 2009-06-04 Basf Plant Science Gmbh Expression Enhancing Intron Sequences
US7803943B2 (en) 2005-03-17 2010-09-28 Biotium, Inc. Methods of using dyes in association with nucleic acid staining or detection and associated technology
US8236757B2 (en) 2005-04-01 2012-08-07 Athenix Corp AXMI-027, AXMI-036 and AXMI-038, a family of delta-endotoxin genes and methods for their use
US20090035765A1 (en) 2005-04-04 2009-02-05 E.I. Du Pont De Nemours And Company Polynucleotides and methods for making plants resistant to fungal pathogens
WO2006119457A1 (en) 2005-05-02 2006-11-09 Athenix Corporation Axmi-028 and axmi-029, family of novel delta-endotoxin genes and methods for their use
EP2431472A1 (en) 2005-07-06 2012-03-21 CropDesign N.V. Plant yield improvement by STE20-like gene expression
US8097769B2 (en) 2005-07-18 2012-01-17 Basf Plant Science Gmbh Yield increase in plants overexpressing the ACCDP genes
US20080234130A1 (en) 2005-08-24 2008-09-25 Pioneer Hi-Bred International, Inc. Compositions providing tolerance to multiple herbicides and methods of use thereof
US7803992B2 (en) 2005-08-24 2010-09-28 Pioneer Hi-Bred International, Inc. Methods and compositions for expressing an herbicide-tolerant polynucleotide
WO2007074405A2 (en) 2005-09-16 2007-07-05 Devgen Nv Dsrna as insect control agent
US20090044298A1 (en) 2005-09-16 2009-02-12 Bayer Cropscience S.A. Transplastomic Plants Expressing Lumen-Targeted Protein
US7449552B2 (en) 2006-04-14 2008-11-11 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7329736B2 (en) 2006-04-14 2008-02-12 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7476781B2 (en) 2006-04-14 2009-01-13 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US20070277263A1 (en) 2006-05-25 2007-11-29 Hexima Ltd. Multi-gene expression vehicle
US7923602B2 (en) 2006-06-14 2011-04-12 Athenix Corp. AXMI-031, AXMI-039, AXMI-040 and AXMI-049, a family of novel delta endotoxin genes and methods for their use
US20110263488A1 (en) 2006-06-15 2011-10-27 Athenix Corp. Family of pesticidal proteins and methods for their use
US7468278B2 (en) 2006-07-21 2008-12-23 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis gene with coleopteran activity
US7510878B2 (en) 2006-07-21 2009-03-31 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis gene with lepidopteran activity
US7521235B2 (en) 2006-07-21 2009-04-21 Pioneer Hi-Bred International, Inc. Unique novel Bacillus thuringiensis gene with Lepidopteran activity
US20120110696A1 (en) 2006-08-07 2012-05-03 Jinrong Wan LysM Receptor-Like Kinases To Improve Plant Defense Response Against Fungal Pathogens
US7858849B2 (en) 2006-12-08 2010-12-28 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US20100024067A1 (en) 2006-12-15 2010-01-28 Cropdesign N.V. Plants having enhanced seed yield-related traits and a method for making the same
US20100017914A1 (en) 2007-03-28 2010-01-21 Syngenta Participations Ag Insecticidal proteins
US8338152B2 (en) 2007-04-10 2012-12-25 E. I. Du Pont De Nemours And Company Delta-8 desaturases and their use in making polyunsaturated fatty acids
US20080295207A1 (en) 2007-04-27 2008-11-27 Monsanto Technology Llc Hemipteran-and Coleopteran Active Toxin Proteins from Bacillus Thuringiensis
US20100257633A1 (en) 2007-06-20 2010-10-07 The Australian National University Method for improving stress resistance in plants and materials therefor
US7772465B2 (en) 2007-06-26 2010-08-10 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis gene with lepidopteran activity
US8338661B2 (en) 2007-07-13 2012-12-25 Basf Plant Science Gmbh Transgenic plants with increased stress tolerance and yield
US20090064373A1 (en) 2007-08-29 2009-03-05 Graziana Taramino Plants with altered root architecture, related constructs and methods involving genes encoding nucleoside diphosphatase kinase (ndk) polypeptides and homologs thereof
US20110061133A1 (en) 2007-09-14 2011-03-10 Basf Plant Science Gmbh Plants having increased yield-related traits and a method for making the same
US20090144852A1 (en) 2007-10-16 2009-06-04 Athenix Corporation Axmi-066 and axmi-076: delta-endotoxin proteins and methods for their use
US8173866B1 (en) 2008-01-11 2012-05-08 Pioneer Hi-Bred International, Inc. Modulation of plant xylan synthases
WO2009091864A2 (en) 2008-01-17 2009-07-23 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides from lygus
US20090192117A1 (en) 2008-01-17 2009-07-30 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides from lygus
US20090188008A1 (en) 2008-01-17 2009-07-23 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides
US20100005543A1 (en) 2008-06-25 2010-01-07 Athenix Corporation Toxin genes and methods for their use
US8334431B2 (en) 2008-07-02 2012-12-18 Athenix Corporation AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184: insecticidal proteins and methods for their use
US8058571B2 (en) 2008-10-08 2011-11-15 Research In Motion Limited Dual-action single-key mechanism
US8084416B2 (en) 2008-12-23 2011-12-27 Athenix Corp. AXMI-150 delta-endotoxin gene and methods for its use
US8319019B2 (en) 2009-01-23 2012-11-27 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
US20110283420A1 (en) 2009-01-28 2011-11-17 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and a method for making the same
US20100197592A1 (en) 2009-02-05 2010-08-05 Athenix Corporation Variant axmi-r1 delta endotoxin genes and methods for their use
US8318900B2 (en) 2009-02-27 2012-11-27 Athenix Corp. Pesticidal proteins and methods for their use
US20100298211A1 (en) 2009-03-11 2010-11-25 Athenix Corporation Axmi-001, axmi-002, axmi-030, axmi-035, and axmi-045: toxin genes and methods for their use
US20120029750A1 (en) 2009-03-12 2012-02-02 Ford Global Technologies, Llc Auto-seek electrical connection for a plug-in hybrid electric vehicle
US20100287669A1 (en) 2009-04-14 2010-11-11 Pioneer Hi-Bred International, Inc. Modulation of acc synthase improves plant yield under low nitrogen conditions
US8304604B2 (en) 2009-04-17 2012-11-06 Dow Agrosciences, Llc. DIG-3 insecticidal Cry toxins
US8334366B1 (en) 2009-04-29 2012-12-18 The United States Of America, As Represented By The Secretary Of Agriculture Mutant lycotoxin-1 peptide sequences for insecticidal and cell membrane altering properties
US20120066794A1 (en) 2009-05-28 2012-03-15 Nirmala Sharma Increased Seed Oil and Abiotic Stress Tolerance Mediated by HSI2
US8304605B2 (en) 2009-06-16 2012-11-06 Dow Agrosciences, Llc. DIG-11 insecticidal cry toxins
US20110023184A1 (en) 2009-07-02 2011-01-27 Nalini Manoj Desai Axmi-205 pesticidal gene and methods for its use
US20110064710A1 (en) 2009-09-11 2011-03-17 Benson Terry A Novel bacillus thuringiensis isolate
US20120272352A1 (en) 2009-10-30 2012-10-25 Syngenta Participations Ag Genes Conferring Drought and Salt Tolerance and Uses Thereof
US20110277181A1 (en) 2009-10-30 2011-11-10 Ei Du Pont De Nemours And Company Drought tolerant plants and related constructs and methods involving genes encoding dtp21 polypeptides
US20120324605A1 (en) 2009-12-16 2012-12-20 Dow Agrosciences Llc Insectcidal protein combinations for controlling fall armyworm and european corn borer, and methods for insect resistance management
US20120311745A1 (en) 2009-12-16 2012-12-06 Dow Agrosciences Llc Combined use of cry1ca and cry1ab proteins for insect resistance management
US20120324606A1 (en) 2009-12-16 2012-12-20 Dow Agrosciences Llc Use of cry 1ab in combination with cry1be for management of resistant insects
US20120317682A1 (en) 2009-12-16 2012-12-13 Dow Agrosciences Llc Combined use of vip3ab and cry1fa for management of resistant insects
US20120317681A1 (en) 2009-12-16 2012-12-13 Thomas Meade COMBINED USE OF CRY1Ca AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20120331589A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc COMBINED USE OF CRY1Da AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20120311746A1 (en) 2009-12-16 2012-12-06 Dow Agroscience Llc Insect resistance management with combinations of cry1be and cry1f proteins
US20120331590A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc Use of cry1da in combination with cry1be for management of resistant insects
US20110191904A1 (en) 2009-12-17 2011-08-04 Rajesh Kumar Plant Genes Associated With Seed Oil Content And Methods Of Their Use
WO2011103248A2 (en) 2010-02-18 2011-08-25 Athenix Corp. AXMI221z, AXMI222z, AXMI223z, AXMI224z, AND AXMI225z DELTA-ENDOTOXIN GENES AND METHODS FOR THEIR USE
WO2011103247A2 (en) 2010-02-18 2011-08-25 Athenix Corp. Axmi218, axmi219, axmi220, axmi226, axmi227, axmi228, axmi229, axmi230, and axmi231 delta-endotoxin genes and methods for their use
US20110301223A1 (en) 2010-06-04 2011-12-08 E.I. Du Pont De Nemours And Company Compositions and methods for insecticidal control of stinkbugs
US20120079622A1 (en) 2010-09-24 2012-03-29 Pioneer Hi-Bred International, Inc. Yield Enhancement in Plants by Modulation of a ZM-LOBDP1 Protein
WO2012038893A1 (en) 2010-09-24 2012-03-29 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and method for making the same
US20120079623A1 (en) 2010-09-27 2012-03-29 Pioneer Hi-Bred International, Inc. Yield Enhancement in Plants by Modulation of a ZM-ZFP1 Protein
WO2012055982A2 (en) 2010-10-27 2012-05-03 Devgen Nv Down-regulating gene expression in insect pests
WO2012058528A2 (en) 2010-10-28 2012-05-03 E. I. Du Pont De Nemours And Company Drought tolerant plants and related constructs and methods involving genes encoding dtp6 polypeptides
WO2012058266A1 (en) 2010-10-29 2012-05-03 Syngenta Participations Ag Overexpression of plant mirnas for parasite control
US20120198586A1 (en) 2010-12-30 2012-08-02 Dow Agrosciences Llc Nucleic acid molecules that target the vacuolar atpase h subunit and confer resistance to coleopteran pests
US20120278954A1 (en) 2011-02-11 2012-11-01 Bowen David J Pesticidal Nucleic Acids and Proteins and Uses Thereof
US20130097728A1 (en) 2011-04-05 2013-04-18 Volker Heinrichs Axmi115 variant insecticidal gene and methods for its use
WO2012139004A2 (en) 2011-04-07 2012-10-11 Monsanto Technology Llc Insect inhibitory toxin family active against hemipteran and/or lepidopteran insects
US20120322660A1 (en) 2011-04-20 2012-12-20 Devgen Nv Down-regulating gene expression in insect pests
US20120297501A1 (en) 2011-04-20 2012-11-22 Devgen Nv Plants resistant to insect pests
US20120304336A1 (en) 2011-05-23 2012-11-29 E.I. Du Pont De Nemours And Company Chloroplast transit peptides and methods of their use
WO2013016617A1 (en) 2011-07-28 2013-01-31 Athenix Corp. Axmi205 variant proteins and methods for their use

Non-Patent Citations (444)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1995, GREENE PUBLISHING AND WILEY-INTERSCIENCE
"GenBank", Database accession no. EU400157
"GenBank", Database accession no. gi|1170109526
"GenBank", Database accession no. gi|1209377
"GenBank", Database accession no. gi|135275135
"GenBank", Database accession no. gi|136141087
"GenBank", Database accession no. gi|136444345
"GenBank", Database accession no. gi|136474758
"GenBank", Database accession no. gi|138446054
"GenBank", Database accession no. gi|142085802
"GenBank", Database accession no. gi|143658948
"GenBank", Database accession no. gi|156316804
"GenBank", Database accession no. gi|156377786
"GenBank", Database accession no. gi|169762636
"GenBank", Database accession no. gi|170109524
"GenBank", Database accession no. gi|238488445
"GenBank", Database accession no. gi|256764986
"GenBank", Database accession no. gi|270056485
"GenBank", Database accession no. gi|294814724
"GenBank", Database accession no. gi|302532087
"GenBank", Database accession no. gi|302766657
"GenBank", Database accession no. gi|302787479
"GenBank", Database accession no. gi|302792467
"GenBank", Database accession no. gi|302823738
"GenBank", Database accession no. gi|302823768
"GenBank", Database accession no. gi|77456557
"GenBank", Database accession no. NP_928713
"GenBank", Database accession no. U37196
"GenBank", Database accession no. U52043
"GenBank", Database accession no. X03182
"GenBank", Database accession no. X98793
"GenBank", Database accession no. XP _001617993
"GenBank", Database accession no. XP_001 885969
"GenBank", Database accession no. YP_001223127
"GenBank", Database accession no. YP_00147861
"GenBank", Database accession no. Z14147
"GenBank", Database accession no. ZP _01077945
"PCR Methods Manual", 1999, ACADEMIC PRESS
"PCR Protocols: A Guide to Methods and Applications", 1990, ACADEMIC PRESS
"PCR Strategies", 1995, ACADEMIC PRESS
"The Pesticide Manual: A World Compendium", THE BRITISH CROP PRODUCTION COUNCIL
A. CRAMERI ET AL., NATURE, vol. 391, 1998, pages 288
ALBERMATHEWS, METHODS ENZYMOL., vol. 154, 1987, pages 511 - 533
ALLSHIRE, SCIENCE, vol. 297, 2002, pages 1818 - 1819
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389
ALTSCHUL, J. MOL. BIOL., vol. 215, 1990, pages 403
AMITAI, MOL MICROBIOL, vol. 47, 2003, pages 61 - 73
ANDREWS ET AL., BIOCHEM. J., vol. 252, 1988, pages 199 - 206
AONO ET AL., PLANT CELL PHYSIOL, vol. 36, 1995, pages 1687
ARNOLD, CURR OPIN BIOTECH, vol. 4, 1993, pages 450 - 455
AYRESPARK, CRITICAL REVIEWS IN PLANT SCIENCE, vol. 13, 1994, pages 219 - 239
BAIM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 5072 - 5076
BALLAS ET AL., NUCLEIC ACIDS RES., vol. 17, 1989, pages 7891 - 7903
BARKLEY ET AL., THE OPERON, 1980, pages 177 - 220
BASS ET AL., SCIENCE, vol. 242, 1988, pages 240 - 245
BEACHY ET AL., ANN. REV. PHYTOPATHOL., vol. 28, 1990, pages 451
BENFEY ET AL., EMBO J., vol. 9, 1990, pages 1685 - 96
BERSTEIN ET AL., NATURE, vol. 409, 2001, pages 363
BOGUSZ ET AL., PLANT CELL, vol. 2, no. 7, 1990, pages 633 - 641
BOMMINENIJAUHAR, MAYDICA, vol. 42, 1997, pages 107 - 120
BONIN: "Ph.D. Thesis", 1993, UNIVERSITY OF HEIDELBERG
BOTELLA ET AL., PLANT MOLEC. BIOL., vol. 24, 1994, pages 757
BOTSTEINSHORTLE, SCIENCE, vol. 229, 1985, pages 1193 - 1201
BRENZEL ET AL., BIOCHEMISTRY, vol. 45, no. 6, 2006, pages 1571 - 8
BRENZEL, BIOCHEMISTRY, vol. 45, no. 6, 2006, pages 1571 - 8
BRETAGNE-SAGNARD ET AL., TRANSGENIC RES., vol. 5, 1996, pages 131 - 137
BRICKER ET AL., BIOCHIM. BIOPHYS ACTA, vol. 1503, 2001, pages 350 - 356
BRIGGS, CURRENT BIOLOGY, vol. 5, no. 2, 1995
BROWN ET AL., CELL, vol. 49, 1987, pages 603 - 612
BUSHNELL ET AL., CAN. J. OF PLANT PATH., vol. 20, no. 2, 1998, pages 137 - 149
BYTEBIER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 5345 - 5349
CAMAREROMUIR, J. AMER. CHEM. SOC., vol. 121, 1999, pages 5597 - 5598
CAMPBELLGOWRI, PLANT PHYSIOL., vol. 92, 1990, pages 1 - 11
CANEVASCINI, PLANT PHYSIOL., vol. 112, no. 2, 1996, pages 513 - 524
CAPANA ET AL., PLANT MOL. BIOL., vol. 25, no. 4, 1994, pages 681 - 691
CARLINIGROSSI-DE-SA, TOXICON, vol. 40, no. 11, 2002, pages 1515 - 1539
CARTER ET AL., NUCL ACIDS RES, vol. 13, 1985, pages 4431 - 4443
CARTER, BIOCHEM J, vol. 237, 1986, pages 1 - 7
CARTER, METHODS IN ENZYMOL, vol. 154, 1987, pages 382 - 403
CHANG ET AL., NAT BIOTECHNOL, vol. 17, 1999, pages 793 - 797
CHANG, METHODS ENZYMOL., vol. 153, 1987, pages 507 - 516
CHATTOPADHYAY ET AL., CRITICAL REVIEWS IN MICROBIOLOGY, vol. 30, no. 1, 2004, pages 33 - 54
CHEN ET AL., PLANT J., vol. 10, 1996, pages 955 - 966
CHONG ET AL., GENE, vol. 192, 1997, pages 271 - 281
CHONG ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22159 - 22168
CHONG ET AL., J. BIOL. CHEM., vol. 273, 1998, pages 10567 - 10577
CHONG ET AL., NUCLEIC ACIDS RES., vol. 26, 1998, pages 5109 - 5115
CHONG ET AL., TRANSGENIC RESEARCH, vol. 9, 2000, pages 71 - 78
CHOTHIA, ANN. REV. BIOCHEM., vol. 53, 1984, pages 537 - 572
CHRISTENSEN ET AL., PLANT MOL. BIOL., vol. 12, 1989, pages 619 - 632
CHRISTENSEN ET AL., PLANT MOL. BIOL., vol. 18, 1992, pages 675 - 689
CHRISTENSENQUAIL, TRANSGENIC RESEARCH, vol. 5, 1996, pages 213 - 218
CHRISTIANS ET AL., NAT BIOTECHNOL, vol. 17, 1999, pages 259 - 264
CHRISTOPHERSON, PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 6314 - 6318
CHRISTOU ET AL., PLANT PHYSIOL., vol. 87, 1988, pages 671 - 674
CHRISTOUFORD, ANNALS OF BOTANY, vol. 75, 1995, pages 407 - 413
COMAI ET AL., J. BIOL. CHEM., vol. 263, no. 29, 1988, pages 15104 - 9
CONG, R. ET AL.: "Proceedings of the 4th Pacific Rim Conferences on Biotechnology of Bacillus thuringiensis and its environmental impact", 2002, pages: 118 - 123
CORDERO ET AL., PHYSIOL. MOL. PLANT PATH., vol. 41, 1992, pages 189 - 200
CORDEROK ET AL., PLANT J., vol. 6, no. 2, 1994, pages 141 - 150
CORNELISSENMELCHERS, PI. PHYSIOL., vol. 101, 1993, pages 709 - 712
COTTON ET AL., J. AM. CHEM. SOC., vol. 121, 1999, pages 1100 - 1101
CRAMERI ET AL., NAT BIOTECHNOL, vol. 14, 1996, pages 315 - 319
CRAMERI ET AL., NAT BIOTECHNOL, vol. 15, 1997, pages 436 - 438
CRAMERI ET AL., NAT MED, vol. 2, 1996, pages 100 - 103
CRAMERI ET AL., NATURE BIOTECH., vol. 15, 1997, pages 436 - 438
CRAMERI ET AL., NATURE, vol. 391, 1998, pages 288 - 291
CRAMERISTEMMER, BIOTECHNIQUES, vol. 18, 1995, pages 194 - 195
CRICKMORE ET AL., BACILLUS THURINGIENSIS TOXIN NOMENCLATURE, 2011, Retrieved from the Internet <URL:lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt>
CROSSWAY ET AL., BIOTECHNIQUES, vol. 4, 1986, pages 320 - 334
CROSSWAY ET AL., MOL GEN. GENET., vol. 202, 1986, pages 179 - 185
CUNNINGHAM, PROC. NATL. ACAD. SCI. U.S.A., vol. 76, 1979, pages 3218 - 3222
CZAPLALANG, J. ECON. ENTOMOL., vol. 83, 1990, pages 2480 - 2485
DALE ET AL., METHODS MOL BIOL, vol. 57, 1996, pages 369 - 374
DASSA ET AL., BIOCHEMISTRY, vol. 46, no. 1, 2007, pages 322 - 30
DASSA, J BIOL CHEM., vol. 279, 2004, pages 32001 - 7
DATTA ET AL., BIOTECHNOLOGY, vol. 8, 1990, pages 736 - 740
DATTA ET AL., PLANT MOL BIOL, vol. 20, 1992, pages 619
DAYHOFF ET AL.: "Atlas of Protein Sequence and Structure", 1978, NATL. BIOMED. RES. FOUND.
DE GREEF, BIO/TECHNOLOGY, vol. 7, 1989, pages 61
DE MAAGD ET AL., APPL. ENVIRON. MICROBIOL., vol. 62, 1996, pages 1537 - 1543
DE WET ET AL.: "The Experimental Manipulation of Ovule Tissues", 1985, LONGMAN, pages: 197 - 209
DEBLOCK ET AL., EMBO J., vol. 6, 1987, pages 2513 - 2518
DEGENKOLB ET AL., ANTIMICROB. AGENTS CHEMOTHER., vol. 35, 1991, pages 1591 - 1595
DELLA-CIOPPA ET AL., PLANT PHYSIOL., vol. 84, 1987, pages 965 - 968
DEUSCHLE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 5400 - 5404
DEUSCHLE ET AL., SCIENCE, vol. 248, 1990, pages 480 - 483
D'HALLUIN ET AL., PLANT CELL, vol. 4, 1992, pages 1495 - 1505
DOMINY ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 235, 2003, pages 209 - 223
DUAN ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 494 - 498
DUFFAUD ET AL., METH. ENZYMOL., vol. 153, 1987, pages 492
ECKELKAMP ET AL., FEBS LETTERS, vol. 323, 1993, pages 73 - 76
EGHTEDARZADEHHENIKOFF, NUCL ACIDS RES, vol. 14, 1986, pages 5115
ELBASHIR ET AL., GENES DEV., vol. 15, 2001, pages 188
ELLEUCHE ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 355, no. 3, 2007, pages 830 - 4
ELLIOT ET AL., PLANT MOLEC. BIOL., vol. 21, 1993, pages 515
ELROY-STEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 6126 - 6130
EMBO J., vol. 8, no. 2, pages 343 - 350
EVANS ET AL., ANNU. REV. PLANT BIOL., vol. 56, 2005, pages 375 - 392
EVANS ET AL., J BIOL CHEM, vol. 275, no. 13, 2000, pages 9091 - 4
EVANS ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 18359 - 18363
EVANS ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 3923 - 3926
EVANS ET AL., J. BIOL. CHEM., vol. 275, 2000, pages 9091 - 9094
EVANS ET AL., PROTEIN SCI., vol. 7, 1998, pages 2256 - 2264
FIGGE ET AL., CELL, vol. 52, 1988, pages 713 - 722
FINERMCMULLEN, IN VITRO CELL DEV. BIOL., vol. 27P, 1991, pages 175 - 182
FIRE ET AL., NATURE, vol. 391, 1998, pages 806
FIRE ET AL., TRENDS GENET., vol. 15, 1999, pages 358
FISHER ET AL., PLANT PHYSIOL., vol. 102, 1993, pages 1045
FRITZ ET AL., NUCL ACIDS RES, vol. 16, 1988, pages 6987 - 6999
FROMM ET AL., BIOTECHNOLOGY, vol. 8, 1990, pages 833 - 839
FUERST ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 2549 - 2553
GAERTNER: "Advanced Engineered Pesticides", 1993
GALLIE ET AL., GENE, vol. 165, no. 2, 1995, pages 233 - 238
GALLIE ET AL., GENE, vol. 60, 1987, pages 217 - 25
GALLIE ET AL.: "Molecular Biology of RNA", 1989, LISS, pages: 237 - 256
GATES ET AL., J MOL BIOL, vol. 255, 1996, pages 373 - 386
GATZ ET AL., MOL. GEN. GENET., vol. 227, 1991, pages 229 - 237
GE ET AL., J. BIOL. CHEM., vol. 266, 1991, pages 17954 - 17958
GEISER ET AL., GENE, vol. 48, 1986, pages 109
GENTILESALVATORE, EUR. J. BIOCHEM., vol. 218, 1993, pages 603 - 621
GHRAYEB ET AL., EMBO J, vol. 3, 1984, pages 2437 - 2442
GILL ET AL., J. BIOL. CHEM., 1995, pages 27277 - 27282
GILL ET AL., NATURE, vol. 334, 1988, pages 721 - 724
GOFF ET AL., SCIENCE, vol. 296, 2002, pages 92 - 100
GOLDENBERGCREIGHTON, J. MOL. BIOL., vol. 165, 1983, pages 407 - 413
GOSSEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
GOSSEN: "Ph.D. Thesis", 1993, UNIVERSITY OF HEIDELBERG
GOTOR, PLANT J., vol. 3, 1993, pages 509 - 18
GRIESS ET AL., PLANT PHYSIOL., vol. 104, 1994, pages 1467
GRUNDSTROM ET AL., NUCL ACIDS RES, vol. 13, 1985, pages 3305 - 3316
GUERINEAU ET AL., MOL. GEN. GENET., vol. 262, 1991, pages 141 - 144
GUERINEAU ET AL., PLANT MOL. BIOL., vol. 15, 1990, pages 127 - 136
GUEVARA-GARCIA, PLANT J., vol. 4, no. 3, 1993, pages 495 - 505
GURA, NATURE, vol. 404, 2000, pages 804 - 808
HALL ET AL., SCIENCE, vol. 297, 2002, pages 2232 - 2237
HAMMOCK ET AL., NATURE, vol. 344, 1990, pages 458
HANDELSMAN ET AL., APPL. ENVIRON. MICROBIOL., vol. 56, 1991, pages 713 - 718
HANSEN ET AL., MOL. GEN GENET., vol. 254, no. 3, 1997, pages 337 - 343
HARLOWLANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
HATTORI ET AL., MOL GEN GENET., vol. 246, 1995, pages 419
HAYES ET AL., BIOCHEM. J., vol. 285, 1992, pages 173
HELLENSMULLINEAUX, TRENDS IN PLANT SCIENCE, vol. 5, 2000, pages 446 - 451
HEPLER ET AL., PROC. NATL. ACAD. SCI., vol. 91, 1994, pages 2176 - 2180
HERRERA ESTRELLA ET AL., EMBO J., vol. 2, 1983, pages 987 - 992
HERRERA ESTRELLA ET AL., NATURE, vol. 303, 1983, pages 209 - 213
HIEI ET AL., THE PLANT JOURNAL, vol. 6, 1994, pages 271 - 282
HIGGINS ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 4680
HILLE ET AL., PLANT MOL. BIOL., vol. 7, 1990, pages 171 - 176
HILLENAND-WISSMAN, TOPICS MOL. STRUC. BIOL., vol. 10, 1989, pages 143 - 162
HINCHLIFFE ET AL., THE OPEN TOXINOLOGY JOURNAL, vol. 3, 2010, pages 101 - 118
HIRE ET AL., PLANT MOL. BIOL., vol. 20, no. 2, 1992, pages 207 - 218
HLAVKA ET AL.: "Handbook of Experimental Pharmacology", vol. 78, 1985, SPRINGER-VERLAG
HOFMANN, EUR. J. BIOCHEM., vol. 173, 1988, pages 85 - 91
HOOYKAAS-VAN SLOGTEREN ET AL., NATURE (LONDON, vol. 311, 1984, pages 763 - 764
HOPPWOODS, MOL. IMMUNOL., vol. 20, 1983, pages 483 - 489
HORLICK ET AL., PROTEIN ENG., vol. 5, 1992, pages 427 - 431
HU ET AL., CELL, vol. 48, 1987, pages 555 - 566
HUSH ET AL., THE JOURNAL OF CELL SCIENCE, vol. 107, 1994, pages 775 - 784
HUTVAGNER ET AL., SCIENCE, vol. 293, 2001, pages 834
IMMUNOLOGY TODAY, vol. 16, 1995, pages 194 - 201
INTERNATIONAL JOURNAL OF EPIDEMIOLOGY, vol. 34, 2005, pages 215 - 220
ISHIDA ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 745 - 750
IWAI ET AL., FEBS LETT, vol. 580, no. 7, 2006, pages 1853 - 8
IWAIPLUCKTHUN, FEBS LETT., vol. 459, 1999, pages 166 - 172
JAYNES ET AL., PLANT SCI., vol. 89, 1993, pages 43
JENUWEIN, SCIENCE, vol. 297, 2002, pages 2215 - 2218
JOBLING ET AL., NATURE, vol. 325, 1987, pages 622 - 625
JOFUKU, K.D.GOLDBERG, R.B., PLANT CELL, vol. 1, 1989, pages 1079 - 1093
JONES ET AL., MOL. GEN. GENET., vol. 210, 1987, pages 86 - 91
JONES ET AL., SCIENCE, vol. 266, 1994, pages 789
JOSHI ET AL., NUCLEIC ACID RES., vol. 15, 1987, pages 9627 - 9639
KABSCHSANDER, BIOPOLYMERS, vol. 22, 1983, pages 2577 - 2637
KAEPPLER ET AL., PLANT CELL REPORTS, vol. 9, 1990, pages 415 - 418
KAEPPLER ET AL., THEOR. APPL. GENET., vol. 84, 1992, pages 560 - 566
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877
KARPLUSSCHULZ, NATURWISSENSCHAFTEN, vol. 72, 1985, pages 212 - 213
KAWALLECK ET AL., PLANT MOLEC. BIOL., vol. 21, 1993, pages 673
KAWAMATA ET AL., PLANT CELL PHYSIOL., vol. 38, no. 7, 1997, pages 792 - 803
KELLERBAUMGARTNER, PLANT CELL, vol. 3, no. 10, 1991, pages 1051 - 1061
KIESELBACH ET AL., FEBS LETT, vol. 480, 2000, pages 271 - 276
KIESELBACH ET AL., PHOTOSYNTHESIS RESEARCH, vol. 78, 2003, pages 249 - 264
KLEIN ET AL., BIOTECHNOLOGY, vol. 6, 1988, pages 559 - 563
KLEIN ET AL., PLANT PHYSIOL., vol. 91, 1988, pages 440 - 444
KLEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 4305 - 4309
KLEINSCHNIDT ET AL., BIOCHEMISTRY, vol. 27, 1988, pages 1094 - 1104
KNULTZON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 2624
KRAMER ET AL., CELL, vol. 38, 1984, pages 879 - 887
KRAMER ET AL., INSECT BIOCHEM. MOLEC. BIOL., vol. 23, 1993, pages 691
KRAMER ET AL., NUCL ACIDS RES, vol. 12, 1984, pages 9441 - 9456
KRAMER ET AL., NUCL ACIDS RES, vol. 16, 1988, pages 7207
KRAMERFRITZ, METHODS ENZYMOL, vol. 154, 1987, pages 350 - 367
KUNKEL ET AL., METHODS ENZYMOL, vol. 154, 1987, pages 367 - 382
KUNKEL, PNAS USA, vol. 82, 1985, pages 488 - 492
KUNKEL: "Nucleic Acids & Molecular Biology", 1987, SPRINGER VERLAG, article "The efficiency of oligonucleotide directed mutagenesis"
KUSTER ET AL., PLANT MOL. BIOL., vol. 29, no. 4, 1995, pages 759 - 772
KWON, PLANT PHYSIOL., vol. 105, 1994, pages 357 - 67
KYTEDOOLITTLE, J MOL BIOL., vol. 157, no. 1, 1982, pages 105 - 32
KYTEDOOLITTLE, J. MOL. BIOL., vol. 157, 1982, pages 105 - 132
LABOW ET AL., MOL. CELL. BIOL., vol. 10, 1990, pages 3343 - 3356
LAM, RESULTS PROBL. CELL DIFFER., vol. 20, 1994, pages 181 - 196
LAMB ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 1436
LAST ET AL., THEOR. APPL. GENET., vol. 81, 1991, pages 581 - 588
LEE ET AL., EMBO J., vol. 7, 1988, pages 1241
LEERICHARDS, J. MOL. BIOL., vol. 55, 1971, pages 379 - 400
LERECIUS ET AL., FEMS MICROBIOL. LETTS., vol. 60, 1989, pages 211 - 218
LI ET AL., PLANT CELL REPORTS, vol. 12, 1993, pages 250 - 255
LI ET AL., PLANT CELL TISS. ORGAN CULT., vol. 89, 2007, pages 159 - 168
LING ET AL., ANAL BIOCHEM, vol. 254, no. 2, 1997, pages 157 - 178
LIU ET AL., J. AGRIC. FOOD CHEM., vol. 58, 2010, pages 12343 - 12349
LIU H ET AL., MOL BIO REP, vol. 37, 2010, pages 677 - 684
LOGEMANN ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 305
LOMMEL ET AL., VIROLOGY, vol. 81, 1991, pages 382 - 385
LYZNIK ET AL., PLANT CELL REP, vol. 21, 2003, pages 925 - 932
MACEJAK ET AL., NATURE, vol. 353, 1991, pages 90 - 94
MACGREGOR, FEBS LETT., vol. 378, 1996, pages 263 - 266
MANDECKI, PNAS USA, vol. 83, 1986, pages 7177 - 7181
MARINEAU ET AL., PLANT MOL. BIOL., vol. 9, 1987, pages 335 - 342
MARRONE ET AL., J. OF ECONOMIC ENTOMOLOGY, vol. 78, 1985, pages 290 - 293
MARSHALL ET AL., THEOR. APPL. GENET., vol. 83, 1992, pages 435
MARTIN ET AL., SCIENCE, vol. 262, 1993, pages 1432
MATHYS ET AL., GENE, vol. 231, 1999, pages 1 - 13
MATSUOKA, PROC NATL. ACAD. SCI. USA, vol. 90, no. 20, 1993, pages 9586 - 9590
MATSUOKA, PROC. NATL. ACAD. SCI. USA, vol. 90, no. 20, 1993, pages 9586 - 9590
MATTON, MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 2, 1989, pages 325 - 331
MCBRIDE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 7301 - 7305
MCCABE ET AL., BIO/TECHNOLOGY, vol. 6, 1988, pages 923 - 926
MCCABE ET AL., BIOTECHNOLOGY, vol. 6, 1988, pages 923 - 926
MCCORMICK ET AL., PLANT CELL REPORTS, vol. 5, 1986, pages 81 - 84
MCDOWELLWOFFENDEN, TRENDS BIOTECHNOL., vol. 21, no. 4, 2003, pages 178 - 83
MCELROY ET AL., PLANT CELL, vol. 2, 1990, pages 163 - 171
MCGURL ET AL., SCIENCE, vol. 225, 1992, pages 1570 - 1573
MCNELLIS ET AL., PLANT J., vol. 14, no. 2, 1998, pages 247 - 257
MEAGHER JL ET AL., GLYCOBIOLOGY, vol. 15, 2005, pages 1033 - 42
MEIJER ET AL., PLANT MOL. BIOL., vol. 16, 1991, pages 807 - 820
MEINKOTHWAHL, ANAL. BIOCHEM., vol. 138, 1984, pages 267 - 284
METHODS ENZYMOL, vol. 154
MIAO ET AL., PLANT CELL, vol. 3, no. 1, 1991, pages 11 - 22
MIKI ET AL., THEOR. APPL. GENET., vol. 80, 1990, pages 449
MILLS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 3543 - 3548
MINDRINOS ET AL., CELL, vol. 78, 1994, pages 1089
MINSHULLSTEMMER, CURR OPIN CHEM BIOL, vol. 3, 1999, pages 284 - 290
MISAWA ET AL., PLANT J., vol. 4, 1993, pages 833 - 840
MISAWA ET AL., PLANT J., vol. 6, 1994, pages 481 - 489
MOGEN ET AL., PLANT CELL, vol. 2, 1990, pages 1261 - 1272
MONALYSIN, PLOS PATHOGENS, vol. 7, 2011, pages 1 - 13
MOORE ET AL., J. MOL. BIOL., vol. 272, 1997, pages 336 - 347
MORGAN ET AL., APPLIED AND ENVIR. MICRO., vol. 67, 2001, pages 2062 - 2069
MUIR ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 6705 - 6710
MULLINS ET AL., J. AM. CHEM. SOC., vol. 116, 1994, pages 5529 - 5533
MUNROE ET AL., GENE, vol. 91, 1990, pages 151 - 158
MURASHIGESKOOG, PHYSIOL. PLANT., vol. 15, 1962, pages 473
MURRAY ET AL., NUCLEIC ACIDS RES., vol. 17, 1989, pages 477 - 498
MYERSMILLER, CABIOS, vol. 4, 1988, pages 11 - 17
NAIMOV ET AL., APPL. ENVIRON. MICROBIOL., vol. 67, 2001, pages 5328 - 5330
NAIMOV ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 74, 2008, pages 7145 - 7151
NAKAMAYEECKSTEIN, NUCL ACIDS RES, vol. 14, 1986, pages 9679 - 9698
NAMBIAR ET AL., SCIENCE, vol. 223, 1984, pages 1299 - 1301
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, no. 3, 1970, pages 443 - 453
NESS ET AL., NAT BIOTECHNOL, vol. 17, 1999, pages 893 - 896
NESS ET AL., NATURE BIOTECHNOL., vol. 20, 2002, pages 1251
NOMURA ET AL., PLANT SCI., vol. 44, 1986, pages 53 - 58
ODELL ET AL., NATURE, vol. 313, 1985, pages 810 - 812
OLESON J. ET AL., J. ECONOMIC ENTOMOLOGY, vol. 98, 2005, pages 1 - 8
OLESON J. ET AL., J. ECONOMIC ENTOMOLOGY, vol. 98, no. 1-8, 2005
OLIVA ET AL., ANTIMICROB. AGENTS CHEMOTHER., vol. 36, 1992, pages 913 - 919
OROZCO ET AL., PLANT MOL BIOL., vol. 23, no. 6, 1993, pages 1129 - 1138
OROZCO, PLANT MOL. BIOL., vol. 23, no. 6, 1993, pages 1129 - 1138
OSJODA ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 745 - 750
OTOMO ET AL., BIOCHEMISTRY, vol. 38, 1999, pages 16040 - 16044
OTOMO ET AL., J. BIOLMOL. NMR, vol. 14, 1999, pages 105 - 114
PARIJS ET AL., PLANTA, vol. 183, 1991, pages 258 - 264
PASZKOWSKI ET AL., EMBO J., vol. 3, 1984, pages 2717 - 2722
PATTEN ET AL., CURR OPIN BIOTECHNOL, vol. 8, 1997, pages 724 - 733
PATTERSON: "Current Protocol in Molecular Biology", vol. 10, 1998, JOHN WILEY & SON INC, pages: 1 - 24
PAUL ET AL., PLANT MOL. BIOL., vol. 19, 1992, pages 611 - 622
PECHY-TARR, ENVIRONMENTAL MICROBIOLOGY, vol. 10, 2008, pages 2368 - 2386
PELTIER ET AL., PLANT CELL, vol. 12, 2000, pages 319 - 341
PEN ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 292
PIETERSEVAN LOON, CURR. OPIN. PLANT BIO., vol. 7, no. 4, 2004, pages 456 - 64
PLANT SCIENCE (LIMERICK, vol. 79, no. 1, pages 69 - 76
PRATT ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 163, 1989, pages 1243
PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 600 - 605
PROUDFOOT, CELL, vol. 64, 1991, pages 671 - 674
PRZIBILLA ET AL., PLANT CELL, vol. 3, 1991, pages 169
PURCELL ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 15, 1993, pages 1406 - 1413
R. HORTON ET AL., GENE, vol. 77, 1989, pages 61 - 68
RAJAGOPAL ET AL., J. BIOL. CHEM., vol. 277, 2002, pages 46849 - 46851
RANG ET AL., APPL. ENVIRON. MICROBIOL., vol. 65, 1999, pages 2918 - 2925
REDOLFI ET AL., NETH. J. PLANT PATHOL., vol. 89, 1983, pages 245 - 254
REGAN, J. BIOL. CHEM., vol. 269, 1994, pages 9
REINES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 1917 - 1921
REZNIKOFF, MOL. MICROBIOL., vol. 6, 1992, pages 2419 - 2422
RIGGS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 5602 - 5606
RINEHART ET AL., PLANT PHYSIOL., vol. 112, no. 3, 1996, pages 1331 - 1341
RIVERA-MADRID ET AL., PROC. NATL. ACAD. SCI., vol. 92, 1995, pages 5620 - 5624
ROHMEIER ET AL., PLANT MOL. BIOL., vol. 22, 1993, pages 783 - 792
RUSSELL ET AL., TRANSGENIC RES., vol. 6, no. 2, 1997, pages 157 - 168
RYAN, ANN. REV. PHYTOPATH., vol. 28, 1990, pages 425 - 449
SAAEM, I. ET AL., NUCLEIC ACIDS RESEARCH, 29 November 2011 (2011-11-29), pages 1 - 8
SAKAMARKHORANA, NUCL ACIDS RES, vol. 14, 1988, pages 6361 - 6372
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOKRUSSELL: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SANAHUJA, PLANT BIOTECH JOURNAL, vol. 9, 2011, pages 283 - 300
SANDHU, CRITICAL REV. BIOTECH., vol. 12, 1992, pages 437 - 462
SANFACON ET AL., GENES DEV., vol. 5, 1991, pages 141 - 149
SANFORD ET AL., PARTICULATE SCIENCE AND TECHNOLOGY, vol. 5, 1987, pages 27 - 37
SANGER ET AL., PLANT MOL. BIOL., vol. 14, no. 3, 1990, pages 433 - 443
SAYERS ET AL., NUCL ACIDS RES, vol. 16, 1988, pages 791 - 802
SAYERS ET AL., NUCL ACIDS RES, vol. 16, 1988, pages 803 - 814
SCHENA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 10421 - 10425
SCHIMMING, EUR. J. BIOCHEM., vol. 204, 1992, pages 13 - 19
SCHNEPF ET AL., J. BIOL. CHEM., vol. 265, 1990, pages 20923 - 20930
SCIENCE, vol. 317, 2007, pages 1548 - 1551
SCOTT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 13638 - 13643
See also references of EP2971000A4
SEVERINOVMUIR, J. BIOL. CHEM., vol. 273, 1998, pages 16205 - 16209
SHAW ET AL., SCIENCE, vol. 233, 1986, pages 478 - 481
SHINGLEDECKER ET AL., GENE, vol. 207, 1998, pages 187 - 195
SHIOTA ET AL., PLANT PHYSIOL, vol. 106, 1994, pages 17
SHIROZA ET AL., J. BACTERIOL., vol. 170, 1988, pages 810
SHOEMAKER ET AL.: "Phytophthora Root Rot Resistance Gene Mapping in Soybean", PLANT GENOME IV CONFERENCE, 1995
SIEBERTZ ET AL., PLANT CELL, vol. 1, 1989, pages 961 - 968
SINGH ET AL., THEOR. APPL. GENET., vol. 96, 1998, pages 319 - 324
SMITH, ANN REV GENET, vol. 19, 1985, pages 423 - 462
SOGAARD ET AL., J. BIOL. CHEM., vol. 268, 1993, pages 22480
SOMSISCH ET AL., MOL. GEN. GENET., vol. 2, 1988, pages 93 - 98
SOMSISCH ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 2427 - 2430
SOMSSICH, CELL, vol. 113, no. 7, 2003, pages 815 - 6
SOONG ET AL., NAT GENET, vol. 25, no. 4, 2000, pages 436 - 439
SOUTHWORTH ET AL., BIOTECHNIQUES, vol. 27, 1999, pages 110 - 120
SOUTHWORTH ET AL., EMBO J., vol. 17, 1998, pages 918 - 926
SRINIVISANROSE, PROTEINS: STRUCT., FUNCT. & GENETICS, vol. 22, 1995, pages 81 - 99
STALKER ET AL., SCIENCE, vol. 242, 1988, pages 419 - 423
STANFORD ET AL., MOL. GEN. GENET., vol. 215, 1989, pages 200 - 208
STEINMETZ ET AL., MOL. GEN. GENET., vol. 200, 1985, pages 220
STEMMER ET AL., GENE, vol. 164, 1995, pages 49 - 53
STEMMER ET AL., TUMOR TARGETING, vol. 4, 1999, pages 1 - 4
STEMMER, BIO/TECHNOLOGY, vol. 13, 1995, pages 549 - 553
STEMMER, NATURE, vol. 370, 1994, pages 389 - 391
STEMMER, PNAS USA, vol. 91, 1994, pages 10747 - 10751
STEMMER, PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 10747 - 10751
STEMMER, SCIENCE, vol. 270, 1995, pages 1510
STEMMER: "The Encyclopedia of Molecular Biology", 1996, VCH PUBLISHERS, article "Sexual PCR and Assembly PCR", pages: 447 - 457
SVAB, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 8526 - 8530
SVABMALIGA, EMBO J., vol. 12, 1993, pages 601 - 606
SVABMALIGA, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 913 - 917
TAVLADORAKI ET AL., NATURE, vol. 366, 1993, pages 469
TAYLOR ET AL., NUCL ACIDS RES, vol. 13, 1985, pages 8749 - 8764
TAYLOR ET AL., NUCL ACIDS RES, vol. 13, 1985, pages 8765 - 8787
TAYLOR ET AL., SEVENTH INT'L SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS, 1994
TEATHERERFLE, J. BACTERIOL., vol. 172, 1990, pages 3837 - 3841
THE ROYAL SOCIETY. PHIL. TRANS. R. SOC. LOND. B., vol. 353, 1998, pages 777 - 1786
THOMPSON ET AL., BIOESSAYS, vol. 10, 1989, pages 108
TIJSSEN: "Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes", 1993, ELSEVIER
TOMES ET AL.: "Plant Cell, Tissue, and Organ Culture: Fundamental Methods", 1995, SPRINGER-VERLAG
TOUBART ET AL., PLANT J., vol. 2, 1992, pages 367
TOYODA ET AL., TRANSGENIC RES., vol. 11, no. 6, 2002, pages 567 - 82
TU ET AL., PLANT MOLECULAR BIOLOGY, vol. 37, 1998, pages 829 - 838
UKNES, PLANT CELL, vol. 4, 1992, pages 645 - 656
URWIN ET AL., PLANTA, vol. 204, 1998, pages 472 - 479
USSUF ET AL., CURR SCI., vol. 80, no. 7, 2001, pages 847 - 853
VAN CAMP ET AL., PLANT PHYSIOL., vol. 112, no. 2, 1996, pages 525 - 535
VAN FRANNKENHUYZEN, J. INVERT. PATH., vol. 101, 2009, pages 1 - 16
VAN HARTINGSVELDT ET AL., GENE, vol. 127, 1993, pages 87
VAN LOON, PLANT MOL. VIROL., vol. 4, 1985, pages 111 - 116
VASCONCELOSOLIVEIRA, TOXICON, vol. 44, no. 4, 2004, pages 385 - 403
VAUCHERET ET AL., PLANT J., vol. 16, 1998, pages 651 - 659
VELTEN ET AL., EMBO J., vol. 3, 1984, pages 2723 - 2730
VICKI CHANDLER: "The Maize Handbook", 1994, SPRINGER-VERLAG
VOLPE ET AL., SCIENCE, vol. 297, 2002, pages 1833 - 1837
WARNER ET AL., PLANT J., vol. 3, 1993, pages 191 - 201
WEISSINGER ET AL., ANN. REV. GENET., vol. 22, 1988, pages 421 - 477
WELLS ET AL., GENE, vol. 34, 1985, pages 315 - 323
WELLS ET AL., PHIL TRANS R SOC LOND A, vol. 317, 1986, pages 415 - 423
WESLEY ET AL., METHODS IN MOLECULAR BIOLOGY, PLANT FUNCTIONAL GENOMICS: METHODS AND PROTOCOLS, vol. 236, 2003, pages 273 - 286
WILLIAMSON, CURR OPIN PLANT BIO., vol. 2, no. 4, 1999, pages 327 - 31
WOHLLEBEN ET AL., GENE, vol. 70, 1988, pages 25 - 37
WOOD ET AL., NAT. BIOTECHNOL., vol. 17, 1999, pages 889 - 892
WU ET AL., BIOCHIM BIOPHYS ACTA, vol. 1387, 1998, pages 422 - 32
WU ET AL., BIOCHIM BIOPHYS ACTA, vol. 1387, 1998, pages 422 - 432
WU ET AL., PROC NATL ACAD SCI USA, vol. 95, no. 16, 1998, pages 9226 - 31
WU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 9226 - 9231
WYBORSKI ET AL., NUCLEIC ACIDS RES., vol. 19, 1991, pages 4647 - 4653
XU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 388 - 393
YAMAMOTO ET AL., PLANT J., vol. 12, no. 2, 1997, pages 255 - 265
YAMAMOTO, PLANT CELL PHYSIOL., vol. 35, no. 5, 1994, pages 773 - 778
YAMAMOTO, PLANT J., vol. 12, no. 2, 1997, pages 255 - 265
YAMAZAKI ET AL., J. AM. CHEM. SOC., vol. 120, 1998, pages 5591 - 5592
YAMIUCHIMINAMIKAWA, FEBS LETT., vol. 260, 1991, pages 127 - 130
YANG, J ET AL., TRANSGENE RES, vol. 15, 2006, pages 583 - 593
YANG, PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 14972 - 14977
YAO ET AL., CELL, vol. 71, 1992, pages 63 - 72
YARRANTON, CURR. OPIN. BIOTECH., vol. 3, 1992, pages 506 - 511
ZAMBRETTI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 3952 - 3956
ZHANG ET AL., ANNALS OF MICROBIOLOGY, vol. 59, 2009, pages 45 - 50
ZHANG ET AL., PNAS USA, vol. 94, 1997, pages 4504 - 4509
ZHANG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 2507 - 2511
ZHANG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 4504 - 4509
ZJAWIONY, J NAT PROD, vol. 67, no. 2, 2004, pages 300 - 310
ZOLLERSMITH, METHODS ENZYMOL, vol. 100, 1983, pages 468 - 500
ZOLLERSMITH, METHODS ENZYMOL, vol. 154, 1987, pages 329 - 350
ZOLLERSMITH, NUCLEIC ACIDS RES, vol. 10, 1982, pages 6487 - 6500

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120276A1 (en) 2014-02-07 2015-08-13 Pioneer Hi Bred International Inc Insecticidal proteins and methods for their use
EP3705489A1 (en) 2014-02-07 2020-09-09 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2015130893A1 (en) 2014-02-28 2015-09-03 E. I. Du Pont De Nemours And Company Synthetic oligoglucosamines for improvement of plant growth and yield
WO2015130890A1 (en) 2014-02-28 2015-09-03 E. I. Du Pont De Nemours And Company Synthetic salt complexes for improvement of plant growth and yield
WO2015134256A1 (en) 2014-02-28 2015-09-11 E. I. Du Pont De Nemours And Company Combinatorial libraries
CN104365250A (en) * 2014-10-31 2015-02-25 山东省农业科学院作物研究所 Soil basic fertility-based efficient application method of nitrogenous fertilizer for wheat in Huang-Huai-Hai wheat growing region
US10472642B2 (en) 2015-02-05 2019-11-12 British American Tobacco (Investments) Limited Method for the reduction of tobacco-specific nitrosamines or their precursors in tobacco plants
US20190330652A1 (en) * 2016-09-09 2019-10-31 Syngenta Pariticpations AG Insecticidal proteins
US11136593B2 (en) * 2016-09-09 2021-10-05 Syngenta Participations Ag Insecticidal proteins
US11661610B2 (en) 2016-09-09 2023-05-30 Syngenta Participations SG Insecticidal proteins
WO2018084936A1 (en) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP4050021A1 (en) 2016-11-01 2022-08-31 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2018140214A1 (en) 2017-01-24 2018-08-02 Pioneer Hi-Bred International, Inc. Nematicidal protein from pseudomonas
EP3691453A4 (en) * 2017-10-02 2021-07-07 Syngenta Participations AG Engineered pesticidal proteins and methods of controlling plant pests
US11805777B2 (en) 2017-10-02 2023-11-07 Syngenta Participations Ag Engineered pesticidal proteins and methods of controlling plant pests
CN113677809A (en) * 2018-10-30 2021-11-19 先正达参股股份有限公司 Insecticidal proteins
EP3874069A4 (en) * 2018-10-30 2022-12-21 Syngenta Participations AG Insecticidal proteins

Also Published As

Publication number Publication date
MX360160B (en) 2018-10-24
US11180775B2 (en) 2021-11-23
WO2014150914A8 (en) 2015-11-26
MX2015013269A (en) 2016-06-07
CA2901316A1 (en) 2014-09-25
RU2015143825A (en) 2017-04-26
BR112015023709A2 (en) 2017-07-18
US10023877B2 (en) 2018-07-17
US20220025393A1 (en) 2022-01-27
EP2971000A4 (en) 2016-11-23
US20180291398A1 (en) 2018-10-11
US20160040184A1 (en) 2016-02-11
US20200131529A1 (en) 2020-04-30
ZA201505705B (en) 2017-11-29
EP2971000A2 (en) 2016-01-20
US10577626B2 (en) 2020-03-03
US11697821B2 (en) 2023-07-11
CN105473605A (en) 2016-04-06
WO2014150914A3 (en) 2014-11-13

Similar Documents

Publication Publication Date Title
US11697821B2 (en) PHI-4 polypeptides and methods for their use
US11325949B2 (en) Insecticidal proteins and methods for their use
US11091771B2 (en) PHI-4 polypeptides and methods for their use
US11780891B2 (en) Insecticidal proteins and methods for their use
US20210254095A1 (en) Novel insecticidal proteins from plants
US10563221B2 (en) Insecticidal proteins and methods for their use
US20190062382A1 (en) Insecticidal proteins and methods for their use
WO2014018853A1 (en) Novel insecticidal proteins and methods for their use

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201480027131.7

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14768504

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2901316

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2014768504

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: IDP00201505691

Country of ref document: ID

Ref document number: 14776212

Country of ref document: US

Ref document number: MX/A/2015/013269

Country of ref document: MX

NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14768504

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2015143825

Country of ref document: RU

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112015023709

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112015023709

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20150915