OA16614A - AXMI115 variant insecticidal gene and methods for its use. - Google Patents

AXMI115 variant insecticidal gene and methods for its use. Download PDF

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Publication number
OA16614A
OA16614A OA1201300426 OA16614A OA 16614 A OA16614 A OA 16614A OA 1201300426 OA1201300426 OA 1201300426 OA 16614 A OA16614 A OA 16614A
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seq
amino acid
sequence
plant
polypeptide
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OA1201300426
Inventor
Duane Lehtinen
Nalini Manoj Desai
Volker Heinrichs
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Athenix Corp.
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Publication of OA16614A publication Critical patent/OA16614A/en

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Abstract

Compositions and methods for conferring pesticidal activital to bacteria, plants, plant cells, tissues and seeds are provided. The toxin coding sequences can be used in DNA constructs or expression cassettes for expression in plants and bacteria. Compositions also include transformed bacteria, plants, plant cells, tissues, and seeds. In particular, polynucleotide sequences and the toxin proteins encoded thereby are provided. Also provided are antibodies specifically binding to those amino acid sequences. In particular, the invention encompasses nucleotide sequences encoding fusion proteins, as well as biologically active variants and fragments thereof, wherein the fusion protein contains the C-terminal portion of SEQ ID NO:43. The fusion protein may also contain the N-terminal portion of SEQ ID NO:45. The invention also includes the nucleotide sequences of SEQ ID NO:47 and 1-14, or a nucleodide sequence encoding the amino acid sequence set forth in SEQ ID NO:48 and 15-31, including biologically active variants and fragments thereof.

Description

This invention relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis is a Gram-positive spore forming soil bacterium characterized by its ability to produce crystalline inclusions that are specifically toxic to certain orders and species of insects, but are harmless to plants and other non-targeted organisme. For this reason, compositions including Bacillus thuringiensis strains or their insecticidal proteïns can be used as environmentally-acceptable insecticides to control agricultural însect pests or insect vectors for a variety of human or animal diseases.
Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis hâve potent insecticidal activity against predominantly Lepidopteran, Hemipteran, Dipteran, and Coleopteran larvae. These proteins also hâve shown activity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari pest orders, as well as other învertebrate orders such as Nemathelminthes, Platyhelminthes,
-l16614 and Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensis family tree. In Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, N.Y.) The crystal protein does not exhibit insecticidal activity until it has been ingested and solubilized in the insect midgut. The ingested protoxin is hydrolyzed by proteases in the insect digestive tract to an active toxic molécule. (Hôfte and Whiteley (1989) Microbiol. Rev. 53:242-255). This toxin binds to apical brush border receptors in the midgut of the target larvae and inserts into the apical membrane creating ion channels or pores, resulting in larval death.
In addition to the endotoxins, B. thuringiensis also produces secreted insecticidal proteins during its végétative growth stage, namely, végétative insecticidal proteins (Vip). Sincethe discovery of the flrst Vip toxin, two major groups of Viptoxins hâve been îdentified in B. thuringiensis. One group of Vip toxins consists of binary toxins which are made of two components, Vip1 and Vip2 (Warren (1997) In N. B. Carozzi and M. G. Koziel (ed.), Advances in insect control: the rôle of transgenic plants. Taylor & Francis, London, United Kingdom). The combination of Vip1 and Vip2 is highly insecticidal to an agriculturally important insect, the western corn rootworm (Diabrotica virgifera), but does not show any insecticidal activity for any lepidopteran insects (Han et al. (1999) Nat. Struct. Biol. 6:932-936). The other group consists of Vip3 toxins, which share no sequence similarity to Vip1 or Vip2, The first-identified Vip3 toxin, Vip3Aa1, is highly insecticidal to several major lepidopteran pests of maize and cotton, including the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea, but shows no activity against the European corn borer Ostrinia nubilalis, a major pest of maize (Estruch et al. (1996) Proc. Natl. Acad. Sci. USA 93:5389-5394). The délétion ofthe vip3Aa1 genefrom a B. thuringiensis strain resulted in a significant réduction of the insecticidal activity of that 8. thuringiensis strain, suggesting that Vip3 contributes to the overall toxicity of B. thuringiensis strains (Donovan et al. (2001) J. Invertebr. Pathol. 78:45-51). It was also observed that Vip3Aa1 kills insects by lysing insect midgut cells (Yu et al. (1997) Appl. Environ. Microbiol. 63:532-536) via cell membrane pore formation (Lee et al. (2003) Appl. Environ. Microbiol. 69:4648-4657).
The intensive use of B. thuringiensis-baseti insecticides has already given rise to résistance in field populations of the diamondback moth, Plutella xylostella (Ferré and Van Rie (2002) Annu. Rev. Entomol. 47:501-533). The most common mechanism of résistance is the réduction of binding of the toxin to its spécifie midgut receptor(s). This may also confer cross-resistance to other toxins that share the same receptor (Ferré and Van Rie (2002)),
SUMMARY OF INVENTION
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Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molécules encoding sequences for pesticidal and insectidal polypeptides, vectors comprising those nucleic acid molécules, and host cells comprising the vectors. Compositions also include the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucléotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisons, including microorganisms and plants. The nucléotide or amino acid sequences may be synthetic sequences that hâve been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise bacteria, plants, plant cells, tissues, and seeds comprising the nucléotide sequence of the invention.
In particular, isolated nucleic acid molécules are provided that encode a pesticidal protein. Additionally, amino acid sequences corresponding to the pesticidal protein are encompassed. In particular, the présent invention provides for an isolated or recombinant nucleic acid molécule comprising a nucléotide sequence encoding a fusion protein, as well as biologlcally active variants and fragments thereof, wherein the fusion protein comprises the C-terminal portion of SEQ ID NO:43. In various embodiments, the fusion protein comprises the N-terminal portion of SEQ ID NO:45. In spécifie embodiments, the nucleic acid molécule encompassed by the présent invention (including vectors, host cells, plants, and seeds comprising the nucleic acid molécule) comprises the nucléotide sequence set forth in SEQ ID NO:47 and 1-14, or a nucléotide sequence encoding the amino acid sequence set forth in SEQ ID NO:48 and 15-31, including biologically active variants and fragments thereof. Nucléotide sequences that are complementary to a nucléotide sequence of the invention, or that hybridize to a sequence of the invention or a complément thereof are also encompassed. Isolated or recombinant fusion proteins encoded by the nucleci acid molécule of the invention are also encompassed herein.
Methods are provided for producing the polypeptides of the invention, and for using those polypeptides for controlling or killing a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest. Methods and kits for detecting the nucleic acids and polypeptides of the invention in a sample are also included.
The compositions and methods of the invention are useful for the production of organisms with enhanced pest résistance or tolérance. These organisms and compositions comprising the organisms are désirable for agricultural purposes. The compositions of the invention are also useful for generating altered or improved proteins that hâve pesticidal activity, or for detecting the presence of pesticidal proteins or nucleic acids in products or organisms.
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a diagram of the fusion constructs.
Figure 2 shows the results of the in vitro leaf disk bioassay. pAG6585 contains optAxmi115v01 (N=14)and pAG6141 contains optAxmi115v02.01.01 (N=8).
DETAILED DESCRIPTION
The présent invention is drawn to compositions and methods for regulating pest résistance or tolérance in organisms, particularly plants or plant cells. By “résistance is intended that the pest (e.g., insect) is killed upon ingestion or other contact with the polypeptides of the invention. By “tolérance is intended an impairment or réduction in the movement, feeding, reproduction, or other functions of the pest. The methods involve transforming organisms with a nucléotide sequence encoding a pesticidal protein of the invention. In particular, the nucléotide sequences of the invention are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are pesticidal nucleic acids and proteins of Bacillus or other species. The sequences fïnd use in the construction of expression vectors for subséquent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the génération of altered pesticidal proteins by methods known in the art, such as domain swapping or DNA shuffling, for example, with members of the Vip1, Vip2, or Vip3 families of toxins. The proteins find use in controlling or killing lepidopteran, hemipteran, coleopteran, dipteran, and nematode pest populations and for producing compositions with pesticidal activity.
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, and Coleoptera orders, or the Nematoda phylum, or a protein that has homology to such a protein. Pesticidal proteins hâve been isolated from organisms including, for example, Bacillus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins include amino acid sequences deduced from the full-length nucléotide 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 nucléotide sequences that confer pesticidal activity. These nucléotide sequences encode polypeptides with homology to known
-416614 toxins. Also provided are the amino acid sequences of the pesticidal proteins. The protein resulting from translation of this gene allows cells to control or kill pests that ingest it.
Isolated Nucleic Acid Molécules, and Variants and Fragments Thereof
One aspect of the invention pertains to isolated or recombinant nucleic acid molécules comprising nucléotide sequences encoding pesticidal proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molécules sufficient for use as hybridization probes to identify nucleic acid molécules encoding proteins with régions of sequence homology. Also encompassed herein are nucléotide sequences capable of hybridizing to the nucléotide sequences of the invention under stringent conditions as defined elsewhere herein. As used herein, the term “nucleic acid molécule” is intended to include DNA molécules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molécules (e.g., mRNA) and analogs of the DNA or RNA generated using nucléotide analogs. The nucleic acid molécule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
An isolated or “recombinant” nucleic acid sequence (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 an in vitro or 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 invention, “isolated when used to refer to nucleic acid molécules excludes isolated chromosomes. For example, in various embodiments, the isolated delta-endotoxin encoding nucleic acid molécule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucléotide sequences that naturally flank the nucleic acid molécule in genomic DNA of the cell from which the nucleic acid is derived. In various embodiments, a delta-endotoxin protein that is substantially free of cellular material includes préparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-delta-endotoxin protein (also referred to herein as a “contaminating protein).
Nucléotide sequences encoding the proteins of the présent invention include the sequence set forth in SEQ ID NO;47 and 1-14, and variants, fragments, and compléments thereof. By “complément is intended a nucléotide sequence that is sufficiently complementary to a given nucléotide sequence such that it can hybridize to the given nucléotide sequence to thereby form a stable duplex. The corresponding amino acid sequences for the pesticidal proteins encoded by these nucléotide sequences are set forth in SEQ ID NO:48 and 15-31.
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Nucleic acid molécules that are fragments of these nucléotide sequences encoding pesticidal proteins are also encompassed by the présent invention. By “fragment is intended a portion of the nucléotide sequence encoding a pesticidal protein. A fragment of a nucléotide sequence may encode a biologically active portion of a pesticidal protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molécules that are fragments of a nucléotide sequence encoding a pesticidal protein comprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1350, 1400 contiguous nucléotides, or up to the number of nucléotides présent in a full-length nucléotide sequence encoding a pesticidal protein disclosed herein, depending upon the intended use. By contiguous nucléotides is intended nucléotide residues that are immediately adjacent to one another. Fragments of the nucléotide sequences of the présent invention will encode protein fragments that retain the biological activity of the pesticidal protein and, hence, retain pesticidal activity. Thus, biologically-active fragments of the polypeptides disclosed herein are also encompassed. By “retains activity is intended that the fragment will hâve at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the pesticidal activity of the pesticidal protein. In various embodiments, the activity may be improved or extended relative to a référencé pesticidal protein (e.g., improved or extended relative to the activity of SEQ ID NO:43 or 45) as defined elsewhere herein. In one embodiment, the pesticidal activity is coleoptericidal activity. In another embodiment, the pesticidal activity is lepidoptericidal activity. In another embodiment, the pesticidal activity is nematocidal activity. In another embodiment, the pesticidal activity is diptericidal activity. In another embodiment, the pesticidal activity is hemiptericidal activity. 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 étal. (1988) Biochem. J. 252:199-206; Marrone étal. (1985) J. of EconomieEntomology 78:290-293; and U.S. Patent No. 5,743,477, ail of which are herein incorporated by référencé in their entirety.
A fragment of a nucléotide sequence encoding a pesticidal protein that encodes a biologically active portion of a protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 contiguous amino acids, or up to the total number of amino acids présent in a full-length pesticidal protein of the invention. In some embodiments, the fragment is a proteolytic cleavage fragment. For example, the proteolytic cleavage fragment may hâve an N-terminal or a C-terminal truncation of at least about 100 amino acids, about 120, about 130, about 140, about 150, or about 160 amino acids relative to SEQ ID NO:48 and 15-31. In some embodiments, the fragments encompassed herein resuit from the removal of the C-terminal crystallization domain, e.g., by proteolysis or by insertion of a stop codon in the coding sequence.
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In other embodiments, the fusion protein comprises a fragment of the C-terminal domain of SEQ ID NO:43 and/or a fragment of the N-terminal domain of SEQ ID NO:45.
Preferred pestïcidal proteins of the présent invention are encoded by a nucléotide sequence sufficiently identical to the nucléotide sequence of SEQ ID NO:47 and 1-14, or the pestïcidal proteins are sufficiently identical to the amino acid sequence set forth in SEQ ID NO:48 and 15-31. In another embodiment, the nucléotide sequence encodes a fusion protein, wherein the N-terminal portion is sufficiently identical to the N-terminal portion of SEQ ID NO:45, or wherein the N-terminal portion is sufficiently identical to the N-terminal portion of SEQ ID NO:45 and the C-terminal portion is sufficiently identical to SEQ ID NO:43. By “sufficiently identical is intended an amino acid or nucléotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignaient programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to détermine corresponding identity of proteins encoded by two nucléotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
To détermine 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 fonction 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) x 100). In one embodiment, the two sequences are the same length. In another embodiment, the percent identity is calculated across the entirety of the reference sequence (i.e., the sequence disclosed herein as any of SEQ ID NO: 1-31, 47 or 48). 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. A gap, i.e. a position in an alignment where a residue is présent in one sequence but not in the other, is regarded as a position with non-identical residues.
The détermination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting 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 étal. (1990) J. Mol. Biol. 215:403. BLAST nucléotide searches can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucléotide sequences homologous to pesticidal-like nucleic acid molécules of the invention. BLAST protein searches can be performed with the BLASTX
-716614 program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to pesticidal protein molécules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul ef al. (1997) Nucleic Acids Res. 25:3389. Alternative^, PSI-Blast can be used to perform an iterated search that detects distant relationships between molécules. 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 étal. (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, CA). 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 4:11 -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, CA, 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.
Unless otherwise stated, GAP Version 10, which uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used to détermine sequence identity or similarity using the following parameters: % identity and % similarity for a nucléotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp 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 “équivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucléotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
The invention also encompasses variant nucleic acid molécules. “Variants” of the pesticidal protein encoding nucléotide sequences include those sequences that encode the pesticidal proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic
-816614 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 nucléotide sequences also include synthetically derived nucléotide sequences that hâve been generated, for example, by using site-directed mutagenesis but which still encode the pestïcidal proteins disclosed in the présent invention as discussed below. Variant proteins encompassed by the présent invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, pestïcidal activity. By “retains activity is intended that the variant will hâve at Ieast about 30%, at Ieast about 50%, at Ieast about 70%, or at Ieast about 80% of the pesticidal activity of the native protein. In some embodiments, the activity is improved or extended relative to a reference protein as defined elsewhere herein. 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 étal. (1985) J. of Economie Entomology 18:290-293; and U.S. Patent No. 5,743,477, ail of which are herein incorporated by reference in their entirety.
The skilled artisan will further appreciate that changes can be introduced by mutation of the nucléotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded pesticidal proteins, without altering the biological activity of the proteins. Thus, variant isolated nucleic acid molécules can be created by introducing one or more nucléotide substitutions, additions, or délétions into the corresponding nucléotide sequence disclosed herein, such that one or more amino acid substitutions, additions or délétions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCRmediated mutagenesis. Such variant nucléotide sequences are also encompassed by the présent invention.
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 pesticidal protein without altering the biological activity, whereas an “essentiar amino acid residue is required for 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 hâve 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), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, méthionine, tryptophan), beta-branched
-916614 side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Amino acid substitutions may be made in nonconserved régions 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 îdentical between ail proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that are îdentical 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 hâve only conservative substitutions between ail proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that hâve only conservative substitutions between ail proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may hâve minor conserved or nonconserved alterations in the conserved residues.
Alternatively, variant nucléotide sequences can be made by introducing mutations randomly along ail or part of the coding sequence, such as by saturation mutagenesis, and the résultant 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.
Using methods such as PCR, hybridization, and the lîke corresponding pesticidal sequences can be identified, such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academie Press, NY).
In a hybridization method, ail or part of the pesticidal nucléotide 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 socalled hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a détectable group such as 32P, or any other détectable 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 pesticidal protein-encoding nucléotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucléotides or amino acid residues in the nucléotide sequence or encoded amino acid sequence can additionaily be used. The probe
-1016614 typically comprises a région of nucléotide 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 nucléotides of nucléotide sequence encoding a pesticidal protein of the invention or a fragment or variant thereof. Methods for the préparation 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 pesticidal sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding pesticidal protein-like sequences and messenger RNAs. To achieve spécifie hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucléotides in length, or at least about 20 nucléotides 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 détermine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA librairies (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, New York).
Thus, the présent invention encompasses probes for hybridization, as well as nucléotide sequences capable of hybridization to ail or a portion of a nucléotide sequence of the invention (e.g., at least about 300 nucléotides, at least about 400, at least about 500,1000,1200,1500, 2000, 2500, 3000, 3500, or up to the full length of a nucléotide sequence disclosed herein). 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 washîng 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 nucléotides in length, preferably less than 500 nucléotides in length.
Typically, stringent conditions will be those in which the sait 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 température is at least about 30°C for short probes (e.g., 10 to 50 nucléotides) and at least about 60°C for long probes (e.g., greater than 50 nucléotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as fomnamide. Exemplary low stringency
-1116614 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 1X to 2X SSC (20X SSC = 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 1X 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 0.1X SSC 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.
Specîficity is typicaily the function of post-hybridization washes, the critical factors being the ionic strength and température of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the équation 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 nucléotides 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 température (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 spécifie sequence and its complément 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 11, 12,13, 14, 15, or 20°C lower than the thermal melting point (Tm). Using the équation, 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 température 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, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-lnterscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
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Isolated Proteins and Variants and Fragments Thereof
Pesticidal proteins are also encompassed within the présent invention. By “pesticidal protein is intended a protein having the amino acid sequence setforth in SEQ ID NO:48 and 15-
31. Fragments, biologically active portions, and variants thereof are also provided, and may be used to practice the methods of the présent invention. An isolated protein or 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.
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:48 and 15-31, and that exhibit pesticidal activity. A biologically active portion of a pesticidal protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. 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 Economie Entomology 78:290-293; and U.S. Patent No. 5,743,477, ail of which are herein incorporated by reference in their entirety. As used here, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO:48 and 15-31. The invention encompasses other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50,100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids in length.
By “variants is intended proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any of SEQ ID NO:48 and 15-31. Variants also include polypeptides encoded by a nucleic acid molécule that hybridizes to the nucleic acid molécule of SEQ ID NO:47 and 1-14, or a complément thereof, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the présent invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining pesticidal activity. In some embodiments, the variants hâve improved activity relative to the native protein. 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 étal. (1988) Biochem. J. 252:199-206; Marrone étal.
-1316614 (1985) J. of Economie Entomology 78:290-293; and U.S. Patent No. 5,743,477, ail of which are herein incorporated by référencé in their entirety.
Bacterial genes, such as the axmi genes of this invention, quite often possess multiple méthionine 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 génération 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 méthionine 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 méthionine. 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 méthionine codons may also lead to génération of pesticidal proteins. These pesticidal proteins are encompassed in the présent invention and may be used in the methods of the présent invention. It will be understood that, when expressed in plants, it will be necessary to alter the alternate start codon to ATG for proper translation.
Antibodies to the polypeptides of the présent invention, 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, NY; U.S. Patent No. 4,196,265).
Altered or Improved Variants
It is recognized that DNA sequences of a pesticidal protein may be altered by various methods, and that these alterations may resuit in DNA sequences encoding proteins with amino acid sequences different than that encoded by a pesticidal protein of the présent invention. This protein may be altered in various ways including amino acid substitutions, délétions, truncations, and insertions of one or more amino acids of SEQ ID NO:48 and 15-31, including up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, or more amino acid substitutions, délétions or insertions within either the C-terminal portion or the N-terminal portion, or both. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a pesticidal protein can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in dîrected évolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the fonction of the protein. Such variants will possess the desired pesticidal activity. However, it is understood
-1416614 that the ability of a pesticidal protein to confer pesticidal activity may be improved by the use of such techniques upon the compositions of this invention. For example, one may express a pesticidal protein in host cells that exhibit high rates of base misincorporation during DNA réplication, such as XL-1 Red (Stratagene, La Jolla, CA). After propagation in such strains, one can isolate the DNA (for example by preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a vector), culture the pesticidal protein mutations in a non-mutagenic strain, and identify mutated genes with pesticidal activity, for example by performing an assay to test for pesticidal activity. Generally, the protein is mîxed and used in feeding assays. See, for example Marrone ef al. (1985) J. of Economie 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. Examples of mutations that resuit in increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.
Alternative^, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, délétions, or alterations introduced by modem 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 détection, or other experimental uses known in the art (3) target sécrétion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic réticulum of eukaryotic cells, the iatter of which often results in glycosylation of the protein.
Variant nucléotide and amino acid sequences of the présent invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different pesticidal protein coding régions can be used to create a new pesticidal protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence régions that hâve 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 of the invention 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
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391; Cramer! étal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336347; Zhang étal. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.
Domain swapping or shuffling is another mechanism for generating altered pesticidal proteins. Domains may be swapped between pesticidal proteins, 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) App/. 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).
Thus, in various embodiments of the présent invention, the nucleic acid sequences encompassed herein (as well as compositions, vectors, host cells, plants, and seed comprising the nucleic acid sequence) comprise a portion of one or more toxin(s) and a portion of one of more different toxin(s). In one embodiment, the nucleic acid sequence comprises a nucléotide sequence encoding the N-terminal portion of AxmiOOS (which is set forth in SEQ ID NO:45) and the C-terminal portion of Axmil 15 (which is set forth in SEQ ID NO:43). In spécifie embodiments, the N-terminal portion of AxmiOOS comprises from about amino acid residues 1 to 173, or from about amino acid residue 1,2,3, 4, 5, 6, 7, 8, 9,10,15, 20, 25, 30, 35, 40, 45, or 50 to about amino acid residue 150, 155, 160, 165, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 185,190, 195, 200, 205, 210, 215, 220, 225, 230, 250, 300, 325, or 350 of AxmiOOS and the C-terminal portion of Axmil 15 comprises from about amino acid residue 174 to about amino acid residue 803 of Axmil 15, or from about amino acid residue 170,171,172, 173,174,175,176, 177, 178, 179, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 250, 300, 325, or 350 to about amino acid residue 600, 650, 700, 750, 760, 770, 780, 790, 795, 796, 797, 798, 799, 800, 801, 802, or 803. One of skill in the art will recognize that minor variants and délétions within each ofthe amino acid sequences can be made and still retain (or improve) activity of the fusion protein. In some embodiments, the nucleic acid sequences of the invention encode an Axmi005/Axmi115 fusion protein with a mutation (relative to the corresponding région of the parent Axmi005 or Axmil 15 protein) at one or more of positions corresponding to the amino acid residues at positions 584, 588, and 771 relative to SEQ ID NO:43 (see, for example, the variant fusion sequences found in SEQ ID NO:18-22). In other embodiments, the nucléotide sequence encompassed herein is set forth in any of SEQ ID NO:47 and 1-14 and the amino acid sequence is set forth in any of SEQ ID NO:48 and 15-31.
In various embodiments, the fusion of Axmi005 with Axmil 15 résulte in an amino acid sequence having improved or extended activity compared to the activity of either AxmiOOS or Axmil 15 alone. By “improved activity is intended an increase in death to at least one pest, or an
-1616614 increase in the noticeable réduction of pest growth, feeding, or normal physiological development relative to the native protein. By “extended” activity is intended activity against a pest that was not demonstrated by both Axmi005 and Axmil 15. For exampie, fusion of a portion of Axmi005 with a portion of Axmil 15 could resuit in a single protein having the activity profile of both AxmiOOS and Axmil 15. In some embodiments, activity against an individual pest is improved in the fusion protein over one or both of Axmi005 and/or Axmil 15.
Vectors
A pesticidal sequence of the invention may be provided in an expression cassette for expression in a plant of interest. By “plant expression cassette” is intended a DNA construct that is capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically these contain a promoter and a coding sequence. Often, such constructs will also contain a 3' untranslated région. Such constructs may 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 réticulum, or Golgi apparatus.
By “signal sequence is intended a sequence that is known or suspected to resuit in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves sécrétion 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 of the présent invention, the signal sequence is located in the native sequence, or may be derived from a sequence of the invention. 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 réticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like.
By “plant transformation vector” is intended a DNA molécule that is necessary for efficient transformation of a plant cell. Such a molécule may consist of one or more plant expression cassettes, and may be organized into more than one vector” DNA molécule. For example, binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode ail requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451 ). “Vector refers to a nucleic acid construct designed for transfer between different host cells. “Expression vector refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell. The cassette will include 5' and/or 3’ regulatory sequences operably linked to a sequence of the
-1716614 invention. By “operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiâtes and médiates 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 régions, contiguous and in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
In various embodiments, the nucléotide sequence of the invention is operably linked to a promoter, e.g., a plant promoter. Promoter refers to a nucleic acid sequence that functions to direct transcription of a downstream coding sequence. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”) are necessary for the expression of a DNA sequence of interest.
Such an expression cassette is provided with a plurality of restriction sites for insertion of the pesticidal sequence to be under the transcriptional régulation of the regulatory régions.
The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation région (i.e., a promoter), a DNA sequence of the invention, and a translational and transcriptional termination région (i.e., termination région) functional in plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is “native” or “homologous to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or “heterologous to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
The termination région may be native with the transcriptional initiation région, 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 DNA sequence of interest, the plant host, or any combination thereof). Convenient termination régions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination régions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Ce//64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Ce//2:1261-1272; Munroe étal. (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, the gene(s) may be optimized for increased expression in the transformed host cell. That is, the genes can be synthesized using host cell-preferred codons for r~
-1816614 improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, U.S. Patent Publication No. 20090137409, and Murray étal. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by référencé.
In one embodiment, the pesticidal protein is targeted to the chloroplast for expression. In this manner, where the pesticidal protein is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the pesticidal protein to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991 ) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:1754417550; Della-Cioppa étal, (1987) PlantPhysiol. 84:965-968; Romer étal. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah étal. (1986) Science 233:478-481.
The pesticidal gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for différences in codon usage between the plant nucléus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by référencé.
Plant Transformation
Methods ofthe invention involve introducing a nucléotide construct into a plant. By “introducing” is intended to présent to the plant the nucléotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not require that a particular method for introducing a nucléotide construct to a plant is used, only that the nucléotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucléotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
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, pollen).
“Transgenic plants or “transformed plants” or stably transformed plants or cells or tissues refers to plants that hâve incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not présent in the untransformed plant cell, as well as those that may be endogenous, or présent in
-1916614 the untransformed plant cell. “Heterologous generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are présent, and hâve been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.
The transgenic plants of the invention express one or more of the novel toxin sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes for insect résistance (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, and Cry1F families; Cry2, such as members ofthe Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.). It will be understood by one of skill in the art that the transgenic plant may comprise any gene împarting an agronomie trait of interest.
Transformation of plant cells can be accomplished by one of several techniques known in the art. The pesticidal gene of the invention may be modifîed to obtain or enhance expression in plant cells. Typically a construct that expresses such a protein would contain a promoter to drive transcription ofthe gene, as well as a 3' untranslated région to allow transcription termination and polyadenylation. The organization of such constructs is well known in the art. In some instances, it may be useful to engîneer the gene such that the resulting peptide is secreted, or otherwise targeted within the plant cell. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic réticulum. It may also be préférable to engîneer the plant expression cassette to contain an intron, such that mRNA processing of the intron is required for expression.
Typically this “plant expression cassette will be inserted into a “plant transformation vector. This plant transformation vector 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 Agrobacfer/um-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 molécules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required forT-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 génération of transgenic plants is desired). Also présent on this plasmid vector are sequences required for bacterial réplication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker
-2016614 gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that médiate 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 virmediated 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 sélection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Expiants 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 régénération medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a sélective 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 14:745750). Expiants 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 présent in any piece of subjected target callus or tissue or group of cells. The ability to kîII 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 génération of transgenic plants.
Transformation protocols as well as protocols for introducing nucléotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Génération of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle accélération, aérosol beam transformation (U.S. Published Application No. 20010026941; U.S.
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Patent No. 4,945,050; International Publication No. WO 91/00915; U.S. Published Application No. 2002015066), Lec1 transformation, and various other non-particle direct-mediated methods to transfer DNA.
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. Sel. 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 plastîd-borne transgene by tissue-preferred expression of a nuclearencoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
Following intégration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate sélection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this sélection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate sélection, one identifies and proliférâtes 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.
The cells that hâve 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 expression of the desired phenotypic characteristic identified. Two or more générations may be grown to ensure that expression ofthe desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the présent invention provides transformed seed (also referred to as “transgenic seed) having a nucléotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
Evaluation of Plant Transformation
Following introduction of heterologous foreign DNA into plant cells, the transformation or intégration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and métabolites associated with the integrated gene.
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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 Manuel. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers spécifie 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 intégration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).
In Northern blot analysis, RNA is isolated from spécifie tissues of transformant, fractionated in a formaldéhyde 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 présent on the pesticidal protein.
Pesticidal Activity in Plants
In another aspect of the invention, one may generate transgenic plants expressing a pesticidal protein that has pesticidal activity. Methods described above by way of example may be utilized to generate transgenic plants, but the manner in which the transgenic plant cells are generated is not critical to this invention. Methods known or described in the art such as Agrobacterium-mediated transformation, biolistic transformation, and non-particle-mediated methods may be used at the discrétion of the expérimenter. Plants expressing a pesticidal protein may be isolated by common methods described in the art, for example by transformation of callus, sélection of transformed callus, and régénération of fertile plants from such transgenic callus. In such process, one may use any gene as a selectable marker so long as its expression in plant cells confers ability to identify or select for transformed cells.
A number of markers hâve been developed for use with plant cells, such as résistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like. Other genes that encode a
-2316614 product involved in chloroplast metabolism may also be used as selectable markers. For example, genes that provide résistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use. Such genes hâve been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromoxynil résistance nitrilase gene); and Sathasivan étal. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone résistance gene). Additionally, the genes disclosed herein are useful as markers to assess transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art. In one embodiment, the presence of the transgene is detected by testing for pesticidal activity.
Fertile plants expressing a pesticidal protein may be tested for pesticidal activity, and the plants showing optimal activity selected for further breeding. Methods are available in the art to assay for pest activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economie Entomology 78:290-293.
The présent invention 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, com (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed râpe, Brassica sp.t alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, omamentals, and conifers.
Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumîs such as cucumber, cantaloupe, and musk melon. Omamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, pétunias, carnation, poinsettia, and chrysanthemum. Preferably, plants of the présent invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed râpe., etc.).
Use in Pesticidal Control
General methods for employing strains comprising a nucléotide sequence of the présent invention, or a variant thereof, in pest control or in engineering other organisms as pesticidal agents are known in the art. See, for example U.S. Patent No. 5,039,523 and EP 0480762A2.
The Bacillus strains containing a nucléotide sequence of the présent invention, or a variant thereof, or the microorganisms that hâve been genetically altered to contain a pesticidal gene of the invention and protein may be used for protecting agricultural crops and products from pests. In one
-2416614 aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).
Alternatively, the pesticide is produced by introducing a pesticidal gene into a cellular host. Expression of the pesticidal gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. In one aspect of this invention, 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 the target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides 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. Alternatively, one may formulate the cells expressing a gene of this invention such as to allow application of the resulting material as a pesticide.
The active ingrédients of the présent invention are normally 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 fertilîzers, weed killers, cryoprotectants, surfactants, détergents, pesticidal soaps, dormant oils, polymers, and/or timerelease or biodégradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be sélective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these préparations, 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. naturel or regenerated minerai substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilîzers. Likewise the formulations may be prepared into edible “baits orfashioned into pest “traps to permit feeding or ingestion by a target pest of the pesticidal formulation.
Methods of applying an active ingrédient of the présent invention or an agrochemical composition of the présent invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the présent invention include leaf application, seed coating and soil application. The number of applications and the rate of application dépend on the intensity of infestation by the corresponding pest.
The composition may be formulated as a powder, dust, pellet, granule, spray, émulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation, sédimentation, or concentration of
-2516614 a culture of cells comprising the polypeptide. In ail such compositions that contain at Ieast one such pesticidal polypeptide, the polypeptide may be présent in a concentration of from about 1% to about 99% by weight.
Lepidopteran, hemipteran, dipteran, or coleopteran pests may be killed or reduced in numbers in a given area by the methods of the invention, 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 Ieast 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 spécifie target pests to be controlled, the spécifie 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 considérations, and/or frequency of application and/or severity of pest infestation.
The pesticide compositions described may be made by formulating either the bacterial cell, the crystal and/or the spore suspension, or the 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 minerai), or water or oîl/water émulsions, 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 ail 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 U.S. Patent No. 6,468,523, herein incorporated by reference.
“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, Lépidoptère, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, Lepidoptera, and Diptera.
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The order Coleoptera includes the suborders Adephaga and Polyphaga. Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea, while suborder Polyphaga includes the superfamilies Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea, Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes the familles Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoidea includes the family Gyrinidae. Superfamily Hydrophiloidea includes the family Hydrophilidae. Superfamily Staphylinoidea includes the familles Silphidae and Staphylinidae. Superfamily Cantharoidea includes the familles Cantharidae and Lampyridae. Superfamily Cleroidea includes the families Cleridae and Dermestidae. Superfamily Elateroidea includes the families Elateridae and Buprestidae. Superfamily Cucujoidea includes the family Coccinellidae. Superfamily Meloidea includes the family Meloidae. Superfamily Tenebrionoidea includes the family Tenebrionidae. Superfamily Scarabaeoidea includes the families Passalidae and Scarabaeidae. Superfamily Cerambycoidea includes the family Cerambycidae. Superfamily Chrysomeloidea includes the family Chrysomelidae. Superfamily Curculionoidea includes the families Curculionidae and Scolytidae.
The order Diptera includes the Suborders Nematocera, Brachycera, and Cyclorrhapha. Suborder Nematocera includes the families Tipulîdae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliîdae, Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschiza and Aschiza. Division Aschiza includes the families Phoridae, Syrphidae, and Conopidae. Division Aschiza includes the Sections Acalyptratae and Calyptratae. Section Acalyptratae includes the families Otitidae, Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptratae includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.
The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae.
Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandîosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum mardis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, twospotted spider mite; Sorqhum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Ouléma melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum mardis', corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carminé spider mite; Tetranychus urticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Ouléma melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodip/osis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfiy; Aceria tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carminé spider mite; Tetranychus urticae, twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sifophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis,
-2816614 velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrînia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; B/issus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Délia platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Râpe: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha armyworm; Plutella xylostella, Diamond-back moth; Délia ssp., Root maggots.
Nematodes include parasitic nematodes such as root-knot, cyst, and lésion 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 pailida (potato cyst nematodes). Lésion nematodes include Pratylenchus spp.
Methods for Increasinq 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 (or susceptible to infestation by) a pest against which said polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a lepidopteran, coleopteran, dipteran, hemipteran, or nematode pest, and said 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%
-2916614 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 spécifie methods, plant yield is increased as a resuit of improved pest résistance of a plant expressing a pesticidal protein disclosed herein. Expression of the pesticidal protein results in a reduced ability of a pest to infest or feed.
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/Veqetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Veqetables Insecticides:
Aldicarb , Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Triflumuron.Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metafiumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Fenamiphos, Pyriproxifen, Fenbutatin-oxid; Fruits/Veqetables Fungicides: Ametoctradin, Azoxystrobin, Benthiavalicarb, Boscalid, Captan, Carbendazim, Chlorothalonil, Copper, Cyazofamid, Cyflufenamid, Cymoxanil, Cyproconazole, Cyprodinil, Difenoconazole, Dimetomorph, Dithianon, Fenamidone, Fenhexamid, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluxapyroxad, Folpet, Fosetyl, Iprodione, Iprovalicarb, Isopyrazam, Kresoxim-methyl, Mancozeb, Mandipropamid, Metalaxyl/mefenoxam, Metiram, Metrafenone, Myclobutanil, Penconazole, Penthiopyrad, Picoxystrobin, Propamocarb, Propiconazole, Propineb, Proquinazid, Prothioconazole, Pyraclostrobin, Pyrimethanil, Quinoxyfen, Spiroxamine, Sulphur, Tebuconazole, Thiophanate-methyl, Trifloxystrobin; Cereals Herbicides: 2.4-D, Amidosulfuron, Bromoxynil, Carfentrazone-E, Chlorotoluron, Chlorsulfuron, Clodinafop-P, Clopyralid, Dicamba, Diclofop-M, Diflufenican, Fenoxaprop, Florasulam, Flucarbazone-NA, Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Flurtamone, Glyphosate, lodosulfuron, loxynil, Isoproturon, MCPA, Mesosulfuron, Metsulfuron, Pendimethalin, Pinoxaden, Propoxycarbazone, Prosulfocarb, Pyroxsulam, Sulfosulfuron, Thifensulfuron, Tralkoxydim, Triasulfuron, Tribenuron, Trifluralin, Tritosulfuron: Cereals Fungicides: Azoxystrobin. Bixafen, Boscalid, Carbendazim, Chlorothalonil, Cyflufenamid, Cyproconazole, Cyprodinil, Dimoxystrobin, Epoxiconazole, Fenpropidin, Fenpropimorph, Fluopyram, Fluoxastrobin, Fluquinconazole, Fluxapyroxad, Isopyrazam, Kresoxim-methyl, Metconazole, Metrafenone, Penthiopyrad, Picoxystrobin, Prochloraz, Propiconazole, Proquinazid, Prothioconazole, Pyraclostrobin, Quinoxyfen,
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Spiroxamine, Tebuconazole, Thiophanate-methyl, Trifloxystrobîn; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Pirimicarb, Methiocarb, Sulfoxaflor: 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, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin; Maize Funqicides: Azoxystrobin, Bixafen, Boscalid, Cyproconazole, Dimoxystrobin, Epoxiconazole, Fenitropan, Fluopyram, Fluoxastrobin, Fluxapyroxad, Isopyrazam, Metconazole, Penthiopyrad, Picoxystrobin, Propiconazole, Prothioconazole, Pyraclostrobin, Tebuconazole, Trifloxystrobîn; 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, Fenobucarb, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Etofenprox, Carbofuran, Benfuracarb, Sulfoxaflor; Rice Funqicides: Azoxystrobin. Carbendazim, Carpropamid, Diclocymet, Difenoconazole, Edifenphos, Ferimzone, Gentamycin, Hexaconazole, Hymexazol, Iprobenfos (IBP), Isoprothiolane, Isotianil, Kasugamycin, Mancozeb, Metominostrobin, Orysastrobin, Pencycuron, Probenazole, Propiconazole, Propineb, Pyroquilon, Tebuconazole, Thiophanate-methyl, Tiadinil, Tricyclazole, Trifloxystrobîn, Validamycin; 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, 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[[(G-Chlorpyridin-S-ylJmethylJfS.Z-difluorethyOaminolfuran-StSHJ-on, Thiodicarb, Avermectin,
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Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor; Cotton Funqicides: Azoxvstrobin. Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam, Fluopyram, Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil, Mancozeb, Maneb, Metominostrobin, Penthiopyrad, Picoxystrobin, Propineb, Prothioconazole, Pyraclostrobin, Quintozene, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxvstrobin: Sovbean Herbicides: Alachlor. Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cvhalothrin. Methomyl, 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; Sovbean Funqicides: Azoxvstrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione, Isotianil, Mancozeb, Maneb, Metconazole, Metominostrobin, Myclobutanil, Penthiopyrad, Picoxystrobin, Propiconazole, Propineb, Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin; Suqarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop; Suqarbeet Insecticides: Imidacloprid. Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3yl)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 Funqicides: Azoxvstrobin. Bixafen, Boscalid, Carbendazim, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flusilazole, Fluxapyroxad, Iprodione, Isopyrazam, Mepiquat-chloride, Metconazole, Metominostrobin, Paclobutrazole, Penthiopyrad., Picoxystrobin, Prochloraz, Prothioconazole, Pyraclostrobin, Tebuconazole, Thiophanate-methyl, Trifloxystrobin, Vinclozolin; Canola Insecticides: Carbofuran. 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)methylj(2,2-difluorethyl)amino]furan-2(5H)-on.
The following examples are offered by way of illustration and not by way of limitation.
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EXPERIMENTAL EXAMPLES
Example 1. Design and testinq of Axmi115 fusion proteins
Axmil 15 is described in U.S. Patent Publication 20100004176 (the amino acid sequence is set forth herein as SEQ ID NO:43). This gene shares 70% sequence homology with Vip3Aa. A codon optimized version of Axmil 15 (also referred to herein as Axmil 15v01 and set forth in SEQ ID NO:42) was cloned and expressed using the E coli expression vector. The protein produced was shown in in vitro bioassay to hâve pesticidal activity against various insect pests including European corn borer (ECB), corn earworm (CEW), fall armyworm (FAW) and black cutworm (BCW).
AxmîOOS is also described in U.S. Patent Publication 20100004176. This gene shares 94% sequence homology with Vip3Aa. A codon optimized version of AxmîOOS (optAxmi005, which is set forth herein as SEQ ID NO:44) was cloned and expressed using the E coli expression vector. The protein produced was shown in in vitro bioassay to hâve pesticidal activity against various insect pests including Helicoverpa zea (Hz), Heliothis virescens (Hv), FAW, BCW, sugarcane borer (SCB), and velvetbean caterpillar (VBC).
The relative activity of Axmil 15 was low against Hz and FAW compared to AxmîOOS. Also as noted above AxmîOOS did not hâve ECB activity. In an attempt to identify the domains responsible for the differential specîficity as well as activity of the two proteins, constructs expressing fusions of optAxmiOOS and a codon-optimized version of Axmil 15 (optAxmil 15v01 ) were made as described below and diagrammed in Figure 1. The protein was expressed in E. coli and tested against ECB, Hz, FAW and BCW in in vitro bioassay. The protein expressed by pAX6307 (Axmil 15v02.01, set forth herein as SEQ ID NO:1) showed enhanced activity when compared with the protein expressed by pAX5477 (Axmil 15v01, set forth herein as SEQ ID NO:42) against all four pests tested.
The gene expressed in pAX6307 (Axmil 15v02.01) was vectored into the plant expression vector pAG6141 in which expression of the protein was driven by the Sugar cane Ubiquitin promoter.
Leaf samples from transgenic plants expressing Axmil 15v01 and Axmil 15v02.01 were tested in laboratory insect bioassay against ECB, Hz, FAW and BCW and in field tests against ECB, Hz and FAW. Results show that the improved Axmil 15v02.01 gene had better efftcacy against all pests tested.
Description of constructs:
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Amino acid sequences derived by in silico translation of the DNA sequence of Vip3Aa, Axmi005, Axmil 15v01, Axmi163, and Axmil 84 were aligned to identify conserved amino acids in ail homologs (Axmil63 and Axmil84 are also described in U.S. Patent Publication 20100004176).
PCR primers were designed to three conserved régions of AxmiOOS and Axmil 15v01 using 5 the sequence of optAxmiOOS found in pAX5478 (which contains a codon optimized version of AxmiOOS, set forth in SEQ ID NO:44) and the sequence of optAxmil 15 found in pAX5477 (which contains a codon optimized version of Axmil 15). Three fusion genes were generated by overlap PCR (see Figure 1).
The DNA of the fusion genes produced by these PCR reactions was cloned into the E. coli 10 expression vector pRSfl B. The resulting expression vectors are shown in Table 1. Protein was expressed using known methods and the E. coli extract was tested in an in vitro bioassay.
Table 1. Fusion gene constructs
Construct name Sequence insert Nucléotide SEQ ID NO: Amino acid SEQ ID NO:
pAX6307 Axmi005/Axmi115 fusion A 1 15
pAX6308 Axmi005/Axmi115 fusion B 2 16
pAX6309 Axmi005/Axmi115 fusion D 3 17
pAX5478 optAxmiOOS 44 45
pAX5477 Axmil 15v01 42 43
pRSflb vector control
ln-vitro bioassay
Crude extracts from E. coli expressed in vectors was assayed against Hz, ECB, FAW, and BCW. The results are shown in Table 2 (stunt) and Table 3 (mortality).
Table 2. Stunt score
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ECB Hz FAW BCW
ave* SD ave* SD ave* SD ave*
PAX6307 (fusion A) 2.2 0.3 pAX6307 1.8 0.3 pAX6307 3 0 pAX6307 0.8
pAX6308 (fusion B) 0.5 0.5 pAX6308 0.3 0.4 pAX6308 1.3 1.3 pAX6308 0
pAX6309 (fusion D) 1.2 0.3 pAX6309 0.2 0.3 pAX6309 0.7 0.7 pAX6309 0
pRSflb (vector control) 0.3 0.4 pRSflb 0 0 pRSflb 0.5 0.5 pRSflb 0
pAX5478 (AxmiOOS) 0 0 pAX5478 1.8 0.3 pAX5478 3 0 pAX5478 1.7
pAX5477 (Axmi115v01) 0.2 0.3 PAX5477 0.5 0.5 pAX5477 1.5 1.5 pAX5477 0.2
*Scoring system:
= no effect observed
1 - mild non-uniform stunting = moderate non-uniform stunting
- moderate to severe uniform stunting = mortality (<100%) with uniform stunting = complété mortality
Table 3. Percent mortality
Hz ECB FAW BCW
pAX6307 (fusion A) 50 50 75 25
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pAX6308 (fusion B) 0 0 0 0
pAX6309 (fusion D) 0 25 25 0
pRSflb (vector control) 0 0 0 0
pAX5478 (optAxmiOOS) 50 0 75 25
pAX5477 (Axmil 15v01) 0 0 0 0
The protein expressed from vector pAX6307 (fusion A) varied by six amino acids and was designated Axmil 15v02.01. The amino acid sequence for this fusion protein is set forth in SEQ ID NO:15.
E coli expression vectors expressing Axmil 15v01 (pAX5476) and Axmil 15v02.01 (pAX6307) had N- terminal 6X His Tags. The two proteins were purified using the nickel binding properties of the 6X His tag. Various concentrations of the purified protein were assayed by in vitro bioassay against ECB, FAW, BCW and Beet Armyworm (BAW). The results show that
Axmil 15v02,01 has enhanced activity compared with Axmil 15v01 in ail cases (Tables 4 and 5).
Table 4. Stunt score
□g/ml BAW FAW ECB BCW
Axmil 15v01 40 4 4 3 0
Axmil 15v01 10 2 3 0 0
Axmil 15v01 1 0 0 0 0
Axmil 15v01 0.1 0 0 0 0
Axmil 15v01 0.01 0 1 0 0
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Axmi115v02 40 4 4 3 3
Axmi115v02 10 4 4 3 1
Axmi115v02 1 4 4 3 0
Axmt115v02 0.1 2 1 2 0
Axmi115v02 0.01 0 2 1 0
Table 5. Mortality score
□g/ml BAW FAW ECB BCW
Axmi115v01 40 75% 0% 0% 0%
Axmi115v01 10 0% 25% 0% 0%
Axmi115v01 1 0% 0% 0% 0%
Axmi115v01 0.1 0% 0% 0% 0%
Axmi115v01 0.01 0% 0% 0% 0%
Axmi115v02 40 75% 50% 0% 0%
Axmi115v02 10 0% 25% 50% 0%
Axmi115v02 1 0% 25% 0% 0%
Axmi115v02 0.1 0% 0% 0% 0%
Axmi115v02 0.01 0% 0% 0% 0%
Plant leaf dise bioassay
Axmi115v01 (SEQ ID NO:42) and Axmi115v02.01 (SEQ ID NO:1) were cloned into plant expression vectors pAG6585 and pAG6141, respectively, and transgenic maize plants were produced. Samples were taken for PCR and Western analysis and for in vitro leaf dise bioassay against Hz, ECB, FAW, and BCW. The bioassay was scored for undamaged, low damage (1- few 10 holes), moderate damage, and heavy damage. Undamaged and light damaged were considered a positive resuit whereas moderate to heavy damage was considered a négative resuit.
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Leaf material from PCR and western positive plants was assayed in in vitro leaf disk bioassay. Figure 2A shows the percent PCR positive plants that gave a bioassay score of undamaged, light damage, moderate damage or heavy damage for each construct. Western blots indicate that the expression level of protein in plants expressing optAxmil 15v02.01 is, in general, higher than plants expressing Axmi115v01.
Additional transgenic plants were produced expressing Axmil 15v02.01. Leaf material from PCR and Western positive plants was assayed in in vitro leaf disk bioassay against Hz, ECB, FAW, and BCW. The results are shown in Figure 2b.
Plant field trials
Plants expressing the genes shown in Table 6 were planted at the Polk County, IA test location. Négative segregates were îdentified and removed using a 1X application of Glyphosate (20 oz./A of Buccaneer 5, Tenkoz, Inc.) when plants were at the V3-V4 leaf stage. Insect pressure resulted from manual infestations of ECB, Hz, and FAW.
Infestations of ECB mimicked the natural occurrence of first and second générations. For ECB, in total, approximately 340 larvae were infested into either the leaf whorls (first génération, ECB1) or leaf axils (second génération, ECB2) of each plant. ECB1 was evaluated by the Guthrie 1-9 rating scale. ECB2 was a measure of the total length of stalk tunneling measured in cm.
Twenty Hz larvae were infested onto the tips of primary ears on each plant. There was also a moderate natural infestation of Hz that augmented these manual infestations. The ear damage was measured in sq. cm.
Approximately 60 FAW larvae were infested into the leaf whorls. Damage was measured in Modified Davis 1-9 rating scale as described below.
The results of these field trials are shown in Table 6.
Table 6. Field trial results
FAW (1-9) Hz (sq.cm) ECB2 (cm)
Mean Score SD Mean Score SD Mean score SD
Axmil 15v02.01 1.20 0.48 0.12 0.15 0.00 0.00
Axmil 15v01 1.92 1.18 1.96 1.40 0.83 N/A
Axmi005 1.75 0.97 4.12 2.06 N/A N/A
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neg. Control 6.42 0.74 7.06 1.61 9.65 1.66
FAW - Modified Davis 1-9 rating scale description.
1. No visible damage or only pinhole lésions présent on whorl leaves.
2. Pinhole and small circular lésions présent on whorl leaves.
3. Small circular lésions and a few small elongated (rectangular- shaped) lésions of up to 1.3 cm (1/2) in length présent on whorl and furl leaves.
4. Several small to mid-sized 1.3 to 2.5 cm (1/2 to 1) in length elongated lésions présent on a few whorl and furl leaves.
5. Several large elongated lésions greater than 2.5 cm (1) in length présent on a few whorl and furl leaves and/or a few small- to mid-sized uniform to irregular shaped holes (basement membrane consumed) eaten from the whorl and/or furl leaves.
6. Several large elongated lésions présent on several whorl and furl leaves and/or several large uniform to irregular shaped holes eaten from furl/whorl leaves.
7. Many elongated lésions of ali sizes présent on several whorl and furl leaves plus several large uniform to irregular shaped holes eaten from the whorl and furl leaves.
8. Many elongated lésions of ail sizes présent on most whorl and furl leaves plus many mid- to large-sized uniform to irregular shaped holes eaten from the whorl and furl leaves.
9. Whorl and furl leaves almost totally destroyed.
Davis, F. M., S. S. Ng, and W.P. Williams. 1992. Visual rating scalesfor screening whorl-stage corn for résistance to fall armyworm. Miss. Agric. Forestry Exp. Stn. Tech. Bull. 186.
ECB - Guthrie 1-9 rating scale description.
1. No visible leaf injury.
2. Small amount of shot-hole injury on a few leaves.
3. Shot-hole injury common on several leaves.
4. Several leaves with shot-holes and elongated lésions.
5. Several leaves with elongated lésions.
6. Several leaves with elongated lésions about 2.5 cm long.
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7. Long lésions common on about one-half of the leaves,
8. Long lésions common on about two-thirds of the leaves,
9. Most leaves with long lésions.
Guthrie, W, D., F. F. Dicke, and C. R. Neiswander. 1960. Leaf and sheath feeding résistance to the European corn borer in eight inbred lines of dent corn. Ohio. Agric. Exp. Sta. Res. Bull. 860.
Example 2. Directed évolution of Axmil 15vQ2.
Directed évolution has been used to improve the potency and activity profile of Axmil 15 against ECB, Hz, FAW, BCW, and VBC. To identify régions of Axmil 15 involved in insect toxicity, a number of Axmil 15/Axmi005 sequence swap variants in the C-terminal part of Axmil 15 were created. Twenty-one blocks of sequence divergence between Axmil 15 and AxmiOOS were designated (see U.S. Patent Publication No. 20100004176 which is herein incorporated by reference in its entirety) and these sequence blocks in Axmil 15 were replaced with the corresponding AxmiOOS sequence blocks. Bioassays of hybrid proteins showed that substitutions in blocks 2, 3, 10 and 18 are linked to increased insect toxicity.
Point mutant libraries were created that targeted positions in blocks 2, 3,10 and 18. These point mutant libraries were assayed against ECB, Hz, FAW, BCW and VBC at 1.5x coverage at the 4 replicate level. Re-assays were carried out at the 4 replicate level, and scale-ups were done at the 16 replicate level. The following point mutants showed improved activity against one or more pests:
Table 7. Activity of Axmil 15 point mutants
nucléotide SEQ ID NO: amino acid SEQ ID NO: Activity improved against Slight improvement in activity against
Block2 L11C7 9 23 FAW Hz, ECB, BCW
Block 2 L11H6 24 FAW Hz, ECB
Block2L11H7 10 25 FAW Hz, ECB, BCW
Block2L11A9 11 26 FAW ECB, BCW
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Block 2 L11F9 27 ECB BCW, FAW
Block 2 L11G10 12 28 Hz, FAW
Block 2 L12C3 13 29 Hz. FAW
Block 18 L12A10 14 30 FAW ECB, VBC
Block 18 L12B10 31 FAW ECB
These variants contain mutations in the C-terminal part. To look for synergistic improvements with Axmil 15v02 (pAX6307), the C-terminal part of several of the above mutants was cloned into Axmil 15v02 (pAX6307). Scale-up assays were carried out and variants with improved activity compared to Axmil 15v02 were identified.
Table 8. Activity of Axmil 15v02 mutants
Gene ECB FAW VBC Hz BCW
Stunt % Mort Stunt %Mort Stunt %Mort Stunt %Mort Stunt %Mort
axmi-115 v02 3.50 14.84 3.75 45.31 4.00 72.27 4.00 16.67 2.08 4.17
115B2L11H6 (v02) - evo27 3.67 13.02 3.67 52.08 4.00 84.38 4.00 35.16 1.50 0.00
115B18L12B10 (v02) - evo28 3.33 15.63 3.67 26.56 4.00 60.94 4.00 1.56 2.25 0.00
115B2L11H7 (v02) 3.33 10.94 3.67 33.33 4.00 56.77 4.00 3.91 2.00 0.00
115B18L12A10 (v02) 3.33 16.15 3.67 27.08 4.00 60.94 4.00 0.78 2.13 0.00
115B2L11F9 (v02) - evo29 3.67 6.88 3.67 54.17 4.00 86.98 4.00 34.38 1.38 3.13
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Variant axmil 15 B2L11H6 (v02) shows improved activity against H. zea, VBC, FAW. It was designated Axmil 15v02(evo27). The nucléotide sequence for Axmil 15v02(evo27) is set forth in SEQ ID NO:4 and the encoded amino acid sequence is set forth in SEQ ID NO:18.
Variant axmil 15 B18L12B10 (v02) shows improvements against ECB. It was designated Axmil 15v02(evo28). The nucléotide sequence for Axmil 15v02(evo28) is set forth in SEQ ID NO:5 and the encoded amino acid sequence is set forth in SEQ ID NO:19.
Variant axmil 15 B2L11F9 (v02) shows improvements against H.zea, VBC, FAW. It was designated Axmil 15v02(evo29). The nucléotide sequence for Axmil 15v02(evo29) is set forth in SEQ ID NO:6 and the encoded amino acid sequence is set forth in SEQ ID NO:20.
Additional mutations were made in the AXMI115v02 sequence in the C-terminal région. Three variants were identified with improved activity relative to AXMI115v02 (Table 9). LC50 and EC50 values were determined for two of these C-terminal mutants (Table 10).
AXMI115v02(EVO31) showed improved mortality against FAW, soybean looper(SBL) and VBC relative to AXMI115v02. The nucléotide sequence for Axmil 15v02(evo31) is set forth in SEQ ID NO:7 and the amino acid sequence is set forth in SEQ ID NO:21.
AXMI115v02(EVO32) showed improved mortality against ECB and H. zea relative to AXMI115v02. The nucléotide sequence for Axmil 15v02(evo32) is set forth in SEQ ID NO:8 and the amino acid sequence is set forth in SEQ ID NO:22.
AXMI115v02(EVO38) showed improved mortality against BCW relative to AXMI115v02. The nucléotide sequence for Axmil 15v02(evo38) is set forth in SEQ ID NO:47 and the amino acid sequence is set forth in SEQ ID NO:48.
Table 9. Activity of Axmil 15v02 C-terminal mutants
Gene ECB FAW VBC Hz BCW
Stunt % Mort Stunt %Mort Stunt %Mort Stunt %Mort Stunt %Moi
Axmil 15v02 3.3 11.5 4.0 16.5 4.0 80.2 4.0 13.8 2.8 1.1
Axmil 15v02(evo31) 3.4 28.6 4.0 20.7 4.0 81.4 4.0 14.3 2.4 0.0
Axmil 15v02(evo32) 3.4 30.0 4.0 18.2 4.0 94.4 4.0 35.0 3.0 0.0
Axmil 15v02(evo38) 0.2 0.0 4.0 15.7 4.0 87.1 4.0 10.5 3.6 6.6
Table 10. LC50 and EC50 for C-terminal mutants
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Gene ECB FAW VBC SBL Hz BCW
LC50 EC50 LC50 EC50 LC50 LC50 LC50 EC50 LC50
Axmi115v02 20 □g/ml 3 □g/ml 6.3 □g/ml 1.3 □g/ml 400 ng/ml 280 ng/ml 339 □g/ml 14.3 □g/ml 7.6 □g/ml
Axmi115v02 (evo31) 18 □g/ml 4.5 □g/ml 2.4 □g/ml 240 ng/ml 120 ng/ml 80 ng/ml 185 □g/ml 12 □g/ml 27.3 □g/ml
Axmi115v02 (evo32) 12.3 □g/ml 4.3 □g/ml 6 □g/ml 400 ng/ml 520 ng/ml 520 ng/ml 42.5 □g/ml 13.3 □g/ml 16.6 □g/ml
SBL - Soybean looper
Example 3. Additional assays for Pesticidal Activity
The nucléotide sequences of the invention can be tested for their ability to produce pesticidal proteins. The ability of a pesticidal protein to act as a pesticide upon a pest is often assessed in a number of ways. One way well known in the art is to perform a feeding assay. In such a feeding assay, one exposes the pest to a sample containing either compounds to be tested or control samples. Often this is performed by placing the material to be tested, or a suitable dilution of such material, onto a material that the pest will ingest, such as an artificial diet. The material to be tested may be composed of a liquid, solid, or slurry. The material to be tested may be placed upon the surface and then allowed to dry. Alternatively, the material to be tested may be mixed with a molten artificial diet, and then dispensed into the assay chamber. The assay chamber may be, for example, a cup, a dish, or a well of a microtiter plate.
Assays for sucking pests (for example aphids) may involve separating the test material from the insect by a partition, ideally a portion that can be pierced by the sucking mouth parts of the sucking insect, to allow ingestion of the test material. Often the test material is mixed with a feeding stimulant, such as sucrose, to promote ingestion of the test compound.
Other types of assays can include microinjection of the test material into the mouth, or gut of the pest, as well as development of transgenic plants, followed by test of the ability of the pest to feed upon the transgenic plant. Plant testing may involve isolation of the plant parts normally consumed, for example, small cages attached to a leaf, or isolation of entire plants in cages containing insects.
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Other methods and approaches to assay pests are known in the art, and can be found, for example in Robertson and Preisler, eds. (1992) Pesticide bioassays with arthropods, CRC, Boca Raton, FL. Alternative^, assays are commonly described in the journals Arthropod Management Tests and Journal of Economie Entomology or by discussion with members of the Entomological Society of America (ESA).
In some embodiments, the DNA régions encoding the toxin région of the pesticidal proteins disclosed herein are cloned into the E. coli expression vector pMAL-C4x behind the malE gene coding for Maltose binding protein (MBP). These in-frame fusions resuit in MBP-Axmi fusion proteins expression in E. coli.
For expression in E. coli, BL21*DE3 are transformed with individual plasmids. Single colonies are inoculated in LB supplemented with carbenicillin and glucose, and grown overnight at 37°C. The following day, fresh medium is inoculated with 1% of overnight culture and grown at 37°C to logarithmic phase. Subsequently, cultures are induced with 0.3mM IPTG overnight at 20°C. Each cell pellet is suspended in 20mM Tris-CI buffer, pH 7.4 + 200mM NaCI + 1mM DTT + protease inhibitors and sonicated. Analysis by SDS-PAGE can be used to confirm expression of the fusion proteins.
Total cell free extracts are then run over amylose column attached to fast protein liquid chromatography (FPLC) for affinity purification of MBP-axmï fusion proteins. Bound fusion proteins are eluted from the resin with 10mM maltose solution. Purified fusion proteins are then cleaved with either Factor Xa ortrypsin to remove the amino terminal MBP tag from the Axmi protein. Cleavage and solubility of the proteins can be determined by SDS-PAGE
Example 4. Construction of synthetic sequences
In one aspect of the invention, synthetic axmi sequences are generated. These synthetic sequences hâve an altered DNA sequence relative to the parent axmi sequence, and encode a protein that is collinear with the parent AXMI protein to which it corresponds, but lacks the Cterminal “crystal domain présent in many delta-endotoxin proteins.
In another aspect of the invention, modified versions of synthetic genes are designed such that the resulting peptide is targeted to a plant organelle, such as the endoplasmic réticulum or the apoplast. Peptide sequences known to resuit in targeting of fusion proteins to plant organelles are known in the art. For example, the N-terminal région of the acid phosphatase gene from the White Lupin Lupinus albus (Genebank ID GL14276838; Miller et al. (2001) Plant Physiology 127: 594606) is known in the art to resuit in endoplasmic réticulum targeting of heterologous proteins. If the resulting fusion protein also contains an endoplasmic rétention sequence comprising the peptide N
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Example 5. Transformation of Maize Cells with the pesticidal protein genes described herein
Maize ears are best collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are preferred for use in transformation. Embryos are plated scutellum side-up on a suitable incubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of 1000x Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50 g/L sucrose; 1 mUL (of 1 mg/mL Stock) 2,4-D). However, media and salts other than DN62A5S are suitable and are known in the art. Embryos are incubated overnight at 25°C in the dark. However, it is not necessary perse to incubate the embryos overnight.
The resulting expiants are transferred to mesh squares (30-40 per plate), transferred onto osmotic media for about 30-45 minutes, then transferred to a beaming plate (see, for example, PCT Publication No. WO/0138514 and U.S. Patent No. 5,240,842).
DNA constructs designed to the genes of the invention in plant cells are accelerated into plant tissue using an aérosol beam accelerator, using conditions essentially as described in PCT Publication No. WO/0138514. After beaming, embryos are incubated for about 30 min on osmotic media, and placed onto incubation media overnight at 25°C in the dark. To avoid unduly damaging beamed expiants, they are incubated for at least 24 hours prior to transfer to recovery media. Embryos are then spread onto recovery period media, for about 5 days, 25°C in the dark, then transferred to a sélection media. Expiants are incubated in sélection media for up to eight weeks, depending on the nature and characteristics of the particular sélection utilized. After the sélection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of régénération is initiated by methods known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.
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Materials
DN62A5S Media
Components Per Liter Source
Chu's N6 Basal Sait Mixture (Prod. No. C 416) 3.98 g/L Phytotechnology Labs
Chu’s N6 Vitamin Solution (Prod. No. C 149) 1 ml_/L (of 10 OOx Stock) Phytotechnology Labs
L-Asparagine 800 mg/L Phytotechnology Labs
Myo-inositol 100 mg/L Sigma
L-Proline 1.4 g/L Phytotechnology Labs
Casamino acids 100 mg/L Fisher Scientific
Sucrose 50 g/L Phytotechnology Labs
2,4-D (Prod. No. D-7299) 1 mL/L (of 1 mg/mL Stock) Sigma
The pH ofthe solution is adjusted to pH 5.8 with 1N KOH/1N KCI, Gelrite (Sigma) is added at a concentration up to 3g/L. and the media is autoclaved. After coolîng to 50°C, 2 ml/L of a 5 mg/ml stock solution of silver nitrate (Phytotechnology Labs) is added.
Example 6. Transformation of genes of the invention in Plant Cells by Aqrobactër/um-Mediated Transformation
Ears are best collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are preferred for use in transformation. Embryos are plated scutellum side-up on a suitable incubation media, and incubated overnight at 25°C in the dark.
However, it is not necessary per se to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors for Ti plasmid mediated transfer for about 5-10 min, and then plated onto co-cultivation media for about 3 days (25°C in the dark). After co-cultivation, expiants are transferred to recovery period media for about five days (at 25°C in the dark). Expiants are incubated in sélection media for up to eight weeks, depending on the nature
-4616614 and characteristics of the particular sélection utilized. After the sélection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of régénération is initiated as known in the art.
Ail publications and patent applications mentioned in the spécification are indicative of the level of skill of those skilled in the art to which this invention pertaîns. Ail publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended daims.

Claims (45)

1. A recombinant nucleic acid molécule comprising a nucléotide sequence encoding a fusion polypeptide having pesticidal activity, wherein the fusion polypeptide comprises a C-terminal portion of SEQ ID NO:43.
2. The recombinant nucleic acid molécule of claim 1, wherein the C-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
3. The recombinant nucleic acid molécule of claim 1 or 2, wherein the fusion polypeptide comprises an N-terminal portion of SEQ ID NO:45.
4. The recombinant nucleic acid molécule of any of daims 1-3, wherein the N-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
5. The recombinant nucleic acid molécule of claim 1, 2, 3, or 4, wherein said nucléotide sequence is selected from the group consisting of:
a) the nucléotide sequence set forth in any of SEQ ID NO:47 and 1-14;
b) a nucléotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:48 and 15-31 ;
c) a nucléotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:48 and 15-31, wherein the pesticidal activity of the fusion polypeptide is improved or extended relative to the pesticidal activity of SEQ ID NO:43 or 45.
6. The recombinant nucleic acid molécule of daims 1, 2, 3, 4, or 5, wherein said nucléotide sequence is a synthetic sequence that has been designed for expression in a plant.
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7. The recombinant nucleic acid molécule of daims 1, 2, 3, 4, 5, or 6, wherein said nucléotide sequence is operably linked to a promoter capable of directing expression of said nucléotide sequence in a plant cell.
8. A vector comprising the recombinant nucleic acid molécule of any of daims 1-7.
9. The vector of daim 8, further comprising a nucleic acid molécule encoding a heterologous polypeptide.
10. A host cell that contains the recombinant nucleic acid of any of daims 1 -7 or the vector of daims 8 or 9.
11. The host cell of claim 10 that is a bacterial host cell.
12. The host cell of claim 10 that is a plant cell.
13. A transgenic plant comprising the host cell of claim 12.
14. The transgenic plant of claim 13, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed râpe.
15. A transgenic seed comprising the nucleic acid molécule of any of daims 1-7 or the vector of daims 8 or 9.
16. A recombinant polypeptide having pesticidal activity, wherein the polypeptide comprises a C-terminal portion of SEQ ID NO:43.
17. The recombinant polypeptide of claim 16, wherein the C-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
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18. The recombinant polypeptide of claim 16 or 17, wherein the polypeptide comprises an N-terminal portion of SEQ ID NO:45.
19. The recombinant polypeptide of any of daims 16-18, wherein the N-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
20. The recombinant polypeptide of daim 16, 17, 18, or 19, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of:
a) the amino acid sequence of any of SEQ ID NO;48 and 15-31 ;
c) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:48 and 15-31, wherein the pesticidal activity ofthe polypeptide is improved or extended relative to the pesticidal activity of SEQ ID NO:43 or 45.
21. The polypeptide of any of daims 16-20 further comprising heterologous amino acid sequences.
22. A composition comprising the polypeptide of any of daims 16-21.
23. The composition of daim 22, wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, émulsion, colloid, and solution.
24. The composition of daim 22, wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sédimentation, or concentration of a culture of bacterial cells.
25. The composition of daim 22, comprising from about 1 % to about 99% by weight of said polypeptide.
26. A method for controlling a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest population comprising contacting said population with a pesticidally-effective amount of the polypeptide of any of daims 16-22 or the composition of daims 23, 24, or 25.
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27. A method for killing a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest, comprising contacting said pest with, or feeding to said pest, a pesticidally-effective amount of the polypeptide of any of daims 16-22 or the composition of daims 23, 24, or 25.
28. A method for producing a polypeptide with pesticidal activity, comprising culturing the host cell of daim 10 under conditions in which the nucleic acid moiecule encoding the polypeptide is expressed.
29. A plant having stably incorporated into its genome a DNA construct comprising a nucleic acid moiecule comprising a nucléotide sequence encoding a fusion polypeptide having pesticidal activity, wherein the fusion polypeptide comprises a C-terminal portion of SEQ ID NO:43.
30. The plant of daim 29, wherein the C-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
31. The plant of daim 29 or 30, wherein the fusion polypeptide comprises an N-terminal portion of SEQ ID NO:45.
32. The plant of any of daims 29-31, wherein the N-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
33. The plant of any of daims 29-32, wherein said nucléotide sequence is selected from the group consisting of:
a) the nucléotide sequence set forth in any of SEQ ID NO:47 and 1-14;
b) a nucléotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:48 and 15-31 ;
c) a nucléotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:48 and 15-31, wherein the pesticidal activity of the fusion polypeptide is improved or extended relative to the pesticidal activity of SEQ ID NO:43 or 45.
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34. The plant of any of daims 29-33, wherein said plant is a plant cell.
35. A method for protecting a plant from a pest, comprising expressing in a plant or cell
5 thereof a nucleic acid molécule comprising a nucléotide sequence encoding a fusion polypeptide having pesticidal activity, wherein the fusion polypeptide comprises a C-terminal portion of SEQ ID NO:43.
36. The method of claim 35, wherein the C-terminal portion corresponds to about amino
10 acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
37. The method of daim 35 or 36, wherein the fusion polypeptide comprises an Nterminal portion of SEQ ID NO:45.
38. The method of any of daims 35-37, wherein the N-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
20
39. The method of any of daims 35-38, wherein said nucléotide sequence is selected from the group consisting of:
a) the nucléotide sequence set forth in any of SEQ ID NO:47 and 1-14;
b) a nucléotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:48 and 15-31 ;
25 c) a nucléotide sequence that encodes a polypeptide comprising an amino acid sequence having at Ieast 95% sequence identity to the amino acid sequence of any of SEQ ID NO:48 and 15-31, wherein the pesticidal activity of the fusion polypeptide is improved or extended relative to the pesticidal activity of SEQ ID NO:43 or 45.
30
40. The method of any of daims 35-39, wherein said plant produces a pesticidal polypeptide having pesticidal activity against a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest.
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41. A method for increasing yield in a plant comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleic acid molécule comprising a nucléotide sequence encoding a fusion polypeptide having pesticidal activity, wherein the fusion polypeptide comprises a C-terminal portion of SEQ ID NO:43.
42. The method of claim 41, wherein the C-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
43. The method of claim 41 or 42, wherein the fusion polypeptide comprises an Nterminal portion of SEQ ID NO:45.
44. The method of any of daims 41-43, wherein the N-terminal portion corresponds to about amino acid position 174 to about amino acid position 803 of SEQ ID NO:43, or a variant or fragment thereof.
45. The method of any of daims 41-44, wherein said nucléotide sequence is selected from the group consisting of:
a) the nucléotide sequence set forth in any of SEQ ID NO:47 and 1-14;
b) a nucléotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:48 and 15-31 ;
c) a nucléotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:48 and 15-31, wherein the pesticidal activity of the fusion polypeptide is improved or extended relative to the pesticidal activity of SEQ ID NO:43 or 45.
OA1201300426 2011-04-05 2012-04-04 AXMI115 variant insecticidal gene and methods for its use. OA16614A (en)

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