RU2772947C2 - Constructed pesticide proteins and methods for controlling plant pests - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a chimeric insecticide protein, which is toxic for a pest insect related to lepidoptera. Polynucleotide, a nucleic acid molecule encoding the specified insecticide protein; a vector and a cell containing the specified nucleic acid molecule; a transgenic plant, a part of the specified plant and a transgenic seed containing the specified polynucleotide; a composition, a collected product, a recycled product containing the specified protein are also disclosed. A method for the production of the specified transgenic plant and transgenic seed, a method for a decrease in a population of an insect pest related to lepidoptera, using the specified protein, a method for reduction in the development of resistance to Vip3A protein, using the specified polynucleotide, are disclosed.

EFFECT: invention allows for effective combating insect pests related to lepidoptera.

21 cl, 8 dwg, 12 tbl, 7 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to engineered pesticidal proteins and the nucleic acid molecules that encode them, as well as compositions and methods for controlling plant pests.

BACKGROUND OF THE INVENTION

[0002] Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming soil bacterium that is distinguished by its ability to produce crystalline inclusions that are particularly toxic to certain orders and species of plant pests, including insects, but harmless to plants and other non-target organisms. For this reason, compositions containing strains of Bacillus thuringiensis or their insecticidal proteins can be used as environmentally acceptable insecticides for the control of insect pests of agricultural importance or insect vectors of various human or animal diseases.

[0003] Crystalline (Cry) proteins from Bacillus thuringiensis have strong insecticidal activity against predominantly Lepidoptera, Diptera and Coleoptera insect pests. These proteins have also shown activity against pests from the orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga and pests from the orders of the subclass Acari, as well as other invertebrate groups such as Nemathelminthes, Platyhelminthes and Sarcomastigorphora (Feitelson, J. 1993. The Bacillus Thuringiensis family tree. In Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, N.Y.). These proteins were initially divided into classes CryI - CryVI, based primarily on their insecticidal activity. The main classes were Lepidoptera specific (I), Lepidoptera and Diptera specific (II), Coleoptera specific (III), Diptera specific (IV), and nematode specific (V) and (VI). The proteins were further divided into subfamilies, the more closely related proteins within each family were assigned letters to indicate the division, such as: CryIA, CryIB, CryIC, etc. Even more closely related proteins within each section have been given names such as: CryIC(a), CryIC(b), etc.

[0004] The expressions "Cry toxin" and "delta-endotoxin" have been used interchangeably with the expression "Cry protein". The current nomenclature of Cry proteins and genes is based on amino acid sequence homology rather than target insect specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In this more common classification, each toxin is given a unique name, including a primary rank (an Arabic numeral), a secondary rank (a capital letter), a tertiary rank (a lowercase letter), and a quaternary rank (another Arabic numeral). In the modern classification in the primary rank, Roman numerals have been replaced by Arabic numerals. For example, "CryIA(a)" according to the old nomenclature is currently called "Cry1Aa" according to the modern nomenclature. According to Ibrahim et al. (2010, Bioeng. Bugs, 1:31-50) Cry toxins have been classified into six major classes according to their insect host specificity and include: group 1 - Lepidoptera (eg Cry1, Cry9 and Cry15); group 2 - Lepidoptera and Diptera (for example, Cry2); group 3 - Coleoptera (Cry3, Cry7 and Cry8); group 4 - Diptera (Cry4, Cry10, Cry11, Cry16, Cry17, Cry19 and Cry20); group 5 - Lepidoptera and Coleoptera (Cry1I); and group 6 - nematodes (Cry6). The Cry1I, Cry2, Cry3, Cry10 and Cry11 (73-82 kDa) toxins are unique in that they appear to be the result of natural truncations of the larger Cry1 and Cry4 (130-140 kDa) proteins.

[0005] Cry proteins are globular protein molecules that accumulate as protoxins in crystalline form during the Bt sporulation step. After being eaten by a pest, the crystals typically dissolve to release protoxins, which can vary in size, for example, from 130-140 kDa for Cry proteins active on many Lepidoptera, such as Cry1 and Cry9, and 60-80 kDa for Cry3 proteins. , active against Coleoptera, and Cry2 proteins, active against Lepidoptera/Dipterans. After dissolution of the crystals in the body of a susceptible insect, the released protoxins are processed by proteases in the insect's alimentary canal, such as trypsin and chymotrypsin, to form the protease-resistant Cry protein toxin core. This proteolytic processing involves the removal of amino acids from different regions of various Cry protoxins. For example, 130-140 kDa Cry protoxins are typically activated by proteolytic removal of the 25-30 amino acid N-terminal peptide and about half of the rest of the C-terminal protein, resulting in the formation of a mature Cry toxin of about 60-70 kDa. The 60-80 kDa protoxins, such as Cry2 and Cry3, are also processed, but not to the same extent as the larger protoxins. Smaller protoxins generally have the same or more amino acids removed from the N-terminus than larger protoxins, but fewer amino acids are removed from the C-terminus. For example, proteolytic activation of members of the Cry2 family typically involves the removal of about 40-50 N-terminal amino acids. Many Cry proteins are quite toxic to certain target insects, but many have narrow spectrums of activity.

[0006] The toxin portions of Cry proteins typically contain five conserved sequence blocks and three conserved domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first domain with a conserved structure, called domain I, typically consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II typically consists of three beta-sheets arranged in a Greek key configuration, while domain III consists of two anti-parallel beta sheets in a 'jelly-roll' pattern (de Maagd et al., 2001, supra). ). Domains II and III are involved in receptor recognition and binding and are therefore considered determinants of toxin specificity.

[0007] The five conserved sequence blocks are numbered CB1 to CB5 from the N-terminus to the C-terminus of the Cry protein (Hofte & Whitely, 1989, Microbiol. Rev. 53:242-255). Conservative block 1 (CB1) contains approximately 29 amino acids, conservative block 2 (CB2) contains approximately 67 amino acids, conservative block 3 (CB3) contains approximately 48 amino acids, conservative block 4 (CB4) contains approximately 10 amino acids, and conservative block 5 (CB5) contains approximately 12 amino acids. The sequences before and after these five conserved blocks are highly variable and are thus referred to as "variable regions" V1-V6. Bt delta-endotoxin domain I typically contains variable region 1, conserved block 1, variable region 2, and the 52 N-terminal amino acids of conserved block 2. Domain II typically contains approximately 15 C-terminal amino acids of conserved block 2, variable region 3, and approximately 10 N -terminal amino acids of conserved block 3. Domain III typically contains about 38 C-terminal amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, and conserved block 5. Among other delta-endotoxins, Lepidoptera-active Cry1 toxins have a variable region 6 with approximately 1-3 amino acids lying in domain III.

[0008] Numerous commercially valuable plants, including widespread crops, are susceptible to attack by plant pests, including insect pests and nematode pests, resulting in significant reductions in crop yields and quality. For example, plant pests are a major factor in yield loss of important global crops. Due to damage caused by invertebrate pests, including insects, the annual loss in the US is approximately $8 billion. Insect pests are also becoming a burden for vegetable and fruit growers, ornamental flower growers, and home gardeners.

[0009] Insect pests are controlled primarily by heavy application of chemical pesticides that act by inhibiting insect growth, preventing feeding or reproduction, or causing death. Biological pest control agents, such as strains of Bacillus thuringiensis expressing pesticide toxins such as Cry proteins, have also been applied to crops with satisfactory results as an alternative or addition to chemical pesticides. The genes encoding some of these Cry proteins have been isolated and their expression in heterologous hosts such as transgenic plants has been shown to provide another tool for the control of economically important insect pests.

[0010] Thus, good insect control can be achieved, but continued use of certain chemical and biological control methods increases the likelihood of insect pests developing resistance to such control measures. This has been partly mitigated through various resistance control practices, but there remains a need to develop new and effective pest control tools. Particularly desirable are controls that can target a wider range of economically important insect pests and/or that provide effective control of insect lines that can or are resistant to existing insect control agents.

SHORT DESCRIPTION

[0011] The present invention provides nucleic acids, polypeptides, compositions, and methods for imparting pesticidal activity (eg, insecticidal activity) to bacteria, plants, plant cells, tissues, and seeds. In particular, the present invention provides novel chimeric pesticidal proteins (eg, chimeric insecticidal proteins), optionally with altered or increased activity compared to the parent molecule.

[0012] In embodiments, the chimeric proteins of the present invention are toxic to economically important insect pests (eg, by inhibiting the insect pest's ability to survive, grow, and/or reproduce), especially insect pests that infect plants. For example, in embodiments, the chimeric insecticidal proteins of the present invention can be used to control one or more economically important Lepidoptera pests, including, but not limited to, ipsilon cutworm (Agrotis ipsilon), corn borer (Ostrinia nuhilalis), grass cutworm (Spodoptera frugiperda), American corn armyworm (Helicoverpa zea), cane moth (Diatraea saccharalis), velvet cutworm caterpillar (Anticarsia gemmatalis), soybean armyworm (Chrysodeixis includes), southwestern corn moth (Diatraea grandiosella), western Bean armyworm (Richia albicosta ), Tobacco armyworm (Heliothis virescens), Oriental corn borer (Ostrinia furnacalis), Cotton bollworm (Helicoverpa armigera), Yellow rice moth (Chilo suppressalis), Pink stem armyworm (Sesamia calamistis), Rice moth (Cnaphalocrocis medinalis), etc. . In embodiments, the chimeric insecticidal protein has activity against the cutworm pest that is resistant to the Vip3A protein and/or the Cry1F protein.

[0013] Accordingly, as one aspect, the present invention provides a chimeric insecticidal protein that is toxic to a Lepidoptera insect pest, comprising in an N-terminal to C-terminal direction: (a) an N-terminal region of a first Cry1 protein , which is optionally the N-terminal region of the BT-0029 protein of SEQ ID NO: 2, or an amino acid sequence substantially identical thereto, fused to (b) the C-terminal region of another Cry1 protein; where the transition position between the first Cry1 protein and another Cry1 protein is in conservative block 3. In illustrative embodiments, the other Cry1 protein is a Cry1F (e.g. Cry1Fa), Cry1G, Cry1I protein (e.g. Cry1Ia or Cry1If such as BT-0022) , Cry1C (for example, Cry1Ca) or Cry1K.

[0014] In embodiments, the chimeric insecticidal protein of the present invention has insecticidal activity against the insect pest Spodoptera frugiperda or insect pest populations with resistance to the Vip3A protein and/or the Cry1F protein.

[0015] As a further aspect, the present invention provides a nucleotide sequence encoding the chimeric insecticidal proteins of the present invention, and expression cassettes and vectors containing it. In embodiments, the polynucleotide is codon-optimized for expression in a plant (eg, a monocot such as maize or a dicot such as soybean).

[0016] As a further aspect, the present invention provides a transgenic cell (e.g., a transgenic plant cell such as a dicot cell or a monocot cell, or a transgenic bacterial cell), a transgenic plant part, a transgenic plant culture, and a transgenic plant seed, which comprise the nucleotide sequence , expression cassette, vector and/or chimeric insecticidal protein of the present invention.

[0017] As another aspect, the present invention encompasses transgenic plants comprising a plant cell, a plant part, a nucleotide sequence, an expression cassette, a vector, and/or a chimeric insecticidal protein of the present invention.

[0018] As a further aspect, there are seeds from which the transgenic plants of the present invention are obtained and seeds obtained with the transgenic plants of the present invention.

[0019] Also provided are harvested products derived from the transgenic plants of the present invention, wherein the harvested product optionally contains a nucleotide sequence, an expression cassette, a vector, and/or a chimeric insecticidal protein of the present invention. Additionally provided are processed products derived from the assembled products of the present invention, where the collected product optionally contains a nucleotide sequence, an expression cassette, a vector and/or a chimeric insecticidal protein of the present invention. In embodiments, the harvested product or processed product contains the chimeric insecticidal protein of the present invention and has improved resistance to an insect pest (eg, Lepidoptera pest).

[0020] As another aspect, the present invention provides an insecticidal composition comprising a chimeric insecticidal protein of the present invention and an agriculturally acceptable carrier.

[0021] In addition, the present invention provides, as a further aspect, a method for producing a transgenic plant with increased resistance to an insect pest (eg, a Lepidoptera pest). In embodiments, the method includes administering to a plant a polynucleotide, an expression cassette, or a vector of the present invention, wherein the chimeric insecticidal protein is expressed in the plant, thereby producing a plant with increased insect resistance. The step of introducing optionally includes: (i) transforming the plant cell with a polynucleotide, expression cassette or vector and regenerating the transgenic plant; or (ii) crossing the first plant containing the polynucleotide, expression cassette or vector with a second plant. In embodiments, the method further comprises obtaining seed from the transgenic plant. In embodiments, the method further comprises generating plant progeny from a transgenic plant, wherein the plant progeny contains a polynucleotide, an expression cassette, or a vector, expresses a chimeric insecticidal protein, and has increased resistance to an insect pest.

[0022] As another aspect, the present invention provides a method for producing a transgenic plant with increased resistance to an insect pest (for example, a Lepidoptera insect pest), the method comprising: (a) planting a seed containing a polynucleotide, a cassette expression or vector of the present invention; and (b) growing a transgenic plant from a seed, wherein the transgenic plant contains a polynucleotide, an expression cassette, or a vector, and produces a chimeric insecticidal protein, and has increased resistance to an insect pest. In embodiments, the method further comprises: (c) harvesting seed from the transgenic plant of (b), wherein the harvested seed contains a polynucleotide, an expression cassette, a vector, and/or a chimeric insecticidal protein. Optionally, the seed has increased resistance to an insect pest (eg a Lepidoptera pest).

[0023] In addition, as another aspect, the present invention provides a method for obtaining a seed. In embodiments, the method includes: (a) obtaining a transgenic plant that contains a polynucleotide, expression cassette, or vector of the present invention; and (b) collecting seed from the transgenic plant of (a), wherein the harvested seed contains a polynucleotide, expression cassette, or vector, and/or a chimeric insecticidal protein of the present invention. Optionally, the seed has increased resistance to an insect pest (eg a Lepidoptera pest).

[0024] The present invention further contemplates a method for producing a hybrid plant seed. In illustrative embodiments, the method comprises: (a) crossing a first inbred plant, which is a transgenic plant containing a polynucleotide, expression cassette, or vector of the present invention, with another inbred plant, which may or may not contain a polynucleotide, expression cassette, or vector according to the present invention; and (b) enabling the formation of a hybrid seed. In embodiments, the hybrid seed contains a polynucleotide, an expression cassette, or a vector, and/or a chimeric insecticidal protein of the present invention. Optionally, the seed has increased resistance to an insect pest (eg a Lepidoptera pest).

[0025] As another aspect, the present invention provides a method for controlling a pest (e.g., a Lepidoptera pest, such as a grass cutworm), the method comprising delivering to the pest's body or environment a composition, containing an effective amount of a chimeric insecticidal protein or insecticidal composition of the present invention. In embodiments, the method is a method for controlling a Lepidoptera insect pest (eg, cutworm) that is resistant to the Vip3A protein and/or the Cry1F protein.

[0026] Accordingly, as another aspect, the present invention provides a method of reducing the development of resistance to the Vip3A protein and/or the Cry1F protein in a population of a target Lepidoptera pest (eg, grass cutworm). In embodiments, the method includes delivering to a target population or its habitat a transgenic plant containing (i) a polynucleotide, expression cassette or vector of the present invention and (ii) a polynucleotide containing a nucleotide sequence encoding a Vip3A protein and/or a nucleotide sequence, encoding protein Cry1F; wherein the chimeric insecticidal protein and the Vip3A protein and/or the Cry1F protein are produced in the transgenic plant.

[0027] The present invention is also directed to methods of using the polynucleotide o in the present invention, for example, in DNA constructs or expression cassettes or vectors for transformation and expression in organisms, including plants and microorganisms such as bacteria. Nucleotide or amino acid sequences can be native or synthetic sequences that have been designed for expression in an organism such as a plant or bacteria. In addition, the present invention is directed to methods for producing the insecticidal proteins of the present invention and to methods for using the polynucleotide sequences and insecticidal proteins, for example, in microorganisms to control insects, or in transgenic plants to provide protection from insect damage.

[0028] Another aspect of the present invention includes insecticidal compositions and formulations containing the chimeric insecticidal proteins of the present invention, and methods of using the compositions or formulations to control insect populations, for example, by applying the compositions or formulations to insect infested areas, or for preventive treatment areas or plants susceptible to insects to provide protection against insect pests. Optionally, the compositions or compositions of the present invention may, in addition to the chimeric insecticidal protein of the present invention, contain other pesticidal agents such as chemical pesticides, other pesticidal proteins, or dsRNA, for example, to increase or enhance the ability of the composition or composition to provide insect control and/ or to control insect resistance to pesticides.

[0029] The compositions and methods of the present invention are useful for controlling insect pests that infect plants, in particular crop plants. The compositions of the present invention are also useful for detecting the presence of a chimeric insecticidal protein or nucleic acid encoding it in commercial products or transgenic organisms.

[0030] The present invention also provides ways of using the chimeric insecticidal proteins, nucleic acids, transgenic plants, plant parts, seeds, and insecticidal compositions of the present invention, for example, to control an insect pest, such as a Lepidoptera pest.

[0031] In embodiments, the present invention provides a method of using a polynucleotide, expression cassette, vector, or host cell of the present invention to produce an insecticidal composition for controlling an insect pest (e.g., a Lepidoptera pest).

[0032] In embodiments, the present invention provides a method of using a polynucleotide, an expression cassette, or a vector of the present invention to produce a transgenic seed, wherein the transgenic seed grows into a transgenic plant with increased resistance to an insect pest.

[0033] As another aspect, the present invention also contemplates the use of a transgenic plant of the present invention to produce a transgenic seed, which is optionally a hybrid seed.

[0034] In embodiments, the present invention provides a method of using a chimeric insecticidal protein, polynucleotide, expression cassette, vector, transgenic plant, or insecticidal composition of the present invention to prevent the development of resistance in a target Lepidoptera insect pest population to the Vip3A protein, and /or Cry1F protein.

[0035] These and other features, aspects and advantages of the present invention will be better understood with reference to the following detailed description and claims.

BRIEF DESCRIPTION OF GRAPHICS

[0036] Figures 1A and 1B show the amino acid sequence alignment of full-length BT-0029 (SEQ ID NO: 2), BT-0022 (SEQ ID NO: 1), Cry1Fa (SEQ ID NO: 8) and Cry1Ka (SEQ ID NO: 12). Domain III core and conserved block 3 (CB3) are indicated. Identical amino acids in aligned sequences are shaded.

[0037] Figures 2A and 2B show the amino acid sequence alignment of full-length BT-0029 (SEQ ID NO: 2) with exemplary BT-0029 chimeras: Bt29-Bt22 (SEQ ID NO: 3), Bt29-1Fa (SEQ ID NO: 9 ) and Bt29-1Ka (SEQ ID NO: 13). Domain III core and conserved block 3 (CB3) are indicated. The domain III core indicated in the figure is derived from the second Cry protein. Identical amino acids in aligned sequences are shaded.

[0038] Figures 3A, 3B, 3C and 3D show a full length Bt29-Bt22 chimera (SEQ ID NO: 3) aligned with a series of Bt29-Bt22, Bt29-Bt22Tr1 (SEQ ID NO: 20), Bt29-Bt22Tr2 chimeras ( SEQ ID NO: 21), Bt29-Bt22Tr3 (SEQ ID NO: 22), Bt29-Bt22Tr4 (SEQ ID NO: 23), Bt29-Bt22Tr5 (SEQ ID NO: 24) and Bt29-Bt22Tr6 (SEQ ID NO: 25) , with C-terminal truncations within the tail of protoxin BT-0029.

SUMMARY OF SEQUENCES IN THE SEQUENCE LISTING

[0039] SEQ ID NO: 1 is the amino acid sequence of the BT-0022 protein.

[0040] SEQ ID NO: 2 is the amino acid sequence of the BT-0029 protein.

[0041] SEQ ID NO: 3 is the amino acid sequence of the Bt29-Bt22 chimera. The sequence BT-0022 is amino acids 459-597.

[0042] SEQ ID NO: 4 is the nucleotide sequence encoding the Bt29-Bt22 chimera under SEQ ID NO: 3.

[0043] SEQ ID NO: 5 is an exemplary maize-optimized sequence encoding the Bt29-Bt22 chimera under SEQ ID NO: 3.

[0044] SEQ ID NO: 6 is an exemplary, maize-optimized sequence encoding the Bt29-Bt22 chimera under SEQ ID NO: 3.

[0045] SEQ ID NO: 7 is an exemplary maize-optimized sequence encoding the Bt29-Bt22 chimera under SEQ ID NO: 3.

[0046] SEQ ID NO: 8 is the amino acid sequence of full length Cry1Fa.

[0047] SEQ ID NO: 9 is the amino acid sequence of the Bt29-1Fa chimera. The Cry1Fa sequence is amino acids 459-597.

[0048] SEQ ID NO: 10 is the nucleotide sequence encoding the Bt29-1Fa chimera under SEQ ID NO: 9.

[0049] SEQ ID NO: 11 is an exemplary maize-optimized sequence encoding the Bt29-1Fa chimera of SEQ ID NO: 9.

[0050] SEQ ID NO: 12 is the amino acid sequence of full length Cry1Ka.

[0051] SEQ ID NO: 13 is the amino acid sequence of the Bt29-1Ka chimera. The Cry1Ka sequence is amino acids 459-597.

[0052] SEQ ID NO: 14 is the nucleotide sequence encoding the Bt29-1Ka chimera under SEQ ID NO: 13.

[0053] SEQ ID NO: 15 is the amino acid sequence of the Bt29-1Kav2 chimera.

[0054] SEQ ID NO: 16 is the nucleotide sequence encoding the Bt29-1Ka chimera under SEQ ID NO: 15.

[0055] SEQ ID NO: 17 is the amino acid sequence of full length Cry1Ca.

[0056] SEQ ID NO: 18 is the amino acid sequence of the Bt29-1Ca chimera. The Cry1Ca sequence is amino acids 459-597.

[0057] SEQ ID NO: 19 is the nucleotide sequence encoding the Bt29-1Ca chimera under SEQ ID NO: 18.

[0058] SEQ ID NO: 20 is the amino acid sequence of the Bt29-Bt22Tr1 protein.

[0059] SEQ ID NO: 21 is the amino acid sequence of the Bt29-Bt22Tr2 protein.

[0060] SEQ ID NO: 22 is the amino acid sequence of the Bt29-Bt22Tr3 protein.

[0061] SEQ ID NO: 23 is the amino acid sequence of the Bt29-Bt22Tr4 protein.

[0062] SEQ ID NO: 24 is the amino acid sequence of the Bt29-Bt22Tr5 protein.

[0063] SEQ ID NO: 25 is the amino acid sequence of the Bt29-Bt22Tr6 protein.

[0064] SEQ ID NO: 26 is the amino acid sequence of the BT29BT22-TL22v1 protein.

[0065] SEQ ID NO: 27 is the amino acid sequence of the BT29BT22-TL22v2 protein.

[0066] SEQ ID NO: 28 is the amino acid sequence of the BT29BT22-TL22v3 protein.

[0067] SEQ ID NO: 29 is the amino acid sequence of the BT29BT22-TL22v4 protein.

[0068] SEQ ID NO: 30 is the amino acid sequence of the BT29BT22-TL22v5 protein.

[0069] SEQ ID NO: 31 is the amino acid sequence of the BT29BT22-TL22v6 protein.

[0070] SEQ ID NO: 32 is the amino acid sequence of the BT291FaTr1 protein.

[0071] SEQ ID NO: 33 is the amino acid sequence of the BT291FaTr2 protein.

[0072] SEQ ID NO: 34 is the amino acid sequence of the BT291FaTr3 protein.

[0073] SEQ ID NO: 35 is the amino acid sequence of the BT291FaTr4 protein.

DETAILED DESCRIPTION

[0074] This description is not intended to be a detailed listing of all the various ways in which the present invention may be implemented, or all features that may be added to the present invention. For example, features illustrated in relation to one embodiment may be included in other embodiments, and features illustrated in relation to a particular embodiment may be removed from that embodiment. Thus, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be omitted or omitted. Moreover, numerous variations and additions to the various embodiments provided herein will be apparent to those skilled in the art in light of the present disclosure, which does not depart from the gist of the present invention. Therefore, the following descriptions are intended to illustrate some specific embodiments of the present invention, and not an exhaustive definition of all their transformations, combinations and variations.

[0075] Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning, which is usually understood by a person skilled in the art to which the present invention relates. The terminology used herein in describing the present invention is only intended to describe specific embodiments and is not intended to limit the present invention.

[0076] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety to explain ideas related to the sentence and/or paragraph in which the reference is made.

[0077] Nucleotide sequences provided herein are presented in a 5'-3' direction from left to right and are represented using the standard code for representing nucleobases as set forth in 37 CFR §§1.821-1.825 and the ST standard. 25. World Intellectual Property Organization (WIPO), for example: adenine (A), cytosine (C), thymine (T) and guanine (G).

[0078] Similarly, amino acids are designated using the WIPO ST.25 standard, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys ; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys ; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), series (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0079] Unless otherwise indicated in the context, it is specifically understood that the various features of the present invention described herein may be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be omitted or omitted. For illustrative purposes, if this specification states that a composition contains components A, B, and C, this specifically implies that any of A, B, or C, or a combination thereof, may be omitted and rejected individually or in any combination.

Definitions.

[0080] As used in the description of the present invention and the attached claims, the singular forms also include the plural forms, unless the context clearly indicates otherwise.

[0081] As used herein, the expression "and/or" refers to and encompasses any and all possible combinations of one or more of the respective listed elements, and no combinations when interpreted as an alternative ("or").

[0082] The expression "approximately" as used herein when referring to a measurable value such as a dosage or a period of time, etc., is intended to cover changes in a specified amount of ±20%, ±10%, ±5%, ±1 %, ±0.5% or even ±0.1%. As used herein, phrases such as "from about X to about Y" mean "from about X to about Y", and phrases such as "from about X to about Y" mean "from about X to about Y".

[0083] As used herein, phrases such as "from X to Y" should be interpreted to include X and Y unless the context indicates otherwise.

[0084] By "activity" of the insecticidal protein of the present invention is meant that the insecticidal protein functions as an orally active insect control agent, has a toxic effect, for example, by inhibiting the ability of an insect pest to survive, grow and/or reproduce (for example, causing morbidity and/or mortality), and/or is capable of disrupting and/or deterring feeding of insects, which may or may not cause death of the insect. Thus, when an insecticidal protein of the present invention is delivered to an insect, the result is typically insect morbidity and/or mortality, and/or the insect reduces or stops feeding on a source that makes the insecticidal protein available to the insect.

[0085] In the context of the present invention, a "chimeric" protein is a protein created by the fusion of all or part of at least two different proteins. In embodiments of the present invention, the chimeric protein is a chimeric Cry protein containing all or part of two different Cry proteins fused together into a single polypeptide. A "chimeric insecticidal protein" is a chimeric protein that has insecticidal activity (as described herein).

[0086] A "coding sequence" is a nucleic acid sequence that is transcribed into an RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. In embodiments, the RNA is then translated to produce a protein.

[0087] As used herein, the term "codon-optimized" nucleotide sequence means a nucleotide sequence of a recombinant, transgenic, or synthetic polynucleotide in which the codons are selected to reflect the predilection for particular codons that may be observed in a host cell or organism. Typically, this is done in such a way as to preserve the amino acid sequence of the polypeptide encoded by the codon-optimized nucleotide sequence. In certain embodiments, the nucleotide sequence is codon-optimized for the cell (eg, animal, plant, fungal, or bacterial cell) in which the construct will be expressed. For example, in a construct that will be expressed in a plant cell, all or parts of the sequence may be codon-optimized for expression in a plant. See, for example, US Pat. No. 6,121,014. In embodiments, the polynucleotides of the present invention are codon-optimized for expression in a plant cell (eg, dicot cell or monocot cell) or bacterial cell.

[0088] "Control" of insect pests means suppressing, through toxic action, the ability of insect pests to survive, grow, feed and/or reproduce, and/or limit insect-related damage or death of a crop plant caused by an insect pest, and/or protecting the maximum potential crop yield caused by the pest when grown in the presence of the pest. "Control" of a pest may or may not mean killing the insect, although in embodiments of the present invention, "control" of an insect means killing the insect.

[0089] The term "comprising", "comprising", or "comprising" as used herein indicates the presence of the recited features, integers, steps, operations, elements, or components, but does not exclude the presence or addition of one or more other features, integers numbers, steps, operations, elements, components and/or groups thereof.

[0090] As used herein, the transition phrase "essentially consisting of" (and its grammatical variants) means that the scope of the claim should be interpreted as covering the specified materials or steps listed in the claim, "and those that are not substantially change the main and new characteristic(s)" of the claimed invention. Thus, the term "substantially consisting of" when used in a claim of the present invention is not intended to be interpreted as equivalent to the term "comprising".

[0091] In the context of the present invention, "corresponding" or "corresponding" means that when the amino acid sequences of modified or homologous proteins align with each other, the amino acids that "correspond" to certain listed positions in the modified or homologous protein are those that align with these positions in the reference protein, but are not necessarily exactly in these numerical positions relative to the particular reference amino acid sequence of the present invention. For example, if SEQ ID NO: 2 (BT-0029) is a reference sequence and aligned with SEQ ID NO: 1 (BT-0022) as in Figure 1, the TLEAVT sequence immediately following Domain III in SEQ ID NO: 1 (BT-0022) "corresponds" to the TFEAEY sequence immediately following domain III in SEQ ID NO: 2 (BT-0029).

[0092] As used herein, the term "Cry protein" means an insecticidal protein of the crystalline delta-endotoxin type from Bacillus thuringiensis. The term "Cry protein" may refer to the protoxin form or any insecticidally active fragment or toxin thereof, including the partially processed form and the mature toxin form (eg, without the N-terminal peptidyl fragment and/or the C-terminal tail of the protoxin).

[0093] As used herein, the expression "delivery" or "deliver" (and grammatical variations) a composition or insecticidal protein means that the composition or insecticidal protein comes into contact with an insect, facilitating oral consumption of the composition or insecticidal protein, resulting in a toxic effect and insect control. A given composition or insecticidal protein can be delivered by a variety of known routes, including, without limitation, expression in a transgenic plant, formulated protein(s) composition(s), spray(s) protein(s) composition(s), matrix with bait or any other protein delivery system known in the art.

[0094] The term "domain" refers to a set of amino acids that are conserved at certain positions along the length of the sequence alignment of evolutionarily related proteins. While amino acids at other positions of the homologues may differ, amino acids that are highly conserved at certain positions indicate amino acids that are likely to be essential for protein structure, stability, or function. Identified by their high degree of conservatism in aligned sequences of a family of protein homologues, they can be used as identifiers to determine whether any given polypeptide belongs to a previously identified group of polypeptides.

[0095] "An effective amount to control insects" means a concentration of an insecticidal protein that suppresses, through toxic action, the ability of insects to survive, grow, feed and/or reproduce, or limits insect-related crop damage or loss. "Insect control effective amount" may or may not mean killing the insect, although in embodiments it means killing the insect.

[0096] As used herein, the term "expression cassette" means a nucleic acid molecule capable of directing the expression of at least one polynucleotide of interest, such as a polynucleotide encoding an insecticidal protein of the present invention, in an appropriate host cell, comprising a promoter operably linked with a polynucleotide of interest that is operably linked to a termination signal. The "expression cassette" also typically contains additional polynucleotides to facilitate proper translation of the polynucleotide of interest. The expression cassette may also contain other polynucleotides that are not associated with the expression of the polynucleotide of interest, but that are present due to the appropriate restriction sites to retrieve the cassette from the expression vector. In embodiments, at least one of the components in the expression cassette may be heterologous (ie, foreign) to at least one of the other components (eg, a heterologous promoter operably linked to the polynucleotide of interest). The expression cassette may also be a sequence that occurs naturally but has been produced in a recombinant form suitable for heterologous expression. However, as a rule, the expression cassette is heterologous with respect to the host, ie. the expression cassette (or even the polynucleotide of interest) does not naturally occur in the host cell and has been introduced into the host cell or its progenitor cell by a transformation process or a crossing process. Expression of the polynucleotide(s) of interest in an expression cassette is usually under the control of a promoter. In the case of a multicellular organism, such as a plant, the promoter may also be specific or preferred for a particular tissue or organ or developmental stage (as described in more detail herein). Once transformed into a plant, the expression cassette or fragment thereof may also be referred to as an "inserted polynucleotide" or "inserted polynucleotide".

[0097] A "gene" is defined herein as a unit of heredity, containing one or more polynucleotides, that occupies a specific location on a chromosome or plasmid, and that contains the genetic instruction for a particular characteristic or trait inherent in an organism.

[0098] As used herein, the term "gut protease" refers to a protease that occurs naturally in the insect GI tract. This protease is usually involved in the digestion of ingested proteins. Examples of alimentary canal proteases include trypsin, which typically cleaves peptides from the C-terminus of lysine (K) or arginine (R) residues, and chymotrypsin, which typically cleaves peptides from the C-terminus of phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0099] As used herein, the term "heterologous" means foreign, exogenous, non-native, and/or not naturally occurring. In embodiments, a "heterologous" polynucleotide or polypeptide is a polynucleotide or polypeptide not naturally associated with the host cell into which it is administered, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence or polypeptide. In embodiments, a nucleotide sequence is heterologous to another sequence to which it is operably linked, for example, a promoter may be heterologous (ie, foreign) to an operably linked coding sequence.

[00100] As used herein, "homologous" means native. For example, a homologous nucleotide sequence or amino acid sequence means a nucleotide sequence or amino acid sequence that is naturally associated with the host cell into which it is inserted, a homologous promoter sequence is a promoter sequence that is naturally associated with a coding sequence, and the like. .

[00101] The terms "increase", "increase", "increased", "increase", "increased", "increase" and "improvement" (and their grammatical variations) and similar terms used in this document describe the increase in pest control plants, for example, by bringing the plant into contact with the polypeptide of the present invention (such as, for example, by transgene expression or methods of topical application). This increase in control may be relative to the level of control of the plant pest in the absence of the polypeptide of the present invention (eg, a plant that does not transgenicly express the polypeptide or is not treated topically with the polypeptide). Thus, in embodiments, the terms "increase", "increase", "increased", "increase", "increase", "increase", and "improve" (and their grammatical variations) and similar terms may indicate an increase of at least at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more compared to a suitable control (e.g. plant, plant part, plant cell that is not in contact with a polypeptide of the present invention).

[00102] The term "insecticidal" as used herein is defined as a toxic biological activity capable of controlling an insect pest, optionally but preferably by killing it.

[00103] A nucleic acid sequence is "isocodonic" with respect to a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.

[00104] In exemplary embodiments, the nucleic acid molecules, polynucleotides or polypeptides of the present invention are "isolated". An "isolated" nucleic acid molecule, polynucleotide or protein, and the like. is a nucleic acid molecule, polynucleotide or protein, etc., which is no longer in its natural environment. An isolated nucleic acid molecule, polynucleotide or protein of the present invention may be in a purified form or may be in a recombinant host such as a transgenic bacterium or a transgenic plant. In embodiments, the isolated nucleic acid molecule, nucleotide sequence, or polypeptide is in purified form, i.e., at least partially separated from at least some other components of a naturally occurring organism or virus, such as structural components of a cell or virus or other polypeptides, or nucleic acids usually found associated with a polynucleotide. In other embodiments, the "isolated" nucleic acid molecule, nucleotide sequence, or polypeptide may exist in a non-natural environment, such as, for example, a recombinant host cell. Thus, for example, in relation to nucleotide sequences, the term "isolated" may mean that a nucleotide sequence is separated from the chromosome and/or cell in which it occurs naturally. A polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs and then inserted into the genetic environment, chromosome and/or cell in which it does not naturally occur (for example, another cell- host, different regulatory sequences, and/or different position in the genome than those found in nature). Accordingly, recombinant nucleic acid molecules, nucleotide sequences, and the polypeptides they encode are "isolated" because they exist through human intervention separately from their natural environment and therefore are not natural products, however, in some embodiments, they can be introduced into a recombinant cell host and exist in it. In exemplary embodiments, the isolated nucleic acid molecule, the isolated nucleotide sequence, and/or the isolated polypeptide are at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% pure. , 80%, 90%, 95%, or more.

[00105] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are often highly conserved portions of domains, but may also include only a portion of a domain, or be located outside a conserved domain (if all amino acids of a motif fall outside a particular domain).

[00106] A "native" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA that occurs naturally in, or is endogenous to, an organism.

[00107] The terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence", "oligonucleotide", and "polynucleotide" are used interchangeably herein unless the context otherwise indicates, and refer to a heteropolymer of nucleotides. These terms include, without limitation, DNA and RNA molecules, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and RNA, plasmid DNA, mRNA, antisense RNA, and RNA/DNA hybrids, any of which may be linear or branched, single stranded. or double-stranded, or combinations thereof. In case dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used to pair antisense sequences, dsRNA and ribozymes. For example, polynucleotides that contain the C-5-propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications can also be made, such as modification of the phosphodiester backbone or the 2'-hydroxy sugar group of the RNA ribose. In embodiments, the terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence", "oligonucleotide", or "polynucleotide" refer to DNA.

[00108] As used herein, "operably linked" or "operably linked" means that the elements are operably linked to each other, and are also usually physically linked. Thus, as used herein, the terms "operably linked" or "operably linked" refer to nucleotide sequences in a single nucleic acid molecule that are operably linked. Thus, the first nucleotide sequence having a functional relationship with the second nucleotide sequence means the situation where the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a nucleotide sequence if the promoter affects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that regulatory sequences (eg, a promoter) need not be contiguous with the nucleotide sequence to which they are operatively linked as long as the regulatory sequences function to direct its expression. Thus, for example, intermediate untranslated but transcribed sequences may be present between a promoter and a nucleotide sequence, and the promoter may still be considered "operably linked" or "operably linked" to that nucleotide sequence.

[00109] As used herein, the term "plant" refers to any plant at any stage of development.

[00110] Any plant (or groupings of plants, eg, into genus or higher order of classification) can be used in the practice of the present invention, including angiosperms or gymnosperms, monocots or dicots.

[00111] Exemplary plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa, including but not limited to Indica and/or Japonica varieties), rape (Brassica napus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tobacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee tree (Cofea spp.), coconut palm (Cocos nucifera), pineapple (Ananas comosus), citrus trees ( Citrus spp.), cocoa tree (Theobroma cacao), tea plant (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig tree (Ficus casica), guava (Psidium guajava), mango (Mangifera indica) , olive (Olea europaea), melon tree (Carica papaya), western cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet species (Beta vulgaris), apple tree (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera ), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), plum (Prunus domestica), pear tree (Pyrus communis), watermelon (Citrullus vulgaris), duckweed (Lemna spp.), oat species (Avena sativa ), barley (Hordium vulgare), vegetables, ornamental plants, conifers, and turf grass species (e.g. for ornamental, recreational or fodder purposes), and biomass grass species (e.g. switchgrass and miscanthus).

[00112] Vegetables include, without limitation, nightshade species (e.g., tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), carrot species (Caucus carota), cauliflower (Brassica oleracea), celery (Apium graveolens), eggplant ( Solanum melongena), asparagus (Asparagus officinalis), okra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), pea species (Lathyrus spp.), members of the Cucurbita genus such as Hubbard's pumpkin (C. hubbard ), butternut squash (C. moschata), common pumpkin (C. rero), gourd (C. crookneck), C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C lundelliana and C. martinezii, and members of the Cucumis genus such as cucumber (Cucumis sativus), cantaloupe (C. cantalupensis) and musk melon (C. melo).

[00113] Ornamental plants include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), rose species (Rosa spp.), tulip species (Tulipa spp.), daffodil species (Narcissus spp.), species petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettia (Euphorbia pulcherima) and chrysanthemum.

[00114] Conifers that can be used in the practice of the present invention include, for example, pines such as fox pine (Pinus taeda), Elliot pine (Pinus elliotii), yellow pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and radiant pine (Pinus radiata); Pseudotsuga Menzies (Pseudotsuga menziesii); Canadian hemlock (Tsuga canadensis); gray spruce (Picea glauca); sequoia evergreen (Sequoia sempervirens); firs such as sweet fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as thuja (Thuja plicata) and Nootka cypress (Chamaecyparis nootkatensis).

[00115] Lawn grass includes, but is not limited to, Zoysia spp., Agrostis spp., Fescue spp., Bluegrass spp., Stenotaphrum spp., Svinora spp., Buffalo grass spp., Weed chaff spp., and Hedgehog spp.

[00116] Also included are plants that serve primarily as laboratory models, such as Arabidopsis.

[00117] A "plant cell" is a structural and physiological unit of a plant containing a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a more highly organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[00118] "Plant cell culture" means the culture of plant units such as, for example, protoplasts, cells in cell culture, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various developmental stages.

[00119] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, eggs, zygotes, seeds, cuttings, cell or tissue cultures, or any other plant parts or products.

[00120] A "plant organ" is a distinct and visually structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[00121] As used herein, the term "plant part" includes, without limitation, embryos, pollen, ovules, seeds, leaves, flowers, twigs, fruits, stems, roots, root tips, anthers, and/or plant cells, including those plant cells which are intact in plants and/or plant parts, plant protoplasts, plant tissues, plant tissue cultures, plant calli, aggregations of plant material, and the like.

[00122] The term "plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any plant tissue in planta or in culture is contemplated. The term includes, without limitation, whole plants, plant organs, plant seeds, tissue culture, and any group of plant cells organized into structural or functional units. The use of this term with or without any specific type of plant tissue as defined above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[00123] A "polynucleotide of interest" refers to any polynucleotide that, when transferred to an organism, such as a plant, confers a desired characteristic on the organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in the manufacturing process, production of commercially valuable enzymes or metabolites, or altered fertility, and the like.

[00124] By "part" or "fragment" of a polypeptide of the present invention is meant an amino acid sequence of reduced length relative to the reference amino acid sequence of a polypeptide of the present invention. Such a portion or fragment according to the present invention may, when appropriate, be incorporated into the larger polypeptide of which it is a part (eg, a labeled or fused protein). In embodiments, the "part" or "fragment" substantially retains insecticidal activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or even 100% activity of the full-length protein or even has greater insecticidal activity than the full-length protein).

[00125] The terms "protein", "peptide", and "polypeptide" are used interchangeably herein.

[00126] The term "promoter" refers to a polynucleotide, typically upstream (5') of a coding polynucleotide, that controls the expression of the coding polynucleotide, allowing recognition by RNA polymerase and other factors necessary for proper transcription.

[00127] As used herein, the term "protoplast" refers to an isolated plant cell without a cell wall or with portions of the cell wall only.

[00128] As used herein, the term "recombinant" refers to a form of a nucleic acid (eg, DNA or RNA) or protein or organism that would not normally occur in nature, and which as such has been created through human intervention. As used herein, a "recombinant nucleic acid molecule" (and similar terms) is a nucleic acid molecule containing a combination of polynucleotides that do not naturally occur together and are the result of human intervention, for example, a nucleic acid molecule that consists of a combination of at least two polynucleotides that are heterologous with respect to each other, or a nucleic acid molecule artificially synthesized and containing a polynucleotide that differs from a polynucleotide that would normally exist in nature, or a nucleic acid molecule that contains a transgene artificially introduced into the genomic DNA of a cell - host, and associated flanking DNA of the host cell genome. An example of a recombinant nucleic acid molecule is a DNA molecule resulting from the insertion of a transgene into the genomic DNA of a plant, which may ultimately result in the expression of the recombinant RNA or protein molecule in that organism. In embodiments, a "recombinant" protein is a protein that does not normally exist in nature or is present in a non-naturally occurring environment and is expressed from a recombinant nucleic acid molecule. As used herein, the term "recombinant plant" is a plant that would not normally exist in nature and is the result of human intervention and contains a recombinant polynucleotide (eg, a transgene or a heterologous nucleic acid molecule inserted into its genome). As a result of this change in the genome, the recombinant plant is clearly different from the wild-type related plant.

[00129] The terms "reduce", "reduced", "decrease", "reduction", "reduce", and "suppress" (and their grammatical variations) and similar terms used herein refer to reduced survival, growth and/or or propagating a plant pest, for example, by bringing the plant into contact with the polypeptide of the present invention (such as, for example, by transgene expression or topical application methods). This decrease in survival, growth and/or reproduction may be relative to the level observed in the absence of the polypeptide of the present invention (eg, a plant that does not transgenicly express the polypeptide or is not subjected to topical treatment with the polypeptide). Thus, in embodiments, the terms "reduce", "reduced", "reduction", "reduction", "reduce" and "suppress" (and their grammatical variants) and similar terms mean a reduction of at least about 5%, 10 %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to a plant that does not come into contact with the polypeptide of the present invention (for example, a plant that does not transgenicly express the polypeptide or is not treated topically with the polypeptide). In exemplary embodiments, the reduction results in no or substantially no (i.e., negligible, e.g., less than about 10%, less than about 5%, or even less than about 1%) detectable survival, growth, and/or reproduction of the pest. plants.

[00130] "Regulatory element" refers to a nucleotide sequence involved in the control of expression of a polynucleotide. Examples of regulatory elements include promoters, termination signals, and nucleotide sequences that facilitate correct translation of a polynucleotide.

[00131] As used herein, the term "selectable marker" means a nucleotide sequence that, when expressed, results in a distinctive phenotype of a plant, plant part, and/or plant cell expressing the marker, and thus allows such transformed plants, plant parts to be distinguished and/or plant cells from those lacking the marker. Such a nucleotide sequence may encode either a selectable or a screenable marker, depending on whether the marker confers a trait for which selection can be made by chemical means, e.g., by applying a selective agent (e.g., antibiotic, herbicide, etc.) , or whether the marker is simply a trait that can be identified through observation or testing, such as by screening (eg, a trait determined at the R-locus).

[00132] As used herein, "specific activity" refers to the amount of protein required to produce an insecticidal effect. Therefore, if the first protein has a higher specific activity than the second protein, this means that in order to obtain an insecticidal effect on the same percentage of insects, a smaller amount of the first protein is needed compared to the amount of the second protein.

[00133] The phrase "substantially identical" in the context of two nucleic acid sequences or two amino acid sequences refers to two or more sequences or subsequences that are at least about 50% identical in nucleotides or amino acid residues when compared and aligned for maximum match, which is determined using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, substantially identical sequences are characterized by at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater nucleotide or amino acid residue identity. In certain embodiments, significant identity exists within a region of sequences that is at least about 50 residues, 100 residues, 150 residues, 200 residues, 250 residues, 300 residues, 350 residues, 400 residues, or more in length. In additional embodiments, sequences are substantially identical when they are identical throughout the length of the coding regions.

[00134] "Identity" or "percent identity" refers to the degree of identity between two nucleic acid sequences or amino acid sequences. When comparing sequences, one sequence usually acts as a reference sequence against which the test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the subsequence are set, and the software parameters of the sequence comparison algorithm are set. Then, using the sequence comparison algorithm, based on the specified program parameters, the percent sequence identity for the tested sequence(s) relative to the reference sequence is calculated.

[00135] When comparing sequences, one sequence typically acts as a reference sequence against which test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the subsequence are set, and the software parameters of the sequence comparison algorithm are set. Then, using the sequence comparison algorithm, based on the specified program parameters, the percent sequence identity for the tested sequence(s) relative to the reference sequence is calculated.

[00136] Optimal alignment of sequences for comparison can be performed, for example, using the local homology search algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), using the Needleman-Wunsch homologous alignment algorithm, J. Mol. Biol. 48: 443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. sci. USA 85: 2444 (1988), by software implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual inspection (see general Ausubel et al., below).

[00137] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). BLAST analysis software is available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm provides for the initial identification of pairs of sequences with the highest similarity (HSP) by identifying short "words" of length W in the requested sequence that either match or satisfy some positive threshold T when aligned with a "word" of the same length in the sequence from the database . T is called the neighbor "word" threshold (Altschul et al., 1990). These initial matches of neighboring "words" act as a seed to begin searches to find longer HSPs containing them. The "word" matches are then extended in both directions along each sequence for as long as the cumulative alignment score can increase. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). In the case of amino acid sequences, a substitution matrix is used to calculate the cumulative score. The extension of "word" matches in each direction is terminated when the cumulative alignment score decreases by X from its maximum value achieved, with the cumulative score falling to zero or below due to the accumulation of one or more negative-scoring residual alignments, or when the end of one is reached. from sequences. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, a threshold of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, BLASTP uses a default word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 substitution matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[00138] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which is a measure of the probability that matches between two nucleotide or amino acid sequences will be observed randomly. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[00139] Another widely used and recognized computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids and multiplied by 100 to obtain percent identity. For example, if two sequences of 580 base pairs had 145 base pairs, they would be 25 percent identical. If the two sequences being compared are of different lengths, then the number of matches is divided by the shorter of the two lengths. For example, if there were 100 identical amino acids in proteins of 200 and 400 amino acids, then they are 50 percent identical, given the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids, then the number of matches is divided by 150 (for nucleic bases) or 50 (for amino acids) and multiplied by 100 to obtain percent identity.

[00140] Two nucleotide sequences can also be considered substantially identical if the two sequences hybridize to each other under stringent conditions. In exemplary embodiments, two nucleotide sequences that are considered substantially identical are hybridized to each other under very stringent conditions.

[00141] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a nucleic acid will selectively hybridize to a target sequence to a definable higher degree than to other sequences (e.g., at least 2-fold higher compared to the non-target sequence) and may optionally substantially eliminate binding to non-target sequences. Stringent conditions are sequence dependent and will vary under different circumstances. By controlling the stringency of the hybridization and/or wash conditions, target sequences can be identified, which can be up to 100% complementary to the reference nucleotide sequence. Alternatively, conditions of moderate or even low stringency may be applied to provide some sequence mismatch in order to determine lower levels of sequence similarity. For example, those skilled in the art will appreciate that to function as a primer or probe, the nucleic acid sequence need only be sufficiently complementary to the target sequence to substantially bind to it, thus forming a stable double stranded structure under the conditions employed. Thus primers or probes can be used under conditions of high, moderate or even low stringency. Similarly, conditions of low or moderate stringency may be preferred for the detection of homologous, orthologous, and/or paralogous sequences having lower degrees of sequence identity than would be identified under very stringent conditions.

[00142] As used herein, the terms "complementary" or "complementarity" (and similar terms) refer to the natural binding of polynucleotides under salt and temperature conditions to permit this, by base pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be partial, in which only some of the nucleotides bind, or it may be complete, in which there is absolute complementarity between single-stranded molecules. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between molecules. As used herein, the term "substantially complementary" (and similar terms) means that two nucleic acid sequences are at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% complementary. , 95%, 96%, 97%, 98%, 99% or more. Alternatively, the term "substantially complementary" (and similar terms) may mean that two nucleic acid sequences can hybridize together under conditions of high stringency (as described herein).

[00143] As used herein, "specifically" or "selectively" hybridizes (and similar terms) refers to the binding, duplexing, or hybridization of a molecule to a particular target nucleic acid sequence under stringent conditions when that sequence is present in a complex mixture (e.g. , total cellular DNA or RNA) with, in fact, the exclusion of non-target nucleic acids or even without detectable binding, duplexing or hybridization with non-target sequences. Specifically or selectively hybridizing sequences are typically at least about 40% complementary, and optionally substantially complementary or even completely complementary (ie, 100% identical).

[00144] For DNA-DNA hybrids, T _m is approximately calculated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-84 (1984): T _m =81.5°C+16.6 (log M)+0.41 (% GC)-0.61 (% formamide) - 500/l; where M is the molarity of the solution of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T _m is the temperature (given ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. T _m reduce approximately 1°C for every 1% mismatch; thus, T _m , hybridization and/or wash conditions can be adjusted to hybridize with sequences with the desired degree of identity. For example, if you are looking for sequences with identity >90%, T _m can be reduced by 10°C. In general, stringent conditions are chosen to be about 5° C. lower than the melting point (T _m ) for the specific sequence and its complementary sequence at certain ionic strength and pH. However, under very stringent conditions, hybridization and/or washing at the melting temperature (T _m ) or 1, 2, 3 or 4°C below the melting temperature (T _m ) can be used; under conditions of moderate stringency, hybridization and/or washing at 6, 7, 8, 9, or 10° C. below the melting temperature (T _m ) can be used; under conditions of low stringency, hybridization and/or washing at 11, 12, 13, 14, 15, or 20° C. below the melting temperature (T _m ) can be used. If the desired degree of mismatch causes a T _m below 45° C. (aqueous solution) or 32° C. (formamide solution), optionally the SSC concentration can be increased so that a higher temperature can be used. A comprehensive guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993); Current Protocols in Molecular Biology, Chapter 2, edited by Ausubel, et al. Greene Publishing and Wiley-Interscience, New York (1995); and Green & Sambrook in Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[00145] Typically, stringent conditions are those where the salt concentration is less than about 1.5 M Na ions, typically the concentration of Na ions (or other salts) is about 0.01-1.0 M at about pH 7 0 - pH 8.3, and the temperature is at least about 30°C for short probes (eg, 10-50 nucleotides) and at least about 60°C for longer probes (eg, more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide or Denhardt's solution (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml water). Illustrative low stringency conditions include hybridization using a buffer solution of 30%-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and washing in 1X-2X SSC (20X SSC=3.0 M NaCl/0 .3 M trisodium citrate) at 50°C-55°C. Illustrative conditions of moderate stringency include hybridization in 40-45% formamide, 1 M NaCl, 1% SDS at 37°C and washing in 0.5X-1X SSC at 55°C-60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C and a wash in 0.1X SSC at 60°C-65°C. An additional non-limiting example of high stringency conditions includes hybridization in 4X SSC, 5X Denhardt's solution, 0.1 mg/ml boiled salmon milt DNA, 25 mM Na phosphate at 65° C. and wash in HDC SSC, 0.1% SDS at 65° FROM. Another example of high stringency hybridization conditions include hybridization in 7% SDS, 0.5 M NaPO ₄ , 1 mM EDTA at 50°C with a wash in 2X SSC, 0.1% SDS at 50°C, alternatively with a wash in 1X SSC , 0.1% SDS at 50°C, alternatively washed in 0.5X SSC, 0.1% SDS at 50°C or alternatively washed in 0.1X SSC, 0.1% SDS at 50°C C, or else washed in 0.1X SSC, 0.1% SDS at 65°C. Those skilled in the art will appreciate that specificity is generally dependent on post-hybridization washes, with ionic strength and temperature of the final wash solution being important factors.

[00146] In this case, nucleic acids that do not hybridize with each other under stringent conditions are essentially identical if the proteins they encode are essentially identical (for example, due to the degeneracy of the genetic code).

[00147] Another indication that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive with the protein encoded by the second nucleic acid. Thus, the protein is typically substantially identical to the second protein, for example, if the two proteins differ only in conservative substitutions.

[00148] As used herein, if a modified polypeptide or fragment (and the like) "substantially retains" insecticidal activity, this means that the modified polypeptide or fragment retains at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or even 100% of the insecticidal activity of the reference protein or is characterized by an even greater insecticidal activity.

[00149] "Synthetic" refers to a nucleotide sequence containing bases or structural feature(s) not found in the natural sequence. For example, an artificial sequence encoding a protein of the present invention is considered synthetic if it is more similar in terms of G+C content and normal codon distribution to the genes of dicotyledonous or monocotyledonous plants.

[00150] As used herein, a protein that is "toxic" to a pest is an orally active insect control that kills the pest, causes reduced growth and/or reproduction of the pest, and/or is capable of disrupting or deter the feeding of the insect, the last two of which may or may not cause the death of the insect. When the protein of the present invention is delivered to an insect or the insect comes into contact with the protein, the result is usually the death of the insect, growth and/or reproduction of the insect is retarded, and/or the insect reduces or stops feeding on a source that makes the toxic protein available to the insect. insect.

[00151] The terms "toxin fragment" and "toxin portion" are used interchangeably herein to refer to a fragment or portion of a longer (e.g., full-length) chimeric insecticidal protein of the present invention, where the "toxin fragment" or "toxin portion" retains insecticidal activity . For example, it is known in the art that native Cry proteins are expressed as protoxins that are processed from the N-terminus and C-terminus to produce a mature toxin. In embodiments, the "toxin fragment" or "toxin portion" of the chimeric insecticidal protein of the present invention is truncated at the N-terminus and/or C-terminus. In embodiments, the "toxin moiety" or "toxin moiety" is truncated at the N-terminus, removing part or all of the N-terminal peptidyl moiety, and optionally contains at least about 400, 425, 450, 475, 500, 510,520, 530, 540 , 550, 560, 570, 580, or 590 contiguous amino acids of a chimeric insecticidal protein specifically described herein, or an amino acid sequence that is substantially identical to it. Thus, in embodiments, the "toxin moiety" or "toxin moiety" of the chimeric insecticidal protein is truncated at the N-terminus (e.g., to exclude part or all of the peptidyl moiety), e.g., N-terminal truncation of one or more amino acids, e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids. In embodiments, the "toxin fragment" or "toxin portion" of the chimeric insecticidal protein is truncated at the C-terminus (e.g., to exclude part or all of the tail of the protoxin), e.g., C-terminal truncation of one or more amino acids, e.g., to 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 , 450, 475, 500, 525, 550, 560 or more amino acids. In embodiments, the "toxin moiety" or "toxin moiety" includes domains 1 and 2 and core domain III (eg, as shown in Figure 2). In embodiments, the "toxin fragment" or "toxin portion" is a mature (ie, processed) toxin (eg, a Cry toxin).

[00152] "Transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism. In specific embodiments, "transformation" means the stable integration of a DNA molecule into the genome of an organism of interest (eg, a plant cell).

[00153] As used herein, the terms "transformed" and "transgenic" refer to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the host genome, or the nucleic acid molecule may also be present as an extrachromosomal molecule. Such an extrachromosomal molecule may be autoreplicating. It is understood that "transformed" or "transgenic" cells, tissues or plants include not only the end product of the transformation process, but also their progeny containing a heterologous nucleic acid molecule. A "non-transformed" or "non-transgenic" host refers to a wild-type organism, such as a bacterium or plant, that does not contain a heterologous nucleic acid molecule.

[00154] The term "vector" refers to a composition for transferring, delivering, or introducing a nucleic acid (or nucleic acids) into a cell. The vector contains a nucleic acid molecule containing the nucleotide(s) sequence(s) to be(s) to be transferred, delivered or administered.

Chimeric insecticidal proteins.

[00155] The present invention provides novel chimeric insecticidal proteins comprising at least one region from a first Cry protein (eg, BT-0029 protein [SEQ ID NO: 2] and substantially identical variants thereof). In embodiments, the present invention provides a chimeric insecticidal protein comprising a region of two or more different Cry proteins. In embodiments, the resulting chimeric insecticidal protein is characterized by increased activity against one or more insect pests (e.g., increased activity or activity against a new target pest) and/or a different mode of action against one or more insect pests compared to one or more insect pests. more (or even all) of the original proteins. In exemplary embodiments, the chimeric insecticidal protein is a chimera containing regions from two different Cry proteins (eg, Cry1) and the chimera has increased insecticidal activity against one or more insect pests compared to both parent proteins.

[00156] In embodiments, the chimeric insecticidal proteins of the present invention may provide novel modes of action against one or more target insect pests. For example, a chimeric insecticidal protein may have insecticidal activity against insect pests or colonies that are normally resistant to the insecticidal activity of another agent (eg, an insecticidal protein, including but not limited to a Bt protein such as a Cry protein or a Vip protein). In embodiments, the parent proteins are not insecticidally active per se, or only weakly active against resistant insect pests or colonies, which may indicate that the chimera has a novel mode of action compared to the parent proteins. For example, if the parent proteins are active against the target insect pest ("susceptible") but not against the resistant counterpart, and the chimeric insecticidal protein is active against the resistant insect pest, this indicates that the chimeric insecticidal protein is toxic to resistant pest due to a new mode of action.

[00157] Accordingly, in embodiments, the present invention provides a chimeric insecticidal protein that is toxic to an insect pest (e.g., a Lepidoptera pest), wherein the chimeric insecticidal protein comprises a region from the BT-0029 protein (SEQ ID NO : 2) or a polypeptide that is substantially identical to a region from the BT-0029 protein. In embodiments, the chimeric insecticidal protein comprises an N-terminal region of a first Cry1 protein, which is optionally a BT-0029 protein (SEQ ID NO: 2), or a polypeptide containing an amino acid sequence that is substantially identical to the N-terminal region of the BT-0029 protein. In embodiments, the N-terminal region of the first Cry protein is fused to the C-terminal region of another Cry protein (eg, another Cry1 protein) to form a chimeric insecticidal protein (eg, a chimeric Cry insecticidal protein).

[00158] In illustrative embodiments, the C-terminal region from another Cry1 protein may be the C-terminal region of another Cry1 protein or a polypeptide containing an amino acid sequence that is substantially identical to the C-terminal region from another Cry1 protein.

[00159] Thus, in an exemplary embodiment, the chimeric insecticidal protein of the present invention comprises, essentially consists or consists, in the N-terminal to C-terminal direction, of the following: (a) the N-terminal region of the first Cry protein (e.g. , Cry1 protein), which is optionally the N-terminal region of the BT-0029 protein or an amino acid sequence that is substantially identical to the N-terminal region of the BT-0029 protein fused to (b) the C-terminal region of another Cry protein (e.g., another protein Cry1).

[00160] In exemplary embodiments, another Cry protein (e.g., Cry1 protein) is selected based on the alignment of the C-terminal region from the other Cry protein and the corresponding region from the first Cry protein (e.g., Cry1 protein), e.g., see illustrative alignments in the figures 1A and 1B. In embodiments, the other Cry protein is selected such that the amino acid sequence of the C-terminal region of the other Cry protein is substantially identical to the corresponding region of the first Cry protein. In exemplary embodiments, the amino acid sequence of the C-terminal region of another Cry protein (e.g., Cry1 protein) is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the corresponding C-terminal region from the first Cry protein. Methods for aligning and determining amino acid sequence identity within an aligned region are well known to those skilled in the art.

[00161] In embodiments, another Cry1 protein includes, but is not limited to, Cry1F protein (e.g., Cry1Fa protein or Cry1Fb protein), Cry1G protein (e.g., Cry1Ga, Cry1Gb or Cry1Gc protein), Cry1I protein (e.g., Cry1Ia protein, Cry1Ib protein, Cry1Ic protein, Cry1Id protein, Cry1Ie protein, Cry1If protein or Cry1Ig protein), Cry1K (eg Cry1Ka protein) or Cry1C (eg Cry1Ca protein). In embodiments, the other Cry1 protein is a Cry1If protein, optionally a BT-0022 protein (SEQ ID NO: 1). In embodiments, the other Cry1 protein is the Cry1Fa protein (SEQ ID NO: 8). In embodiments, the other Cry1 protein is the Cry1K protein (SEQ ID NO: 12). In embodiments, the other Cry1 protein is the Cry1Ca protein (SEQ ID NO: 17).

[00162] The terms "N-terminal region" and "C-terminal region" do not necessarily indicate that most of the N-terminal or C-terminal amino acids (e.g., N-terminus or C-terminus), respectively, from the full-length protein are included in region. For example, it is well known to those skilled in the art that Cry protoxins undergo both N-terminal and C-terminal processing to produce a mature (ie, processed) toxin. Thus, in embodiments, the "N-terminal region" and/or the "C-terminal region" excludes some or all of the processed portions of the protoxin such that the chimeric insecticidal protein contains the mature toxin protein (e.g., domains I, II, and III of the protein Cry) without part or all of the N-terminal peptidyl fragment and/or the C-terminal tail of the protoxin, or a polypeptide that is substantially identical to the mature protein of the toxin. In embodiments, the chimeric insecticidal protein contains a peptidyl moiety and/or a protoxin tail. In embodiments, the chimeric insecticidal protein does not contain a peptidyl moiety or protoxin tail, i. corresponds to the mature processed toxin.

[00163] In embodiments, the N-terminal region of the first Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39 or 40 to approximately position 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 , 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474 , 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 SEQ ID NO: 2 (full length BT-0029) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00164] In embodiments, the N-terminal region of the first Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 1 to about position 430, 431, 432, 433, 434, 435, 436, 437, 438 , 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 , 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488 , 489, 490 or 491 SEQ ID NO: 2.

[00165] In an exemplary embodiment, the N-terminal region of the first Cry1 protein contains, essentially consists of, or consists of an amino acid sequence corresponding to an amino acid sequence from about position 1 to about amino acid position 458 of SEQ ID NO: 2.

[00166] In illustrative embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 up to approximately position 580, 581, 582, 583, 584, 585, 586, 587, 588 589 590 591 592 593 594 595 596 587 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613, 614, 615, 616, 617, 618, 619, or 620 SEQ ID NO: 8 (full length Cry1Fa) and any combination of lower and higher positions, as if each such combination were specifically set forth herein m document.

[00167] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 464 to about position 580, 581, 582, 583, 584, 585, 586, 587, 588 , 589, 590, 591, 592, 593, 594, 595, 596, 587, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613 , 614, 615, 616, 617, 618, 619 or 620 SEQ ID NO: 8.

[00168] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445 , 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460 , 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485 , 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 up to approximately position 602 of SEQ ID NO: 8.

[00169] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 464 to about position 602 of SEQ ID NO: 8.

[00170] In exemplary embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, or 535 up to approximately position 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632 , 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654 or 655 SEQ ID NO: 1 (full size BT-0022) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00171] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 498 to about position 615, 616, 617, 618, 619, 620, 621, 622, 623 , 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648 , 649, 650, 651, 652, 653, 654 or 655 SEQ ID NO: 1.

[00172] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480 , 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505 , 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530 , 531, 532, 533, 534, or 535 until approximately position 636 of SEQ ID NO: 1.

[00173] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 498 to about position 636 of SEQ ID NO: 1.

[00174] In illustrative embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, or 537 to approximately position 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656 or 657 SEQ ID NO: 12 (full size Cry1Ka) and any combination of lower and higher high provisions, as if each such combination were specifically set forth in this document.

[00175] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 500 to about position 590, 591, 592, 593, 594, 595, 596, 597, 598 , 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623 , 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648 , 649, 650, 651, 652, 653, 654, 655, 656 or 657 SEQ ID NO: 12.

[00176] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 , 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507 , 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532 , 533, 534, 535, 536, 537 to approximately position 597 of SEQ ID NO: 12.

[00177] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 , 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507 , 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532 , 533, 534, 535, 536, 537 to approximately position 610 of SEQ ID NO: 12.

[00178] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 500 to about position 597 of SEQ ID NO: 12.

[00179] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 500 to about position 610 of SEQ ID NO: 12.

[00180] In illustrative embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, or 467 to approximately position 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616 or 617 of SEQ ID NO: 17 ( full length Cry1Ca) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00181] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 467 to about position 580, 581, 582, 583, 584, 585, 586, 587, 588 , 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613 , 614, 615, 616 or 617 SEQ ID NO: 17.

[00182] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445 , 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460 , 461, 462, 463, 464, 465, 466, or 467 up to approximately position 617 of SEQ ID NO: 17.

[00183] In embodiments, the C-terminal region of the second Cry1 protein contains, essentially consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 467 to about position 617 of SEQ ID NO: 17.

[00184] As is well known in the art, Bt Cry proteins have 5 conserved sequence domains (conservative block [CB] 1-5]) separated by more variable regions (Hofte & Whitely, 1989, Microbiol. Rev. 53:242 -255), and three domains with a conserved structure (domains I, II and III) (de Maagd et al., 2001, Trends Genetics 17:193-199). Figures 1A and 1B show the alignment of the Cry proteins BT-0029, BT-0022, Cry1Fa and Cry1Ka with marked CB3 and domain III. Those skilled in the art can use the well-known sequence and structure information available for Cry proteins to design a chimeric Cry insecticidal protein according to the present invention, for example, to select the appropriate transition region(s) between two Cry proteins. In embodiments, the transition region is located in a conservative block, for example, in CB3. The term "in a conservative block" includes positions at each end of the conservative block. To illustrate this, the term "in CB3" includes the position immediately before (for example, between the methionine and phenylalanine residues within the VPMFSW sequence in the BT-0029 sequence in Figure 2A) and immediately after (for example, between the threonine and asparagine residues within the RRTNVG sequence in sequence BT-0029 in Figure 2A) of conserved block 3. Figures 2A and 2B show exemplary chimeric insecticidal proteins in accordance with the present invention with a transition region in CB3.

[00185] With reference to Figure 2A, in embodiments, the transition is located at a position corresponding to the position immediately before amino acid residue 1 or immediately after amino acid residue 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51, or any combination thereof in CB3. In embodiments, the transition is located within the region (including residues at each end of the region) corresponding to the region from amino acid residue 2 to amino acid residue 7, 8, 9 or 10 CB3, within the region corresponding to the region from amino acid residue 11 to amino acid residue 14, 15, 16, 17 or 18 CB3, within the region corresponding to the region from amino acid residue 19 to amino acid residue 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 CB3, and/or within the region, corresponding to the region from amino acid residue 37, 38 or 39 to amino acid residue 51, and any combination of such regions. In exemplary embodiments, the transition is located at a position within the region corresponding to amino acid residue 19 to amino acid residue 51, amino acid residue 19 to amino acid residue 33, amino acid residue 19 to amino acid residue 28, amino acid residue 19 to amino acid residue 27, or from amino acid residue 19 to amino acid residue 26 in CB3. In embodiments, the transition position is located at a specific site between amino acid residue 18 and amino acid residue 19 in CB3 (eg, specifically between valine and isoleucine in the DPDVITQ sequence in BT-0029, SEQ ID NO: 2).

[00186] Chimeric insecticidal proteins can also be defined in relation to the structural domains derived from each of the original molecules. For example, in embodiments, the N-terminal region of the first Cry protein contains domain I of the first Cry protein and all or substantially all of domain II. In embodiments, the C-terminal region of the other Cry protein contains all or substantially all of domain III of the other Cry protein. Those skilled in the art will appreciate that there is some variability in the exact location of the ends of the structural domains of the Cry protein and the linker regions between the domains, such as the location of the end of the linker region between domains II and III and the start of domain III, although the core of domain III is easily identified by those skilled in the art. region (for example, in accordance with the location of the main domain III, as shown in figure 1A and figure 2A). In embodiments, the junction between the N-terminal region of the first Cry protein and the C-terminal region derived from another Cry protein is located at CB3.

[00187] In illustrative embodiments, the C-terminal region of another Cry protein (e.g., Cry1 protein) is selected based on the alignment of domain III (e.g., core domain III as shown in Figure 1A) from the other Cry protein and the corresponding domain III sequence from the first a Cry protein (eg, a Cry1 protein such as BT-0029), for example, see illustrative alignments in Figures 1A and 1B with Domain III specifically noted. In embodiments, the other Cry protein is selected such that the domain III amino acid sequence of the other Cry protein is substantially identical to the corresponding domain III region of the first Cry protein (eg, BT-0029). In illustrative embodiments, the amino acid sequence of the domain III region of another Cry protein (e.g., Cry1 protein) by at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the corresponding domain III region from the first Cry protein. Methods for aligning and determining amino acid sequence identity within an aligned region are well known to those skilled in the art.

[00188] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, essentially consists of, or consists of (a) an amino acid sequence of any of amino acids 1-597 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-610 of SEQ ID NO: 15, or amino acids 1-617 of SEQ ID NO: 18, or a toxin fragment of any of SEQ ID NO: 3, 9, 13, 15, or 18; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00189] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, essentially consists of, or consists of (a) an amino acid sequence of any of amino acids 1-603 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-615 of SEQ ID NO:15 or SEQ ID NO:18, or their toxin fragment; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00190] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, essentially consists of, or consists of (a) an amino acid sequence of any of amino acids 1-607 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-620 of SEQ ID NO: 15 or SEQ ID NO: 18, or their toxin fragment; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00191] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, essentially consists of, or consists of (a) an amino acid sequence of any of amino acids 1-610 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-620 of SEQ ID NO: 15 or SEQ ID NO: 18, or their toxin fragment; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00192] As will be understood by those skilled in the art, native Cry toxins are expressed as protoxins that are processed to produce a mature toxin, processed by cleavage of the N-terminal peptidyl fragment and the C-terminal tail of the protoxin. The N-terminal peptidyl fragment and/or the C-terminal tail of the protoxin can function to increase the stability and/or insecticidal activity of the Cry toxin. In embodiments of the present invention, the chimeric insecticidal protein contains all or part of the N-terminal peptidyl fragment and/or tail of the protoxin. In embodiments, the chimeric insecticidal protein does not contain a complete N-terminal peptidyl fragment and/or a complete protoxin tail. In embodiments, the chimeric insecticidal protein does not contain an N-terminal peptidyl fragment and/or a protoxin tail, i. corresponds to the mature processed toxin.

[00193] In embodiments, the N-terminal peptidyl fragment is derived from a Cry protein (eg, from a first Cry protein, which is optionally a Cry1 protein). In other embodiments, the N-terminal peptidyl fragment is heterologous to the first Cry protein, eg, is not derived from the Cry protein and/or is partially or wholly synthetic. In embodiments, the peptidyl fragment contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids, optionally from the first Cry protein, including without limitation the first Cry1 protein (e.g., BT-0029, SEQ ID NO: 2) . In embodiments, the peptidyl fragment contains amino acids from about amino acid 1 to about amino acid 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the first Cry protein, e.g., the Cry1 protein, such as BT-0029.

[00194] In embodiments, the chimeric insecticidal protein of the present invention comprises a Cry Bt protoxin tail region at the C-terminus, including modifications of native Cry protoxin tails that are substantially identical to the native Cry protoxin tail. In embodiments, the tail of the protoxin is derived from the Lepidoptera-active Cry protein. In embodiments, the tail of the protoxin is not derived from the Cry protein and/or is partially or fully synthetic. In embodiments, the Cry protein is heterologous to the first Cry protein and/or another Cry protein. In embodiments, the protoxin tail is derived from a Cry1 protein, such as BT-0029, BT-0022, Cry1F (eg Cry1Fa), Cry1I (eg Cry1Ia or Cry1If), Cry1K (eg Cry1Ka) or Cry1C (eg Cry1Ca), or it is a polypeptide that is essentially identical to the protoxin tail (or fragment thereof) of any of the above. The protoxin tail region may contain the entire protoxin tail of the Cry protein, or any portion thereof. In embodiments, the protoxin tail region contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 38, 40, 45, 50 or more contiguous amino acids from a Cry protein protoxin tail (eg, a Cry1 protoxin tail such as BT-0029), for example, as shown for various Cry proteins in Figure 1A. In embodiments, the protoxin tail contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 38, 40, 45, 50, or more contiguous amino acids starting at amino acid position 598 of SEQ ID NO: 2 (BT-0029) or the corresponding region from another Cry protein, such as a Cry1 protein (e.g., BT-0022 protein, Cry1I, Cry1F, Cry1K, or Cry1C). In embodiments, the protoxin tail contains amino acids 598-1169 or 598-652 or 598-636 or 598-622 or 598-610 or 598-607 or 598-603 or 598-600 of SEQ ID NO: 2 (BT-0029) or the corresponding region from another Cry protein (see, for example, the alignment of BT-0029 with BT-0022, Cry1Fa and Cry1Ka in Figures 1A and 1B). In some embodiments, the implementation of the chimeric insecticidal protein contains any of SEQ ID NO: 20-25 or SEQ ID NO: 32-35. In other embodiments, the protoxin tail contains amino acids 637-715 or 637-691 or 637-675 or 637-661 or 637-649 or 637-646 or 637-642 of SEQ ID NO: 1 (BT- 0022). In yet other embodiments, the implementation of the chimeric insecticidal protein of the present invention contains, essentially consists of or consists of any of SEQ ID NO: 26-31.

[00195] Accordingly, in embodiments, the chimeric insecticidal protein of the present invention comprises, essentially consists of, or consists of (a) the amino acid sequence of any of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO : 15, SEQ ID NO: 18, SEQ ID NO: 20-35, or a toxin fragment thereof; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a). In optional embodiments, the chimeric insecticidal protein of the present invention comprises, essentially consists of, or consists of an amino acid sequence of any of the amino acids of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 20-35.

[00196] Those skilled in the art will appreciate that the chimeric insecticidal proteins of the present invention may additionally contain other functional domains and/or peptide tags, such as a peptide tag at the N-terminus and/or C-terminus. For example, it may be useful to express a chimeric insecticidal protein with a peptide tag that can be recognized by a commercially available antibody (eg, a FLAG motif), or with a peptide tag that facilitates purification (eg, by adding a poly-His tag) and/or detection. Alternatively, an epitope can be introduced into the chimeric protein to facilitate the generation of antibodies that specifically recognize the modified chimeric protein to distinguish the modified chimeric protein from the unmodified chimera and/or parent protein(s). For example, one or more amino acids can be changed in the native sequence antigenic loop to create a new epitope. In one embodiment, the antigenic loop is located in a non-conserved region outside of domain I of the native Cry protein. In embodiments, the antigenic loop is not a loop involved in the recognition of a receptor in the insect alimentary canal by the Cry protein and/or is not involved in the activation of the Cry protein by a protease. In other embodiments, the implementation of the chimeric protein can be modified to improve its stability, for example, by fusing a maltose binding protein (MBP) or glutathione-S-transferase with a polypeptide. As another alternative, the fusion protein may contain a reporter group.

[00197] Chimeric insecticidal proteins that are modified by introducing or removing a protease processing site at the appropriate position(s) to allow or eliminate proteolytic cleavage by the protease of insects, plants, and/or microorganisms are also within the scope of the present invention. In embodiments, the modified chimeric insecticidal protein substantially retains insecticidal activity. In embodiments, the stability and/or insecticidal activity of such modified chimeric proteins is improved relative to a chimeric insecticidal protein that does not contain such modification to introduce/remove a protease cleavage site.

[00198] Thus, the present invention encompasses polypeptides having amino acid sequences that are substantially identical to those specifically disclosed herein and toxin fragments thereof. It will be understood that the chimeric insecticidal proteins specifically disclosed herein will typically undergo modifications in amino acid sequence and substantially retain biological activity (eg, insecticidal activity). Such modifications include insertions, deletions (including truncations at either end), and substitutions of one or more amino acids, 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, deletions and/or insertions.

[00199] To identify polypeptides substantially identical to the chimeric insecticidal proteins specifically disclosed herein, amino acid substitutions may be based on any characteristic known in the art, including the relative similarity or difference of amino acid side chain substituents, such as their hydrophobicity. , hydrophilicity, charge, size, etc.

[00200] For example, when identifying amino acid sequences encoding insecticidal polypeptides other than those specifically disclosed herein, the amino acid hydrophobicity index can be considered. The importance of amino acid hydrophobicity indexes in ensuring consistent biological function of a protein is generally known in the art (see Kyte and Doolittle, (1982) J. Mol. Biol. 157:105; incorporated herein by reference in its entirety). The relative hydrophobic characteristic of an amino acid is thought to contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[00201] Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, Id.), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[00202] Accordingly, the hydrophobicity index of an amino acid (or amino acid sequence) can be taken into account when modifying the chimeric polypeptides specifically disclosed herein.

[00203] It is also known in the art that amino acid substitutions can be made based on hydrophilicity. US Pat. No. 4,554,101 states that the greatest average local hydrophilicity of a protein, which is determined by the hydrophilicity of its adjacent acids, correlates with the biological property of the protein.

[00204] As detailed in US Pat. No. 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (.+-.3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+I); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[00205] Thus, the hydrophilicity of an amino acid (or amino acid sequence) can be taken into account when identifying additional insecticidal polypeptides beyond those specifically disclosed herein.

[00206] Chimeric insecticidal proteins of the present invention, including modifications and fragments of chimeric polypeptide toxins specifically disclosed herein, can be obtained by any suitable method known in the art, usually by modification of nucleic acid coding sequences. Methods for manipulating and modifying nucleic acids to achieve the desired modification are well known in the art. In addition, gene editing techniques can also be used to produce the chimeric insecticidal protein of the present invention or to make further modifications to it.

[00207] As another approach, the modifiable polypeptide can be expressed in a host cell that exhibits a high rate of base erroneous insertion during DNA replication, such as XL-1 Red (Stratagene, La Jolla, CA). After propagation in such strains, DNA can be isolated (for example, by obtaining plasmid DNA or by amplifying by PCR and cloning the resulting PCR fragment into a vector), expressing mutant protein sequences in a non-mutating strain, and detecting mutated insecticidal genes. activity, for example, by performing an assay to test for insecticidal activity. In exemplary methods, the protein is mixed and used in feeding assays. See, for example, Marrone et al. (1985) J. of Economic Entomology 78:290-293. Such analyzes may include bringing plants into contact with one or more pests and determining the ability of the plants to survive or kill the pests. Examples of mutations that lead to increased toxicity can be found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.

[00208] In embodiments, the chimeric insecticidal protein (including substantially similar polypeptides and toxin fragments) of the present invention is isolated. In embodiments, the chimeric insecticidal protein (including substantially similar polypeptides and toxin fragments) of the present invention is a recombinant protein.

[00209] The chimeric insecticidal proteins of the present invention have insecticidal activity against a Lepidoptera pest. In embodiments, the chimeric insecticidal protein has activity against one or more of the following non-limiting examples of a Lepidoptera pest: Ostrinia spp. such as O. nubilalis (corn borer) and/or O. furnacalis (eastern corn borer); Plutella spp. such as P. xylostella (cabbage moth); Spodoptera spp. such as S. frugiperda (fall armyworm), S. littoralis (Egyptian cotton cutworm), S. ornithogalli (yellow cutworm), S. praefica (western yellow cut cutworm), S. eridania (southern cut cutworm), and/or S. exigua (small cutworm); Agrotis spp. such as A. ipsilon (upsilon cutworm), A. segetum (common winter cutworm), A. gladiaria (fall cutworm of the genus Agrotis) and/or A. orthogonia (rectangular cutworm); Striacosta spp. such as S. albicosta (Western Fall Armyworm); Helicoverpa spp. such as H. zea (corn bollworm), H. punctigera (Australian cotton bollworm) and/or H. armigera (cotton bollworm); Heliothis spp. such as H. virescens (tobacco leaf roller); Diatraea spp. such as D. grandiosella (southwestern corn moth) and/or D. saccharalis (sugar cane stalk grinder); Trichoplusia spp. such as T. ni (Cabbage cutworm); Sesamia spp. such as S. nonagroides (Mediterranean corn borer) and/or S. calamistis (pink stalk armyworm); Pectinophora spp. such as P. gossypiella (pink bollworm); Cochylis spp. such as C. hospes (striped sunflower moth); Manduca spp. such as M. sexta (tobacco hawk hawk) and/or M. quinquemaculata (5-spot hawk hawk); Elasmopalpus spp. such as E. lignosellus (small corn stalk grinder); Pseudoplusia spp. such as P. includens (soy worm); Anticarsia spp. such as A. gemmatalis (velvet bean cutworm); Plathypena spp. such as P. scabra (clover cutworm); Pieris spp. such as P. brassicae (cabbage whitefish), Papaipema spp. such as P. nebris (Papaipema nebris grinder); Pseudaleiia spp. such as P. unipuncta (short cutworm); Peridroma spp. such as P. saucia (daisy cutworm); Keiferia spp. such as K. lycopersicella (tomato pinworm); Artogeia spp. such as A. rapae (turnip whitefish); Phthorimaea spp. such as P. operculella (potato moth); Chrysodeixis spp. such as C. includes (soy worm); Feltia spp. such as F. ducens (cutworm Feltia jaculifera); Chilo spp. such as C. suppressalis (yellow rice moth), Cnaphalocrocis spp. such as C. medinalis (rice moth), or any combination of the above.

[00210] Optionally, the chimeric insecticidal protein has increased activity against one or more Lepidoptera pests compared to one or more parent molecules (eg, the first Cry protein and another Cry protein). In embodiments, the chimeric insecticidal protein has increased activity against one or more Lepidoptera pests compared to BT-0029. In embodiments, the chimeric insecticidal protein has increased activity against one or more Lepidoptera pests compared to BT-0022, Cry1Fa, Cry1Ia, Cry1If, Cry1Ka, or Cry1C.

[00211] In embodiments, the chimeric insecticidal protein has increased activity against the cutworm (Spodoptera frugiperda) compared to one or more parent molecules (eg, a first Cry protein and another Cry protein). In embodiments, the chimeric insecticidal protein has increased activity against fall armyworm compared to BT-0029. In embodiments, the chimeric insecticidal protein has increased activity against fall armyworm compared to BT-0022, Cry1Fa, Cry1Ia, Cry1If, Cry1Ka, and/or Cry1Ca. In accordance with the above embodiments, the chimeric insecticidal protein may optionally have insecticidal activity against an insect pest or cutworm colony that is resistant to another insecticidal agent, including another insecticidal protein (such as, for example, Bt protein). In embodiments, the chimeric insecticidal protein has increased activity against a bollworm colony that is resistant to a Vip3A protein (e.g., Vip3Aa, including but not limited to the maize transformant MIR162) or a Cry1F protein (e.g., Cry1Fa, including but not limited to the maize transformant TC1507). In embodiments, the chimeric insecticidal protein has increased activity against a resistant bollworm colony over one or more parent molecules, e.g., BT-0029, BT-0022, Cry1Fa, Cry1Ia, Cry1If Cry1Ka and/or Cry1Ca.

[00212] The present invention also encompasses antibodies that specifically bind to the chimeric insecticidal protein of the present invention. The antibody may optionally be a monoclonal antibody or a polyclonal antiserum. In embodiments, the antibody is selective for the chimeric protein and does not bind to one or more parent molecules (eg, BT-0029, BT-0022, Cry1Fa, etc.) and can be used to distinguish between the chimeric protein and the parent protein. Such antibodies can be obtained using standard immunological techniques for obtaining polyclonal antisera and optionally immortalizing the antibody-producing cells of the immunized host to obtain sources of monoclonal antibodies. Techniques for producing antibodies to any substance of interest are well known, such as those described in Harlow and Lane (1988. Antibodies a laboratory manual, pp. 726. Cold Spring Harbor Laboratory) and in Coding (Monoclonal Antibodies: Principles & practice. 1986. Academic Press, Inc., Orlando, FL). The present invention also encompasses an insecticidal protein that cross-reacts with an antibody, in particular a monoclonal antibody raised against one or more chimeric insecticidal proteins of the present invention.

[00213] The antibodies of the present invention are useful, for example, in immunological assays to determine the amount or presence of a chimeric insecticidal protein of the present invention or an antigenically related polypeptide, for example, in a biological sample. Such assays are also useful in preparing compositions containing one or more chimeric insecticidal proteins of the present invention or an antigenically related polypeptide, with quality control. In addition, antibodies can be used to evaluate the efficiency of recombinant production of one or more chimeric insecticidal proteins of the present invention or an antigenically related polypeptide, as well as to screen expression libraries for the presence of a nucleotide sequence encoding one or more chimeric insecticidal proteins of the present invention or an antigenically related polypeptide. polypeptide. Antibodies are further useful as affinity ligands for the purification or isolation of any one or more proteins of the present invention or antigenically related polypeptide.

Nucleic acids, expression cassettes and vectors.

[00214] As a further aspect, the present invention provides nucleic acids encoding polypeptides of the present invention, including modified polypeptides and toxin fragments as described herein.

[00215] According to some embodiments, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that contains, essentially consists of, or consists of the following: (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20-35, or a toxin fragment thereof; (b) a nucleotide sequence encoding an amino acid sequence that is substantially identical to the amino acid sequence of (a); (c) a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleotide sequence from (a) or (b); or (d) a nucleotide sequence that differs from the nucleotide sequences of (a), (b) or (c) due to the degeneracy of the genetic code.

[00216] In embodiments, the nucleic acid molecule comprises a nucleotide sequence that contains, essentially consists of, or consists of the following: (a) the nucleotide sequence under SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 16 or a toxin-encoding fragment thereof; (b) a nucleotide sequence that is essentially identical to the nucleotide sequence of (a); (c) a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleotide sequence from (a) or (b); or (d) a nucleotide sequence that differs from the nucleotide sequences of (a), (b) or (c) due to the degeneracy of the genetic code. Optionally, the nucleotide sequence comprises, essentially consists of, or consists of the nucleotide sequence under SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 16.

[00217] In embodiments, the nucleotide sequence is a partially or wholly synthetic sequence, e.g., which has been codon-optimized for expression in a host organism, e.g., a bacterial host or plant host (e.g., a transgenic monocot host or a transgenic dicot host). host plant). Non-limiting examples of nucleotide sequences that are codon-optimized for expression in the maize plant include SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 11.

[00218] In illustrative embodiments, for expression in transgenic plants, the nucleotide sequences of the present invention are modified and/or optimized. For example, although in many cases the genes of microorganisms can be expressed in plants at high levels and without modification, the low level of expression in transgenic plants may be due to nucleotide sequences of microorganisms having codons that are not preferred by plants. As known in the art, living organisms have certain codon frequency preferences, and therefore the codons of the nucleotide sequences described in this invention can be changed in accordance with plant-specific preferences, while maintaining the amino acids encoded by them. In addition, it is known in the art that high levels of expression in plants, such as corn plants, are best achieved with coding sequences that have at least about 35% GC content, or at least about 45%, or at least about 50% or at least about 60%. Microbial nucleotide sequences that have low GC content may be poorly expressed in plants. Although certain nucleotide sequences may be appropriately expressed in both monocot and dicot species, the sequences can be modified to accommodate codon preferences and GC content preferences for monocots or dicots, as these preferences have been shown to differ (Murray et al Nucl Acids Res 17:477-498 (1989)). In addition, in embodiments, the nucleotide sequence is modified to remove illegitimate splicing sites that may cause transcript truncation. Such modifications of the nucleotide sequences can be performed using well-known methods of site-directed mutagenesis, PCR and construction of synthetic genes using the methods described, for example, in US patent No. 5625136; 5500365 and 6013523.

[00219] In some embodiments, implementation of the present invention provides synthetic coding sequences or polynucleotide obtained according to the procedure disclosed in US patent No. 5625136. This procedure uses maize-preferred codons, i.e. one codon that most often codes for a given amino acid in maize. A maize-preferred codon encoding a particular amino acid can be determined, for example, based on known maize gene sequences. For example, maize codon usage data for 28 genes from maize plants are found in Murray et al., Nucleic Acids Research 17:477-498 (1989). It is known that codons optimized for expression in a plant of one species will also function in plants of other species, but probably not to the same extent as in the plant species for which the codons have been optimized. Similarly, nucleotide sequences can be optimized for expression in any plant. It should be understood that all or any portion of the nucleotide sequence may be optimized or synthetic. That is, the polynucleotide may contain a nucleotide sequence that is partly a native sequence and partly a codon-optimized sequence.

[00220] In illustrative embodiments, the polynucleotide of the present invention is an isolated polynucleotide. In embodiments, the implementation of the polynucleotide of the present invention is a recombinant polynucleotide.

[00221] In embodiments, the present invention further provides a nucleic acid molecule comprising a polynucleotide operably linked to a promoter (eg, a heterologous promoter). Promoters may include, for example, constitutive, inducible, transiently regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters. In specific aspects, a promoter useful in the present invention is a promoter capable of initiating transcription of a nucleotide sequence in a plant cell, such as a monocot (eg maize or rice) or dicot (eg soybean, cotton) plant cell.

[00222] In embodiments, the heterologous promoter is a promoter that allows expression in a plant (eg, expressed in a monocot or expressed in a dicot). For example, without limitation, a promoter for expression in a plant can be selected from: ubiquitin, orange cestrum virus, maize TrpA, OsMADS 6, maize histone H3, bacteriophage T3 gene 9 5'-UTR, maize sucrose synthetase 1, maize alcohol dehydrogenase 1 , maize light-harvesting complex, maize heat shock protein, maize mtl, pea carboxylase RuBP small subunit, rice actin, rice cyclophilin, Ti-plasmid mannopine synthase, Ti-plasmid nopaline synthase, petunia chalcone isomerase, bean glycine-rich protein 1, potato patatin, lectin , 35S CaMV and small subunit S-E9 RuBP carboxylase.

[00223] While many promoters from dicots have been shown to function in monocots and vice versa, in embodiments, dicot promoters are selected for expression in dicots and monocot promoters are selected for expression in monocots. However, there are no restrictions on the origin of the selected promoters; it is sufficient that they are functional in relation to directing the expression of nucleotide sequences in the desired cell.

[00224] The choice of promoter may vary depending on the temporal and spatial requirements for expression, as well as depending on the host cell to be transformed. Thus, for example, the expression of the nucleotide sequences of the present invention can be carried out in any plant and/or plant part, (for example, in leaves, in pedicels or stems, in ears, in inflorescences (for example, ears, panicles, cobs, etc. .), in roots, seeds and/or seedlings, etc.). For example, when expression is desired in a particular tissue or organ, a tissue-specific or tissue-preferred promoter (eg, root-specific/-preferred promoter) can be used. In contrast, if expression in response to a stimulus is desired, a promoter induced by certain stimuli or chemicals can be used. If constant expression at a relatively stable level in all cells of the plant is desired, a constitutive promoter may be selected.

[00225] Promoters useful in accordance with the present invention include, without limitation, promoters that drive the expression of a nucleotide sequence constitutively, promoters that drive expression upon induction, and promoters that drive expression in a tissue-specific manner or in a developmentally specific manner. These various types of promoters are known in the art.

[00226] Suitable constitutive promoters include, for example, the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. W004081173 A2); maize Ubi 1 promoter (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); 19S CaMV (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant J November; 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); maize histone H3 (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); actin 2 (An et al., Plant J. 10(1); 107-121, 1996), the constitutive CT2 root tip promoter (SEQ ID NO: 1535; see also PCT Application No. IL/2005/000627) and synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those described in US Pat. Nos. 5,659,026, 5,608,149; 5608144; 5604121; 5569597: 5466785; 5399680; 5268463 and 5608142.

[00227] Tissue-specific or tissue-preferred promoters useful for expression of the polypeptides of the present invention in plants, optionally in maize, include those that drive expression in the root, heartwood, leaf, or pollen. Suitable tissue-specific promoters include, without limitation, leaf-specific promoters [such as are described, for example, in Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. sci. USA 90:9586-9590, 1993], seed-preferred promoters (e.g., from seed-specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil nut albumin promoters (Pearson et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), glutelin (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), zein (Matzke et al., Plant Mol Biol, 143). 323-32 1990), napA (Stalberg, et al., Planta 199: 515-519, 1996), Wheat SPA ( Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-876, 1992)], endosperm-specific promoters (e.g., LMW and HMW wheat, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat gliadins a, b and g (EMBO3: 1409-15, 1984), barley ltrl promoter, hordein B1, C, D barley (Theor App l Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), barley DOF (Mena et al., The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP 99106056.7), synthetic promoter (Vicente-Carbajosa et al ., Plant J. 13: 629-640, 1998), rice prolamin NRP33 promoter, rice globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), REB/OHP alpha-globulin -1 rice (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR family gene promoter (Plant J 12:235 -46, 1997), sorghum gamma-kafirin (Plant Mol. Biol 32:1029-35, 1996)], germ-specific promoters (e.g. rice OSH1; Sato et al., Proc. Nati. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998), specific for flower promoters, e.g., AtPRP4, chalcone synthase (chsA) (Van der Meer, et al. Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240 -245; 1989), apetala-3 and prom tors specific to plant reproductive tissues (eg, OsMADS promoters; US Patent Publication No. 2007/0006344).

[00228] Examples of promoters suitable for preferential expression in green tissue include a variety of promoters that regulate genes involved in photosynthesis, and many of these have been cloned from both monocots and dicots. One such promoter is the PEPC promoter of the maize phosphoenolcarboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Another promoter for root-specific expression is that described by de Framond (FEBS 290:103-106 (1991) or US Pat. No. 5,466,785). Another promoter useful in the present invention is the stalk-specific promoter described in US Pat. No. 5,625,136, which naturally drives the expression of the maize trpA gene.

[00229] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5'UTR promoter and other promoters disclosed in US Pat. No. 7,579,516. Other promoters useful in accordance with the present invention include, but are not limited to, the RuBP carboxylase small subunit S-E9 promoter and the Kunitz trypsin inhibitor (Kti3) gene promoter.

[00230] In some embodiments of the present invention, inducible promoters may be used. Thus, for example, chemically regulated promoters can be used to modulate gene expression in a plant through the use of an exogenous chemical regulator. Regulation of the expression of the nucleotide sequences of the present invention by chemically regulated promoters allows the synthesis of the polypeptides of the present invention only when the crop plants are treated with inducing chemicals. Depending on the purpose, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressive promoter, where application of the chemical suppresses gene expression. Examples of such technology for chemical induction of gene expression are described in detail in EP 0332104 and US Pat. No. 5,614,395.

[00231] Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds used as preemergence herbicides, and the o promoter. Tobacco PR-1a that is activated by salicylic acid (e.g. PR1a systems), steroid dependent promoters (see e.g. glucocorticoid inducible promoter in Schenaetal. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) and tetracycline-inducible and tetracycline-repressed promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and US Pat. (1997) Plant J. 11:605-612), and promoters of the ecdysone inducible system.

[00232] Other non-limiting examples of inducible promoters include ABA and turgor pressure inducible promoters, auxin binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), UDP-glucosoflavonoid glycosyltransferase promoter (Ralston et al (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150) and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant Mol Biol 29:1293-1298 Martinez et al (1989) J Mol Biol 208:551-565 and Quigley et al (1989) J Mol Evol 29:412-421). Also included are benzenesulfonamide-inducible (US Pat. No. 5,364,780) and alcohol-inducible (International Patent Application Publication Nos. WO 97/06269 and WO 97/06268) systems and glutathione-S-transferase gene promoters. Similarly, any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol can be used. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically inducible promoters useful in directing the expression of the nucleotide sequences of the present invention in plants are disclosed in US Pat.

[00233] Another category of promoters useful in the present invention are wound inducible promoters. Numerous promoters have been described that are expressed at sites of injury as well as sites of infection by a phytopathogen. Ideally, such a promoter should be activated locally at the sites of insect invasion, and thus the insecticidal proteins accumulate exclusively in cells where insecticidal protein synthesis is required to kill the attacking insect pest. Examples of promoters of this type include those described by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993) and Warner et al. Plant J. 3:191-201 (1993).

[00234] In embodiments, the nucleic acid of the present invention may comprise, consist essentially of, or consist of an expression cassette, or may be contained in an expression cassette.

[00235] The expression cassette containing the polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette can also be a naturally occurring sequence that has been produced in recombinant form suitable for heterologous expression. However, as a rule, the expression cassette is heterologous with respect to the host, ie. the particular nucleic acid sequence in the expression cassette is not naturally occurring in the host cell and had to be introduced into the host cell or an ancestor of the host cell by a transformation event.

[00236] In addition to the promoters operably linked to the nucleotide sequences of the present invention, the expression cassette of the present invention may also contain other regulatory elements. Regulatory elements include, but are not limited to, enhancers, introns, translational leader sequences, termination signals, and polyadenylation signal sequences. Examples of suitable transcription termination signals are available and known in the art (eg tml from CaMV, E9 from rbcS). In the context of the present invention, any available terminator known to function in plants can be used.

[00237] Many other sequences can be introduced into the expression cassettes described in the present invention. They include sequences that are shown to enhance expression, such as intron sequences (eg from Adhl and bronzel) and viral leader sequences (eg from TMV, MCMV and AMV).

[00238] For more efficient translation initiation, sequences adjacent to the initiating methionine can be modified. For example, they can be modified to include sequences known to be effective in plants. Joshi has proposed a suitable plant consensus sequence (NAR 15:6643-6653 (1987)), and Clonetech has proposed an additional translation initiator consensus sequence (catalog 1993/1994, page 210). These consensus sequences are suitable for use with the nucleotide sequences of the present invention. The sequences are inserted into constructs containing nucleotide sequences upstream and including the ATG (with the second amino acid remaining unmodified) or alternatively upstream and including the GTC located after the ATG (with the possibility of modifying the second amino acid of the transgene).

[00239] In embodiments, it may be preferable to target expression of the polypeptides of the present invention at a specific cellular location in a plant cell. In some cases, localization to the cytosol may be desirable, while in other cases, localization to a particular subcellular organelle may be preferred. Any mechanism for targeting gene products, for example in plants, can be used to practice the present invention, and such mechanisms are known to exist in plants, and the sequences regulating the functioning of such mechanisms have been described in some detail. Sequences have been characterized that allow targeting of gene products to other compartments of the cell. For example, amino-terminal sequences can target the protein of interest to a cell compartment such as the vacuole, mitochondria, peroxisome, protein bodies, endoplasmic reticulum, chloroplast, starch granule, amyloplast, apoplast, or plant cell wall (e.g., Unger et. al. Plant). Molec Biol 13: 411-418 (1989) Rogers et al (1985) Proc Natl Acad Sci USA 82: 6512-651 US Pat. be an N-terminal signal sequence from waxy, an N-terminal signal sequence from gamma-zein, a starch-binding domain, a C-terminal starch-binding domain, a chloroplast-targeting sequence that ensures delivery of the mature protein to the chloroplast (Comai et. al (1988) J Biol Chem 263: 15104-15109; van den Broeck, et al (1985) Nature 313: 358-363; US Pat. aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). In addition, amino-terminal sequences, together with carboxy-terminal sequences, cause gene products to be targeted to the vacuole and can be used in the present invention (Shinshi et. al. (1990) Plant Molec. Biol. 14: 357-368). In one embodiment, the selected signal sequence includes a known cleavage site, and any amino acids after the cleavage site(s) required for cleavage are considered in the fusion design. In some cases, this requirement can be met by adding a small number of amino acids between the cleavage site and the ATG of the transgene, or alternatively by substituting some amino acids within the transgene sequence. These design techniques are well known in the art and are equally applicable to any cellular compartment.

[00240] It will be understood that the targeting mechanisms described above in a cell can be used not only in combination with promoters cognate to them, but also in combination with heterologous promoters, so that the specific task of targeting in the cell is carried out under the transcriptional control of a promoter characterized by an expression pattern that is different from that of the promoter from which the targeting signal is derived.

[00241] The expression cassette of the present invention may also include a nucleotide sequence for a selectable marker that can be used to select a transformed plant, plant part, or plant cell. Many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[00242] Examples of selectable markers include, without limitation, the nucleotide sequence encoding neo or nptII, which confer resistance to kanamycin, G418, and the like. (Potrykus et al. (1985) Mol. Gen. Genet. 199: 183-188); a nucleotide sequence encoding bar which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) that confers glyphosate resistance (Hinchee et al. (1988) Biotech. 6:915-922); a nucleotide sequence encoding a nitrilase, such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea, or other ALS inhibitory chemicals (European Patent Application No. 154204); a nucleotide sequence encoding methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding dalapon dehalogenase which confers resistance to dalapon; a nucleotide sequence encoding mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)), which confers the ability to metabolize mannose (US Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyltryptophan; or a nucleotide sequence encoding hph which confers resistance to hygromycin. One skilled in the art will be able to select an appropriate selectable marker for use in the expression cassette of the present invention.

[00243] Additional selectable markers include, without limitation, a nucleotide sequence encoding β-glucuronidase or uidA (GUS), which encodes an enzyme for which various chromogenic substrates are known; the nucleotide sequence of the R-locus, which encodes a product that regulates the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al, "Molecular cloning of the maize R-nj allele by transposon-tagging with Ac," pp. 263-282, in Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, an enzyme for which various chromogenic substrates are known (eg, PADAC, chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE, which encodes catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et at (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme for which chromogenic substrates exist; a nucleotide sequence encoding a luciferase (lux) that allows detection by bioluminescence (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which can be used in the detection of calcium-sensitive bioluminescence (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding a green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One skilled in the art can select an appropriate selectable marker for use in the expression cassette of the present invention.

[00244] In some embodiments, the expression cassette of the present invention may also contain polynucleotides that encode other desired features in addition to the chimeric insecticidal proteins of the present invention. Examples of such other polynucleotides include those that encode a polypeptide or dsRNA for other desired feature(s) of interest(s). Such expression cassettes containing "packaged" traits can be used, for example, to produce plants, plant parts or plant cells having the desired trait bundled phenotype (ie, molecular bundling). Such bagged combinations in plants can also be created using other methods, including, without limitation, crossbreeding plants using any conventional technique (ie, breeding bag). When "packaging" by genetic transformation of plants, the nucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant containing one or more of the desired traits can be used as a target for the introduction of additional traits by subsequent transformation. Additional nucleotide sequences can be administered according to the co-transformation protocol simultaneously with the nucleotide sequence, nucleic acid molecule, nucleic acid construct or composition of the present invention provided by any combination of expression cassettes. For example, if two nucleotide sequences are to be introduced, they can be inserted into separate cassettes (trans) or they can be inserted into one cassette (cis). Expression of polynucleotides may be driven by the same promoter or by different promoters. In addition, it is known that polynucleotides can be "packaged" at the desired location in the genome using a site-specific recombination system. See, for example, International Patent Application Publications Nos. WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855 and WO 99/25853.

[00245] In exemplary embodiments, the expression cassette may also contain an additional sequence encoding one or more double-stranded RNA (dsRNA) polypeptides or molecules that provide an agronomic trait of interest (e.g., an agronomic trait that primarily benefits a seed company, grower, or grain processor). The polypeptide of interest may be any polypeptide encoded by the nucleotide sequence of interest. Non-limiting examples of polypeptides of interest that are suitable for production in plants include those that confer agronomically important traits such as herbicide tolerance (also sometimes referred to as "herbicide tolerance"), virus resistance, resistance to pathogenic bacteria, resistance to insect resistance, nematode resistance or fungal resistance. See, for example, US Pat. Nos. 5,569,823; 5304730; 5495071; 6,329,504 and 6,337,431. In embodiments, the polypeptide of interest can also be one that increases plant vigor or yield (including traits that enable a plant to grow at different temperatures, soil conditions, and levels of sunlight and precipitation), or one that which makes it possible to identify a plant exhibiting a trait of interest (eg, selectable marker, seed coat color, etc.). A variety of polypeptides of interest, as well as methods for introducing these polypeptides into a plant, are described, for example, in US patent No. 4761373; 4769061; 4810648; 4940835; 4975374; 5013659; 5162602; 5276268; 5304730; 5495071; 5554798; 5561236; 5569823; 5767366; 5879903, 5928937; 6084155; 6329504 and 6337431; and also in US Patent Application Publication No. 2001/0016956. See also on the World Wide Web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

[00246] In some embodiments, implementation of the present invention can also be used polynucleotides that confer resistance/tolerance to a herbicide that inhibits growth cone or meristem, such as imidazolinone or a sulfonylurea. Exemplary polynucleotides from this category encode mutant ALS and AHAS enzymes as described, for example, in US Pat. US Pat. No. 4,975,374 relates to plant cells and plants containing a nucleic acid encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides known to inhibit GS, such as phosphinothricin and methionine sulfoximine. US Pat. No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. Resistance is imparted by an altered acetyl-coenzyme A-carboxylase (ACCase).

[00247] Polypeptides encoded by nucleotide sequences conferring glyphosate resistance are also suitable for the present invention. See, for example, US Pat. No. 4,940,835 and US Pat. No. 4,769,061. US Pat. No. 5,554,798 discloses glyphosate-tolerant transgenic maize plants that are conferred by an altered 5-enolpyruvil-3-phosphoshikimate synthase (EPSP) gene.

[00248] Also suitable are polynucleotides encoding resistance to phosphonic compounds such as ammonium glufosinate or phosphinothricin, as well as pyridineoxy- or phenoxypropionic acids and cyclohexanones. See European Patent Application No. 0242246. See also U.S. Patent Nos. 5,879,903, 5,276,268, and 5,561,236.

[00249] Other suitable polynucleotides include those encoding resistance to herbicides that inhibit photosynthesis, such as triazine and benzonitrile (nitrilase gene). See US Pat. No. 4,810,648. Additional suitable polynucleotides encoding herbicide resistance include those encoding resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides, and bromoxynil. Also suitable are polynucleotides that confer resistance to inhibitors of the protox enzyme or that confer increased resistance to plant diseases; increased tolerance to adverse environmental conditions (types of abiotic stress), including, without limitation, drought, excessive cooling, excessive heating, or excessive soil salinity, or extreme acidity or alkalinity; and changes in the structure or development of plants, including changes in the timing of development. See, for example, US Patent Application Publication No. 2001/0016956 and US Patent No. 6,084,155.

[00250] Additional suitable polynucleotides include those encoding pesticidal (eg, insecticidal) polypeptides. These polypeptides can be obtained in amounts sufficient to control, for example, insect pests (ie, in amounts that provide control of insects). In embodiments, the polypeptide is a Lepidoptera-active, Coleoptera-active, Hemiptera-active, and/or Diptera-active polypeptide, or any combination thereof. It is believed that the amount of pesticidal polypeptide produced in a plant to control insects or other pests may vary depending on the cultivar, pest type, environmental factors, and the like. Polynucleotides useful for conferring additional resistance to insects or pests include, for example, those encoding toxins identified in Bacillus organisms. Polynucleotides containing nucleotide sequences encoding Bacillus thuringiensis (Bt) Cry proteins from several subspecies have been cloned and the recombinant clones have been found to be toxic to Lepidoptera, Diptera and Coleoptera larvae. Examples of such insecticidal Bt proteins include Cry proteins such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, Cry9C and the like, as well as vegetative insecticidal proteins such as Vip1, Vip2, Vip3, etc., and any combination of the above Bt insecticidal proteins. A complete list of Bt proteins can be found on the Internet in the Bacillus thuringiensis toxin nomenclature database maintained by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

[00251] In embodiments, the additional polypeptide is an insecticidal polypeptide derived from a source other than Bt, including, but not limited to, alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus spp insecticidal protein. (such as X. nematophila or X. bovienii), Photorhabdus spp. insecticidal protein. (such as P. luminescens or P. asymobiotica), the insecticidal protein of Brevibacillus spp. (such as B. laterosporous), the insecticidal protein of Lysinibacillus spp. (such as L. sphearicus), insecticidal protein of Chromobacterium spp. (such as C. subtsugae or C. piscinae), the insecticidal protein of Yersinia spp. (such as Y. entomophaga), the insecticidal protein of Paenibacillus spp. (such as P. propylaea), insecticidal protein of Clostridium spp. (such as C. bifermentans), Pseudomonas spp. (such as P. fluorescens) and lignin.

[00252] Polypeptides suitable for production in plants further include those polypeptides that improve the conversion of harvested plants or plant parts into a commercially applicable product or otherwise facilitate it, including, for example, providing increased or altered content or distribution of carbohydrates, improved fermentability properties, increased oil content, increased protein content, improved digestibility, or increased nutraceutical content, such as increased phytosterol content, increased tocopherol content, increased stanol content, or increased vitamin content. Polypeptides of interest also include, for example, those that cause or contribute to the reduction of the content of an undesirable component in the harvested crop, for example, phytic acid or sugar degrading enzymes. By "causing" or "facilitating" is meant that the polypeptide of interest can directly or indirectly contribute to the presence of the trait of interest (eg, by increasing cellulose digestion with the heterologous cellulase enzyme).

[00253] In some embodiments, the implementation of the polypeptide improves the digestibility of food or feed. Xylanases are hemicellulose-cleaving enzymes that enhance the breakdown of plant cell walls, resulting in better utilization of plant nutrients by animals. This leads to increased growth rates and feed conversion. You can also reduce the viscosity of feeds containing xylan. The heterologous production of xylanases in plant cells can also assist in the conversion of lignocellulose to fermentable sugars during industrial processing.

[00254] Numerous xylanases from microorganisms that are fungi and bacteria have been identified and characterized (see, for example, US patent No. 5437992; Coughlin et al. (1993) "Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases" Espoo, Souminen and Reinikainen, eds (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135, US Patent Publication No. 2005/0208178, and PCT Publication WO 03/16654). In particular, three specific xylanases (XYL-I, XYL-II and XYL-III) have been identified in T. reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio /Technology 10:1461 and Xu et al (1998) Appl Microbiol Biotechnol 49:718).

[00255] In other embodiments, a polypeptide useful in the present invention may be a polysaccharide degrading enzyme. Plants of the present invention that produce such an enzyme can be used to obtain, for example, fermentable feedstock for biological processing. In some embodiments, enzymes useful for the fermentation process include alpha-amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycosyltransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolysing enzyme, and others. glucoamylase.

[00256] Polysaccharide degrading enzymes include starch degrading enzymes such as alpha-amylases (EC 3.2.1.1), glucuronidases (EC 3.2.1.131); exo-1,4-alpha-D-glucanases such as amyloglucosidases and glucoamylase (EC 3.2.1.3), beta-amylases (EC 3.2.1.2), alpha-glucosidases (EC 3.2.1.20) and other exo-amylases; starch degrading enzymes such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41) and the like; b) cellulases such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-beta-D-glucanase (EC 3.2.1.39), beta-glucosidase (EC 3.2.1.21); c) L-arabinases such as endo-1,5-alpha-L-arabinase (EC 3.2.1.99), alpha-arabinosidases (EC 3.2.1.55) and the like; d) galactanases such as endo-1,4-beta-D-galactanase (EC 3.2.1.89), endo-1,3-beta-D-galactanase (EC 3.2.1.90), alpha-galactosidase (EC 3.2.1.22) , beta-galactosidase (EC 3.2.1.23), etc.; e) mannanases such as endo-1,4-beta-D-mannanase (EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), alpha-mannosidase (EC 3.2.1.24) and the like; f) xylanases such as endo-1,4-beta-xylanase (EC 3.2.1.8), beta-D-xylosidase (EC 3.2.1.37), 1,3-beta-D-xylanase, and the like; and g) other enzymes such as alpha-L-fucosidase (EC 3.2.1.51), alpha-L-rhamnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulanase (EC 3.2.1.7), etc. P. In one embodiment, the alpha-amylase is a synthetic alpha-amylase, Amy797E, as described in US Pat. No. 8,093,453.

[00257] Additional enzymes that can be used in accordance with the present invention include proteases, such as fungal and bacterial proteases. Fungal proteases include, without limitation, those derived from Aspergillus, Trichoderma, Mucor, and Rhizopus, such as A. niger, A. awamori, A. oryzae, and M. miehei. In some embodiments, the polypeptides of the present invention may be cellobiohydrolase (CBH) enzymes (EC 3.2.1.91). In one embodiment, the cellobiohydrolase enzyme may be CBH1 or CBH2.

[00258] Other enzymes useful in accordance with the present invention include, without limitation, hemicellulases such as mannases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (eg E.C. 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha-1,6-glucosidases (eg E.C. 3.2.1.20); esterases such as ferulic acid esterase (EC 3.1.1.73) and acetylxylanesterase (EC 3.1.1.72); and cutinase (e.g. E.C. 3.1.1.74).

[00259] Double-stranded RNA (dsRNA) molecules useful in the present invention include, without limitation, those that repress gene expression in a target pest (eg, insect). In embodiments, the dsRNA is targeted to a gene of a Lepidoptera, Coleoptera, Hemiptera, or Diptera insect pest, or any combination of the above. As used herein, the phrase "gene suppression" is intended to refer collectively to any well known means of reducing the levels of a protein produced by transcription of a gene into mRNA and further translation of the mRNA. It is also understood that gene suppression means a decrease in the expression of a protein from a gene or coding sequence, including post-transcriptional gene suppression and transcriptional suppression. Post-transcriptional gene suppression is due to the homology of all or part of the mRNA transcribed from the gene or coding sequence that is the target of suppression and the corresponding double-stranded RNA used for suppression, and means a significant and measurable decrease in the amount of available mRNA available in the cell for binding by ribosomes. The transcribed RNA can be in sense orientation to provide an effect called cosuppression, in antisense orientation to provide an effect called antisense suppression, or both orientations to produce dsRNA to provide an effect called RNA interference (RNAi). Transcriptional suppression is due to the presence in the cell of dsRNA, a gene suppressor agent characterized by significant sequence identity with the DNA promoter sequence or its complementary strand, to provide an effect called trans promoter suppression. Gene suppression can be effective against a native plant gene associated with a trait, for example to provide plants with reduced levels of the protein encoded by the native gene or increased levels of an altered metabolite. Gene suppression may also be effective against target genes in plant pests that may be ingested or come into contact with plant material containing gene suppression agents specifically designed to suppress or suppress one or more homologous or complementary sequences in pest cells. Such suppression target genes may encode an essential protein whose primary function is selected from the group consisting of muscle formation, juvenile hormone production, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, cell membrane potential maintenance, amino acid biosynthesis, amino acid degradation formation of sperm, synthesis of pheromones, perception of pheromones, formation of antennae, formation of wings, formation of limbs, development and differentiation, formation of eggs, maturation of larvae, formation of digestive enzymes, synthesis of hemolymph, maintenance of a constant composition of hemolymph, transmission of nerve impulses, cell division, energy metabolism , development and differentiation, respiration and apoptosis.

[00260] In embodiments, nucleic acids of the present invention may further comprise, consist essentially of, or consist of a vector. In embodiments, the expression polynucleotides and cassettes of the present invention are contained in a vector. Vectors for use in the transformation of plants and other organisms are well known in the art. Non-limiting examples of general classes of vectors include plasmid, phage vector, phagemid vector, cosmid vector, fosmid, bacteriophage, artificial chromosome, or viral vector. In embodiments, the vector is a plant vector, for example, for use in plant transformation. In embodiments, the vector is a bacterial vector, for example, for use in the transformation of bacteria. Suitable vectors for plants, bacteria and other organisms are known in the art.

Transgenic plants, plant parts, plant cells, seeds.

[00261] The present invention also encompasses a transgenic host cell, other than a human cell, which contains a polynucleotide, nucleic acid molecule, expression cassette, vector, or polypeptide of the present invention. A transgenic host cell other than a human cell may include, without limitation, a plant cell (including a monocot cell and/or a dicot cell), a yeast cell, a bacterial cell, or an insect cell. Therefore, in some embodiments, the present invention provides a bacterial cell selected from the genus Bacillus, Brevibacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteuria, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius , Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc or Alcaligenes. Thus, for example, as insect biological control agents, the chimeric insecticidal proteins of the present invention can be obtained by expressing the polynucleotide a encoding them in a bacterial cell. For example, in some embodiments, a Bacillus thuringiensis cell is provided that contains a polynucleotide encoding a chimeric insecticidal protein of the present invention.

[00262] In embodiments, the transgenic plant cell is a monocot plant cell or a dicot plant cell. In additional embodiments, the dicot cell is a soybean cell, a sunflower cell, a tomato cell, a cabbage cultivar cell, a cotton cell, a sugar beet cell, and a tobacco cell. In additional embodiments, the monocot cell is a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugar cane cell, and a wheat cell. In embodiments of the present invention, a population of dicotyledonous plant cells or monocotyledonous plant cells comprising a polynucleotide expressing a chimeric insecticidal protein of the present invention is provided. In embodiments, the cells are collectively located near the apoplast formation and grown under natural sunlight. In embodiments, the transgenic plant cell cannot be regenerated into a whole plant.

[00263] In embodiments of the present invention, the insecticidal protein of the present invention is expressed in a higher organism, such as a plant. In this case, transgenic plants expressing effective amounts of the insecticidal protein protect themselves from plant pests such as insect pests. When an insect begins to feed on such a transgenic plant, it also takes up the expressed insecticidal protein. This may keep the insect from further burrowing into the plant tissue, or may even harm or kill the insect. In embodiments, a polynucleotide of the present invention is inserted into an expression cassette, which is then stably integrated into the plant genome. In other embodiments, the implementation of the polynucleotide is included in a non-pathogenic self-replicating virus.

[00264] In some embodiments of the present invention, the transgenic plant cell comprising the nucleic acid molecule or polypeptide of the present invention is a cell of a plant part, plant organ, or plant culture (each as described herein), including, without limitation, root, leaf , seed, flower, fruit, pollen cell, plant organ or culture, and the like, or callus cell or culture.

[00265] The transgenic plant or plant cell of the present invention may be a monocot or dicot plant or plant cell, and includes, but is not limited to, corn (maize), soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanuts, vegetables (including sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, carrots, eggplant, cucumber , radish, spinach, potato, tomato, asparagus, onion, garlic, melon, pepper, celery, large-fruited pumpkin, nutmeg pumpkin, common pumpkin, etc.), fruit crops (including apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, etc.) and a specialized plant or plant cell (such as Arabidopsis), or a woody plant or plant cell (such as coniferous and/or deciduous trees). In embodiments, the plant or plant cell of the present invention is a crop plant or plant cell, such as a plant or plant cell of maize, sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley, oilseed rape, etc.

[00266] The present invention further provides a transgenic plant portion of the present invention. Optionally, the plant part contains a chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it.

[00267] The present invention further provides the seed of the transgenic plant of the present invention or the seed from which the transgenic plant of the present invention is obtained. Optionally, the seed contains a chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it.

[00268] Additional embodiments of the present invention include harvested products derived from the transgenic plants, plant parts, or seeds of the present invention, as well as a processed product derived from the harvested product. The harvested product may be the whole plant or any part of the plant as disclosed herein. Thus, in some embodiments, non-limiting examples of a harvested product include a seed, fruit, flower, or part thereof (eg, anther, stigma, and the like), leaf, stem, and the like. In other embodiments, the processed product includes, without limitation, flour, meal, oil, starch, grain, and the like obtained from the harvested seed or other plant part of the present invention. Optionally, the harvested product or processed product contains a chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it.

[00269] In other embodiments, the present invention provides an extract from a transgenic plant or plant part of the present invention, wherein the extract optionally comprises a chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it. Extracts from plants or plant parts can be obtained according to procedures well known in the art (see de la Torre et al., Food, Agric. Environ. 2(1):84-89 (2004); Guidet, Nucleic Acids Res. 22(9): 1772-1773 (1994); Lipton et al., Food Agric Immun 12:153-164 (2000)).

[00270] The chimeric insecticidal protein can function in a plant part, plant cell, plant organ, seed, harvested product, processed product or extract, and the like as an insect control agent. In other words, the chimeric insecticidal protein can continue to perform the insecticidal function it had in the transgenic plant. The nucleic acid can function to express the chimeric insecticidal protein. As an alternative to encoding an insecticidal protein of the present invention, a nucleic acid may function to detect a transgenic plant part, plant cell, plant organ, seed, harvested product, processed product, or extract of the present invention.

[00271] In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the present invention is hemizygous for a polynucleotide or expression cassette of the present invention. In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the present invention is homozygous for a polynucleotide or expression cassette of the present invention.

[00272] In embodiments, the transgenic plant, plant part, plant cell, plant organ, seed, harvested product, processed product, or extract has increased resistance to one or more insect pests (e.g., a lepidoptera pest such as grass cutworm). ) compared to a suitable control that does not contain a nucleic acid encoding an insecticidal protein of the present invention.

Plant transformation.

[00273] Plant transformation procedures are well known and accepted in the art and are described in the literature in every respect. Non-limiting examples of plant transformation methods include transformation by bacteria-mediated nucleic acid delivery (e.g., by Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid microneedle-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated uptake of nucleic acids, or any other electrical, chemical, physical (mechanical), or biological mechanism , which results in the introduction of a nucleic acid into a plant cell, including any combination thereof. General guidelines for a variety of plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy- Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[00274] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA boundary sequence are generally suitable, while for direct gene transfer (eg, particle bombardment, etc.) any vector is suitable, it is possible to use linear DNA containing only the construct of interest. In the case of direct gene transfer, single DNA transformation or co-transformation can be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). In the case of both direct gene transfer and Agrobacterium-mediated gene transfer, transformation is usually (but not necessarily) performed with a selectable marker, which may be a positive selection agent (e.g., phosphomannose isomerase) that confers antibiotic resistance (e.g., kanamycin, hygromycin or methotrexate) or a herbicide (eg glyphosate or glufosinate). However, the choice of selectable marker is not critical to the present invention.

& Willmitzer (1988) Nucleic Acids Res. 16:9877).[00275] Agrobacterium mediated transformation is a method widely used for plant transformation due to its high transformation efficiency and due to its broad applicability to many different species. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying the foreign DNA of interest into the appropriate Agrobacterium strain, which may depend on the set of vir genes of the Agrobacterium host strain located either on a co-resident Ti plasmid or on the chromosome (Uknes et al (1993) Plant Cell 5:159-169). Transfer of a recombinant binary vector into Agrobacterium can be accomplished using a three-parent crossing procedure using Escherichia coli carrying the recombinant binary vector to an E. coli helper strain carrying a plasmid that is capable of mobilizing the recombinant binary vector in the target Agrobacterium strain. Alternatively, a recombinant binary vector can be transferred into Agrobacterium by nucleic acid transformation (

& Willmitzer (1988) Nucleic Acids Res. 16:9877).

[00276] Dicots as well as monocots can be transformed using Agrobacterium. Methods for Agrobacterium mediated transformation of rice include well known methods for transformation of rice such as those described in any of the following: European Patent Application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), the disclosures of which are incorporated herein by reference as if they were set forth in their entirety. In the case of maize transformation, the preferred method is that described or in Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996), or Frame et al. (Plant Physiol 129(1): 13-22, 2002), the disclosures of which are incorporated herein by reference as if they were set forth in their entirety. These methods are also described by way of example in c. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or construct to be expressed are preferably cloned into a vector which is suitable for the transformation of Agrobactehum tumefaciens, eg pBin19 (Bevan et al, Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, such as model plants, such as Arabidopsis, or crops, such as, for example, tobacco plants, for example, by dipping crushed leaves or chopped leaves in an Agrobacteria solution and then culturing them in a suitable medium. Plant transformation by Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or known in particular from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[00277] Transformation of a plant with recombinant Agrobacterium typically involves co-culturing Agrobacterium with plant explants and is carried out according to methods well known in the art. The transformed tissue is regenerated on a selective medium containing an antibiotic or herbicide resistance marker between the T-DNA boundary sequences of the binary plasmid.

[00278] As previously discussed, another method for transforming plants, plant parts, and plant cells involves incorporating inert or biologically active particles into plant tissues and cells. See, for example, US Pat. When using inert particles, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, the cell or cells may be surrounded by a vector such that the vector is carried into the cell following the particle. Biologically active particles (eg, dried yeast cell, dried bacterium or bacteriophage, each of which contains one or more nucleic acids to be introduced) can also be introduced into plant tissue.

[00279] In other embodiments, a polynucleotide of the present invention can be directly introduced into the plastid genome by transformation. The main advantage of plastid transformation is that plastids are usually able to express bacterial genes without significant modification, while plastids are able to express multiple open reading frames under the control of a single promoter. The plastid transformation technology is described in detail in US Pat. (1994) Proc. Natl Acad. sci. USA 91, 7301-7305. The basic technique for transforming chloroplasts involves introducing the cloned plastid DNA flanking the selectable marker, together with the gene of interest, into a suitable target tissue, eg using biolistics or protoplast transformation (eg, calcium chloride or PEG mediated transformation). Flanking regions of 1-1.5 kb, called targeting sequences, promote homologous recombination with the plastid genome and thus provide replacement or modification of specific regions of the plastome. First, point mutations in the 16S rRNA and rps12 genes of chloroplasts conferring resistance to spectinomycin or streptomycin can be used as a selectable transformation marker (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers allows the creation of a plastid-targeting vector for the introduction of foreign genes (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606). A significant increase in transformation frequency was achieved by replacing the recessive rRNA or r-protein genes conferring antibiotic resistance with a dominant selectable marker, the bacterial gene aadA, which encodes the aminoglycoside-3'-adenyltransferase enzyme that detoxifies spectinomycin (Svab, Z., and Maliga, P (1993) Proc Natl Acad Sci USA 90, 913-917). This marker has previously been successfully used to transform the plastid genome of the green alga Chlamydomonas reinhardtii at high frequency (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the present invention. As a rule, it takes about 15-20 cycles of cell division after transformation to achieve a state in which all plastids are the same. Plastid expression, in which genes are inserted by homologous recombination into all of the few thousand copies of the circular plastid genome present in each plant cell, takes advantage of the huge copy number over nuclear-expressed genes, allowing expression levels that can easily be exceed 10% of total soluble vegetable proteins. In one embodiment, a polynucleotide of the present invention can be inserted into a plastid-targeting vector and introduced into the plastid genome of the desired host plant by transformation. Thus, it is possible to obtain plants that are homoplastic to plastid genomes containing the nucleotide sequence of the present invention, capable of expressing a polynucleotide at a high level.

[00280] Methods for selecting transformed transgenic plants, plant cells, or plant tissue cultures are conventional in the art and can be used in the methods of the present invention provided herein. For example, the recombinant vector of the present invention may also include an expression cassette containing a selectable marker nucleotide sequence that can be used to select a transformed plant, plant part, or plant cell.

[00281] Additionally, as is well known in the art, whole transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture, or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I.R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

[00282] In addition, the above-described properties of genes provided by genetic engineering techniques in the transgenic seeds and plants, plant parts or plant cells of the present invention can be transmitted by sexual reproduction or vegetative growth and, therefore, can be maintained and inherited. descendant plants. As a rule, the maintenance and inheritance use known agricultural methods designed to suit specific purposes, such as harvesting, sowing or cultivation.

[00283] Therefore, a polynucleotide can be introduced into a plant, plant part, or plant cell using any of a number of methods well known in the art, as described above. Therefore, no particular method is followed for introducing one or more polynucleotides into a plant, but rather any method that ensures the stable integration of one or more polynucleotides into the plant genome can be used. If more than one polynucleotide is to be introduced, then the respective polynucleotides can be assembled as parts of a single nucleic acid molecule or as separate nucleic acid molecules and can be arranged within the same or different nucleic acid molecules. Accordingly, the introduction of polynucleotides into a cell of interest can be done during a single transformation event, during separate transformation events, or, for example, into plants, as part of a breeding protocol.

[00284] Once the desired polynucleotide has been introduced into a particular plant species by transformation, it can be transferred within that species or transferred to other varieties of the same species, particularly including commercial varieties, using conventional propagation techniques.

insecticidal compositions.

[00285] In some embodiments, the present invention provides an insecticidal composition comprising a chimeric insecticidal protein of the present invention in an agriculturally acceptable carrier. As used herein, the expression "agriculturally acceptable carrier" may include a natural or synthetic, organic or inorganic material that is combined with an active protein to facilitate its application to a plant or part thereof or in a plant or part thereof. Examples of agriculturally acceptable carriers include, without limitation, powders, dusts, pellets, granules, aerosols, emulsions, colloids, and solutions. Agriculturally acceptable carriers further include, without limitation, inerts, dispersants, surfactants, tackifiers, tackifiers, adhesives, binders, or combinations thereof that can be used in agricultural formulations. Such compositions can be applied in any manner by which pesticidal proteins or other pest control agents are brought into contact with pests. Accordingly, the compositions can be applied to the surfaces of plants or plant parts, including seeds, leaves, flowers, stems, tubers, roots, and the like. In other embodiments, a plant producing an insecticidal protein of the present invention in planta is an agriculturally acceptable carrier for the expressed insecticidal protein. In embodiments, the compositions and agriculturally acceptable carriers of the present invention exclude transgenic plants.

[00286] In further embodiments, the insecticidal composition comprises a bacterial cell or transgenic bacterial cell of the present invention, wherein the bacterial cell or transgenic bacterial cell produces an insecticidal protein of the present invention. Such an insecticidal composition can be obtained by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, precipitating or concentrating a Bacillus thuringiensis (Bt) cell culture, including a Bt transgenic culture. In embodiments, the composition of the present invention may contain at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30% , at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least 99% by weight of a polypeptide of the present invention. In additional embodiments, the implementation of the composition contains from about 1% to about 99% by weight of the insecticidal protein of the present invention.

[00287] The insecticidal proteins of the present invention can be used in combination with other pest control agents to increase the target pest spectrum and/or to prevent or control insect resistance. In addition, the use of the insecticidal proteins of the present invention in combination with an insecticidal agent that has a different mode of action or targets a different receptor in the insect alimentary canal is particularly useful in preventing and/or controlling insect resistance.

[00288] Therefore, in some embodiments, the present invention provides a composition that provides control of one or more pests of plants (for example, an insect pest, such as a Lepidoptera pest, a Coleoptera pest, a pest hemipteran and/or dipteran pest), wherein the composition comprises a first pest control agent that is a chimeric insecticidal protein of the present invention and at least a second pest control agent that is different from the first agent. for pest control. In other embodiments, the composition is a formulation for topical application to a plant. In still other embodiments, the composition is a transgenic plant. In additional embodiments, the implementation of the composition is a combination of the composition and the transgenic plant to which it is applied. In some embodiments, the composition comprises a first pest control agent, which is a chimeric insecticidal protein of the present invention, if the transgenic plant contains a second pest control agent. In other embodiments, the formulation contains a second pest control agent if the transgenic plant contains a first pest control agent that is a chimeric insecticidal protein of the present invention.

[00289] In some embodiments, the second pest control agent may be one or more of a chemical pesticide, such as an insecticide, a Bacillus thuringiensis (Bt) insecticidal protein, and/or an insecticidal agent from a source other than Bt, including, but not limited to, an insecticidal protein Xenorhabdus, Photorhabdus insecticidal protein, Brevibacillus laterosporus insecticidal protein, Bacillus sphaericus insecticidal protein, protease inhibitor (both serine and cysteine types), lectin, alpha-amylase, peroxidase, cholesterol oxidase, or double-stranded RNA (dsRNA) molecule.

[00290] In other embodiments, the second pest control agent is one or more chemical pesticides, which are optionally a seed coat. Non-limiting examples of chemical pesticides include pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrocyclic lactones, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimics. In other embodiments, the chemical pesticide is one or more of abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, binphenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl , хромафенозида, клотианидина, цифлутрина, бета-цифлутрина, цигалотрина, лямбда-цигалотрина, циперметрина, циромазина, дельтаметрина, диафентиурона, диазинона, дифлубензурона, диметоата, диофенолана, эмамектина, эндосульфана, эсфенвалерата, этипрола, фенотикарба, феноксикарба, фенпропатрина, фенпроксимата, фенвалерата , fipronil, flonicamid, flucitrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonofos, halofenoside, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methalidophos, metidathione, methomyl, methoprene, methoxychlorine, methoxyfenoside, nithiazine, novaluron, noviflumuron (XDE-007), oxamil, paraty ona, parathion-methyl, permethrin, phorate, fosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalil, pyriproxyfen, rotenone, spinosad, spiromesifen (BSN 2060), sulprofos, tebufenoside, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos, тиаметоксама, тиодикарба, тиосултап-натрия, тралометрина, трихлорфона и трифлумурона, алдикарба, оксамила, фенамифоса, амитраза, хинометионата, хлоробензилата, цигексатина, дикофола, диенохлора, этоксазола, феназаквина, оксида фенбутатина, фенпропатрина, фенпироксимата, гекситиазокса, пропаргита, пиридабена и тебуфенпирада . In still other embodiments, the chemical pesticide is selected from one or more of cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, fenvalerate, tralometrin, phenoticarb, methomyl, oxamil, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin , emamectin, endosulfan, ethiprol, fipronil, flufenoxuron, triflumuron, diofenolan, pyriproxyfen, pimetrosine and amitraz.

[00291] In additional embodiments, the second pest control agent may be one or more of any number of Bacillus thuringiensis insecticidal proteins, including, but not limited to, the Cry protein, a vegetative insecticidal protein (VIP), and insecticidal chimeric variants of any of the insecticidal proteins listed above. . In other embodiments, the second pest control agent is a Cry protein selected from: Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg , Cry1Bh Cry1Bi Cry1Ca Cry1Cb Cry1Da Cry1Db Cry1Dc Cry1Dd Cry1Ea Cry1Eb Cry1Fa Cry1Fb Cry1Ga Cry1Gb Cry1Gc Cry1Ha Cry1Hb Cry1Hc Cry1Ia Cry1Ib Cry1Ife Cry1Ic Cry1Ja Cry1Jb Cry1Jc Cry1Jd Cry1Ka Cry1La Cry1Ma Cry1Na Cry1Nb Cry2Aa Cry2Ab Cry2Ac Cry2Ad Cry2Ae Cry2Af Cry2Ag Cry2Ah Cry2Ai Cry2Aj . , Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8 Bc Cry8Ca Cry8Da Cry8Db Cry8Ea Cry8Fa Cry8Ga Cry8Ha Cry8Ia Cry8Ib Cry8Ja Cry8Ka Cry8Kb Cry8La Cry8Ma Cry8Na Cry8Pa Cry8Qa Cry8Ra Cry8Sa Cry8Ta Cry9Aary CryB9Ba Cry9Da, Cry9Db, Cry9Dc, Cry9Ea, Cry9Eb, Cry9Ec, Cry9Ed, Cry9Ee, Cry9Fa, Cry9Ga, Cry10Aa, Cry11Aa, Cry11Ba, Cry11Bb, Cry12Aa,Cry13Aa, Cry14Aa, Cry11Ab, Cry15Aa, Cry16Aa, Cry17Aa, Cry18Aa, Cry18Ba, Cry18Ca, Cry19Aa, Cry19Ba, Cry19Ca, Cry20Aa, Cry20Ba, Cry21Aa, Cry21Ba, Cry21Ca, Cry21Da, Cry21Ea, Cry21Fa, Cry21Ga, Cry21Ha, Cry22Aa, Cry22Ab, Cry22Ba, Cry22Bb, Cry23Aa, Cry24Aa, Cry24Ba, Cry24Ca, Cry25Aa, Cry26Aa, Cry27Aa, Cry28Aa, Cry29Aa, Cry29Ba, Cey30Aa, Cry30Ba, Cry30Ca, Cry30Da, Cry30Db, Cry30Ea, Cry30Fa, Cry30Ga,Cry31Aa, Cry31Ab, Cry31Ac, Cry31Ad, Cry32Aa, Cry32Ab, Cry32Ba, Cry32Ca, Cry32Cb, Cry32Da, Cry32Ea, Cry32Eb, Cry32Fa, Cry32Ga, Cry32Ha, Cry32Hb, Cry32Ia, Cry32Ja, Cry32Ka, Cry32La, Cry32Ma, Cry32Mb, Cry32Na, Cry320a, Cry32Pa, Cry32Qa, Cry32Ra, Cry32Sa, Cry32Ta, Cry32Ua, Cry33Aa, Cry34A a, Cry34Ab, Cry34Ac, Cry34Ba, Cry35Aa, Cry35Ab, Cry35Ac, Cry35Ba, Cry36As, Cry37Aa, Cry38Aa, Cry39Aa, Cry40Aa, Cry40Ba, Cry40Ca, Cry40Da, Cry41Aa, Cry41Ab, Cry41Ba, Cry42Aa, Cry43Aa, Cry43Ba, Cry43Ca, Cry43Cb, Cry43Cc, Cry44Aa, Cry45Aa, Cry46Aa Cry46Ab, Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry30Aa, Cry30Ba, Cry51Aa, Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry36Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba or any combination of the above. In embodiments, the Cry protein is Cry1Fa, for example, as shown in maize transformant TC 1507.

[00292] In additional embodiments, the second pest control agent is one or more Vip3 vegetative insecticidal proteins selected from Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3Aa12, Vip3Aa14, Vip3Aa15, Vip3Aa16, Vip3Aa17, Vip3Aa18, Vip3Aa19, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2, Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Ab1, Vip3Ab2, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, Vip3Af2, Vip3Af3, Vip3Ag1, Vip3Ag2, Vip3Ag3 HM117633, Vip3Ag4, Vip3Ag5, Vip3Ah1, Vip3Ba1, Vip3Ba2, Vip3Bb1, Vip3Bb2 , Vip3Bb3, or any combination of the above. In embodiments, the Vip3 protein is Vip3Aa (US Patent No. 6,137,033), for example, as shown in the MIR162 maize transformant (US Patent No. 8,232,456; US Patent No. 8,455,720 and US Patent No. 8,618,272).

[00293] In embodiments, the second pest control may be obtained from sources other than B. thuringiensis. For example, the second pest control agent can be alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, insecticidal protein Xenorhabdus spp. (such as X. nematophila or X. bovienii), Photorhabdus spp. insecticidal protein (such as P. luminescens or P. asymobiotica), Brevibacillus spp. insecticidal protein (such as P. laterosporous), Lysinibacillus spp. insecticidal protein. (such as L. sphearicus), insecticidal protein of Chromobacterium spp. (such as C. subtsugae or C. piscinae), the insecticidal protein of Yersinia spp. (such as Y. entomophaga), the insecticidal protein of Paenibacillus spp. (such as P. propylaea), insecticidal protein of Clostridium spp. (such as C. bifermentans), Pseudomonas spp. (such as P. fluorescens) and lignin. In other embodiments, the second agent may be at least one insecticidal protein derived from an insecticidal toxin complex (Tc) from Photorhabdus, Xenorhabus, Serratia, or Yersinia. In other embodiments, the insecticidal protein may be an ADP-ribosyltransferase derived from an insecticidal bacterium such as Photorhabdus ssp. In other embodiments, the insecticidal protein may be a VIP protein, such as VIP1 or VIP2 from B. cereus. In still other embodiments, the insecticidal protein may be a binary toxin derived from insecticidal bacteria, such as ISP1A and ISP2A from B. laterosporous or BinA and BinB from L. sphaericus. In still other embodiments, the implementation of the insecticidal protein may be engineered or may be a hybrid or chimera of any previous insecticidal proteins.

[00294] In some embodiments, the second pesticidal agent may be non-proteinaceous, such as an interfering RNA molecule such as dsRNA, which may be expressed transgenic or applied as part of a composition (eg, using topical application methods). Typically, the interfering RNA contains at least an RNA fragment corresponding to the target gene, a spacer sequence, and a second RNA fragment that is complementary to the first, whereby a double-stranded RNA structure can be formed. RNA interference (RNAi) occurs when the body recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of approximately 19-24 nucleotides in length, called small interfering RNA (siRNA). The siRNAs then spread or spread throughout the body, including through cell membranes, where they hybridize with mRNA (or other RNAs) and cause RNA hydrolysis. Interfering RNAs are recognized by the RNA interference silencing complex (RISC) into which an effector strand (or "guide strand") RNA is introduced. This guide strand acts as a template for recognition and destruction of duplex sequences. This process is repeated each time a siRNA hybridizes to its complementary target RNA, effectively preventing the translation of such mRNAs, and thus "silencing" the expression of the specific genes from which the mRNAs were transcribed. Interfering RNAs are known in the art for insect control (see, for example, WO 2013/192256, incorporated herein by reference). An interfering RNA for use in insect control provides a non-naturally occurring double-stranded RNA that uses native RNAi pathways in the insect to trigger target gene silencing, which can result in feeding and/or growth failure and can lead to insect death. pest. The interfering RNA molecule may confer insect resistance to the same target pest as the protein of the present invention, or may target a different pest. The target plant pest can feed by chewing, sucking, or piercing. Interfering RNAs are known in the art to be useful in insect control. In embodiments, a dsRNA useful for insect control is described in U.S. Provisional Applications Nos. 62/371259, 62/371261, or 62/371262, filed Aug. US Nos. 92388223, 9340797, or 8946510. In embodiments, dsRNA suitable for insect control is described in US patent applications Nos. In other embodiments, the interfering RNA may confer resistance to a plant pest other than an insect, such as a pest nematode or pest virus.

[00295] In still further embodiments, the first insect control agent, which is the chimeric insecticidal protein of the present invention, and the second pest control agent are co-expressed in the transgenic plant. Such co-expression of more than one pesticidal component in the same transgenic plant can be achieved by producing a plant containing and expressing nucleic acid sequences encoding insect control agents using genetic engineering techniques. For example, co-expression of more than one pesticide in the same transgenic plant can be achieved by obtaining a single recombinant vector containing the coding sequences for more than one pesticide in a so-called "molecular package" and genetically engineering the plant to contain and express all these pesticides in the transgenic plant. Such molecular packages can also be obtained using minichromosomes, which are described, for example, in US patent No. 7235716. Alternatively, a plant, parent 1, can be genetically engineered to express the chimeric insecticidal protein of the present invention. The second plant, parent 2, can be genetically engineered to express the second pest control agent. Crossing parent 1 with parent 2 produces progeny plants that express both insect control agents from parents 1 and 2.

[00296] In other embodiments, the present invention provides a trait-packaged transgenic plant resistant to plant pest infestation, comprising a nucleic acid sequence (e.g., DNA) encoding a dsRNA for suppressing a major gene in a target pest organism, and a nucleic acid sequence (e.g., DNA) encoding a chimeric insecticidal protein of the present invention that exhibits insecticidal activity against the target pest. It has been previously reported that dsRNAs are ineffective against certain Lepidoptera pests (Rajagopol et al. 2002. J. Biol. Chem. 277:468-494) probably due to high midgut pH which impairs dsRNA resistance. . Therefore, in some embodiments where the target pest is a Lepidoptera pest, the chimeric insecticidal protein of the present invention acts to temporarily lower the pH in the midgut, which allows stabilization of the co-uptake dsRNA, making the dsRNA effective in silencing the target genes.

[00297] Transgenic plants or seeds containing and/or expressing an insecticidal protein of the present invention can also be treated with an insecticide or insecticidal seed coating as described in US Pat. Nos. 5,849,320 and 5,876,739. for seeds and the transgenic plant or seed of the present invention are active against the same target insect, e.g., a Lepidoptera pest (e.g., cutworm), this combination is useful (i) in a method of further enhancing the activity of the composition of the present invention in relation to the target insect; and/or (ii) in a method for preventing the development of resistance to the composition of the present invention by providing another mechanism of action in relation to the target insect. Thus, in embodiments of the present invention, there is provided a method for improving population control of Lepidoptera insects, comprising providing a transgenic plant or seed of the present invention and applying to the plant or seed an insecticide or an insecticidal seed coating intended for the transgenic plant or seed according to the present invention.

[00298] Even if the insecticide or insecticidal seed coating is active against another insect, the insecticide or insecticidal seed coating is useful for extending the range of insect control, for example, by adding an insecticide or insecticidal seed coating that is active in against insects related to beetles, to the transgenic seed of the present invention, which in some embodiments, the implementation is characterized by activity against insects related to Lepidoptera, and the resulting coated transgenic seed provides control of insect pests related to both Lepidoptera and Coleoptera.

Methods for obtaining and using chimeric insecticidal proteins, nucleic acids and transgenic plants.

[00299] The present invention also encompasses methods for producing a transgenic plant that is resistant to insects (eg, resistant to insects related to Lepidoptera). In exemplary embodiments, the method includes introducing into a plant a polynucleotide, an expression cassette, or a vector of the present invention containing a nucleotide sequence that encodes a chimeric insecticidal protein of the present invention (including toxin fragments and modified forms that are substantially identical to the polypeptides specifically disclosed herein. ), wherein the nucleotide sequence is expressed in the plant to produce the chimeric insecticidal protein of the present invention, thereby conferring insect resistance on the plant and producing an insect-resistant transgenic plant (e.g., compared to a suitable control plant, such as a plant that does not contains a polynucleotide, expression cassette or vector of the present invention and/or does not express a polypeptide of the present invention).

[00300] In embodiments, a method of introducing a polynucleotide a, expression cassette, or vector of the present invention into a plant comprises first transforming a plant cell using the polynucleotide a, expression cassette, or vector, and regenerating a transgenic plant from it, wherein the transgenic plant comprises the polynucleotide, cassette expression or vector and expresses the chimeric insecticidal protein of the present invention.

[00301] Alternatively or additionally, the step of introducing may include crossing the first plant containing the polynucleotide, expression cassette, or vector with a second plant (e.g., a plant other than the first plant, e.g., a plant that does not contain the polynucleotide, expression cassette, or vector) and optionally obtaining a progeny plant that contains a polynucleotide, an expression cassette or a vector and expresses a chimeric insecticidal protein of the present invention, thereby resulting in increased resistance to at least one insect pest. Thus, a transgenic plant of the present invention encompasses a plant that is the direct result of a transformation event and its descendant (of any generation) that contains a polynucleotide, an expression cassette, or a vector and optionally expresses a chimeric insecticidal protein resulting in increased resistance to at least one pest insect.

[00302] The present invention further provides a method for detecting a transgenic plant of the present invention, comprising detecting the presence of a polynucleotide, an expression cassette, a vector, or a chimeric insecticidal protein of the present invention in a plant (or plant cell, plant part, or the like derived therefrom) ) and thereby defining a plant as a transgenic plant of the present invention based on the presence of a polynucleotide, expression cassette, vector or chimeric insecticidal protein of the present invention.

[00303] The present invention further provides a method for producing a transgenic plant with increased resistance to at least one insect pest (e.g., at least one Lepidoptera pest), comprising: planting a seed containing a polynucleotide, an expression cassette, or a vector according to the present invention, and growing a transgenic plant from this seed, where the transgenic plant contains a polynucleotide, an expression cassette or a vector and produces a chimeric insecticidal protein.

[00304] In embodiments, transgenic plants obtained using the methods of the present invention comprise a polynucleotide, an expression cassette, or a vector of the present invention. In embodiments, a transgenic plant obtained using the methods of the present invention contains a chimeric insecticidal protein of the present invention and optionally has increased resistance to at least one insect pest.

[00305] The methods for producing a transgenic plant described herein optionally include the additional step of collecting seed from the transgenic plant, wherein the seed contains a polynucleotide, expression cassette, or vector and produces a chimeric insecticidal protein. Optionally, the seed provides an additional transgenic plant that contains a polynucleotide, an expression cassette, or a vector and produces a chimeric insecticidal protein and thus has increased resistance to at least one insect pest.

[00306] The present invention further provides for plant parts, plant cells, plant organs, plant cultures, seeds, plant extracts, harvested products, and processed products of transgenic plants obtained using the methods of the present invention.

[00307] As a further aspect, the present invention also provides a method for producing a seed, the method comprising obtaining a transgenic plant that contains a polynucleotide, an expression cassette, or a vector of the present invention, and collecting seed from a transgenic plant, where the seed contains a polynucleotide, an expression cassette , a vector and produces a chimeric insecticidal protein. Optionally, the seed provides an additional transgenic plant that contains a polynucleotide, an expression cassette, or a vector and produces a chimeric insecticidal protein and thus has increased resistance to at least one insect pest. In exemplary embodiments, the step of obtaining a transgenic plant includes sowing a seed that produces a transgenic plant.

[00308] The present invention further provides a method for producing a hybrid plant seed, the method comprising crossing a first inbred plant, which is a transgenic plant containing a polynucleotide, expression cassette or vector of the present invention and optionally expressing a chimeric insecticidal protein of the present invention, with another inbred plant (eg, an inbred plant that does not contain a polynucleotide, expression cassette, or vector of the present invention) and allowing hybrid seed to form. Optionally, the method further comprises collecting the hybrid seed. In embodiments, the hybrid seed comprises a polynucleotide, expression cassette or vector of the present invention and, in embodiments, may further comprise a chimeric insecticidal protein of the present invention and have increased resistance to an insect pest. In embodiments, the hybrid seed provides a transgenic plant that contains a polynucleotide, expression cassette, or vector of the invention, expresses a chimeric insecticidal protein of the invention, and has increased resistance to at least one insect pest.

[00309] In some embodiments, the transgenic plant of the present invention is resistant to at least one Lepidoptera insect pest (as described herein). In embodiments, the transgenic plant provides control of an insect pest or cutworm colony that is resistant to a Vip3A protein (e.g., a Vip3Aa protein, e.g., which is expressed in the MIR162 maize transformant) and/or Cry1F (e.g., a Cry1Fa protein, which is expressed in the TC1507 maize transformant ).

[00310] In additional embodiments, a method for controlling at least one insect pest (e.g., at least one Lepidoptera insect pest, such as a bollworm) comprises providing a chimeric insecticidal protein of the present invention. In embodiments, the method comprises delivering (eg, oral delivery) to the insect pest or its habitat an effective amount of the chimeric insecticidal protein of the present invention. Typically, for purposes of effectiveness, the polypeptide is taken up by the insect by the oral route. However, the chimeric insecticidal protein can be delivered to the insect using many known methods. Methods for orally delivering a protein to an insect include, but are not limited to, providing the protein (1) to the transgenic plant, wherein the insect eats (ingests) one or more parts of the transgenic plant, thereby consuming the polypeptide expressed in the transgenic plant; (2) in formulated protein(s) composition(s) that can be applied to, for example, insect growth media or incorporated into insect culture media; (3) in a protein composition(s) that can be applied to a surface, for example by spraying the surface of a plant part, and which is then ingested by the insect by virtue of the fact that the insect eats one or more of the sprayed plant parts; (4) in a bait matrix; or (5) with any other protein delivery system known in the art. Thus, any method of oral delivery to an insect body can be used to deliver the toxic proteins of the present invention. In some specific embodiments, the chimeric insecticidal protein of the present invention is delivered to the insect by the oral route, such as when the insect ingests one or more parts of the transgenic plant of the present invention.

[00311] In other embodiments, the insecticidal protein of the present invention is delivered to an insect by the oral route, wherein the insect consumes one or more plant parts sprayed with the composition comprising the insecticidal protein of the present invention. Delivery of the composition of the present invention to the surface of the plant can be accomplished by any method of applying compounds, compositions, formulations, and the like. on the surface of plants known to those skilled in the art. Some non-limiting examples of delivery to or contact with a plant or part thereof include spraying, dusting, sprinkling, spraying, misting, misting, spreading, soaking, soil injection, soil application, wetting (e.g., root treatment, soil ), dipping, watering, coating, soaking in leaves or stems, inter-row application or seed treatment, etc. and their combinations. These and other procedures for bringing a plant or part thereof into contact with a compound(s), composition(s), or composition(s) are well known to those skilled in the art.

[00312] In further embodiments, the present invention provides a method for controlling a Lepidoptera insect pest that is resistant to a Vip3A protein (e.g., a Vip3Aa protein, e.g., which is expressed by the maize transformant MIR162) and/or a Cry1F protein (e.g., a Cry1Fa protein, e.g. , which is expressed by the maize transformant TC1507), comprising delivering to the Lepidoptera insect or its habitat an effective amount of the chimeric insecticidal protein or composition of the present invention. In exemplary embodiments, the resistant insect pest is a resistant insect pest or armyworm colony.

[00313] In other embodiments, the present invention provides a method for preventing the development of resistance in a target lepidoptera insect pest population to a Vip3A protein (e.g., the Vip3Aa protein, e.g., which is expressed by the maize transformant MIR162) and/or Cry1F (e.g., the Cry1Fa protein , for example, which is expressed by the maize transformant TC1507) expressed in a transgenic plant, the method comprising delivery to the target population of a transgenic plant containing a polynucleotide that contains a nucleotide sequence encoding a Vip3A protein and/or a nucleotide sequence encoding a Cry1F protein; and a polynucleotide, expression cassette or vector of the present invention expressing the chimeric insecticidal protein of the present invention. In some embodiments, the target Lepidoptera pest is the Fall Armyworm. According to the above embodiments, the transgenic plant may comprise a selection package of two or more insecticidal traits, a molecular package of two or more insecticidal traits, or a combination thereof.

[00314] In some embodiments, the present invention encompasses a method of providing a means to control a pest (e.g., a Lepidoptera pest such as Fall Armyworm) to a farmer, the method comprising supplying or selling plant material, such as seed, to the farmer. wherein the plant material contains a polynucleotide, an expression cassette, a vector capable of expressing the chimeric insecticidal protein of the present invention. In embodiments, the plant material comprises a chimeric insecticidal protein of the present invention and optionally has increased resistance to at least one insect pest. In embodiments, the plant material is a seed, and the plant grown from the seed contains a polynucleotide, expression cassette, or vector of the invention, expresses a chimeric insecticidal protein of the invention, and has increased resistance to at least one insect pest.

[00315] In addition to providing compositions, the present invention provides methods for producing a chimeric insecticidal protein that is toxic to a Lepidoptera pest. Such a method includes culturing a transgenic host cell, other than a human cell, that contains a polynucleotide, an expression cassette, or a vector of the present invention that expresses a chimeric insecticidal protein of the present invention, under conditions under which the host cell produces a chimeric insecticidal protein that is toxic to pest related to Lepidoptera. In some embodiments, the non-human transgenic host cell is a plant cell. In some other embodiments, the plant cell is a maize cell. In other embodiments, the conditions under which the plant cell or maize cell is grown include natural sunlight. In other embodiments, the non-human transgenic host cell is a bacterial cell. In still other embodiments, the non-human transgenic host cell is a yeast cell.

[00316] In some embodiments, the methods of the present invention control at least one Lepidoptera insect pest, including, without limitation, one or more of the following: Ostrinia spp., such as O. nubilalis (corn borer) and/or O. furnacalis (eastern corn borer); Plutella spp. such as P. xylostella (cabbage moth); Spodoptera spp. such as S. frugiperda (fall armyworm), S. littoralis (Egyptian cotton cutworm), S. ornithogalli (yellow cutworm), S. praefica (western yellow cut cutworm), S. eridania (southern cut cutworm), and/or S. exigua (small cutworm); Agrotis spp. such as A. ipsilon (upsilon cutworm), A. segetum (common winter cutworm), A. gladiaria (fall cutworm of the genus Agrotis) and/or A. orthogonia (rectangular cutworm); Striacosta spp. such as S. albicosta (Western Fall Armyworm); Helicoverpa spp., such as H. zea (corn bollworm), H. punctigera (Australian cotton bollworm) and/or H. armigera (cotton bollworm); Heliothis spp. such as H. virescens (tobacco leaf roller); Diatraea spp. such as D. grandiosella (southwestern corn moth) and/or D. saccharalis (sugar cane stalk grinder); Trichoplusia spp. such as T. ni (Cabbage cutworm); Sesamia spp. such as S. nonagroides (Mediterranean corn borer) and/or S. calamistis (pink stalk armyworm); Pectinophora spp. such as P. gossypiella (pink bollworm); Cochylis spp. such as C. hospes (striped sunflower moth); Manduca spp. such as M. sexta (tobacco hawk hawk) and/or M. quinquemaculata (5-spot hawk hawk); Elasmopalpus spp. such as E. lignosellus (small corn stalk grinder); Pseudoplusia spp. such as P. includens (soy worm); Anticarsia spp. such as A. gemmatalis (velvet bean cutworm); Plathypena spp. such as P. scabra (clover cutworm); Pieris spp. such as P. brassicae (cabbage whitefish), Papaipema spp. such as P. nebris (Papaipema nebris grinder); Pseudaletia spp. such as P. unipuncta (short cutworm); Peridroma spp. such as P. saucia (daisy cutworm); Keiferia spp. such as K. lycopersicella (tomato pinworm); Artogeia spp. such as A. rapae (turnip whitefish); Phthorimaea spp. such as P. operculella (potato moth); Chrysodeixis spp. such as C. includes (soy worm); Feltia spp. such as F. ducens (cutworm Feltia jaculifera); Chilo spp. such as C. suppressalis (yellow rice moth), Cnaphalocrocis spp. such as C. medinalis (rice moth), or any combination of the above. In embodiments, the methods of the present invention provide control of an insect pest or bollworm colony that is resistant to a Vip3A protein (e.g., a Vip3Aa protein, e.g., which is expressed in the MIR162 maize transformant) and/or Cry1F (e.g. expressed in the maize transformant TC1507).

[00317] The present invention also contemplates the use of the chimeric insecticidal proteins, nucleic acids, transgenic plants, plant parts, seeds, and insecticidal compositions of the present invention, for example, to control an insect pest, such as a Lepidoptera pest (as described herein). document).

[00318] In embodiments, the present invention provides a method of using a polynucleotide, expression cassette, vector, or host cell of the present invention to produce an insecticidal composition for controlling an insect pest (e.g., a Lepidoptera pest).

[00319] In embodiments, the present invention provides a method of using a polynucleotide, an expression cassette, or a vector of the present invention to produce a transgenic seed, wherein the transgenic seed grows into a transgenic plant with increased resistance to an insect pest.

[00320] As another aspect, the present invention also contemplates the use of a transgenic plant of the present invention to produce a transgenic seed, which is optionally a hybrid seed.

[00321] In embodiments, the present invention provides a method of using a chimeric insecticidal protein, polynucleotide, expression cassette, vector, transgenic plant, or insecticidal composition of the present invention to prevent the development of resistance in a target Lepidoptera insect pest population to the Vip3A protein, and /or Cry1F protein (each as described herein).

[00322] Statements of the present invention include, without limitation, the following:

[00323] 1. A chimeric insecticidal protein that is toxic to a Lepidoptera insect pest, comprising in N-terminal to C-terminal direction: (a) an N-terminal region of the first Cry1 protein, which is an N-terminal region BT-0029 protein under SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to it, fused to (b) the C-terminal region of another Cry1 protein; where the transition position between the first Cry1 protein and another Cry1 protein is in the conservative block 3.

[00324] 2. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is a Cry1F, Cry1G, Cry1I, or Cry1K protein.

[00325] 3. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is a Cry1Fa protein.

[00326] 4. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is a Cry1Ia or Cry1If protein.

[00327] 5. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is the BT-0022 protein of SEQ ID NO: 1.

[00328] 6. The chimeric insecticidal protein of claim 1, wherein the C-terminal region of the other Cry1 protein contains an amino acid sequence corresponding to the amino acid sequence from position 464 to 602 of SEQ ID NO: 8 or an amino acid sequence that is at least 80% identical her.

[00329] 7. The chimeric insecticidal protein of claim 1, wherein the C-terminal region of the other Cry1 protein contains an amino acid sequence corresponding to the amino acid sequence from position 498 to 636 of SEQ ID NO: 1 or an amino acid sequence that is at least 80% identical her.

[00330] 8. Chimeric insecticidal protein according to any one of paragraphs. 1-7, where the N-terminal region of the first Cry1 protein contains an amino acid sequence corresponding to the amino acid sequence from position 1 to 458 of SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to it.

[00331] 9. Chimeric insecticidal protein according to any one of paragraphs. 1-8, wherein the chimeric insecticidal protein further comprises a peptidyl moiety at the N-terminus that is cleaved from the protoxin upon ingestion by a Lepidoptera insect pest.

[00332] 10. Chimeric insecticidal protein according to any one of paragraphs. 1-9, wherein the chimeric insecticidal protein further comprises at the C-terminus a protoxin tail from the Cry protein, which is cleaved from the protoxin upon ingestion by a Lepidoptera insect pest.

[00333] 11. Chimeric insecticidal protein according to any one of paragraphs. 1-10, where the chimeric insecticidal protein contains (a) an amino acid sequence from position 1 to 597 of SEQ ID NO: 3 or a toxin fragment thereof, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a) .

[00334] 12. Chimeric insecticidal protein according to any one of paragraphs. 1-11, where the chimeric insecticidal protein contains (a) the amino acid sequence under SEQ ID NO: 3 or its toxin fragment, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a).

[00335] 13. Chimeric insecticidal protein according to any one of paragraphs. 1-10, where the chimeric insecticidal protein contains (a) the amino acid sequence from position 1 to 597 of SEQ ID NO: 9 or its toxin fragment, or (b)

an amino acid sequence that is at least 80% identical to the amino acid sequence of (a).

[00336] 14. Chimeric insecticidal protein according to any one of paragraphs. 1-10 or claim 13, where the chimeric insecticidal protein contains (a) the amino acid sequence under SEQ ID NO: 9 or its toxin fragment, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a) .

[00337] 15. Chimeric insecticidal protein according to any one of paragraphs. 1-14, where the chimeric insecticidal protein contains the amino acid sequence under SEQ ID NO: 3 or SEQ ID NO: 9.

[00338] 16. Chimeric insecticidal protein according to any one of paragraphs. 1-15, wherein the chimeric insecticidal protein is characterized by insecticidal activity against one or more of Spodoptera frugiperda (grass cutworm), Chrysodeixis includes (soy cutworm), Diatraea saccharalis (sugar cane stalk grinder), Diatraea grandiosella (southwestern corn moth), and Agrotis ipsilon (upsilon scoop).

[00339] 17. Chimeric insecticidal protein according to any one of paragraphs. 1-16, where the chimeric insecticidal protein is characterized by insecticidal activity against an insect pest or Spodoptera frugiperda colony with resistance to the Vip3A protein and/or the Cry1F protein.

[00340] 18. A polynucleotide containing a nucleotide sequence encoding a chimeric insecticidal protein according to any one of paragraphs. 1-17.

[00341] 19. The polynucleotide of claim 18, wherein the polynucleotide is codon-optimized for expression in a plant.

[00342] 20. A polynucleotide according to claim 18 or claim 19, wherein the polynucleotide comprises a nucleotide sequence that comprises (a) the nucleotide sequence under SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 14 or a toxin-coding fragment thereof; (b) a nucleotide sequence that is substantially identical to the nucleotide sequence from (a); (c) a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleotide sequence from (a) or (b); or (d) a nucleotide sequence that differs from the nucleotide sequence of (a), (b) or (c) due to the degeneracy of the genetic code.

[00343] 21. A polynucleotide according to claim 18 or claim 19, wherein the nucleotide sequence comprises the nucleotide sequence under SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 14.

[00344] 22. A nucleic acid molecule containing a polynucleotide according to any one of paragraphs. 18-21 operably linked to a heterologous promoter.

[00345] 23. The nucleic acid molecule of claim 22, wherein the heterologous promoter is a promoter that allows expression in a plant.

[00346] 24. A vector containing a polynucleotide according to any one of paragraphs. 18-21 or a nucleic acid molecule according to claim 22 or claim 23.

[00347] 25. A transgenic cell containing a polynucleotide according to any one of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23, or a vector according to claim 24.

[00348] 26. The transgenic cell of claim 25, wherein the cell is a transgenic bacterial cell.

[00349] 27. The transgenic cell of claim 25, wherein the cell is a transgenic plant cell.

[00350] 28. The transgenic plant cell of claim 27, wherein the plant cell is: (i) a monocot cell, optionally a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell, or a wheat cell; or (ii) a dicot cell, optionally a soybean cell, a sunflower cell, a tomato cell, a cabbage cultivar cell, a cotton cell, a sugar beet cell, or a tobacco cell.

[00351] 29. A transgenic plant comprising a transgenic plant cell according to claim 27 or claim 28.

[00352] 30. The transgenic plant of claim 29, wherein the plant is: (i) a monocotyledonous plant, optionally a barley plant, a maize plant, an oat plant, a rice plant, a sorghum plant, a sugar cane plant, or a wheat plant; or (ii) a dicotyledonous plant, optionally a soybean plant, a sunflower plant, a tomato plant, a cabbage plant, a cotton plant, a sugar beet plant, or a tobacco plant.

[00353] 31. The transgenic plant of claim 28 or claim 29, wherein the transgenic plant contains an additional nucleotide sequence encoding a second insect control agent.

[00354] 32. A transgenic plant part from a transgenic plant according to any one of paragraphs. 29-31, where the transgenic plant part contains a chimeric insecticidal protein.

[00355] 33. Transgenic seed of a transgenic plant according to any one of paragraphs. 29-31.

[00356] 34. The collected product obtained from the transgenic plant according to any one of paragraphs. 29-31, the transgenic plant part of claim 32, or the transgenic seed of claim 33, wherein the harvested product contains the chimeric insecticidal protein.

[00357] 35. A processed product obtained from the harvested product of claim 34, wherein the processed product is flour, meal, oil, starch, or a product derived from any of the above.

[00358] 36. An insecticidal composition containing a chimeric insecticidal protein according to any one of paragraphs. 1-17 and an agriculturally acceptable carrier.

[00359] 37. The insecticidal composition of claim 36 wherein the agriculturally acceptable carrier is a powder, dust, pellet, granule, aerosol, emulsion, colloid, or solution.

[00360] 38. The insecticidal composition of claim 36 or claim 37, wherein the composition is a dried, lyophilized, homogenized, extracted, filtered, centrifuged and/or precipitated composition and/or is a bacterial culture concentrate.

[00361] 39. An insecticidal composition according to any one of paragraphs. 36-38, where the composition contains a transgenic bacterial cell that produces a chimeric insecticidal protein.

[00362] 40. An insecticidal composition according to any one of paragraphs. 36-39, wherein the composition contains from about 1% to about 99% by weight of the chimeric insecticidal protein.

[00363] 41. An insecticidal composition according to any one of paragraphs. 36-40 wherein the composition comprises a second insect control agent.

[00364] 42. A method for producing a transgenic plant with increased resistance to a Lepidoptera pest, the method comprising introducing into the plant a polynucleotide according to any one of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23, or a vector according to claim 24, wherein the chimeric insecticidal protein is expressed in a plant, thereby obtaining a transgenic plant with increased resistance to an insect pest.

[00365] 43. The method of claim 42, wherein the step of introducing comprises (i) transforming a plant cell with a polynucleotide, nucleic acid molecule, or vector and regenerating a transgenic plant, or (ii) crossing a first plant containing a polynucleotide, nucleic acid molecule, or vector, with second plant.

[00366] 44. The method of claim 42 or claim 43, wherein the method further comprises obtaining a progeny plant from a transgenic plant, wherein the progeny plant contains a polynucleotide, nucleic acid molecule, or vector and is characterized by increased resistance to an insect pest.

[00367] 45. A method of obtaining a transgenic plant with increased resistance to a Lepidoptera pest, the method comprising: (a) sowing a seed containing a polynucleotide according to any one of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23, or a vector according to claim 24; and (b) growing a transgenic plant from seed, wherein the transgenic plant contains a polynucleotide, nucleic acid molecule, or vector and produces a chimeric insecticidal protein.

[00368] 46. The method of claim 45, wherein the method further comprises (c) collecting seed from the transgenic plant of (b), wherein the harvested seed contains the chimeric insecticidal protein.

[00369] 47. The method of obtaining a seed, the method includes (a) obtaining a transgenic plant that contains a polynucleotide according to any one of paragraphs. 18-21, the nucleic acid molecule of claim 22 or claim 23, or the vector of claim 24; and (b) collecting seed from the transgenic plant of (a), wherein the harvested seed contains the chimeric insecticidal protein.

[00370] 48. The method of obtaining a hybrid plant seed, the method includes: (a) crossing the first inbred plant, which is a transgenic plant containing a polynucleotide according to any one of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23, or a vector according to claim 24, with another inbred plant, and (b) allowing the formation of a hybrid seed.

[00371] 49. A method of controlling a Lepidoptera pest, the method comprising delivering to the body of the pest or its habitat a composition comprising an effective amount of a chimeric insecticidal protein according to any one of paragraphs. 1-17 or an insecticidal composition according to any one of paragraphs. 36-41.

[00372] 50. A method for controlling a Lepidoptera insect pest resistant to the Vip3A protein and/or Cry1F protein, the method comprising delivering to the organism of the resistant Lepidoptera pest or to its habitat an insecticidal protein according to any from paragraphs. 1-17 or an insecticidal composition according to any one of paragraphs. 36-41.

[00373] 51. A method of reducing the development of resistance to the Vip3A protein and/or the Cry1F protein in a target Lepidoptera insect pest population, the method comprising delivering to the target population or its habitat a transgenic plant containing (i) a polynucleotide at any of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23 or a vector according to claim 24, and (ii) a polynucleotide containing a nucleotide sequence encoding a Vip3A protein and/or a nucleotide sequence encoding a Cry1F protein; wherein the chimeric insecticidal protein and the Vip3A protein and/or the Cry1F protein are produced in the transgenic plant.

[00374] 52. The method according to any one of paragraphs. 42-51, wherein the Lepidoptera pest includes one or more of Spodoptera frugiperda (grass cutworm), Chrysodeixis includes (soybean cutworm), Diatraea saccharalis (sugar cane stalk grinder), Diatraea grandiosella (southwestern corn moth) and Agrotis ipsilon (upsilon cutworm).

[00375] 53. The method according to any one of paragraphs. 42-52, where the Lepidoptera insect pest includes Spodoptera frugiperda (grass cutworm), which is resistant to the Vip3A protein and/or the Cry1F protein.

[00376] 54. The method according to any one of paragraphs. 42-53, wherein the plant is: (i) a monocotyledonous plant, optionally a barley plant, a maize plant, an oat plant, a rice plant, a sorghum plant, a sugar cane plant, or a wheat plant; or (ii) a dicot plant, optionally a soybean plant, a sunflower plant, a tomato plant, a cabbage plant, a cotton plant, a sugar beet plant, or a tobacco plant.

[00377] 55. The method according to any one of paragraphs. 42-54, where the plant is a maize plant.

[00378] Hereinafter, the present invention will be described with reference to the following examples. Those skilled in the art will appreciate that these examples do not limit the scope of the claims of the present invention, but rather are intended to be illustrative of certain embodiments. Other embodiments of the present invention may be practiced without departing from the spirit and scope of the present invention, the scope of which is defined by the present disclosure and the appended claims.

EXAMPLES

Example 1 Detection of the chimeric protein BT-0029, Bt29-Bt22, with insecticidal activity against the grass armyworm

[00379] Two Lepidoptera active proteins from Bacillus thuringiensis (Bt), BT-0022 (SEQ ID NO: 1; closest known member of the Cry family is Cry1If) and BT-0029 (SEQ ID NO: 2; closest known member of the Cry family -Cry1Gb) have been disclosed in PCT/US16/038947.

[00380] As shown in Table 1 below, the insect spectrum data for BT-0022 and BT-0029 indicate that BT-0022 has no activity against Fall Armyworm (FAW, Spodoptera frugiperda), while BT-0029 has only weak activity against FAW. Other Lepidoptera insect pests tested were: Corn borer (ECB; Ostrinia nubilalis), Upsilon cutworm (BCW; Agrotis ipsilon), Corn cutworm (CEW; Helicoverpa zea), Sugar cane stalk grinder (SCB; Diatraea saccharalis ), southwestern corn moth (SWCB; Diatraea grandiosella), western bean armyworm (WBCW; Striacosta albicosta), soybean armyworm (SBL; Pseudoplusia includens), velvet bean armyworm (VBC; Anticarsia gemmatalis) and tobacco leafworm (TBW; Heliothis virescens ).

[00381] A protein engineering approach was used in an attempt to increase the activity of BT-0029 against FAW. Using BT-0029 as a template, six engineered proteins were generated by replacing domain III (DIII) from BT-0029 with domain III from another Cry protein. Table 2 illustrates six chimeric proteins with information about their domain composition. Table 3 provides sequence information for full length proteins.

[00382] Encoding engineered cDNA proteins were synthesized at Genewiz (South Plainfield, NJ) and cloned into a Bacillus thuringiensis (Bt) expression vector containing the Cry1 Ac promoter and no terminator. Plasmid DNA was introduced into the Bt strain AB227 using an electroporation-mediated transformation procedure. All six engineered proteins were obtained as crystalline proteins in Bt. Crystalline proteins were purified from cultures and dissolved in buffer containing 50 mm Na ₂ CO ₃ , pH 11.0, 2 mm DTT. Each soluble protein was then evaluated in a biological assay using insects.

[00383] Briefly, the insecticidal activity of the chimeric proteins was examined by an insect bioassay based on an artificial diet in which dissolved crystalline proteins were placed on the surface of the artificial insect diet at a final concentration of 2-3.2 μg/cm ² . Buffer (50 mM Na ₂ CO ₃ , pH 11.0, 2 mM DTT) used to dissolve Bt crystalline proteins served as a negative control. The full-length Cry1Fa protein (SEQ ID NO: 8) was used as a positive control for FAW activity. Each chimeric protein was tested in duplicate. Insect mortality was assessed on the 7th day.

[00384] The results are shown in Table 4. When the chimeric proteins were tested in an insect bioassay, the Bt29-Bt22 chimera (SEQ ID NO: 3) showed strong insecticidal activity against FAW, while the other two chimeric proteins (Bt29-Bt67 and Bt29-3A) showed only minor activity against the pest. The Bt29-1Ab chimera did not show any detectable activity against FAW.

[00385] Insufficient Bt29-Bt51 and Bt29-8D chimera protein was obtained in this test to assess insecticidal activity.

Example 2. Insecticidal activity of chimeric proteins BT-0029 against various Lepidoptera pests

[00386] The four chimeric proteins described in Example 1 (Bt29-3A, Bt29-Bt22, Bt29-1Ab, and Bt29-Bt67) were tested for their insecticidal activity against the following Lepidoptera pests using art-known artificial diet bioassay methods: sugarcane stalk grinder (SCB; Diatraea saccharlis), southwestern corn moth (SWCB; Diatraea grandiosella), soybean armyworm (SBL; Pseudoplusia includens), corn cutworm (CEW; Helicoverpa zea), tobacco leaf roller (TBW; Heliothis virescens), epsilon cutworm (BCW; Agrotis ipsilon) and corn borer (ESB; Ostrinia nubilalis).

[00387] As shown in Table 5, all four chimeric proteins tested retained strong SBL activity of native BT-0029. Bt29-Bt22 and Bt29-Bt67 had weak SCB activity comparable to native BT-0029, while Bt29-3A and Bt29-lAb essentially lost all SCB activity. Slightly increased SWCB activity was observed with Bt29-Bt67 compared to full length BT-0029. Fairly consistent with the results in Table 1 for native BT-0029, only weak BCW activity was observed for Bt29-3A, Bt29-Bt22, and Bt29-Bt67, and none of the chimeras showed activity against CEW, TBW, or ECB.

Example 3. Bt29-1F has insecticidal activity against grass armyworm

[00388] To explore additional chimeric proteins, a second set of BT-0029 chimeric proteins was developed and tested (Table 6). Bt29-1Fa is a chimeric protein in which domain III from BT-0029 has been replaced with domain III from Cry1Fa. The full length amino acid sequence of Cry1Fa is shown as SEQ ID NO: 8 (see also Genbank accession number AAB00376.1). The amino acid sequence of the Bt29-1Fa chimera is shown as SEQ ID NO: 9, where the Cry1Fa sequence is amino acids 459-597.

[00389] Bt29-1Ka is a chimeric protein in which domain III from BT-0029 has been replaced with domain III from Cry1Ka. The full length amino acid sequence of Cry1Ka is shown as SEQ ID NO: 12 (see also Genbank accession number AAB00376.1). The amino acid sequence of the Bt29-1Ka chimera is also shown as SEQ ID NO: 13, where the Cry1Ka sequence is amino acids 459-597. The second variant of the Bt29-1Ka chimeric protein (herein referred to as Bt29-1Kav2) was generated by moving the junction between the Cry1Ka sequence and the BT-0029 sequence at the C-terminus of domain III from amino acid 598 (the transition in the first Bt chimera 29-1Ka) to amino acid 610. The amino acid sequence of the Bt29-1Kav2 chimera is shown as SEQ ID NO: 15, where the Cry1Ka sequence is amino acids 459-609.

[00390] Bt29-1Ca is a chimeric protein in which domain III from Bt-0029 has been replaced with domain III from Cry1Ca. The full length amino acid sequence of Cry1Ca is shown as SEQ ID NO: 17 (see also Genbank accession number AF362020.1). The amino acid sequence of the Bt29-1Ca chimera is shown as SEQ ID NO: 18, where the Cry1Ca sequence is amino acids 467-617.

[00391] The insecticidal activity of Bt29-1Fa (SEQ ID NO: 9), Bt29-1Ka (SEQ ID NO: 13, Bt29-1Kav2 (SEQ ID NO: 16) and Bt29-1Ca (SEQ ID NO: 18) chimeras was studied with using an insect bioassay based on an artificial diet in which dissolved proteins were placed on the surface of an artificial insect diet at a final concentration of 2 µg/cm ² Buffer (50 mM Na ₂ CO ₃ , pH 10.5, 2 mM DTT) used to dissolve Bt crystal proteins served as a negative control Full-length Cry1Fa protein (SEQ ID NO: 8) was used as a positive control for FAW activity Each chimeric protein was tested in triplicate Insecticidal activity was measured as effective mortality per 7 day 1 (larvae that are stunted and die are scored effectively dead).

[00392] As shown in Table 7, the chimeric protein designated Bt29-1Fa showed strong activity against FAW.

[00393] Insufficient Bt29-1Ka protein was generated in this test to be evaluated in a biological assay. Surprisingly, the protein derived from the Bt29-1Kav2 chimera was active against the grass cutworm (see Table 7).

Example 4 Bt29-Bt22 and Bt29-1Fa have insecticidal activity against Fall Armyworm resistant to Cry1F

[00394] To determine whether the toxicity of the BT-0029 chimeric proteins to FAW depends on the mode of action (MOA) other than Cry1Fa, two chimeric proteins, Bt29-Bt22 and Bt29-1Fa, were prepared as described in Example 1 and crystalline proteins were isolated and purified. Purified crystals were dissolved in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT) and the purity of the dissolved protein preparation was monitored using the Bio-Rad Experion System (BioRad, Hercules, CA).

[00395] The purified proteins were tested for efficacy against a FAW strain that is resistant to the Cry1Fa insecticidal toxin. Diet coated assays were performed with various doses of each purified toxin (125, 500 and 2000 ng/cm ² ), essentially as described in Example 1. Vip3A protein (positive control) was dissolved in PBS and other proteins in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT). The two negative control treatments were PBS and buffer 1. A FAW population that is susceptible to Cry1Fa or Vip3A was also tested, ie. susceptible FAW line. Each protein was tested in duplicate. Insecticidal activity was assessed as effective mortality at day 7 (larvae that are stunted and die are counted as effectively dead).

[00396] As shown in Table 8, susceptible FAW larvae were controlled with Cry1Fa, Vip3A, Bt29-Bt22 and Bt29-1Fa even at the lowest concentration. In contrast, the Cry1F-resistant FAW line was not controlled with any tested dose of Cry1Fa demonstrating that the line was resistant to this toxin (Table 9). Surprisingly, both Bt29-Bt22 and Bt29-1Fa were very effective in controlling Cry1F resistant FAW even at the very low dose of 125 ng/cm ² as shown in Table 9, indicating that the mode of action of the two chimeric proteins in relation to resistant FAW differs from the mode of action of the Cry1Fa protein.

Example 5 Bt29-Bt22 and Bt29-1Fa have insecticidal activity against Vip3A resistant Fall Armyworm

[00397] To determine whether the toxicity of the chimeric BT-0029 proteins depends on MOA other than the Vip3A protein, the Bt29-Bt22 and Bt29-1Fa proteins were prepared as described in Example 1, and the crystalline proteins were isolated and purified. Purified crystals were dissolved in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT) and the purity of the dissolved protein preparation was monitored using the Bio-Rad Experion System (BioRad, Hercules, CA).

[00398] The purified proteins were tested for efficacy against a FAW strain that is resistant to the Vip3A insecticidal toxin. Diet coated assays were performed with various doses of each purified toxin (125, 500 and 2000 ng/cm ² ), essentially as described in Example 1. Vip3A protein was dissolved in PBS and other proteins in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT). The two negative control treatments were PBS and buffer 1. Cry1Fa proteins were used as a positive control for the Vip3A resistant FAW line. Each protein was tested in duplicate. Insecticidal activity was assessed as effective mortality at day 7 (larvae that are stunted and die are assessed as effectively killed).

[00399] The Vip3A resistant FAW line was not controlled with any tested dose of Vip3A demonstrating that the line was resistant to this toxin (Table 10). In contrast, both Bt29-Bt22 and Bt29-1Fa showed a high degree of efficacy against Vip3A-resistant FAW, indicating that the mode of action of the two chimeric proteins against Vip3A-resistant FAW differs from that of the Vip3A protein.

Example 6 Truncations in the Protoxin Tail Region

[00400] It is well known that the protoxin form of the Cry Bt proteins undergoes processing at both the N- and C-terminus to produce the mature toxin. Six C-terminally truncated forms of the Bt29-Bt22 chimera (SEQ ID NO: 3), which contains the tail region of the protoxin BT-0029, were prepared as shown in Figure 3. All truncated variants, referred to herein as Bt29-Bt22Tr1 ( SEQ ID NO: 20), Bt29-Bt22Tr2 (SEQ ID NO: 21), Bt29-Bt22Tr3 (SEQ ID NO: 22), Bt29-Bt22Tr4 (SEQ ID NO: 23), Bt29-Bt22Tr5 (SEQ ID NO: 24) , Bt29-Bt22Tr6 (SEQ ID NO: 25), domain III of BT-0022 (ending in PVTA), and at least the first 6 amino acids of the tail of the protoxin BT-0029 (TFEAEY) were retained. Those skilled in the art will appreciate that the end of domain III and the start of the protoxin tail have not been precisely defined for BT-0029 or the chimeras described herein. The core domain III shown in Figures 1A and 1B is based on alignment with other Cry proteins in which the domain III region has been defined.

[00401] All truncated forms of Bt29-Bt22Tr1, -Tr2, -Tr4, and -Tr5 exhibited FAW activity as measured by growth inhibition (not mortality) in insect bioassays when tested as E. coli lysates. The Bt29-Bt22Tr1 and -Tr4 forms were the most promising, showing the highest activity in this assay (growth inhibition of 67% and 75%, respectively).

[00402] A second full length Bt29-Bt22 chimera was generated in which the protoxin tail region was derived from BT-0022 (Cry1I) instead of BT-0029 (Cry1G). Six C-terminally truncated forms of this full-length chimera, shown herein under SEQ ID NOs: 26-31, were prepared and tested against Fall Armyworm and Fall Armyworm. Cry1Fa was used as a positive control. The results are shown in Table 11.

[00403] Four truncated forms of Bt29-1Fa (SEQ ID NO: 9) that contain the tail region of the protoxin BT-0029, represented herein as Bt29-1FaTr1 (SEQ ID NO: 32), Bt29-1FaTr2 (SEQ ID NO: 33), Bt29-1FaTr3 (SEQ ID NO: 34) and Bt29-1FaTr4 (SEQ ID NO: 35) were generated in an E. coli expression system and the lysates were tested against the grass armyworm. The results are shown in Table 12.

Example 7 Expression and Activity of Bt29-Bt22 and Bt29-Cry1Fa Chimeric Proteins in Maize Plants

[00404] Prior to expression in plants, synthetic polynucleotides containing the nucleotide sequence encoding the Bt29-Bt22 and Bt29-1Fa chimera were synthesized using an automated gene synthesis platform (Genscript, Inc., Piscataway, NJ). In this example, a first and second expression cassette containing a maize ubiquitin promoter (Ubi1) operably linked to a chimeric sequence encoding Bt29-Bt22 or Bt29-1Fa that is operably linked to a maize ubiquitin terminator was prepared and a third expression cassette was prepared containing the Ubi1 promoter. , operably linked to a phosphomannose isomerase (PMI) coding sequence that is operably linked to the Ubi terminator. Expression of PMI allows positive selection of transgenic plants for mannose. For plant transformation, the first and third expression cassettes and the second and third expression cassettes were cloned into a suitable vector for Agrobacterium mediated maize transformation.

[00405] Transformation of immature maize germs was performed primarily as described in Negrotto et al., 2000, Plant Cell Reports 19: 798 803. Briefly, Agrobacterium LBA4404 (pSB1) containing an expression vector expressing Bt29-Bt22 or Bt29 Cry1Fa, grown on YEP solid medium (yeast extract (5 g/l), peptone (10 g/l), NaCl (5 g/l), 15 g/l agar, pH 6.8) for 2-4 days at 28°C. Approximately 0.8×10 ⁹ Agrobacterium cells were suspended in LS-inf medium supplemented with 100 μM As. The bacteria were pre-induced in this medium for about 30-60 minutes.

[00406] Immature embryos of the maize inbred line were excised from 8-12 day old cobs and transferred to LS-inf + 100 μM As liquid medium. The embryos were rinsed once with fresh infection medium. The Agrobacterium solution was then added and the embryos were vortexed for 30 seconds and left to settle with the bacteria for 5 minutes. The embryos were then transferred with the scutellum side up onto LSAs medium and cultured in the dark for two to three days. Subsequently, about 20-25 embryos per petri dish were transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark at about 28° C. for 10 days.

[00407] Immature embryos forming embryogenic callus were transferred to LSD1M0.5S medium. Selection of cultures on this medium was carried out for about 6 weeks, with a subcultivation step being performed after about 3 weeks. Surviving calluses were transferred to Reg1 medium supplemented with mannose. After culturing in light (16 hours light/8 hours dark), the green tissues were then transferred to Reg2 medium without growth regulators and incubated for approximately 1-2 weeks. Seedlings were transferred to Magenta GA-7 containers (Magenta Corp, Chicago, IL) containing Reg3 medium and grown in the light. Approximately 2-3 weeks later, the plants were tested by PCR for the presence of the PMI genes as well as the chimeric Bt cry gene. Plants that showed positive results in PCR analysis were transferred to the greenhouse for further evaluation.

[00408] Transgenic plants were evaluated for copy number (determined by Taqman assay), protein expression level (determined by ELISA), and potency against insect species of interest in leaf-cut bioassays. Specifically, plant tissue (leaf or stigma) was excised from single copy transformants (stages V3-V4) and infected with newborn larvae of the target pest, then incubated at room temperature for 5 days. Leaf disks from transgenic plants expressing each Bt chimeric protein were tested against the grass cutworm (Spodoptera frugiperda, FAW).

[00409] The results confirm that the transgenic plants express the chimeric proteins of the present invention and are active against insect pests. Protein expression in transgenic transformants for the Bt29-Bt22 chimera was approximately 25-125 ng/mg total soluble protein (TSP) and for the Bt29-1Fa chimera was approximately 25-290 ng/mg TSP. Approximately 90% and 92% of the transgenic transformants expressing the Bt29-Bt22 and Bt29-1Fa chimeras, respectively, resulted in 100% mortality and growth suppression of the grass cutworm larvae.

[00410] The examples presented above clearly illustrate the advantages of the present invention. While the present invention has been described in terms of specific details of some of its embodiments, such details are not intended to be considered limitations on the scope of the claimed invention, except and to the extent that they are included in the appended claims of the present invention.

Claims (32)

1. A chimeric insecticidal protein that is toxic to a Lepidoptera insect pest, comprising the amino acid sequence of SEQ ID NO: 3 or amino acids 1 to 597 of SEQ ID NO: 3. 2. The chimeric insecticidal protein according to claim 1, wherein the chimeric insecticidal protein comprises SEQ ID NO: 3. 3. The chimeric insecticidal protein according to claim 1 or 2, wherein the chimeric insecticidal protein has insecticidal activity against the Vip3A protein or Cry1F protein resistant insect pest Spodoptera frugiperda . 4. Polynucleotide encoding insecticidal protein according to claims. 1-3, containing (a) a nucleotide sequence encoding a chimeric insecticidal protein according to any one of paragraphs. 1-3; or (b) a nucleotide sequence according to (a) that is codon-optimized for expression in a plant. 5. A polynucleotide according to claim 4, wherein the polynucleotide contains the nucleotide sequence of SEQ ID NO: 4. 6. Nucleic acid molecule to ensure the expression of the insecticidal protein according to paragraphs. 1-3, containing a polynucleotide according to claim 4 or 5, functionally linked to a heterologous promoter that provides expression in the plant. 7. A vector for the expression of an insecticidal protein, containing a nucleic acid molecule according to claim 6. 8. Transgenic cell to obtain an insecticidal protein according to any one of paragraphs. 1-3 containing a polynucleotide according to claim 4 or 5, where the transgenic cell is a bacterial cell or a plant cell. 9. Transgenic plant cell according to claim 8, where the plant cell is: (a) a monocot cage, optionally a barley cage, a maize cage, an oat cage, a rice cage, a sorghum cage, a sugarcane cage, or a wheat cage; or (b) a dicot cell, optionally a soybean cell, a sunflower cell, a tomato cell, a cabbage cultivar cell, a cotton cell, a sugar beet cell, or a tobacco cell. 10. A transgenic plant for increasing resistance to Lepidoptera insect pests, containing a transgenic plant cell according to claim 9, where the plant is: (a) a monocotyledonous plant, optionally a barley plant, a maize plant, an oat plant, a rice plant, a sorghum plant, a sugar cane plant, or a wheat plant; or (b) a dicotyledonous plant, optionally a soybean plant, a sunflower plant, a tomato plant, a cabbage plant, a cotton plant, a sugar beet plant, or a tobacco plant. 11. A transgenic plant for increasing resistance to Lepidoptera insect pests according to claim 10, wherein the transgenic plant further comprises a nucleotide sequence encoding a second insect control agent. 12. A plant part from a transgenic plant for increasing resistance to Lepidoptera pests according to claim 10, wherein the transgenic plant part contains a chimeric insecticidal protein. 13. Transgenic seed of a transgenic plant to increase resistance to insect pests according to claim 10, related to Lepidoptera, where the seed contains a chimeric insecticidal protein according to any one of paragraphs. 1-3. 14. A harvested product with resistance to lepidoptera insect pests and obtained from a transgenic plant according to claim 10, where the harvested product contains a chimeric insecticidal protein. 15. Lepidoptera pest resistant processed product obtained from the harvested product of claim 14, wherein the processed product is flour, meal, oil, starch, or a product derived from any of the above. 16. Insecticidal composition, toxic to insect pests related to Lepidoptera, containing a chimeric insecticidal protein according to any one of paragraphs. 1-3 and an agriculturally acceptable carrier. 17. A method for producing a transgenic plant with increased resistance to a Lepidoptera pest, the method comprising: (a) introducing into the plant by transformation of the plant cell a polynucleotide according to claim 4 or 5, wherein the chimeric insecticidal protein is expressed in the plant, thereby obtaining a transgenic plant with increased resistance to an insect pest, and optionally obtaining a progeny plant from the transgenic plant where the progeny plant contains a polynucleotide and is characterized by increased resistance to insect pests; or (b) sowing a seed containing the polynucleotide and growing a transgenic plant from the seed, wherein the transgenic plant contains the polynucleotide and produces the chimeric insecticidal protein. 18. The method of claim 17, wherein the method further comprises harvesting seed from the transgenic plant of (b), wherein the harvested seed contains the chimeric insecticidal protein. 19. A method for obtaining a seed of a transgenic plant, the method comprising: (a) obtaining a transgenic plant containing a polynucleotide according to claim 4 or 5; and (b) enabling the formation of transgenic seed. 20. A method for reducing a population of a Lepidoptera insect pest, the method comprising delivering to the body of the pest or to its habitat a composition containing an effective amount of a chimeric insecticidal protein according to any one of paragraphs. 1-3, where the Lepidoptera pest is susceptible or resistant to the Vip3 protein or the Cry1F protein. 21. A method for reducing the development of resistance to the Vip3A protein or the Cry1F protein in a population of a target Lepidoptera pest, the method comprising delivering a transgenic plant to the target population or its habitat, containing: (i) a polynucleotide according to claim 4 or 5; and (ii) a polynucleotide containing a nucleotide sequence encoding a Vip3A protein or a nucleotide sequence encoding a Cry1F protein; wherein the chimeric insecticidal protein and the Vip3A protein or the Cry1F protein are produced in the transgenic plant.

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