Insecticide protein and method of using the same

Abstract

The present invention relates to insecticidal proteins and methods of using them. Compositions and methods for controlling harmful organisms are provided. These methods involve transforming organisms with nucleic acid sequences encoding insecticidal proteins. Specifically, these nucleic acid sequences can be used to prepare plants and microorganisms with insecticidal activity. Therefore, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions are insecticidal nucleic acids and proteins of bacterial species. These sequences have uses in constructing expression vectors that are subsequently transformed into organisms of interest, including plants, acting as probes for isolating other homologous (or partially homologous) genes. These insecticidal proteins can be used to control, inhibit growth or kill Lepidoptera, Coleoptera, Diptera, Fungi, Hemiptera and nematode pest populations and can be used to produce compositions with insecticidal activity.

Description

[0001] This application is a divisional application of Chinese patent application 201680016008.4 “Insecticide protein and method of use thereof” filed on January 7, 2016.

[0002] References to sequence listings submitted electronically

[0003] The sequence listing with the file name "5914-PCT_seq_list.txt" was created on November 19, 2015, is 542 kilobytes in size, is in a computer readable form, and is filed with this specification. The sequence listing is part of this specification and is incorporated herein by reference in its entirety.

[0004] Government support

[0005] The Government has certain rights in this invention under Agreement No. LB09005376. Technical Field

[0006] The present disclosure relates to the field of molecular biology. Provided are novel genes encoding pesticidal proteins. These pesticidal proteins and nucleic acid sequences encoding them can be used to prepare pesticidal formulations and can be used to produce transgenic pest-resistant plants. Background Art

[0007] The use of microbial agents (such as fungi, bacteria or other insect species) to control insect pests with agricultural significance provides an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. In general, the use of biological pesticides has a lower risk of pollution and environmental damage, and biological pesticides provide a stronger target specificity than traditional broad-spectrum chemical insecticides. In addition, biological pesticides often have lower production costs and can therefore increase the economic yield of various crops.

[0008] Certain species of Bacillus microorganisms are known to have pesticidal activity against a range of insect pests, including Lepidoptera, Diptera, Coleoptera, Hemiptera, etc. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biological control agents discovered to date. Insect pathogenicity is also attributed to strains of Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus, and Bacillus cereus. Microbial insecticides, particularly those obtained from Bacillus strains, play an important role in agriculture as an alternative to chemical control of pests.

[0009] By carrying out genetic engineering to produce pest-killing proteins from Bacillus, crop plants with enhanced insect resistance have been developed.For example, corn and cotton plants have been genetically engineered to produce pest-killing proteins separated from Bt strains.Now, these genetically engineered crops are widely used in agriculture, and provide farmers with an environmentally friendly alternative to replace traditional insect control methods.Although they have been proven to be very successful commercially, these genetically engineered insect-resistant crop plants only provide resistance to economically important insect pests in a narrow range.In some cases, insects can produce resistance to different insecticide compounds, which just causes the need to identify the alternative biological control agents for pest control.

[0010] Thus, there remains a need for new pesticidal proteins having a diverse range of insecticidal activity against insect pests, for example, insecticidal proteins active against a variety of insects of the Lepidoptera and Coleoptera orders, including but not limited to insect pests that have developed resistance to existing insecticides. Summary of the invention

[0011] Provided are compositions and methods for imparting pesticidal activity to bacteria, plants, plant cells, tissues and seeds. The compositions comprise nucleic acid molecule coding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising those vectors. The compositions also include these pesticidal polypeptide sequences and antibodies against those polypeptides. These nucleic acid sequences can be used in DNA constructs or expression cassettes for conversion and expression in a variety of organisms (including microorganisms and plants). These nucleotide or amino acid sequences can be synthetic sequences that have been designed for expression in organisms including, but not limited to, microorganisms or plants. The compositions also include transformed bacteria, plants, plant cells, tissues, and seeds.

[0012] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-45-1 (PIP-45-1) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-45-1 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-45-1 polypeptide of SEQ ID NO: 1 are provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-45-1 polypeptides of SEQ ID NO: 1, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0013] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-45-2 (PIP-45-2) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-45-2 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules capable of encoding the PIP-45-2 polypeptide of SEQ ID NO: 2 are provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-45-2 polypeptides of SEQ ID NO: 2, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0014] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-64-1 (PIP-64-1) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-64-1 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-64-1 polypeptide of SEQ ID NO: 53 and amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof are provided. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-64-1 polypeptides of SEQ ID NO: 53 and amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0015] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-64-2 (PIP-64-2) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-64-2 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-64-2 polypeptide of SEQ ID NO: 54 are provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-64-2 polypeptides of SEQ ID NO: 54, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0016] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-74-1 (PIP-74-1) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-74-1 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-74-1 polypeptide of SEQ ID NO: 73 and amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof are provided. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-74-1 polypeptides of SEQ ID NO: 73 and amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0017] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-74-2 (PIP-74-2) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-74-2 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-74-2 polypeptide of SEQ ID NO: 74 are provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-74-2 polypeptides of SEQ ID NO: 74, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0018] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-75 (PIP-75) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-75 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-75 polypeptide of SEQ ID NO: 79 are provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-75 polypeptides of SEQ ID NO: 79, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0019] In particular, isolated or recombinant nucleic acid molecules encoding Pseudomonas insecticidal protein-77 (PIP-77) polypeptides are provided, including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In addition, amino acid sequences corresponding to these PIP-77 polypeptides are contemplated. Isolated or recombinant nucleic acid molecules encoding the PIP-77 polypeptide of SEQ ID NO: 88 are provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Also contemplated are nucleic acid sequences that are complementary to the nucleic acid sequences of the embodiments or that hybridize to the sequences of the embodiments. Also provided are isolated or recombinant PIP-77 polypeptides of SEQ ID NO: 88, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0020] Provided are methods for producing insecticidal polypeptides, and using these polypeptides to control or kill Lepidoptera, Coleoptera, nematodes, fungi, and / or dipteran pests. The transgenic plants of the embodiments express one or more insecticidal sequences disclosed herein. In various embodiments, the transgenic plants further comprise one or more other insect resistance genes, for example, one or more other genes for controlling Coleoptera, Lepidoptera, Hemiptera or nematode pests. It will be appreciated by those skilled in the art that the transgenic plants may comprise any gene that confers an agronomic trait of interest.

[0021] Also included are methods for detecting these nucleic acids and polypeptides of the embodiments in a sample. Kits for detecting the presence of the insecticidal polypeptides of the present disclosure or detecting the presence of nucleotide sequences encoding the insecticidal polypeptides of the present disclosure in a sample are provided. The kits can be provided with all reagents and control samples necessary to perform the method for detecting the intended agent, as well as instructions for use.

[0022] These compositions and methods of the embodiments can be used to produce organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising these organisms are desirable for agricultural purposes. The compositions of the embodiments can also be used to produce altered or improved proteins with pesticidal activity, or can be used to detect the presence of the insecticidal polypeptides of the present disclosure or nucleic acids encoding them in products or organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1a-1mThe amino acid sequence alignment of the following is shown: PIP-45Aa-1 (SEQ ID NO: 1), PIP-45Ab-1 (SEQ ID NO: 3), PIP-45Ac-1 (SEQ ID NO: 5), PIP-45Ad-1 (SEQ ID NO: 7), PIP-45Ae-1 (SEQ ID NO: 9), PIP-45Af-1 (SEQ ID NO: 11), PIP-45Ba-1 (SEQ ID NO: 13), PIP-45Bb-1 (SEQ ID NO: 15), PIP-45Bc-1 (SEQ ID NO: 17), PIP-45Bd-1 (SEQ ID NO: 19), PIP-45Be-1 (SEQ ID NO: 21), PIP-45Bf-1 (SEQ ID NO: 23), PIP-45Bg-1 (SEQ ID NO: 25), PIP-45Bh-1 (SEQ ID NO: 26), PIP-45 NO: 27), PIP-45Bi-1 (SEQ ID NO: 29), PIP-45Bj-1 (SEQ ID NO: 31), PIP-45Bk-1 (SEQ ID NO: 33), PIP-45Bl-1 (SEQ ID NO: 232), PIP-45Bm-1 (SEQ ID NO: 234), PIP-45Ca-1 (SEQ ID NO: 35), PIP-45Cb-1 (SEQ ID NO: 37), PIP-45Cc-1 (SEQ ID NO: 39), PIP-45Cd-1 (SEQ ID NO: 41), PIP-45Ce-1 (SEQ ID NO: 43), PIP-45Cf-1 (SEQ ID NO: 236), PIP-45Da-1 (SEQ ID NO: 45), PIP-45Db-1 (SEQ ID NO: 47), PIP-45Ea-1 (SEQ ID NO: 49), and PIP-45Ga-1 (SEQ ID NO: 51). Amino acid diversity between PIP-45-1 polypeptide homologues is indicated by shading.

[0024] Figure 2a-2lAn alignment of the amino acid sequences of PIP-45Aa-2 (SEQ ID NO: 2), PIP-45Ab-2 (SEQ ID NO: 4), PIP-45Ac-2 (SEQ ID NO: 6), PIP-45Ad-2 (SEQ ID NO: 8), PIP-45Ae-2 (SEQ ID NO: 10), PIP-45Af-2 (SEQ ID NO: 12), PIP-45Ba-2 (SEQ ID NO: 14), PIP-45Bb-2 (SEQ ID NO: 16), PIP-45Bc-2 (SEQ ID NO: 18), PIP-45Bd-2 (SEQ ID NO: 20), PIP-45Be-2 (SEQ ID NO: 22), PIP-45Bf-2 (SEQ ID NO: 24), PIP-45Bg-2 (SEQ ID NO: 26), PIP-45Bh-2 (SEQ ID NO: 27), PIP-45Bh-2 (SEQ ID NO: 28), PIP-45Bh-2 (SEQ ID NO: 29). NO: 28), PIP-45Bi-2 (SEQ ID NO: 30), PIP-45Bj-2 (SEQ ID NO: 32), PIP-45Bk-2 (SEQ ID NO: 34), PIP-45Bl-2 (SEQ ID NO: 233), PIP-45Bm-2 (SEQ ID NO: 235), PIP-45Ca-2 (SEQ ID NO: 36), PIP-45Cb-2 (SEQ ID NO: 38), PIP-45Cc-2 (SEQ ID NO: 40), PIP-45Cd-2 (SEQ ID NO: 42), PIP-45Ce-2 (SEQ ID NO: 44), PIP-45Cf-2 (SEQ ID NO: 237), PIP-45Da-2 (SEQ ID NO: 46), PIP-45Db-2 (SEQ ID NO: 48), PIP-45Ea-2 (SEQ ID NO: 50), and PIP-45Ga-2 (SEQ ID NO: 52). Amino acid diversity between PIP-45-2 polypeptide homologues is indicated by shading.

[0025] Figure 3a-3bAlignment of the amino acid sequences of PIP-64Aa-1 (SEQ ID NO: 53), PIP-64Ba-1 (SEQ ID NO: 238), PIP-64Ca-1 (SEQ ID NO: 56), PIP-64Ea-1 (SEQ ID NO: 58), PIP-64Eb-1 (SEQ ID NO: 60), PIP-64Ec-1 (SEQ ID NO: 62), PIP-64Ga-1 (SEQ ID NO: 64), PIP-64Ha-1 (SEQ ID NO: 65), PIP-64Hb-1 (SEQ ID NO: 67), PIP-64Hc-1 (SEQ ID NO: 69), and PIP-64Hd-1 (SEQ ID NO: 71) is shown. Amino acid diversity between PIP-64-1 polypeptide homologs is indicated by shading.

[0026] Figure 4a-4b Alignment of the amino acid sequences of PIP-64Aa-2 (SEQ ID NO: 54), PIP-64Ab-2 (SEQ ID NO: 55), PIP-64Ba-2 (SEQ ID NO: 239), PIP-64Ca-2 (SEQ ID NO: 57), PIP-64Ea-2 (SEQ ID NO: 59), PIP-64Eb-2 (SEQ ID NO: 61), PIP-64Ec-2 (SEQ ID NO: 63), PIP-64Ha-2 (SEQ ID NO: 66), PIP-64Hb-2 (SEQ ID NO: 68), PIP-64Hc-2 (SEQ ID NO: 70), and PIP-64Hd-2 (SEQ ID NO: 72) are shown. Amino acid diversity between PIP-64-2 polypeptide homologs is indicated by shading.

[0027] Figure 5a-5b An alignment of the amino acid sequences of PIP-74Aa-1 (SEQ ID NO: 73), PIP-74Ab-1 (SEQ ID NO: 75), and PIP-74Ca-1 (SEQ ID NO: 77) is shown. Amino acid diversity between PIP-74-1 polypeptide homologs is indicated by shading.

[0028] Figure 6An alignment of the amino acid sequences of PIP-74Aa-2 (SEQ ID NO: 74), PIP-74Ab-2 (SEQ ID NO: 76), and PIP-74Ca-2 (SEQ ID NO: 78) is shown. Amino acid diversity between PIP-74-2 polypeptide homologues is indicated by shading.

[0029] Figure 7 An alignment of the amino acid sequences of PIP-75Aa (SEQ ID NO: 79), PIP-75Ba (SEQ ID NO: 80), PIP-75Da (SEQ ID NO: 81), PIP-75Ea (SEQ ID NO: 82), PIP-75Ga (SEQ ID NO: 83), PIP-75Gb (SEQ ID NO: 84), PIP-75Gc (SEQ ID NO: 85), PIP-75Gd (SEQ ID NO: 86), PIP-75Ge (SEQ ID NO: 87) is shown. Amino acid diversity between PIP-75 polypeptide homologues is indicated by shading.

[0030] Figure 8a-8bAn alignment of the amino acid sequences of PIP-77Aa (SEQ ID NO: 88), PIP-77Ab (SEQ ID NO: 89), PIP-77Ac (SEQ ID NO: 90), PIP-77Ad (SEQ ID NO: 91), PIP-77Ae (SEQ ID NO: 92), PIP-77Af (SEQ ID NO: 240), PIP-77Ba (SEQ ID NO: 93), PIP-77Bb (SEQ ID NO: 94), PIP-77Bc (SEQ ID NO: 95), PIP-77Bd (SEQ ID NO: 96), PIP-77Be (SEQ ID NO: 97), PIP-77Bf (SEQ ID NO: 98), PIP-77Bg (SEQ ID NO: 99), PIP-77Bh (SEQ ID NO: 241), PIP-77Bi (SEQ ID NO: 242), PIP-77Ca (SEQ ID NO: 243), PIP-77Bh (SEQ ID NO: 244), PIP-77Bh (SEQ ID NO: 245), PIP-77Bh (SEQ ID NO: 246), PIP-77Bh (SEQ ID NO: 247), PIP-77Bh (SEQ ID NO: 248), PIP-77Bh (SEQ ID NO: 249), PIP-77Bc (SEQ ID NO: 300), PIP-77Bd (SEQ ID NO: 311), PIP-77Bh (SEQ ID NO: 312), PIP-77Bh (SEQ ID NO: 313), PIP-77Bh (SEQ ID NO: 314), PIP-77Bh (SEQ ID NO: 315), PIP-77Bh (SEQ ID NO: 316), PIP-77Bh (SEQ ID NO: 317), PIP-77Bh (SEQ ID NO: NO: 100), PIP-77Ea (SEQ ID NO: 101), PIP-77Eb (SEQ ID NO: 102), PIP-77Ec (SEQ ID NO: 103), PIP-77Ed (SEQ ID NO: 104), PIP-77Ee (SEQ ID NO: 105), PIP-77Ef (SEQ ID NO: 106), PIP-77Eg (SEQ ID NO: 107), PIP-77Eh (SEQ ID NO: 243), PIP-77Ei (SEQ ID NO: 244), and PIP-77Ej (SEQ ID NO: 245). Amino acid diversity between PIP-77 polypeptide homologs is indicated by shading. DETAILED DESCRIPTION

[0031] It is to be understood that the present disclosure is not limited to the specific methods, protocols, cell lines, genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

[0032] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "a protein" includes one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless expressly stated otherwise.

[0033] The present disclosure relates to compositions and methods for controlling harmful organisms. These methods relate to transforming organisms with nucleic acid sequences encoding insecticidal polypeptides disclosed herein. In particular, the nucleic acid sequences of the embodiments can be used to prepare plants and microorganisms with pesticidal activity. Therefore, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are pesticidal nucleic acids and proteins of bacterial species. These nucleic acid sequences can be used to construct expression vectors for subsequent transformation into organisms of interest, as probes for isolating other homologous (or partially homologous) genes, and for producing altered insecticidal polypeptides by methods known in the art (such as site-directed mutagenesis, domain exchange or DNA shuffling). The insecticidal polypeptides disclosed herein can be used to control or kill Lepidoptera, Coleoptera, Diptera, Fungi, Hemiptera and nematode pest populations and can be used to produce compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species, including, but not limited to, diamondback moth, e.g., Helicoverpa zea Boddie; soybean armyworm, e.g., Pseudoplusia includens Walker; and velvet bean caterpillar, e.g., Anticarsia gemmatalis Hübner, and Coleoptera species, including, but not limited to, Western corn rootworm (Diabrotica virgifera) - WCRW, Southern corn rootworm (Diabrotica undecimpunctata howardi) - SCRW, and Northern corn rootworm (Diabrotica barberi) - NCRW.

[0034] "Pesticide toxin" or "pesticide protein" is used herein to refer to a toxin or a protein homologous to such a protein that has toxic activity against one or more pests including, but not limited to, members of the orders Lepidoptera, Diptera, Hemiptera, and Coleoptera or Nematoda. Pesticide proteins have been isolated from organisms including, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans, and Paenibacillus popilliae. Pesticide proteins include, but are not limited to, insecticide proteins from Pseudomonas species, such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7: 1-13 [Monalysin; (2011), PLoS Pathogens, 7: 1-13]); Pseudomonas protegens strains CHA0 and Pf-5 (formerly fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10: 2368-2386; GenBank Accession No. EU400157 [Pechy-Tarr, (2008), Environmental Microbiology, 10: 2368-2386; GenBank Accession No. EU400157]); Pseudomonas Taiwanensis (Liu, et al. al., (2010) J. Agric. Food Chem., 58: 12343-12349 [Liu et al., (2010), Journal of Agricultural and Food Chemistry, 58: 12343-12349]) and Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59: 45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168 [Zhang et al., (2009), Annals of Microbiology, 59: 45-50 and Li et al., (2007), Plant Cell, Tissue and Organ Culture, 89: 159-168]);Insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3: 101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67: 2062-2069); U.S. Pat. No. 6,048,838 and U.S. Pat. No. 6,379,946; PIP-1 polypeptides from U.S. Pat. No. US 2014-0007292 A1; U.S. Pat. No. US AfIP-1A and / or AfIP-1B polypeptides of 2014-0033361; PHI-4 polypeptides of U.S. Serial No. 13 / 839702; PIP-47 polypeptides of PCT Serial No. PCT / US 14 / 51063; PHI-4 polypeptides of U.S. Patent Publication US 20140274885 or PCT Patent Publication WO 2014 / 150914; PIP-72 polypeptides of PCT Serial No. PCT / US 14 / 55128; Insecticidal proteins of U.S. Serial Nos. 61 / 863761 and 61 / 863763;and delta-endotoxins, including but not limited to: Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38 Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71 and Cry72 class delta-endotoxin genes and Bacillus thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes of Bacillus thuringiensis insecticidal proteins include, but are not limited to, Cry1Aa1 (Accession No. AAA22353); Cry1Aa2 (Accession No. AAA22552); Cry1Aa3 (Accession No. BAA00257); Cry1Aa4 (Accession No. CAA31886); Cry1Aa5 (Accession No. BAA04468); Cry1Aa6 (Accession No. AAA86265); Cry1Aa7 (Accession No. AAD46139); Cry1Aa8 (Accession No. I26149); Cry1Aa9 (Accession No. BAA77213); Cry1Aa10 (Accession No. AAD55382); Cry1Aa11 (Accession No. CAA70856); Cry1Aa12 (Accession No. AAD46139); Cry1Aa13 (Accession No. AAD46139); Cry1Aa14 (Accession No. AAD46139); Cry1Aa15 (Accession No. AAD46139); Cry1Aa16 (Accession No. I26149); Cry1Aa17 (Accession No. AAD46139); Cry1Aa18 (Accession No. I26149); Cry1Aa19 (Accession No. BAA77213); Cry1Aa10 (Accession No. AAD55382); Cry1Aa11 (Accession No. CAA70856); Cry1Aa12 (Accession No. AAD46139); Cry1Aa1 AP80146); Cry1Aa13 (Accession No. AAM44305); Cry1Aa14 (Accession No. AAP40639); Cry1Aa15 (Accession No. AAY66993); Cry1Aa16 (Accession No. HQ439776); Cry1Aa17 (Accession No. HQ439788); Cry1Aa18 (Accession No. HQ439790); Cry1Aa19 (Accession No. HQ685121); Cry1Aa20 (Accession No. JF340156); Cry1Aa21 (Accession No. JN651496); Cry1Aa22 (Accession No. KC158223); Cry1Ab1 (Accession No. AAA22330); Cry1Ab2 (Accession No. AAA22613);Cry1Ab3 (Accession No. AAA22561); Cry1Ab4 (Accession No. BAA00071); Cry1Ab5 (Accession No. CAA28405); Cry1Ab6 (Accession No. AAA22420); Cry1Ab7 (Accession No. CAA31620); Cry1Ab8 (Accession No. AAA22551); Cry1Ab9 (Accession No. CAA38701); Cry1Ab10 (Accession No. A29125); Cry1Ab11 (Accession No. I12419); Cry1Ab12 (Accession No. AAC64003); Cry1Ab13 (Accession No. AAN76494); Cry1Ab14 (Accession No. AAG1 6877); Cry1Ab15 (Accession No. AAO13302); Cry1Ab16 (Accession No. AAK55546); Cry1Ab17 (Accession No. AAT46415); Cry1Ab18 (Accession No. AAQ88259); Cry1Ab19 (Accession No. AAW31761); Cry1Ab20 (Accession No. ABB72460); Cry1Ab21 (Accession No. ABS18384); Cry1Ab22 (Accession No. ABW87320); Cry1Ab23 (Accession No. HQ439777); Cry1Ab24 (Accession No. HQ439778); Cry1Ab25 (Accession No. HQ685122) ; Cry1Ab26 (Accession No. HQ847729); Cry1Ab27 (Accession No. JN135249); Cry1Ab28 (Accession No. JN135250); Cry1Ab29 (Accession No. JN135251); Cry1Ab30 (Accession No. JN135252); Cry1Ab31 (Accession No. JN135253); Cry1Ab32 (Accession No. JN135254); Cry1Ab33 (Accession No. AAS93798); Cry1Ab34 (Accession No. KC156668); Cry1Ab-like (Accession No. AAK14336); Cry1Ab-like (Accession No. AAK14337); Cry1Ab Cry1Ac1 (Accession No. AAA22331); Cry1Ac2 (Accession No. AAA22338); Cry1Ac3 (Accession No. CAA38098); Cry1Ac4 (Accession No. AAA73077); Cry1Ac5 (Accession No. AAA22339); Cry1Ac6 (Accession No. AAA86266); Cry1Ac7 (Accession No. AAB46989); Cry1Ac8 (Accession No. AAC44841); Cry1Ac9 (Accession No. AAB49768); Cry1Ac10 (Accession No. CAA05505);Cry1Ac11 (Accession No. CAA10270); Cry1Ac12 (Accession No. I12418); Cry1Ac13 (Accession No. AAD38701); Cry1Ac14 (Accession No. AAQ06607); Cry1Ac15 (Accession No. AAN07788); Cry1Ac16 (Accession No. AAU87037); Cry1Ac17 (Accession No. AAX18704); Cry1Ac18 (Accession No. AAY88347); Cry1Ac19 (Accession No. ABD37053); Cry1Ac20 (Accession No. ABB89046); Cry1Ac21 (Accession No. AAY66992); Cry1Ac2 2 (accession number ABZ01836); Cry1Ac23 (accession number CAQ30431); Cry1Ac24 (accession number ABL01535); Cry1Ac25 (accession number FJ513324); Cry1Ac26 (accession number FJ617446); Cry1Ac27 (accession number FJ617447); Cry1Ac28 (accession number ACM90319); Cry1Ac29 (accession number DQ438941); Cry1Ac30 (accession number GQ227507); Cry1Ac31 (accession number GU446674); Cry1Ac32 (accession number HM061081); Cry1Ac33 (accession number G Q866913); Cry1Ac34 (Accession No. HQ230364); Cry1Ac35 (Accession No. JF340157); Cry1Ac36 (Accession No. JN387137); Cry1Ac37 (Accession No. JQ317685); Cry1Ad1 (Accession No. AAA22340); Cry1Ad2 (Accession No. CAA01880); Cry1Ae1 (Accession No. AAA22410); Cry1Af1 (Accession No. AAB82749); Cry1Ag1 (Accession No. AAD46137); Cry1Ah1 (Accession No. AAQ14326); Cry1Ah2 (Accession No. ABB76664); Cry 1Ah3 (Accession No. HQ439779); Cry1Ai1 (Accession No. AAO39719); Cry1Ai2 (Accession No. HQ439780); Cry1A-like (Accession No. AAK14339); Cry1Ba1 (Accession No. CAA29898); Cry1Ba2 (Accession No. CAA65003); Cry1Ba3 (Accession No. AAK63251); Cry1Ba4 (Accession No. AAK51084); Cry1Ba5 (Accession No. ABO20894); Cry1Ba6 (Accession No. ABL60921); Cry1Ba7 (Accession No. HQ439781); Cry1Bb1 (Accession No. AAA22344);Cry1Bb2 (Accession No. HQ439782); Cry1Bc1 (Accession No. CAA86568); Cry1Bd1 (Accession No. AAD10292); Cry1Bd2 (Accession No. AAM93496); Cry1Be1 (Accession No. AAC32850); Cry1Be2 (Accession No. AAQ52387); Cry1Be3 (Accession No. ACV96720); Cry1Be4 (Accession No. HM070026); Cry1Bf1 (Accession No. CAC50778); Cry1Bf2 (Accession No. AAQ52380); Cry1Bg1 (Accession No. AAO39720); Cry1Bh1 (Accession No. HQ589 331); Cry1Bi1 (Accession No. KC156700); Cry1Ca1 (Accession No. CAA30396); Cry1Ca2 (Accession No. CAA31951); Cry1Ca3 (Accession No. AAA22343); Cry1Ca4 (Accession No. CAA01886); Cry1Ca5 (Accession No. CAA65457); Cry1Ca6[1] (Accession No. AAF37224); Cry1Ca7 (Accession No. AAG50438); Cry1Ca8 (Accession No. AAM00264); Cry1Ca9 (Accession No. AAL79362); Cry1Ca10 (Accession No. AAN16462); Cry1Ca11 (Accession No. AAX53094); Cry1Ca12 (Accession No. HM070027); Cry1Ca13 (Accession No. HQ412621); Cry1Ca14 (Accession No. JN651493); Cry1Cb1 (Accession No. M97880); Cry1Cb2 (Accession No. AAG35409); Cry1Cb3 (Accession No. ACD50894); Cry1Cb-like (Accession No. AAX63901); Cry1Da1 (Accession No. CAA38099); Cry1Da2 (Accession No. I76415); Cry1Da3 (Accession No. HQ439784); Cry1Db1 (Accession No. CAA80234); Cry 1Db2 (Accession No. AAK48937); Cry1Dc1 (Accession No. ABK35074); Cry1Ea1 (Accession No. CAA37933); Cry1Ea2 (Accession No. CAA39609); Cry1Ea3 (Accession No. AAA22345); Cry1Ea4 (Accession No. AAD04732); Cry1Ea5 (Accession No. A15535); Cry1Ea6 (Accession No. AAL50330); Cry1Ea7 (Accession No. AAW72936); Cry1Ea8 (Accession No. ABX11258); Cry1Ea9 (Accession No. HQ439785); Cry1Ea10 (Accession No. ADR00398);Cry1Ea11 (Accession No. JQ652456); Cry1Eb1 (Accession No. AAA22346); Cry1Fa1 (Accession No. AAA22348); Cry1Fa2 (Accession No. AAA22347); Cry1Fa3 (Accession No. HM070028); Cry1Fa4 (Accession No. HM439638); Cry1Fb1 (Accession No. CAA80235); Cry1Fb2 (Accession No. BAA25298); Cry1Fb3 (Accession No. AAF21767); Cry1Fb4 (Accession No. AAC10641); Cry1Fb5 (Accession No. AAO13295); Cry1Fb6 (Accession No. AC D50892); Cry1Fb7 (Accession No. ACD50893); Cry1Ga1 (Accession No. CAA80233); Cry1Ga2 (Accession No. CAA70506); Cry1Gb1 (Accession No. AAD10291); Cry1Gb2 (Accession No. AAO13756); Cry1Gc1 (Accession No. AAQ52381); Cry1Ha1 (Accession No. CAA80236); Cry1Hb1 (Accession No. AAA79694); Cry1Hb2 (Accession No. HQ439786); Cry1H-like (Accession No. AAF01213); Cry1Ia1 (Accession No. CAA44633); Cry1Ia2 (Accession No. AAA22354); Cry1Ia3 (Accession No. AAC36999); Cry1Ia4 (Accession No. AAB00958); Cry1Ia5 (Accession No. CAA70124); Cry1Ia6 (Accession No. AAC26910); Cry1Ia7 (Accession No. AAM73516); Cry1Ia8 (Accession No. AAK66742); Cry1Ia9 (Accession No. AAQ08616); Cry1Ia10 (Accession No. AAP86782); Cry1Ia11 (Accession No. CAC85964); Cry1Ia12 (Accession No. AAV53390); Cry1Ia13 (Accession No. ABF832 02); Cry1Ia14 (Accession No. ACG63871); Cry1Ia15 (Accession No. FJ617445); Cry1Ia16 (Accession No. FJ617448); Cry1Ia17 (Accession No. GU989199); Cry1Ia18 (Accession No. ADK23801); Cry1Ia19 (Accession No. HQ439787); Cry1Ia20 (Accession No. JQ228426); Cry1Ia21 (Accession No. JQ228424); Cry1Ia22 (Accession No. JQ228427); Cry1Ia23 (Accession No. JQ228428); Cry1Ia24 (Accession No. JQ228429);Cry1Ia25 (Accession No. JQ228430); Cry1Ia26 (Accession No. JQ228431); Cry1Ia27 (Accession No. JQ228432); Cry1Ia28 (Accession No. JQ228433); Cry1Ia29 (Accession No. JQ228434); Cry1Ia30 (Accession No. JQ317686); Cry1Ia31 (Accession No. JX944038); Cry1Ia32 (Accession No. JX944039); Cry1Ia33 (Accession No. JX944040); Cry1Ib1 (Accession No. AAA82114); Cry1Ib2 (Accession No. ABW88019); Cry1Ib3 (Accession No. Cry1Ib4 (Accession No. HM051227); Cry1Ib5 (Accession No. HM070028); Cry1Ib6 (Accession No. ADK38579); Cry1Ib7 (Accession No. JN571740); Cry1Ib8 (Accession No. JN675714); Cry1Ib9 (Accession No. JN675715); Cry1Ib10 (Accession No. JN675716); Cry1Ib11 (Accession No. JQ228423); Cry1Ic1 (Accession No. AAC62933); Cry1Ic2 (Accession No. AAE71691); Cry1Id1 (Accession No. AAD44366); Cry1 Id2 (Accession No. JQ228422); Cry1Ie1 (Accession No. AAG43526); Cry1Ie2 (Accession No. HM439636); Cry1Ie3 (Accession No. KC156647); Cry1Ie4 (Accession No. KC156681); Cry1If1 (Accession No. AAQ52382); Cry1Ig1 (Accession No. KC156701); Cry1I-like (Accession No. AAC31094); Cry1I-like (Accession No. ABG88859); Cry1Ja1 (Accession No. AAA22341); Cry1Ja2 (Accession No. HM070030); Cry1Ja3 (Accession No. JQ228425); Cry 1Jb1 (Accession No. AAA98959); Cry1Jc1 (Accession No. AAC31092); Cry1Jc2 (Accession No. AAQ52372); Cry1Jd1 (Accession No. CAC50779); Cry1Ka1 (Accession No. AAB00376); Cry1Ka2 (Accession No. HQ439783); Cry1La1 (Accession No. AAS60191); Cry1La2 (Accession No. HM070031); Cry1Ma1 (Accession No. FJ884067); Cry1Ma2 (Accession No. KC156659); Cry1Na1 (Accession No. KC156648); Cry1Nb1 (Accession No. KC156678);Cry1-like (Accession No. AAC31091); Cry2Aa1 (Accession No. AAA22335); Cry2Aa2 (Accession No. AAA83516); Cry2Aa3 (Accession No. D86064); Cry2Aa4 (Accession No. AAC04867); Cry2Aa5 (Accession No. CAA10671); Cry2Aa6 (Accession No. CAA10672); Cry2Aa7 (Accession No. CAA10670); Cry2Aa8 (Accession No. AAO13734); Cry2Aa9 (Accession No. AAO13750); Cry2Aa10 (Accession No. AAQ04263); Cry2Aa11 (Accession No. AAQ523 84); Cry2Aa12 (Accession No. ABI83671); Cry2Aa13 (Accession No. ABL01536); Cry2Aa14 (Accession No. ACF04939); Cry2Aa15 (Accession No. JN426947); Cry2Ab1 (Accession No. AAA22342); Cry2Ab2 (Accession No. CAA39075); Cry2Ab3 (Accession No. AAG36762); Cry2Ab4 (Accession No. AAO13296); Cry2Ab5 (Accession No. AAQ04609); Cry2Ab6 (Accession No. AAP59457); Cry2Ab7 (Accession No. AAZ66347); Cry2Ab8 ( Cry2Ab15 (Accession No. HM037126); Cry2Ab16 (Accession No. GQ866914); Cry2Ab17 (Accession No. HQ439789); Cry2Ab18 (Accession No. JN135255); Cry2Ab19 (Accession No. JN135256); Cry2Ab20 (Accession No. ABM21764); Cry2Ab21 (Accession No. ACG76120); Cry2Ab22 (Accession No. ACG76121); Cry2Ab23 (Accession No. HM037126); Cry2Ab24 (Accession No. GQ866914); Cry2Ab25 (Accession No. HQ439789); Cry2Ab26 (Accession No. JN135255); Cry2Ab27 (Accession No. ACG76121); Cry2Ab28 (Accession No. ACG76122); Cry2Ab29 (Accession No. ACG76123); Cry2Ab30 (Accession No. ACG76124); Cry2Ab31 (Accession No. ACG76125 35256); Cry2Ab20 (Accession No. JN135257); Cry2Ab21 (Accession No. JN135258); Cry2Ab22 (Accession No. JN135259); Cry2Ab23 (Accession No. JN135260); Cry2Ab24 (Accession No. JN135261); Cry2Ab25 (Accession No. JN415485); Cry2Ab26 (Accession No. JN426946); Cry2Ab27 (Accession No. JN415764); Cry2Ab28 (Accession No. JN651494); Cry2Ac1 (Accession No. CAA40536); Cry2Ac2 (Accession No. AAG35410);Cry2Ac3 (Accession No. AAQ52385); Cry2Ac4 (Accession No. ABC95997); Cry2Ac5 (Accession No. ABC74969); Cry2Ac6 (Accession No. ABC74793); Cry2Ac7 (Accession No. CAL18690); Cry2Ac8 (Accession No. CAM09325); Cry2Ac9 (Accession No. CAM09326); Cry2Ac10 (Accession No. ABN15104); Cry2Ac11 (Accession No. CAM83895); Cry2Ac12 (Accession No. CAM83896); Cry2Ad1 (Accession No. AAF09583); Cry2Ad2 (Accession No. ABC86927); Cry2Ad3 (Accession No. CAK29504); Cry2Ad4 (Accession No. CAM32331); Cry2Ad5 (Accession No. CAO78739); Cry2Ae1 (Accession No. AAQ52362); Cry2Af1 (Accession No. ABO30519); Cry2Af2 (Accession No. GQ866915); Cry2Ag1 (Accession No. ACH91610); Cry2Ah1 (Accession No. EU939453); Cry2Ah2 (Accession No. ACL80665); Cry2Ah3 (Accession No. GU073380); Cry2Ah4 (Accession No. KC156702); Cry2A i1 (accession number FJ788388); Cry2Aj (accession number); Cry2Ak1 (accession number KC156660); Cry2Ba1 (accession number KC156658); Cry3Aa1 (accession number AAA22336); Cry3Aa2 (accession number AAA22541); Cry3Aa3 (accession number CAA68482); Cry3Aa4 (accession number AAA22542); Cry3Aa5 (accession number AAA50255); Cry3Aa6 (accession number AAC43266); Cry3Aa7 (accession number CAB41411); Cry3Aa8 (accession number AAS79487); Cry3Aa9 (Accession No. AAW05659); Cry3Aa10 (Accession No. AAU29411); Cry3Aa11 (Accession No. AAW82872); Cry3Aa12 (Accession No. ABY49136); Cry3Ba1 (Accession No. CAA34983); Cry3Ba2 (Accession No. CAA00645); Cry3Ba3 (Accession No. JQ397327); Cry3Bb1 (Accession No. AAA22334); Cry3Bb2 (Accession No. AAA74198); Cry3Bb3 (Accession No. I15475); Cry3Ca1 (Accession No. CAA42469); Cry4Aa1 (Accession No. CAA68485);Cry4Aa2 (Accession No. BAA00179); Cry4Aa3 (Accession No. CAD30148); Cry4Aa4 (Accession No. AFB18317); Cry4A-like (Accession No. AAY96321); Cry4Ba1 (Accession No. CAA30312); Cry4Ba2 (Accession No. CAA30114); Cry4Ba3 (Accession No. AAA22337); Cry4Ba4 (Accession No. BAA00178); Cry4Ba5 (Accession No. CAD30095); Cry4Ba-like (Accession No. ABC47686); Cry4Ca1 (Accession No. EU646202); Cry4Cb1 (Accession No. FJ40320 8); Cry4Cb2 (accession number FJ597622); Cry4Cc1 (accession number FJ403207); Cry5Aa1 (accession number AAA67694); Cry5Ab1 (accession number AAA67693); Cry5Ac1 (accession number I34543); Cry5Ad1 (accession number ABQ82087); Cry5Ba1 (accession number AAA68598); Cry5Ba2 (accession number ABW88931); Cry5Ba3 (accession number AFJ04417); Cry5Ca1 (accession number HM461869); Cry5Ca2 (accession number ZP_04123426); Cry5Da1 (accession number HM 461870); Cry5Da2 (Accession No. ZP_04123980); Cry5Ea1 (Accession No. HM485580); Cry5Ea2 (Accession No. ZP_04124038); Cry6Aa1 (Accession No. AAA22357); Cry6Aa2 (Accession No. AAM46849); Cry6Aa3 (Accession No. ABH03377); Cry6Ba1 (Accession No. AAA22358); Cry7Aa1 (Accession No. AAA22351); Cry7Ab1 (Accession No. AAA21120); Cry7Ab2 (Accession No. AAA21121); Cry7Ab3 (Accession No. ABX24522); Cry 7Ab4 (accession number EU380678); Cry7Ab5 (accession number ABX79555); Cry7Ab6 (accession number ACI44005); Cry7Ab7 (accession number ADB89216); Cry7Ab8 (accession number GU145299); Cry7Ab9 (accession number ADD92572); Cry7Ba1 (accession number ABB70817); Cry7Bb1 (accession number KC156653); Cry7Ca1 (accession number ABR67863); Cry7Cb1 (accession number KC156698); Cry7Da1 (accession number ACQ99547); Cry7Da2 (accession number HM572236);Cry7Da3 (Accession No. KC156679); Cry7Ea1 (Accession No. HM035086); Cry7Ea2 (Accession No. HM132124); Cry7Ea3 (Accession No. EEM19403); Cry7Fa1 (Accession No. HM035088); Cry7Fa2 (Accession No. EEM19090); Cry7Fb1 (Accession No. HM572235); Cry7Fb2 (Accession No. KC156682); Cry7Ga1 (Accession No. HM572237); Cry7Ga2 (Accession No. KC156669); Cry7Gb1 (Accession No. KC156650); Cry7Gc1 (Accession No. KC156651); 6654); Cry7Gd1 (Accession No. KC156697); Cry7Ha1 (Accession No. KC156651); Cry7Ia1 (Accession No. KC156665); Cry7Ja1 (Accession No. KC156671); Cry7Ka1 (Accession No. KC156680); Cry7Kb1 (Accession No. BAM99306); Cry7La1 (Accession No. BAM99307); Cry8Aa1 (Accession No. AAA21117); Cry8Ab1 (Accession No. EU044830); Cry8Ac1 (Accession No. KC156662); Cry8Ad1 (Accession No. KC156684); Cry8Ba1 (Accession No. Cry8Ca1 (Accession No. AAA21119); Cry8Ca2 (Accession No. AAR98783); Cry8Ca3 (Accession No. EU625349); Cry8Ca4 (Accession No. ADB54826); Cry8Da1 (Accession No. BAC07226); Cry8Da2 (Accession No. BD133574); Cry8Da3 (Accession No. BD133575); Cry8Db1 (Accession No. BAF93483); Cry8Ea1 (Accession No. AAQ73470); Cry8E Cry8a2 (Accession No. EU047597); Cry8Ea3 (Accession No. KC855216); Cry8Fa1 (Accession No. AAT48690); Cry8Fa2 (Accession No. HQ174208); Cry8Fa3 (Accession No. AFH78109); Cry8Ga1 (Accession No. AAT46073); Cry8Ga2 (Accession No. ABC42043); Cry8Ga3 (Accession No. FJ198072); Cry8Ha1 (Accession No. AAW81032); Cry8Ia1 (Accession No. EU381044); Cry8Ia2 (Accession No. GU073381); Cry8Ia3 (Accession No. HM044664);Cry8Ia4 (Accession No. KC156674); Cry8Ib1 (Accession No. GU325772); Cry8Ib2 (Accession No. KC156677); Cry8Ja1 (Accession No. EU625348); Cry8Ka1 (Accession No. FJ422558); Cry8Ka2 (Accession No. ACN87262); Cry8Kb1 (Accession No. HM123758); Cry8Kb2 (Accession No. KC156675); Cry8La1 (Accession No. GU325771); Cry8Ma1 (Accession No. HM044665); Cry8Ma2 (Accession No. EEM86551); Cry8Ma3 (Accession No. HM2 10574); Cry8Na1 (Accession No. HM640939); Cry8Pa1 (Accession No. HQ388415); Cry8Qa1 (Accession No. HQ441166); Cry8Qa2 (Accession No. KC152468); Cry8Ra1 (Accession No. AFP87548); Cry8Sa1 (Accession No. JQ740599); Cry8Ta1 (Accession No. KC156673); Cry8-like (Accession No. FJ770571); Cry8-like (Accession No. ABS53003); Cry9Aa1 (Accession No. CAA41122); Cry9Aa2 (Accession No. CAA41425); Cry9Aa3 (Accession No. G Cry9Ca1 (Accession No. CAA85764); Cry9Ca2 (Accession No. AAQ52375); Cry9Da1 (Accession No. BAA19948); Cry9Da2 (Accession No. AAB97923); Cry9Da3 (Accession No. GQ249293); Cry9Da 4 (accession number GQ249297); Cry9Db1 (accession number AAX78439); Cry9Dc1 (accession number KC156683); Cry9Ea1 (accession number BAA34908); Cry9Ea2 (accession number AAO12908); Cry9Ea3 (accession number ABM21765); Cry9Ea4 (accession number ACE88267); Cry9Ea5 (accession number ACF04743); Cry9Ea6 (accession number ACG63872); Cry9Ea7 (accession number FJ380927); Cry9Ea8 (accession number GQ249292); Cry9Ea9 (accession number JN651495);Cry9Eb1 (Accession No. CAC50780); Cry9Eb2 (Accession No. GQ249298); Cry9Eb3 (Accession No. KC156646); Cry9Ec1 (Accession No. AAC63366); Cry9Ed1 (Accession No. AAX78440); Cry9Ee1 (Accession No. GQ249296); Cry9Ee2 (Accession No. KC156664); Cry9Fa1 (Accession No. KC156692); Cry9Ga1 (Accession No. KC156699); Cry9-like (Accession No. AAC63366); Cry10Aa1 (Accession No. AAA22614); Cry10Aa2 (Accession No. E0061 4); Cry10Aa3 (Accession No. CAD30098); Cry10Aa4 (Accession No. AFB18318); Cry10A-like (Accession No. DQ167578); Cry11Aa1 (Accession No. AAA22352); Cry11Aa2 (Accession No. AAA22611); Cry11Aa3 (Accession No. CAD30081); Cry11Aa4 (Accession No. AFB18319); Cry11Aa-like (Accession No. DQ166531); Cry11Ba1 (Accession No. CAA60504); Cry11Bb1 (Accession No. AAC97162); Cry11Bb2 (Accession No. HM068615); Cry1 2Aa1 (Accession No. AAA22355); Cry13Aa1 (Accession No. AAA22356); Cry14Aa1 (Accession No. AAA21516); Cry14Ab1 (Accession No. KC156652); Cry15Aa1 (Accession No. AAA22333); Cry16Aa1 (Accession No. CAA63860); Cry17Aa1 (Accession No. CAA67841); Cry18Aa1 (Accession No. CAA67506); Cry18Ba1 (Accession No. AAF89667); Cry18Ca1 (Accession No. AAF89668); Cry19Aa1 (Accession No. CAA68875); Cry19Ba1 (Accession No. Cry19Ca1 (Accession No. AFM37572); Cry20Aa1 (Accession No. AAB93476); Cry20Ba1 (Accession No. ACS93601); Cry20Ba2 (Accession No. KC156694); Cry20-like (Accession No. GQ144333); Cry21Aa1 (Accession No. I32932); Cry21Aa2 (Accession No. I66477); Cry21Ba1 (Accession No. BAC06484); Cry21Ca1 (Accession No. JF521577); Cry21Ca2 (Accession No. KC156687); Cry21Da1 (Accession No. JF521578);Cry22Aa1 (Accession No. I34547); Cry22Aa2 (Accession No. CAD43579); Cry22Aa3 (Accession No. ACD93211); Cry22Ab1 (Accession No. AAK50456); Cry22Ab2 (Accession No. CAD43577); Cry22Ba1 (Accession No. CAD43578); Cry22Bb1 (Accession No. KC156672); Cry23Aa1 (Accession No. AAF76375); Cry24Aa1 (Accession No. AAC61891); Cry24Ba1 (Accession No. BAD32657); Cry24Ca1 (Accession No. CAJ43600); Cry25Aa 1 (Accession No. AAC61892); Cry26Aa1 (Accession No. AAD25075); Cry27Aa1 (Accession No. BAA82796); Cry28Aa1 (Accession No. AAD24189); Cry28Aa2 (Accession No. AAG00235); Cry29Aa1 (Accession No. CAC80985); Cry30Aa1 (Accession No. CAC80986); Cry30Ba1 (Accession No. BAD00052); Cry30Ca1 (Accession No. BAD67157); Cry30Ca2 (Accession No. ACU24781); Cry30Da1 (Accession No. EF095955); Cry30Db1 (Accession No. BAE80088); Cry30Ea1 (Accession No. ACC95445); Cry30Ea2 (Accession No. FJ499389); Cry30Fa1 (Accession No. ACI22625); Cry30Ga1 (Accession No. ACG60020); Cry30Ga2 (Accession No. HQ638217); Cry31Aa1 (Accession No. BAB11757); Cry31Aa2 (Accession No. AAL87458); Cry31Aa3 (Accession No. BAE79808); Cry31Aa4 (Accession No. BAF32571); Cry31Aa5 (Accession No. BAF32572); Cry31Aa6 (Accession No. BAI44 026); Cry31Ab1 (Accession No. BAE79809); Cry31Ab2 (Accession No. BAF32570); Cry31Ac1 (Accession No. BAF34368); Cry31Ac2 (Accession No. AB731600); Cry31Ad1 (Accession No. BAI44022); Cry32Aa1 (Accession No. AAG36711); Cry32Aa2 (Accession No. GU063849); Cry32Ab1 (Accession No. GU063850); Cry32Ba1 (Accession No. BAB78601); Cry32Ca1 (Accession No. BAB78602); Cry32Cb1 (Accession No. KC156708);Cry32Da1 (Accession No. BAB78603); Cry32Ea1 (Accession No. GU324274); Cry32Ea2 (Accession No. KC156686); Cry32Eb1 (Accession No. KC156663); Cry32Fa1 (Accession No. KC156656); Cry32Ga1 (Accession No. KC156657); Cry32Ha1 (Accession No. KC156661); Cry32Hb1 (Accession No. KC156666); Cry32Ia1 (Accession No. KC156667); Cry32Ja1 (Accession No. KC156685); Cry32Ka1 (Accession No. KC156688); Cry32 Cry32La1 (Accession No. KC156689); Cry32Ma1 (Accession No. KC156690); Cry32Mb1 (Accession No. KC156704); Cry32Na1 (Accession No. KC156691); Cry32Oa1 (Accession No. KC156703); Cry32Pa1 (Accession No. KC156705); Cry32Qa1 (Accession No. KC156706); Cry32Ra1 (Accession No. KC156707); Cry32Sa1 (Accession No. KC156709); Cry32Ta1 (Accession No. KC156710); Cry32Ua1 (Accession No. KC156655); Cry33Aa1 (Accession No. KC156656); Cry34Aa1 (Accession No. AAG50341); Cry34Aa2 (Accession No. AAK64560); Cry34Aa3 (Accession No. AAT29032); Cry34Aa4 (Accession No. AAT29030); Cry34Ab1 (Accession No. AAG41671); Cry34Ac1 (Accession No. AAG50118); Cry34Ac2 (Accession No. AAK64562); Cry34Ac3 (Accession No. AAT29029); Cry34Ba1 (Accession No. AAK64565); Cry34Ba2 (Accession No. AAT29033); Cry34Ba3 (Accession No. AAT2 9031); Cry35Aa1 (Accession No. AAG50342); Cry35Aa2 (Accession No. AAK64561); Cry35Aa3 (Accession No. AAT29028); Cry35Aa4 (Accession No. AAT29025); Cry35Ab1 (Accession No. AAG41672); Cry35Ab2 (Accession No. AAK64563); Cry35Ab3 (Accession No. AY536891); Cry35Ac1 (Accession No. AAG50117); Cry35Ba1 (Accession No. AAK64566); Cry35Ba2 (Accession No. AAT29027); Cry35Ba3 (Accession No. AAT29026);Cry36Aa1 (Accession No. AAK64558); Cry37Aa1 (Accession No. AAF76376); Cry38Aa1 (Accession No. AAK64559); Cry39Aa1 (Accession No. BAB72016); Cry40Aa1 (Accession No. BAB72018); Cry40Ba1 (Accession No. BAC77648); Cry40Ca1 (Accession No. EU381045); Cry40Da1 (Accession No. ACF15199); Cry41Aa1 (Accession No. BAD35157); Cry41Ab1 (Accession No. BAD35163); Cry41Ba1 (Accession No. HM461871); Cry Cry41Ba2 (Accession No. ZP_04099652); Cry42Aa1 (Accession No. BAD35166); Cry43Aa1 (Accession No. BAD15301); Cry43Aa2 (Accession No. BAD95474); Cry43Ba1 (Accession No. BAD15303); Cry43Ca1 (Accession No. KC156676); Cry43Cb1 (Accession No. KC156695); Cry43Cc1 (Accession No. KC156696); Cry43-like (Accession No. BAD15305); Cry44Aa (Accession No. BAD08532); Cry45Aa (Accession No. BAD22577); Cry46Aa ( Accession number BAC79010); Cry46Aa2 (Accession number BAG68906); Cry46Ab (Accession number BAD35170); Cry47Aa (Accession number AAY24695); Cry48Aa (Accession number CAJ18351); Cry48Aa2 (Accession number CAJ86545); Cry48Aa3 (Accession number CAJ86546); Cry48Ab (Accession number CAJ86548); Cry48Ab2 (Accession number CAJ86549); Cry49Aa (Accession number CAH56541); Cry49Aa2 (Accession number CAJ86541); Cry49Aa3 (Accession number CAJ86546); Cry49Ab (Accession number CAJ86548); Cry49Ab2 (Accession number CAJ86549); 43); Cry49Aa4 (Accession No. CAJ86544); Cry49Ab1 (Accession No. CAJ86542); Cry50Aa1 (Accession No. BAE86999); Cry50Ba1 (Accession No. GU446675); Cry50Ba2 (Accession No. GU446676); Cry51Aa1 (Accession No. ABI14444); Cry51Aa2 (Accession No. GU570697); Cry52Aa1 (Accession No. EF613489); Cry52Ba1 (Accession No. FJ361760); Cry53Aa1 (Accession No. EF633476); Cry53Ab1 (Accession No. FJ361759);Cry54Aa1 (Accession No. ACA52194); Cry54Aa2 (Accession No. GQ140349); Cry54Ba1 (Accession No. GU446677); Cry55Aa1 (Accession No. ABW88932); Cry54Ab1 (Accession No. JQ916908); Cry55Aa2 (Accession No. AAE33526); Cry56Aa1 (Accession No. ACU57499); Cry56Aa2 (Accession No. GQ483512); Cry56Aa3 (Accession No. JX025567); Cry57Aa1 (Accession No. ANC87261); Cry58Aa1 (Accession No. ANC87260); Cry5 9Ba1 (Accession No. JN790647); Cry59Aa1 (Accession No. ACR43758); Cry60Aa1 (Accession No. ACU24782); Cry60Aa2 (Accession No. EAO57254); Cry60Aa3 (Accession No. EEM99278); Cry60Ba1 (Accession No. GU810818); Cry60Ba2 (Accession No. EAO57253); Cry60Ba3 (Accession No. EEM99279); Cry61Aa1 (Accession No. HM035087); Cry61Aa2 (Accession No. HM132125); Cry61Aa3 (Accession No. EEM19308); Cry62Aa1 (Accession No. GU810818); Cry60Ba2 (Accession No. EAO57253); Cry60Ba3 (Accession No. EEM99279); Cry61Aa1 (Accession No. HM035087); Cry61Aa2 (Accession No. HM132125); Cry61Aa3 (Accession No. EEM19308); Cry62Aa1 (Accession No. Accession number HM054509); Cry63Aa1 (Accession number BAI44028); Cry64Aa1 (Accession number BAJ05397); Cry65Aa1 (Accession number HM461868); Cry65Aa2 (Accession number ZP_04123838); Cry66Aa1 (Accession number HM485581); Cry66Aa2 (Accession number ZP_04099945); Cry67Aa1 (Accession number HM485582); Cry67Aa2 (Accession number ZP_04148882); Cry68Aa1 (Accession number HQ113114); Cry69Aa1 (Accession number HQ401006); Cry69 Aa2 (Accession No. JQ821388); Cry69Ab1 (Accession No. JN209957); Cry70Aa1 (Accession No. JN646781); Cry70Ba1 (Accession No. ADO51070); Cry70Bb1 (Accession No. EEL67276); Cry71Aa1 (Accession No. JX025568); Cry72Aa1 (Accession No. JX025569); Cyt1Aa (GenBank Accession No. X03182); Cyt1Ab (GenBank Accession No. X98793); Cyt1B (GenBank Accession No. U37196); Cyt2A (GenBank Accession No. Z14147);and Cyt2B (GenBank accession number U52043). ;

[0035] Examples of delta-endotoxins also include, but are not limited to: Cry1A proteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; DIG-3 or DIG-11 toxins (N-terminal deletions of α-helix 1 and / or α-helix 2 variants of cry proteins (e.g., Cry1A, Cry3A)) of U.S. Pat. Nos. 8,304,604, 8,304,605, and 8,476,226; Cry1B of U.S. Patent Application Serial No. 10 / 525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A / F chimeras of U.S. Pat. Nos. 7,070,982, 6,962,705, and 6,713,063; ,064,249 Cry2 proteins such as Cry2Ab proteins; Cry3A proteins, including but not limited to: engineered hybrid insecticidal proteins (eHIPs) produced by fusing a unique combination of variable and conserved regions of at least two different Cry proteins (U.S. Patent Application Publication No. 2010 / 0017914); Cry4 proteins; Cry5 proteins; Cry6 proteins; Cry8 proteins of U.S. Patent Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; Cry9 proteins such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families; Naimov, et al. al., (2008) Applied and Environmental Microbiology, 74: 7145-7151 [Naimov et al., (2008), Applied and Environmental Microbiology, 74: 7145-7151] Cry15 protein; U.S. Patent Nos. 6,127,180, 6,624,145 and 6,340,593 Cry22, Cry34Ab1 proteins; U.S. Patent Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229 CryET33 and cryET34 proteins; U.S. Patent Publication Nos. 2006 / 0191034, 2012 / 0278954, and PCT Publication No. WO CryET33 and CryET34 homologs of 2012 / 139004; Cry35Ab1 protein of U.S. Patent Nos. 6,083,499, 6,548,291 and 6,340,593; Cry46 protein, Cry51 protein, Cry binary toxin; TIC901 or related toxins; TIC807 of U.S. Patent Application Publication No. 2008 / 0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006 / 033867; TIC853 toxin of U.S. Patent No. 8,513,494, AXMI-027, AXMI-036, and AXMI-038 of U.S. Patent No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Patent No. 7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006 / 083891; WO AXMI-010 of WO2005 / 038032; AXMI-003 of WO2005 / 021585; AXMI-008 of U.S. Patent Application Publication No. 2004 / 0250311; AXMI-006 of U.S. Patent Application Publication No. 2004 / 0216186; AXMI-007 of U.S. Patent Application Publication No. 2004 / 0210965; AXMI-009 of U.S. Patent Application No. 2004 / 0210964; AXMI-014 of U.S. Patent Application Publication No. 2004 / 0197917; AXMI-004 of U.S. Patent Application Publication No. 2004 / 0197916; WO AXMI-028 and AXMI-029 of 2006 / 119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004 / 074462; AXMI-150 of U.S. Patent No. 8,084,416; AXMI-205 of U.S. Patent Application Publication No. 2011 / 0023184; U.S. Patent Application Publication No. 2011 / 0263 488; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064; AXMI-R1 and related proteins of U.S. Patent Application Publication No. 2010 / 0197592; WO AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of 2011 / 103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011 / 103247;AXMI-115, AXMI-113, AXMI-005, AXMI-163, and AXMI-184 of U.S. Patent No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of U.S. Patent Application Publication No. 2010 / 0298211; AXMI-066 and AXMI-076 of U.S. Patent Application Publication No. 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI143, AXMI144, AXMI145, AXMI146, AXMI147, AXMI148, AXMI149, AXMI150, AXMI151, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI159, AXMI160, AXMI161, AXMI162, AXMI163, AXMI164, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI169, AXMI161, AXMI163, AXMI164, AXMI165, AXMI166 MI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI16 5. AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXM 104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI113, AXMI114, AXMI115, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI128, AXMI129, AXMI130, AXMI131, AXMI132, AXMI133, AXMI134, AXMI135, AXMI136, AXMI137, AXMI138, AXMI139, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI145 0, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137, Cry proteins such as Cry1A and Cry3A with modified proteolytic sites of U.S. Patent No. 8,319,019;Cry1Ac, Cry2Aa and Cry1Ca toxin proteins from Bacillus thuringiensis strain VBTS 2528 of U.S. Patent Application Publication No. 2011 / 0064710. Other Cry proteins are well known to those skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011) at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / , which can be accessed on the World Wide Web using the "www" prefix). The insecticidal activity of Cry proteins is well known to those skilled in the art (for review, see van Frannkenhuyzen, (2009) J. Invert. Path. 101: 1-16). The use of Cry proteins as transgenic plant traits is well known to those skilled in the art, and Cry transgenic plants (including but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt) have obtained regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC [Sanahuja, (2011), Plant Biotechnology Journal, 9: 283-300 and CERA, (2010), Center for Environmental Risk Assessment of Transgenic Crops Database (CERA), ILSI Research Foundation, Washington DC]). More than one pesticidal protein known to those skilled in the art may also be present in plants such as Vip3Ab and Cry1Fa (US 2012 / 0317682); Cry1BE and Cry1F (US 2012 / 0311746); Cry1CA and Cry1AB (US 2012 / 0311745); Cry1F and CryCa (US 2012 / 0317681);Cry1DA and Cry1BE (US 2012 / 0331590); Cry1DA and Cry1Fa (US 2012 / 0331589); Cry1AB and Cry1BE (US 2012 / 0324606); Cry1Fa and Cry2Aa and Cry1I and Cry1E (US 2012 / 0324605); Cry34Ab / 35Ab and Cry6Aa (US 20130167269); Cry34Ab / VCry35Ab and Cry3Aa (US 20130167268);As well as Cry3A and Cry1Ab or Vip3Aa (US20130116170). Pesticide proteins also include insecticide lipases, including lipid acyl hydrolases of U.S. Patent No. 7,491,869, and cholesterol oxidases, such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15: 1406-1413 [Purcell et al., (1993), Biochemistry and Biophysics Research Communications 15: 1406-1413]). Pesticide proteins also include VIP (vegetative insecticide protein) toxins of U.S. Patent Nos. 5,877,012, 6,107,2796,137,033, 7,244,820, 7,615,686, and 8,237,020. Other VIP proteins are well known to those skilled in the art (see, lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html, which can be accessed on the World Wide Web using the "www" prefix). Pesticide proteins also include toxin complex (TC) proteins obtainable from organisms such as Xenorhabdus, Photorhabdus, and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have "stand-alone" insecticidal activity and other TC proteins enhance the activity of stand-alone toxins produced by the same given organism. The toxicity of "stand-alone" TC proteins (e.g., from Photorhabdus, Xenorhabdus, or Paenibacillus) can be enhanced by one or more TC protein "enhancers" derived from source organisms of different genera. There are three major types of TC proteins. As described herein, class A proteins ("Protein A") are stand-alone toxins. Class B proteins ("Protein B") and class C proteins ("Protein C") enhance the toxicity of class A proteins. Examples of class A proteins are TcbA, TcdA, XptA1, and XptA2. Examples of class B proteins are TcaC, TcdB, XptB1Xb, and XptC1Wi. Examples of class C proteins are TccC, XptC1Xb, and XptB1Wi. Pesticide proteins also include spider, snake, and scorpion venom proteins. Examples of spider peptides include, but are not limited to, the lycotoxin-1 peptide and its mutants (U.S. Patent No. 8,334,366). ;

[0036] In some embodiments, the insecticidal polypeptides disclosed herein include amino acid sequences deduced from the full-length nucleic acid sequences disclosed herein and amino acid sequences shorter than the full-length sequences due to the use of alternative downstream start sites or due to processing to produce shorter proteins with pesticidal activity. Processing can occur in the organism expressing the protein or in the pest after ingestion of the protein.

[0037] Thus, novel isolated or recombinant nucleotide sequences are provided herein that confer pesticidal activity. Also provided are amino acid sequences of pesticidal polypeptides of the present disclosure. Proteins produced by translation of these insecticidal polypeptide genes allow cells to control or kill pests that have ingested the protein.

[0038] Nucleic acid molecules, variants and fragments thereof

[0039] One aspect of the present disclosure relates to isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding the insecticidal polypeptides of the present disclosure or biologically active portions thereof; together with nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins having regions of sequence homology. As used herein, the term "nucleic acid molecule" refers to DNA molecules (e.g., recombinant DNA, eDNA, genomic DNA, plasmid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0040] "Isolated" nucleic acid molecules (or DNA) are used herein to refer to nucleic acid sequences (or DNA) that are no longer in their natural environment, e.g., in vitro. "Recombinant" nucleic acid molecules (or DNA) are used herein to refer to nucleic acid sequences (or DNA) that are in a recombinant bacterial or plant host cell. In some embodiments, an "isolated" or "recombinant" nucleic acid does not contain sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived (preferably protein coding sequences). For the purposes of this disclosure, "isolated" or "recombinant" when used to refer to nucleic acid molecules does not include isolated chromosomes. For example, in various embodiments, a recombinant nucleic acid molecule encoding an insecticidal polypeptide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0041] In some embodiments, the isolated nucleic acid molecules encoding the insecticidal polypeptides of the present disclosure have one or more changes in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments, changes in the native or genomic nucleic acid sequence include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to amino acid substitutions, insertions, deletions and / or additions compared to the native or genomic sequence; removal of one or more introns; deletion of one or more upstream or downstream regulatory regions; and deletion of the 5' and / or 3' untranslated regions associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the insecticidal polypeptide is a non-genomic sequence.

[0042] The present disclosure encompasses polynucleotides encoding PIP-45-1 polypeptides. Various polynucleotides encoding PIP-45-1 polypeptides are contemplated. One source of polynucleotides encoding a PIP-45-1 polypeptide or related protein is a bacterial strain comprising SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 The polynucleotide of SEQ ID NO: 152, SEQ ID NO: 220 or SEQ ID NO: 222. One source of polynucleotides encoding a PIP-45-1 polypeptide or related protein is from a strain of Pseudomonas, Thalassuspira, Paracoccus or Cellulovibrio. One source of polynucleotides encoding a PIP-45-1 polypeptide or related protein is from a Pseudomonas strain selected from, but not limited to, Pseudomonas brenneri, Pseudomonas monteilii, Pseudomonas gessardii, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas poae, Pseudomonas trivialis, Pseudomonas libanensis, Pseudomonas fluorescens, and Pseudomonas asplenii.

[0043] In some embodiments, the nucleic acid molecule encoding the PIP-45-1 polypeptide is a non-genomic nucleic acid sequence. As used herein, a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" or "non-genomic polynucleotide" refers to a nucleic acid molecule having one or more changes in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, changes in a native or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of a nucleic acid sequence for expression in a plant; changes in a nucleic acid sequence that introduce at least one amino acid substitution, insertion, deletion and / or addition compared to a native or genomic sequence; removal of one or more introns associated with a genomic nucleic acid sequence; insertion of one or more heterologous introns; removal of one or more upstream or downstream regulatory regions associated with a genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of a 5' and / or 3' untranslated region associated with a genomic nucleic acid sequence; insertion of a heterologous 5' and / or 3' untranslated region; and modification of a polyadenylation site. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0044] In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. As used herein, the term "about", when used in conjunction with sequence identity, means ± 0.5%. In some embodiments, the sequence homology is to the full length sequence of the PIP-45-1 polypeptide.

[0045] In some embodiments, the polynucleotide encodes a polypeptide having the sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234, or SEQ ID NO: 355. : The invention relates to a PIP-45-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to ID NO:236.

[0046] In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 99.1% or more sequence identity compared to SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 99.4% or more sequence identity compared to SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 99.6% or more sequence identity compared to SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 87% or more sequence identity compared to SEQ ID NO: 21. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 88% or more sequence identity compared to SEQ ID NO: 23. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 99.1% or more sequence identity compared to SEQ ID NO: 27. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 99.8% or more sequence identity compared to SEQ ID NO: 29. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 92.3% or more sequence identity compared to SEQ ID NO: 31. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 91.1% or more sequence identity compared to SEQ ID NO: 33. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 95.4% or more sequence identity compared to SEQ ID NO: 35. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 93% or more sequence identity compared to SEQ ID NO: 39. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 97.5% or more sequence identity compared to SEQ ID NO: 43. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 70% or more sequence identity compared to SEQ ID NO: 45. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 94% or more sequence identity compared to SEQ ID NO: 234. In some embodiments, the polynucleotide encodes a PIP-45-1 polypeptide having at least 96% or greater sequence identity compared to SEQ ID NO:236.

[0047] The present disclosure encompasses polynucleotides encoding PIP-45-2 polypeptides. Various polynucleotides encoding PIP-45-2 polypeptides are contemplated. One source of polynucleotides encoding a PIP-45-2 polypeptide or related protein is a bacterial strain comprising SEQ ID NO: 109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 NO: 153, SEQ ID NO: 221 or SEQ ID NO: 223. One source of polynucleotides encoding a PIP-45-2 polypeptide or related protein is from a strain of Pseudomonas, Thalassuspira, Paracoccus or Cellulovibrio. One source of polynucleotides encoding a PIP-45-2 polypeptide or related protein is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas buxiformis, Pseudomonas monnieri, Pseudomonas geissii, Pseudomonas proteans, Pseudomonas putida, Pseudomonas thaliana, Pseudomonas vulgaris, Pseudomonas lebanonii, Pseudomonas fluorescens and Pseudomonas aspleniformis.

[0048] In some embodiments, the nucleic acid molecule encoding a PIP-45-2 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0049] In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-45-2 polypeptide. In some embodiments, the polynucleotide encodes a polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:235, or SEQ ID NO: 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to NO:237.

[0050] In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 99.2% or more sequence identity compared to SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 98.5% or more sequence identity compared to SEQ ID NO: 18. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 96% or more sequence identity compared to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 80% or more sequence identity compared to SEQ ID NO: 22. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 81% or more sequence identity compared to SEQ ID NO: 24. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 99.5% or more sequence identity compared to SEQ ID NO: 28. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 98.5% or more sequence identity compared to SEQ ID NO: 30. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 92% or greater sequence identity compared to SEQ ID NO: 32. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 91.5% or greater sequence identity compared to SEQ ID NO: 34. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 36. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 90% or greater sequence identity compared to SEQ ID NO: 40. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 94% or greater sequence identity compared to SEQ ID NO: 44. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 46. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 91% or greater sequence identity compared to SEQ ID NO: 235. In some embodiments, the polynucleotide encodes a PIP-45-2 polypeptide having at least 93.5% or greater sequence identity compared to SEQ ID NO:237.

[0051] The present disclosure encompasses polynucleotides encoding PIP-64-1 polypeptides. A variety of polynucleotides encoding PIP-64-1 polypeptides are contemplated. One source of polynucleotides encoding PIP-64-1 polypeptides or related proteins is a bacterial strain comprising a polynucleotide of SEQ ID NO: 160, SEQ ID NO: 165 or SEQ ID NO: 224 encoding the PIP-64-1 polypeptides of SEQ ID NO: 53, SEQ ID NO: 58 and SEQ ID NO: 238. One source of polynucleotides encoding PIP-64-1 polypeptides or related proteins is from a strain of Pseudomonas, Enterobacter or Alcaligenes. One source of polynucleotides encoding a PIP-64-1 polypeptide or related protein is from a strain of Pseudomonas or Alcaligenes selected from, but not limited to, Pseudomonas buchneri, Pseudomonas geissii, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas entomogenes, and Alcaligenes faecalis.

[0052] In some embodiments, the nucleic acid molecule encoding a PIP-64-1 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0053] In some embodiments, the polynucleotide encodes a PIP-64-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO: 238 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of the protein, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of the PIP-64-1 polypeptide. In some embodiments, the polynucleotide encodes a PIP-64-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO: 238.

[0054] In some embodiments, the polynucleotide encodes a PIP-64-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 53. In some embodiments, the polynucleotide encodes a PIP-64-1 polypeptide having at least 99.7% or greater sequence identity compared to SEQ ID NO: 58. In some embodiments, the polynucleotide encodes a PIP-64-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 238.

[0055] The present disclosure encompasses polynucleotides encoding a PIP-64-2 polypeptide. A variety of polynucleotides encoding a PIP-64-2 polypeptide are contemplated. One source of polynucleotides encoding a PIP-64-2 polypeptide or related protein is a bacterial strain comprising a polynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 166 or SEQ ID NO: 225 encoding a PIP-64-2 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 and SEQ ID NO: 239, respectively. One source of polynucleotides encoding a PIP-64-2 polypeptide or related protein is from a strain of Pseudomonas, Enterobacter or Alcaligenes. One source of polynucleotides encoding a PIP-64-2 polypeptide or related protein is from a strain of Pseudomonas or Alcaligenes selected from, but not limited to, Pseudomonas burschii, Pseudomonas geiseri, Pseudomonas fluorescens, Pseudomonas brassicae, Pseudomonas entomogenes, and Alcaligenes faecalis.

[0056] In some embodiments, the nucleic acid molecule encoding a PIP-64-2 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0057] In some embodiments, the polynucleotide encodes a PIP-64-2 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of the protein, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of the PIP-64-2 polypeptide. In some embodiments, the PIP-64-2 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239.

[0058] In some embodiments, the polynucleotide encodes a PIP-64-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 54. In some embodiments, the polynucleotide encodes a PIP-64-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 55. In some embodiments, the polynucleotide encodes a PIP-64-2 polypeptide having at least 91% or greater sequence identity compared to SEQ ID NO: 59. In some embodiments, the polynucleotide encodes a PIP-64-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 239.

[0059] The present disclosure encompasses polynucleotides encoding a PIP-74-1 polypeptide. A variety of polynucleotides encoding a PIP-74-1 polypeptide are contemplated. One source of polynucleotides encoding a PIP-74-1 polypeptide or related protein is a bacterial strain comprising a polynucleotide of SEQ ID NO: 180, SEQ ID NO: 182 or SEQ ID NO: 184 encoding a PIP-74-1 polypeptide of SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77, respectively. One source of polynucleotides encoding a PIP-74-1 polypeptide or related protein is from a Pseudomonas strain. One source of polynucleotides encoding a PIP-74-1 polypeptide or related protein is from a Pseudomonas strain selected from, but not limited to, Pseudomonas rhodesiae and Pseudomonas orientalis.

[0060] In some embodiments, the nucleic acid molecule encoding the PIP-74-1 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0061] In some embodiments, the polynucleotide encodes a PIP-74-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of the protein, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of the PIP-74-1 polypeptide. In some embodiments, the polynucleotide encodes a PIP-74-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77.

[0062] In some embodiments, the polynucleotide encodes a PIP-74-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 73. In some embodiments, the polynucleotide encodes a PIP-74-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 75. In some embodiments, the polynucleotide encodes a PIP-74-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 77.

[0063] The present disclosure encompasses polynucleotides encoding PIP-74-2 polypeptides. A variety of polynucleotides encoding PIP-74-2 polypeptides are contemplated. One source of polynucleotides encoding PIP-74-2 polypeptides or related proteins is a bacterial strain comprising polynucleotides of SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185 encoding the PIP-74-2 polypeptides of SEQ ID NO: 74, SEQ ID NO: 76 and SEQ ID NO: 78, respectively. One source of polynucleotides encoding PIP-74-2 polypeptides or related proteins is from a Pseudomonas strain. One source of PIP-74-2 polypeptides or related proteins is from a Pseudomonas strain selected from, but not limited to, Pseudomonas rosea and Pseudomonas orientalis.

[0064] In some embodiments, the nucleic acid molecule encoding the PIP-74-2 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0065] In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of the protein, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of the PIP-74-2 polypeptide. In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78.

[0066] In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 74. In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 76. In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 78.

[0067] The present disclosure encompasses polynucleotides encoding PIP-75 polypeptides. A variety of polynucleotides encoding PIP-75 polypeptides are contemplated. One source of polynucleotides encoding PIP-75 polypeptides or related proteins is a bacterial strain comprising a polynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 encoding the PIP-75 polypeptides of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87. One source of polynucleotides encoding PIP-75 polypeptides or related proteins is from a strain of Pseudomonas, Enterobacter or Serratia. One source of PIP-75 polypeptides or related proteins is from a strain of Pseudomonas, Enterobacter or Serratia selected from, but not limited to, Pseudomonas antarctica, Pseudomonas orientalis, Enterobacter affluvium, Serratia plymuthica, and Serratia liquefaciens.

[0068] In some embodiments, the nucleic acid molecule encoding a PIP-75 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0069] In some embodiments, the polynucleotide encodes a PIP-75 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-75 polypeptide. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87.

[0070] In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 75% or more sequence identity compared to SEQ ID NO: 79. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 75% or more sequence identity compared to SEQ ID NO: 80. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 86% or more sequence identity compared to SEQ ID NO: 81. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 75% or more sequence identity compared to SEQ ID NO: 84. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 75% or more sequence identity compared to SEQ ID NO: 85. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 75% or more sequence identity compared to SEQ ID NO: 86. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least 75% or more sequence identity compared to SEQ ID NO: 87.

[0071] The present disclosure encompasses polynucleotides encoding PIP-77 polypeptides. Various polynucleotides encoding PIP-77 polypeptides are contemplated. One source of polynucleotides encoding a PIP-77 polypeptide or related protein is a bacterial strain comprising SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310 NO: 228 or SEQ ID NO: 231. One source of polynucleotides encoding PIP-77 polypeptides or related proteins is from a strain of Pseudomonas, Enterobacter, Shewanella, Haemophilus or Aeromonas. One source of PIP-77 polypeptides or related proteins is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas chlororaphis, Pseudomonas brassicae, Pseudomonas fluorescens and Pseudomonas rhodocae.

[0072] In some embodiments, the nucleic acid molecule encoding a PIP-77 polypeptide is a non-genomic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0073] In some embodiments, the polynucleotide encodes a PIP-77 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-77 polypeptide. In some embodiments, the polynucleotide encodes a polypeptide having the sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 103. 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to NO:245.

[0074] In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 93% or more sequence identity compared to SEQ ID NO: 88. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 97% or more sequence identity compared to SEQ ID NO: 89. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 99% or more sequence identity compared to SEQ ID NO: 90. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 97% or more sequence identity compared to SEQ ID NO: 92. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 87% or more sequence identity compared to SEQ ID NO: 93. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 86% or more sequence identity compared to SEQ ID NO: 94. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 85% or more sequence identity compared to SEQ ID NO: 95. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 84% or greater sequence identity compared to SEQ ID NO: 96. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 85% or greater sequence identity compared to SEQ ID NO: 97. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 83% or greater sequence identity compared to SEQ ID NO: 98. In some embodiments, the polynucleotide encodes a PIP-77 polypeptide having at least 79% or greater sequence identity compared to SEQ ID NO: 100.

[0075] These polynucleotide sequences are isolated from Pseudomonas or other bacterial hosts and are therefore suitable for expressing the encoded insecticidal polypeptides in other bacterial hosts, including but not limited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas and Rhizobium bacterial host cells. The polynucleotides can also be used as probes for isolating homologous or substantially homologous polynucleotides encoding the insecticidal polypeptides or related proteins of the present disclosure. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from Pseudomonas or other related bacteria.

[0076] The polynucleotide encoding the insecticidal polypeptide can also be synthesized de novo from the polypeptide sequence. The sequence of the polynucleotide gene can be deduced from the polypeptide sequence using the genetic code. Computer programs such as "BackTranslate" (GCG TMPackage, Acclerys, Inc., San Diego, California) can be used to convert a peptide sequence into a corresponding nucleotide sequence encoding the peptide. In addition, the synthetic polynucleotide sequences of the present disclosure can be designed so that they are expressed in plants. U.S. Patent No. 5,500,365 describes a method for synthesizing plant genes to improve the expression level of proteins encoded by the synthesized genes. The method involves modification of the structural gene sequence of an exogenous transgene so that it is more effectively transcribed, processed, translated and expressed by the plant. The characteristics of genes that express well in plants include eliminating sequences that may cause undesirable intron splicing or polyadenylation in the coding region of the gene transcript, while substantially retaining the amino acid sequence of the toxic portion of the insecticidal protein. A similar method for obtaining enhanced transgenic expression in monocotyledons is disclosed in U.S. Patent No. 5,689,052.

[0077] "Complementary sequence" is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence so that it can hybridize with the given nucleic acid sequence to form a stable duplex. "Polynucleotide sequence variant" is used herein to refer to nucleic acid sequences other than the degeneracy of the genetic code that encode the same polypeptide.

[0078] In some embodiments, the nucleic acid molecule encoding the insecticidal polypeptide of the present disclosure is a non-genomic nucleic acid sequence. As used herein, a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" refers to a nucleic acid molecule having one or more changes in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, changes in a native or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of a nucleic acid sequence for expression in a plant; changes in the nucleic acid sequence by introducing at least one amino acid substitution, insertion, deletion and / or addition compared to a native or genomic sequence; removal of one or more introns associated with a genomic nucleic acid sequence; insertion of one or more heterologous introns; removal of one or more upstream or downstream regulatory regions associated with a genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of a 5' and / or 3' untranslated region associated with a genomic nucleic acid sequence; insertion of a heterologous 5' and / or 3' untranslated region; and modification of a polyadenylation site. In some embodiments, the non-genomic nucleic acid molecule is a cDNA.

[0079] Also provided are nucleic acid molecules encoding transcription and / or translation products that are subsequently spliced ​​to ultimately produce functional insecticidal polypeptides. Splicing can be completed in vitro or in vivo, and can involve cis or trans splicing. The substrate for splicing can be a polynucleotide (e.g., RNA transcript) or a polypeptide. An example of cis splicing of a polynucleotide is that an intron inserted into a coding sequence is removed and the exon regions of the two flanks are spliced ​​to produce an insecticidal polypeptide coding sequence of the present disclosure. An example of trans splicing is that a polynucleotide is encoded by separating the coding sequence into two or more fragments, which can be transcribed separately, and then spliced ​​to form a full-length pest-killing coding sequence. The use of a splicing enhancer sequence that can be introduced into a construct can promote splicing in the cis or trans splicing of a polypeptide (U.S. Patent Nos. 6,365,377 and 6,531,316). Therefore, in some embodiments, a polynucleotide does not directly encode a full-length insecticidal polypeptide of the present disclosure, but encodes one or more fragments of an insecticidal polypeptide of the present disclosure. These polynucleotides can be used to express functional insecticidal polypeptides of the present disclosure by a mechanism involving splicing, wherein splicing can occur at the level of polynucleotides (e.g., introns / exons) and / or polypeptides (e.g., inteins / exons). This can be used, for example, to control the expression of pesticidal activity, since functional pesticidal polypeptides are only expressed if all necessary fragments are expressed in an environment that allows the splicing process to produce a functional product. In another example, the introduction of one or more insertion sequences into a polynucleotide can promote recombination with low homology polynucleotides; the use of introns or inteins of the insertion sequence facilitates the removal of intervening sequences, thereby restoring the function of the encoded variant.

[0080] The embodiments also encompass nucleic acid molecules that are fragments of these nucleic acid sequences encoding insecticidal polypeptides. "Fragments" as used herein refer to portions of nucleic acid sequences encoding insecticidal polypeptides of the present disclosure. Fragments of nucleic acid sequences can encode biologically active portions of insecticidal polypeptides of the present disclosure, or they can be fragments that can be used as hybridization probes or PCR primers using the methods disclosed below. Nucleic acid molecules that are fragments of nucleic acid sequences encoding insecticidal polypeptides of the present disclosure contain at least about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or 260 consecutive nucleotides or up to the number of nucleotides present in the full-length nucleic acid sequence encoding the insecticidal polypeptides of the present disclosure disclosed herein, depending on the intended use. "Contiguous nucleotides" when used herein refer to nucleotide residues that are immediately adjacent to each other. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the insecticidal polypeptides of the present disclosure and thus retain insecticidal activity. "Retaining insecticidal activity" is used herein to mean having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or more of the insecticidal activity of a full-length native polypeptide. In one embodiment, the insecticidal activity is Lepidoptera activity. In one embodiment, the insecticidal activity is against a species of the order Coleoptera. In one embodiment, the insecticidal activity is against a species of the genus Diabrotica. In one embodiment, the insecticidal activity is against one or more insect pests of the corn rootworm complex: western corn rootworm, Diabrotica virgifera virgifera; northern corn rootworm, D. barberi; southern corn rootworm or spotted cucumber beetle; spotted cucumber beetle, and Mexican corn rootworm, D. virgifera zeae. In one embodiment, the insecticidal activity is against western corn rootworm, Diabrotica virgifera virgifera.

[0081] In some embodiments, a fragment of a nucleic acid sequence encoding an insecticidal polypeptide of the disclosure, or encoding a biologically active portion of a protein, will encode at least about 15, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 consecutive amino acids or up to the total number of amino acids present in the full-length insecticidal polypeptide of these embodiments. In some embodiments, the fragment is an N-terminal and / or C-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids from the N-terminus and / or C-terminus by proteolysis, insertion of a start codon, deletion of the codon encoding the deleted amino acid with the concomitant insertion of a stop codon, or insertion of a stop codon in the coding sequence.

[0082] The present disclosure provides isolated or recombinant polynucleotides encoding any of the insecticidal polypeptides disclosed herein. One of ordinary skill in the art will readily appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the insecticidal polypeptides disclosed herein. Table 1 is a codon table providing synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at each position of the nucleic acids of the present disclosure, where arginine is specified by a codon, the codon can be changed to any of the above corresponding codons without changing the encoded polypeptide. It should be understood that the U in the RNA sequence corresponds to the T in the DNA sequence.

[0083] Table 1

[0084]

[0085] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequence, thereby resulting in changes in the amino acid sequence of the encoded insecticidal polypeptide without changing the biological activity of the protein. Thus, variant nucleic acid molecules can be produced by introducing one or more nucleotide substitutions, additions and / or deletions into the corresponding nucleic acid sequence disclosed herein, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0086] Alternatively, variant nucleic acid sequences can be prepared by randomly introducing mutations (such as by saturation mutagenesis) along all or part of the coding sequence, and the resulting mutants can be screened for the ability to confer pesticidal activity to identify mutants that retain activity. After mutagenesis, this encoded protein can be expressed recombinantly, and the activity of this protein can be determined using standard assay techniques.

[0087] The polynucleotides and fragments thereof disclosed herein are optionally used as substrates for various recombination and recursive recombination reactions, in addition to standard cloning methods described by, for example, Ausubel, Berger, and Sambrook, i.e., to produce additional pesticidal polypeptide homologues and fragments thereof having desired properties. Various such reactions are known, including those developed by the inventors and their colleagues. Methods for producing variants of any nucleic acid listed herein (these methods include recursive recombination of this polynucleotide with a second (or more) polynucleotide to form a variant polynucleotide library) are also embodiments of the disclosure, as are the libraries produced, the cells comprising these libraries, and any recombinant polynucleotides produced by these methods. In addition, such methods optionally include selecting variant polynucleotides from these libraries based on pesticidal activity, as where such recursive recombination is performed in vitro or in vivo.

[0088] Various diversity generation schemes, including nucleic acid recursive recombination schemes are available and are fully described in the art. The programs can be used alone and / or in combination to produce one or more variants of nucleic acids or nucleic acid groups, and variants of encoded proteins. Individually or in their entirety, these programs provide robust and widely applicable methods for producing diverse nucleic acids and nucleic acid collections (including, for example, nucleic acid libraries), which can be used, for example, to engineer or rapidly evolve nucleic acids, proteins, pathways, cells and / or organisms with new and / or improved characteristics.

[0089] Although clear distinctions and classifications are made in the ensuing discussion, it should be understood that these techniques are generally not mutually exclusive. In fact, various methods can be used alone or in combination, in parallel or in series, so as to obtain different sequence variants.

[0090] The result of any diversity generation procedure described herein can be the generation of one or more nucleic acids, which can select or screen for nucleic acids having or conferring desired properties or nucleic acids encoding proteins having or conferring desired properties. After diversification by one or more methods that can be obtained by those skilled in the art here or otherwise, any nucleic acid produced can be selected for a desired activity or property, for example, pesticidal activity or, such activity at a desired pH, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable form, by any assay in the art, see, for example, the following discussion of insecticidal activity screening. Various related (or even unrelated) properties can be evaluated in series or in parallel as appropriate by the practitioner.

[0091] Descriptions of various diversity generation procedures for generating modified nucleic acid sequences, such as those encoding polypeptides or fragments thereof having pesticidal activity, can be found in the following publications and the references cited therein: Soong, et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Targeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang, et al., (1999) Nat Biotechnol 18:893-896. Chang et al., (1999) Nature Biotechnology, 17: 793-797; Minshull and Stemmer, (1999) Curr Opin Chem Biol 3: 284-290; Christians, et al., (1999) Nat Biotechnol 17: 259-264; Crameri, et al., (1998) Nature 391: 288-291; Crameri, et al., (1997) Nat Biotechnol 17: 259-264; 15:436-438 [Crameri et al., (1997), Nature Biotechnology, 15:436-438]; Zhang, et al., (1997) PNAS USA 94:4504-4509 [Zhang et al., (1997), Proceedings of the National Academy of Sciences of the United States of America, 94:4504-4509]; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733 [Patten et al., (1997), Current Opinion in Biotechnology, 8:724-733]; Crameri, et al., (1996) Nat Med 2:100-103 [Crameri et al., (1996), Nature Medicine, 2:100-103]; Crameri, et al., (1996) Nat Biotechnol 14: 315-319 [Crameri et al., (1996), Nature Biotechnology, 14: 315-319]; Gates, et al., (1996) J Mol Biol 255: 373-386 [Gates et al., (1996), Journal of Molecular Biology, 255: 373-386]; Stemmer, (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457 [Stemmer, (1996), "Sexual PCR and Assembly PCR", In: The Encyclopedia of Molecular Biology, VCH Publishers, New York, pp. 447-457]; Crameri and Stemmer, (1995) BioTechniques 18: 194-195 [Crameri and Stemmer, (1995), Biotechnology, 18: 194-195]; Stemmer, et al., (1995) Gene, 164: 49-53 [Stemmer et al., (1995), Gene, 164: 49-53]; Stemmer, (1995) Science 270: 1510 [Stemmer, (1995), Science, 270: 1510]; Stemmer, (1995) Bio / Technology 13: 549-553 [Stemmer, (1995), Bio / Technology, 13: 549-553]; Stemmer, (1994) Nature 370: 389-391and Stemmer, (1994)PNAS USA 91: 10747-10751 [Stemmer, (1994), Nature, 370: 389-391 and Stemmer, (1994), Proceedings of the National Academy of Sciences of the United States of America, 91: 10747-10751].

[0092] Mutational methods for generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith, (1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 19:423-462). 229: 1193-1201 [Botstein and Shortle, (1985), Science, 229: 1193-1201]; Carter, (1986) Biochem J 237: 1-7 and Kunkel, (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin) [Carter, (1986), Journal of Biological Chemistry, 237: 1-7 and Kunkel, (1987), "The efficiency of oligonucleotide directed mutagenesis", in: Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin])); mutagenesis using uracil-containing templates (Kunkel, (1985) PNAS USA 82: 488-492; Kunkel, et al. al., (1987) Methods Enzymol 154: 367-382 and Bass, et al., (1988) Science 242:240-245 [Kunkel, (1985) Proc. Natl. Acad. Sci. USA, 82:488-492; Kunkel et al., (1987) Methods in Enzymology, 154:367-382 and Bass et al., (1988) Science, 242:240-245]); oligonucleotide-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Res. 10:6487-6500 [Zoller and Smith, (1983), Methods in Enzymology, 100:468-500; Zoller and Smith, (1987), Methods in Enzymology, 154:329-350 (1987); Zoller and Smith, (1982), Nucleic Acids Res, 10:6487-6500]); phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1986) Nucl Acids Res 15 ... al., (1988) Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814 [Taylor et al., (1985), Nucleic Acids Res, 13:8749-8764; Taylor et al., (1985), Nucleic Acids Res, 13:8765-8787 (1985); Nakamaye and Eckstein, (1986), Nucleic Acids Res, 14:9679-9698; Sayers et al., (1988), Nucleic Acids Res, 16:791-802 and Sayers et al., (1988), Nucleic Acids Res, 16:803-814]); mutagenesis using gapped double-stranded DNA (Kramer, et al., (1984) Nucl Acids Res 12: 9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154: 350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207and Fritz, et al., (1988) Nucl Acids Res 16:6987-6999 [Kramer et al., (1984), Nucleic Acids Res, 12:9441-9456; Kramer and Fritz, (1987), Methods in Enzymology, 154:350-367; Kramer et al., (1988), Nucleic Acids Res, 16:7207 and Fritz et al., (1988), Nucleic Acids Res, 16:6987-6999]).

[0093] Other suitable methods include point mismatch repair (Kramer, et al., (1984) Cell 38:879-887), mutagenesis using a repair-deficient host strain (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115 [Eghtedarzadeh and Henikoff, (1986), Nucleic Acids Res., 14:5115]), restriction selection and restriction purification (Wells, et al., (1986) Phil Trans R Soc Lond A 317:415-423 [Wells et al., (1986), Philosophical Transactions of the Royal Society of London A, 317:415-423]), mutagenesis by total gene synthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 and et al., (1985) Nucl Acids Res 13:3305-3316 [Nambiar et al., (1984), Science, 223:1299-1301; Sakamar and Khorana, (1988), Nucleic Acids Res, 14:6361-6372; Wells et al., (1985), Gene, 34:315-323 and et al., (1985), Nucleic Acids Research, 13:3305-3316]), double-strand break repair (Mandecki, (1986), PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455 [Mandecki, (1986), Proceedings of the National Academy of Sciences of the United States of America, 83:7177-7181 and Arnold, (1993), Current Opinion in Biotechnology, 4:450-455]). Additional details of many of the above methods can be found in Methods in Enzymology, Vol. 154, which also describes useful controls for troubleshooting problems with various mutagenesis methods.

[0094] Additional details regarding various diversity generating methods can be found in the following U.S. patents, PCT publications and applications, and EPO publications: U.S. Patent No. 5,723,323, U.S. Patent No. 5,763,192, U.S. Patent No. 5,814,476, U.S. Patent No. 5,817,483, U.S. Patent No. 5,824,514, U.S. Patent No. 5,976,862, U.S. Patent No. 5,605,793, U.S. Patent No. 5,811,238, U.S. Patent No. 5,830,721, U.S. Patent No. 5,834,252, U.S. Patent No. 5,837,458, WO 1995 / 22625, WO 1996 / 33207, WO 1997 / 20078, WO 1997 / 35966, WO 1999 / 41402, WO 1999 / 41383, WO 1999 / 41369、WO 1999 / 41368、EP 752008、EP 0932670、WO 1999 / 23107、WO 1999 / 21979、WO 1998 / 31837、WO 1998 / 27230、WO 1998 / 27230、WO 2000 / 00632、WO 2000 / 09679、WO 1998 / 42832、WO 1999 / 29902、WO 1998 / 41653、WO 1998 / 41622、WO1998 / 42727、WO 2000 / 18906、WO 2000 / 04190、WO 2000 / 42561、WO 2000 / 42559, WO 2000 / 42560, WO 2001 / 23401 and PCT / US 01 / 06775.

[0095] The nucleotide sequences of the embodiments can also be used to separate corresponding sequences from other organisms, particularly other bacteria, particularly Pseudomonas species and more particularly Pseudomonas putida, Pseudomonas chrysogenum or Pseudomonas chlororaphis strains. In this way, such sequences (based on their sequence homology to sequences described herein) can be identified using methods such as PCR, hybridization, etc. The embodiments encompass sequences selected based on the sequence identity to the entire sequence or its fragments illustrated herein. These sequences include sequences as orthologs of disclosed sequences. The term "orthologs" refers to genes derived from a common ancestral gene and found in different species due to speciation. When its nucleotide sequence and / or its encoded protein sequence have substantial identity as defined elsewhere herein, genes found in different species are considered to be orthologs. The functions of orthologs are usually highly conserved between species.

[0096] In the PCR method, oligonucleotide primers can be designed for use in PCR reactions to amplify the corresponding DNA sequence from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafter "Sambrook". See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, NY); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, NY). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene specific primers, vector specific primers, partially mismatched primers, and the like.

[0097] In order to identify potential insecticidal polypeptides from bacterial collections, bacterial cell lysates can be screened with antibodies produced against the insecticidal polypeptides disclosed herein using Western blot and / or ELISA methods. This type of assay can be performed in a high throughput manner. Positive samples can be further analyzed by various techniques such as antibody-based protein purification and identification. Methods for generating antibodies are well known in the art, as described below.

[0098] Alternatively, mass spectrometry-based protein identification methods can be used for homologues of insecticidal polypeptides using protocols in the literature (Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc. [Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc.]). Specifically, the MS data of a given cell lysate or a desired molecular weight enriched sample (excised from an SDS-PAGE gel of the relevant molecular weight band) is combined with the sequence information of the insecticidal polypeptides of the present disclosure using an LC-MS / MS-based protein identification method. Any match in the peptide sequence indicates the possibility of having a homologue in the sample. Additional techniques (protein purification and molecular biology) can be used to isolate the protein and identify the sequence of the homologue.

[0099] In hybridization methods, all or part of the pesticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. So-called hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides, and can be labeled with a detectable group (such as 32P or any other detectable marker, such as other radioisotopes, fluorescent compounds, enzymes or enzyme cofactors). Probes for hybridization can be prepared by labeling synthetic oligonucleotides based on the nucleic acid sequence encoding the insecticidal polypeptide disclosed herein. Degenerate primers can be used in addition, which are designed based on conservative nucleotides or amino acid residues in the nucleic acid sequence or the encoded amino acid sequence. Such probes typically comprise a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleic acids of a nucleic acid sequence encoding an insecticidal polypeptide of the disclosure, or a fragment or variant thereof. Methods for preparing probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra, which is incorporated herein by reference.

[0100] For example, the complete nucleic acid sequence disclosed herein encoding the insecticidal polypeptide of the present disclosure, or one or more portions thereof, can be used as a probe capable of specifically hybridizing with the corresponding nucleic acid sequence and messenger RNA encoding the insecticidal polypeptide-like sequence. To achieve specific hybridization under different conditions, such probes include sequences that are unique and preferably at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes can be used to amplify the corresponding pesticidal sequence from a selected organism by PCR. This technique can be used to isolate additional coding sequences from a desired organism, or as a diagnostic test for determining the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of electroplated DNA libraries (plaques or colonies); see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual [Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual] (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0101] Hybridization of such sequences can be carried out under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" are used herein to refer to conditions in which the degree to which a probe hybridizes to its target sequence will be detectably higher (e.g., at least 2 times higher than background) than the degree to which it hybridizes to other sequences. Stringent conditions are sequence-dependent and will vary in different situations. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can also be adjusted to allow certain mismatches in the sequence so that a lower degree of similarity is detected (heterologous detection). Typically, the probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

[0102] Typically, stringent conditions will be those under which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides), and at least about 60° C. for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents (e.g., formamide). Exemplary low stringency conditions include hybridization at 37° C. using a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1× to 2× SSC (20×SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary moderate stringency conditions include hybridization at 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and washing in 0.5× to 1× SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization at 50% formamide, 1 M NaCl, 1% SDS at 37° C., and washing in 0.1× SSC at 60° C. to 65° C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours.

[0103] Specificity typically depends on the function of post-hybridization washes, the key factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, Tm can be estimated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138: 267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. For every 1% of mismatch, Tm is reduced by about 1°C; therefore, Tm, hybridization and / or washing conditions can be adjusted to hybridize with sequences of desired identity. For example, if sequences with ≥90% identity are sought, the Tm can be reduced by 10°C. Stringent conditions are generally selected to be about 5°C below the thermal melting point Tm of the specific sequence and its complement at a defined ionic strength and pH. However, very stringent conditions can hybridize and / or wash at 1°C, 2°C, 3°C or 4°C below the thermal melting point (Tm); moderately stringent conditions can hybridize and / or wash at 6°C, 7°C, 8°C, 9°C or 10°C below the thermal melting point (Tm); and low stringency conditions can hybridize and / or wash at 11°C, 12°C, 13°C, 14°C, 15°C or 20°C below the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, one of ordinary skill in the art will understand that variations in the stringency of hybridization and / or wash solutions are essentially described. If the desired degree of mismatching results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used.Comprehensive guides to the hybridization of nucleic acids are found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, NY); and Ausubel, et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley Scientific, New York). See Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0104] Proteins and their variants and fragments

[0105] One aspect of the disclosure is an isolated insecticidal polypeptide.

[0106] The present disclosure encompasses PIP-45-1 polypeptides. As used herein, "Pseudomonas insecticidal protein-45-1," "PIP-45-1 polypeptide," or "PIP-45-1 protein" interchangeably refer to a polypeptide that has insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and is sufficiently homologous to the protein of SEQ ID NO: 1. A variety of PIP-45-1 polypeptides are contemplated. One source of a PIP-45-1 polypeptide or related protein is a bacterial strain comprising SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 NO: 220 or SEQ ID NO: 222. One source of PIP-45-1 polypeptide or related protein is from a strain of Pseudomonas, Thalassuspira, Paracoccus or Cellulovibrio. One source of PIP-45-1 polypeptide or related protein is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas buxiformis, Pseudomonas monnieri, Pseudomonas geissii, Pseudomonas proteans, Pseudomonas putida, Pseudomonas thaliana, Pseudomonas vulgaris, Pseudomonas lebanonii, Pseudomonas fluorescens and Pseudomonas asplenii.

[0107] In some embodiments, the PIP-45-1 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-45-1 polypeptide.

[0108] In some embodiments, the PIP-45-1 polypeptide is identical to SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 355. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to ID NO: 236.

[0109] In some embodiments, the PIP-45-1 polypeptide has at least 99.1% or more sequence identity compared to SEQ ID NO: 1. In some embodiments, the PIP-45-1 polypeptide has at least 99.4% or more sequence identity compared to SEQ ID NO: 17. In some embodiments, the PIP-45-1 polypeptide has at least 99.6% or more sequence identity compared to SEQ ID NO: 19. In some embodiments, the PIP-45-1 polypeptide has at least 87% or more sequence identity compared to SEQ ID NO: 21. In some embodiments, the PIP-45-1 polypeptide has at least 88% or more sequence identity compared to SEQ ID NO: 23. In some embodiments, the PIP-45-1 polypeptide has at least 99.1% or more sequence identity compared to SEQ ID NO: 27. In some embodiments, the PIP-45-1 polypeptide has at least 99.8% or more sequence identity compared to SEQ ID NO: 29. In some embodiments, the PIP-45-1 polypeptide has at least 92.3% or more sequence identity compared to SEQ ID NO: 31. In some embodiments, the PIP-45-1 polypeptide has at least 91.1% or more sequence identity compared to SEQ ID NO: 33. In some embodiments, the PIP-45-1 polypeptide has at least 95.4% or more sequence identity compared to SEQ ID NO: 35. In some embodiments, the PIP-45-1 polypeptide has at least 93% or more sequence identity compared to SEQ ID NO: 39. In some embodiments, the PIP-45-1 polypeptide has at least 97.5% or more sequence identity compared to SEQ ID NO: 43. In some embodiments, the PIP-45-1 polypeptide has at least 70% or more sequence identity compared to SEQ ID NO: 45.

[0110] The present disclosure encompasses PIP-45-2 polypeptides. As used herein, "Pseudomonas insecticidal protein-45-2," "PIP-45-2 polypeptide," or "PIP-45-2 protein" interchangeably refer to a polypeptide that has insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and is sufficiently homologous to the protein of SEQ ID NO: 2. A variety of PIP-45-2 polypeptides are contemplated. One source of a PIP-45-2 polypeptide or related protein is a bacterial strain comprising SEQ ID NO: 109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 NO: 221 or SEQ ID NO: 223. One source of PIP-45-2 polypeptide or related protein is from a strain of Pseudomonas, Thalassuspira, Paracoccus or Cellulovibrio. One source of PIP-45-2 polypeptide or related protein is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas buxiformis, Pseudomonas monnieri, Pseudomonas geissii, Pseudomonas proteans, Pseudomonas putida, Pseudomonas thaliana, Pseudomonas vulgaris, Pseudomonas lebanonii, Pseudomonas fluorescens and Pseudomonas asplenii.

[0111] In some embodiments, the PIP-45-2 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-45-2 polypeptide. In some embodiments, the PIP-45-2 polypeptide is identical to SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 36. 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to NO:237.

[0112] In some embodiments, the PIP-45-2 polypeptide has at least 99.2% or more sequence identity compared to SEQ ID NO: 2. In some embodiments, the PIP-45-2 polypeptide has at least 98.5% or more sequence identity compared to SEQ ID NO: 18. In some embodiments, the PIP-45-2 polypeptide has at least 96% or more sequence identity compared to SEQ ID NO: 20. In some embodiments, the PIP-45-2 polypeptide has at least 80% or more sequence identity compared to SEQ ID NO: 22. In some embodiments, the PIP-45-2 polypeptide has at least 81% or more sequence identity compared to SEQ ID NO: 24. In some embodiments, the PIP-45-2 polypeptide has at least 99.5% or more sequence identity compared to SEQ ID NO: 28. In some embodiments, the PIP-45-2 polypeptide has at least 98.5% or more sequence identity compared to SEQ ID NO: 30. In some embodiments, the PIP-45-2 polypeptide has at least 92% or more sequence identity compared to SEQ ID NO: 32. In some embodiments, the PIP-45-2 polypeptide has at least 91.5% or more sequence identity compared to SEQ ID NO: 34. In some embodiments, the PIP-45-2 polypeptide has at least 70% or more sequence identity compared to SEQ ID NO: 36. In some embodiments, the PIP-45-2 polypeptide has at least 90% or more sequence identity compared to SEQ ID NO: 40. In some embodiments, the PIP-45-2 polypeptide has at least 94% or more sequence identity compared to SEQ ID NO: 44. In some embodiments, the PIP-45-2 polypeptide has at least 70% or more sequence identity compared to SEQ ID NO: 46.

[0113] The present disclosure encompasses PIP-64-1 polypeptides. As used herein, "Pseudomonas insecticidal protein-64-1," "PIP-64-1 polypeptide," or "PIP-64-1 protein" interchangeably refers to a polypeptide having insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and sufficiently homologous to the protein of SEQ ID NO: 53. A variety of PIP-64-1 polypeptides are contemplated. One source of a PIP-64-1 polypeptide or related protein is a bacterial strain comprising a polynucleotide of SEQ ID NO: 160, SEQ ID NO: 165, or SEQ ID NO: 224 encoding a PIP-64-1 polypeptide of SEQ ID NO: 53, SEQ ID NO: 58, and SEQ ID NO: 238. One source of a PIP-64-1 polypeptide or related protein is from a strain of Pseudomonas, Enterobacter, or Alcaligenes. One source of PIP-64-1 polypeptides or related proteins is from a strain of Pseudomonas or Alcaligenes selected from, but not limited to, Pseudomonas burschii, Pseudomonas geiseri, Pseudomonas fluorescens, Pseudomonas brassicae, Pseudomonas entomogenes, and Alcaligenes faecalis.

[0114] In some embodiments, the PIP-64-1 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO: 238 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of a PIP-64-1 polypeptide. In some embodiments, the PIP-64-1 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO: 238.

[0115] In some embodiments, the PIP-64-1 polypeptide has at least 75% or greater sequence identity compared to SEQ ID NO: 53. In some embodiments, the PIP-64-1 polypeptide has at least 99.7% or greater sequence identity compared to SEQ ID NO: 58. In some embodiments, the PIP-64-1 polypeptide has at least 75% or greater sequence identity compared to SEQ ID NO: 238.

[0116] The present disclosure encompasses PIP-64-2 polypeptides. As used herein, "Pseudomonas insecticidal protein-64-2," "PIP-64-2 polypeptide," or "PIP-64-2 protein" interchangeably refers to a polypeptide having insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and sufficiently homologous to the protein of SEQ ID NO: 54. A variety of PIP-64-2 polypeptides are contemplated. One source of a PIP-64-2 polypeptide or related protein is a bacterial strain comprising a polynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 166, or SEQ ID NO: 225 encoding a PIP-64-2 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59, and SEQ ID NO: 239, respectively. One source of a PIP-64-2 polypeptide or related protein is from a strain of Pseudomonas, Enterobacter, or Alcaligenes. One source of PIP-64-2 polypeptides or related proteins is from a strain of Pseudomonas or Alcaligenes selected from, but not limited to, Pseudomonas burschii, Pseudomonas geiseri, Pseudomonas fluorescens, Pseudomonas brassicae, Pseudomonas entomogenes, and Alcaligenes faecalis.

[0117] In some embodiments, the PIP-64-2 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of the protein, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of the PIP-64-2 polypeptide. In some embodiments, the PIP-64-2 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239.

[0118] In some embodiments, the PIP-64-2 polypeptide has at least 70% or greater sequence identity compared to SEQ ID NO: 54. In some embodiments, the PIP-64-2 polypeptide has at least 70% or greater sequence identity compared to SEQ ID NO: 55. In some embodiments, the PIP-64-2 polypeptide has at least 91% or greater sequence identity compared to SEQ ID NO: 59. In some embodiments, the PIP-64-2 polypeptide has at least 70% or greater sequence identity compared to SEQ ID NO: 239.

[0119] The present disclosure encompasses PIP-74-1 polypeptides. As used herein, "Pseudomonas insecticidal protein-74-1," "PIP-74-1 polypeptide," or "PIP-74-1 protein" interchangeably refers to a polypeptide having insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and being sufficiently homologous to the protein of SEQ ID NO: 73. A variety of PIP-74-1 polypeptides are contemplated. One source of a PIP-74-1 polypeptide or related protein is a bacterial strain comprising a polynucleotide of SEQ ID NO: 180, SEQ ID NO: 182, or SEQ ID NO: 184 encoding a PIP-74-1 polypeptide of SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO: 77, respectively. One source of a PIP-74-1 polypeptide or related protein is from a strain of Pseudomonas. One source of PIP-74-1 polypeptide or related protein is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas rhodesii and Pseudomonas orientalis.

[0120] In some embodiments, the PIP-74-1 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of a PIP-74-1 polypeptide. In some embodiments, the PIP-74-1 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77.

[0121] In some embodiments, the PIP-74-1 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 73. In some embodiments, the PIP-74-1 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 75. In some embodiments, the PIP-74-1 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 77.

[0122] The present disclosure encompasses PIP-74-2 polypeptides. As used herein, "Pseudomonas insecticidal protein-74-2," "PIP-74-2 polypeptide," or "PIP-74-2 protein" interchangeably refers to a polypeptide having insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and sufficiently homologous to the protein of SEQ ID NO: 74. A variety of PIP-74-2 polypeptides are contemplated. One source of a PIP-74-2 polypeptide or related protein is a bacterial strain comprising polynucleotides of SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185 encoding the PIP-74-2 polypeptides of SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 78, respectively. One source of a PIP-74-2 polypeptide or related protein is from a strain of Pseudomonas. One source of PIP-74-2 polypeptide or related protein is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas rhodesii and Pseudomonas orientalis.

[0123] In some embodiments, the PIP-74-2 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full length sequence of a PIP-74-2 polypeptide. In some embodiments, the PIP-74-2 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78.

[0124] In some embodiments, the PIP-74-2 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 74. In some embodiments, the PIP-74-2 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 76. In some embodiments, the PIP-74-2 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 78.

[0125] The present disclosure encompasses PIP-75 polypeptides. As used herein, "Pseudomonas insecticidal protein-75", "PIP-75 polypeptide" or "PIP-75 protein" interchangeably refers to a polypeptide having insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and being sufficiently homologous to the protein of SEQ ID NO: 79. A variety of PIP-75 polypeptides are contemplated. One source of PIP-75 polypeptides or related proteins is a bacterial strain comprising a polynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 encoding the PIP-75 polypeptides of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, respectively. One source of PIP-75 polypeptide or related protein is from a strain of Pseudomonas, Enterobacter or Serratia. One source of PIP-75 polypeptide or related protein is from a strain of Pseudomonas, Enterobacter or Serratia selected from, but not limited to, Pseudomonas antarctica, Pseudomonas orientalis, Enterobacter affluvium, Serratia plymuthica, and Serratia liquefaciens.

[0126] In some embodiments, the PIP-75 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-75 polypeptide. In some embodiments, the PIP-75 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87.

[0127] In some embodiments, the PIP-75 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 79. In some embodiments, the PIP-75 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 80. In some embodiments, the PIP-75 polypeptide has at least 86% or more sequence identity compared to SEQ ID NO: 81. In some embodiments, the PIP-75 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 84. In some embodiments, the PIP-75 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 85. In some embodiments, the PIP-75 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 86. In some embodiments, the PIP-75 polypeptide has at least 75% or more sequence identity compared to SEQ ID NO: 87.

[0128] The present disclosure encompasses PIP-77 polypeptides. As used herein, "Pseudomonas insecticidal protein-77," "PIP-77 polypeptide," or "PIP-77 protein" interchangeably refer to a polypeptide that has insecticidal activity against one or more insect pests of the order Lepidoptera and / or Coleoptera, and is sufficiently homologous to the protein of SEQ ID NO: 88. A variety of PIP-77 polypeptides are contemplated. One source of a PIP-77 polypeptide or related protein is a bacterial strain comprising SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 230 encoding the PIP-77 polypeptide of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 245, respectively. A polynucleotide of IDNO: 231. One source of PIP-77 polypeptide or related protein is from a strain of Pseudomonas, Enterobacter, Shewanella, Haemophilus or Aeromonas. One source of PIP-77 polypeptide or related protein is from a strain of Pseudomonas selected from, but not limited to, Pseudomonas chlororaphis, Pseudomonas brassicae, Pseudomonas fluorescens and Pseudomonas rhodesii.

[0129] In some embodiments, the PIP-77 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-77 polypeptide. In some embodiments, the PIP-77 polypeptide is identical to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 103. 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to NO:245.

[0130] In some embodiments, the PIP-77 polypeptide has at least 93% or more sequence identity compared to SEQ ID NO: 88. In some embodiments, the PIP-77 polypeptide has at least 97% or more sequence identity compared to SEQ ID NO: 89. In some embodiments, the PIP-77 polypeptide has at least 99% or more sequence identity compared to SEQ ID NO: 90. In some embodiments, the PIP-77 polypeptide has at least 97% or more sequence identity compared to SEQ ID NO: 92. In some embodiments, the PIP-77 polypeptide has at least 87% or more sequence identity compared to SEQ ID NO: 93. In some embodiments, the PIP-77 polypeptide has at least 86% or more sequence identity compared to SEQ ID NO: 94. In some embodiments, the PIP-77 polypeptide has at least 85% or more sequence identity compared to SEQ ID NO: 95. In some embodiments, the PIP-77 polypeptide has at least 84% or more sequence identity compared to SEQ ID NO: 96. In some embodiments, the PIP-77 polypeptide has at least 85% or more sequence identity compared to SEQ ID NO: 97. In some embodiments, the PIP-77 polypeptide has at least 83% or more sequence identity compared to SEQ ID NO: 98. In some embodiments, the PIP-77 polypeptide has at least 80% or more sequence identity compared to SEQ ID NO: 100. In some embodiments, the PIP-77 polypeptide has at least 85% or more sequence identity compared to SEQ ID NO: 241. In some embodiments, the PIP-77 polypeptide has at least 83% or more sequence identity compared to SEQ ID NO: 242. In some embodiments, the PIP-77 polypeptide has at least 96% or more sequence identity compared to SEQ ID NO: 245.

[0131] As used herein, the term "protein", "peptide molecule", or "polypeptide" includes any molecule comprising five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules can be modified, including post-translational modifications such as but not limited to disulfide bond formation, glycosylation, phosphorylation or oligomerization. Therefore, as used herein, the term "protein", "peptide molecule" or "polypeptide" includes any protein modified by any biological or non-biological process. The term "amino acid" refers to all naturally occurring L-amino acids.

[0132] In some embodiments, the PIP-45-1 polypeptide has a calculated molecular weight of between about 40 kDa and about 80 kDa, between about 50 kDa and about 70 kDa, between about 60 kDa and about 65 kDa, between about 61 kDa and about 64 kDa, between about 62 kDa and about 63 kDa, and between about 62.25 kDa and about 62.75 kDa. As used herein, the term "about" used in the context of the molecular weight of an insecticidal polypeptide means ± 0.25 kilodaltons.

[0133] In some embodiments, the PIP-45-2 polypeptide has a calculated molecular weight of between about 40 kDa and about 80 kDa, between about 50 kDa and about 64 kDa, between about 55 kDa and about 60 kDa, between about 56.5 kDa and about 59 kDa, and between about 57.25 kDa and about 58 kDa.

[0134] In some embodiments, the PIP-64-1 polypeptide has a calculated molecular weight of between about 20 kDa and about 40 kDa, between about 25 kDa and about 32 kDa, between about 26 kDa and about 31 kDa, between about 27 kDa and about 30 kDa, between about 28 kDa and about 29 kDa, and between about 28.1 kDa and about 28.7 kDa.

[0135] In some embodiments, the PIP-64-2 polypeptide has a calculated molecular weight of between about 20 kDa and about 40 kDa, between about 25 kDa and about 32 kDa, between about 26 kDa and about 31 kDa, between about 27 kDa and about 30 kDa, and between about 28.25 kDa and about 29 kDa.

[0136] In some embodiments, the PIP-74-1 polypeptide has a calculated molecular weight of between about 40 kDa and about 80 kDa, between about 50 kDa and about 70 kDa, between about 55 kDa and about 73 kDa, between about 57 kDa and about 61 kDa, between about 58 kDa and about 60 kDa, and between about 58.75 kDa and about 59.25 kDa. As used herein, the term "about" used in the context of the molecular weight of an insecticidal polypeptide means ± 0.25 kilodaltons.

[0137] In some embodiments, the PIP-74-2 polypeptide has a calculated molecular weight of between about 35 kDa and about 65 kDa, between about 45 kDa and about 51.5 kDa, between about 47.5 kDa and about 49.5 kDa, and between about 48.25 kDa and about 48.75 kDa.

[0138] In some embodiments, the PIP-75 polypeptide has a calculated molecular weight of between about 6 kDa and about 14 kDa, between about 8 kDa and about 13.5 kDa, between about 9 kDa and about 12 kDa, between about 9.5 kDa and about 11.5 kDa, and between about 10.4 kDa and about 10.8 kDa.

[0139] In some embodiments, the PIP-77 polypeptide has a calculated molecular weight of between about 7 kDa and about 13 kDa, between about 8 kDa and about 12 kDa, between about 9 kDa and about 11 kDa, between about 9.5 kDa and about 10.3 kDa, and between about 9.75 kDa and about 10.25 kDa.

[0140] In some embodiments, the insecticidal polypeptides of the present disclosure have modified physical properties. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "physical properties of interest" and "properties of interest" are used interchangeably to refer to the physical properties of the protein being studied and / or modified. Examples of physical properties include, but are not limited to: net surface charge and charge distribution on the protein surface, net hydrophobicity and hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobic density, total count of surface ionized groups, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, but are not limited to: solubility, folding, stability, and digestibility. In some embodiments, the insecticidal polypeptides of the present disclosure increase the digestibility of proteolytic fragments in the insect gut. Models of digestion by simulating gastric fluid are known to those skilled in the art (Fuchs, RL and JD Astwood. Food Technology 50:83-88, 1996; Astwood, JD, et al Nature Biotechnology 14:1269-1273, 1996; Fu TJ et al J. Agric Food Chem. 50:7154-7160, 2002).

[0141] In some embodiments, variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present disclosure have biological activity, i.e., they still have the biological activity required of the native protein (i.e., pesticidal activity). In some embodiments, the variant will have at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80% or more of the insecticidal activity of the native protein. In some embodiments, variants may have improved activity over the native protein.

[0142] Bacterial genes usually have multiple methionine start codons near the start of an open reading frame. Often, the translation initiation at one or more of these start codons will result in the production of a functional protein. These start codons can include ATG codons. However, bacteria (such as Bacillus species) also recognize this codon GTG as a start codon, and the protein that starts translation at the GTG codon includes methionine at the first amino acid. In a few cases, the translation in the bacterial system can be started at the TTG codon, although TTG encodes methionine in this event. In addition, it is often not determined which of these codons are used naturally in bacteria. Therefore, it should be understood that the use of one of these alternative methionine codons may also result in the production of insecticidal proteins. These pest-killing proteins are encompassed in the present disclosure, and can be used in these methods of the present disclosure. It should be understood that when expressed in plants, it will be necessary to change the alternative start codon to ATG for correct translation.

[0143] In another aspect, the insecticidal polypeptides of the disclosure can be expressed as precursor proteins with intervening sequences that catalyze multi-step post-translational protein splicing. Protein splicing involves the excision of the intervening sequence from the polypeptide and the concomitant ligation of the flanking sequences to produce a new polypeptide (Chong, et al., (1996) J. Biol. Chem., 271: 22159-22168). This intervening sequence or protein splicing element, called the intein, catalyzes its own excision by three coordinated reactions at the N-terminal and C-terminal splice sites: acyl rearrangement of the N-terminal cysteine ​​or serine; transesterification between the two termini to form a branched ester or thioester intermediate, and peptide bond cleavage coupled with cyclization of the intein's C-terminal asparagine to release the intein (Evans, et al., (2000) J. Biol. Chem., 275:9091-9094). Elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., US Patent Number 5,496,714; Comb, et al., US Patent Number 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res. 26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273:10567-10577; Cotton, et al., (1999) J. Am. Chem. Soc. 121:1100-1101; Evans, et al., (1998) J. Am. Chem. Soc. 121:1100-1101). al., (1999) J. Biol. Chem. 274: 18359-18363; Evans, et al., (1999) J. Biol. Chem. 274: 3923-3926; Evans, et al., (1998) Protein Sci. 7: 2256-2264; Evans, et al. al., (2000) J. Biol. Chem. 275: 9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459: 166-172; Mathys, et al., (1999) Gene 231: 1-13;Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95: 3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA 95: 6705-6710; Otomo, et al., (1999) Biochemistry 38: 16040-16044; Otomo, et al., (1999) J. Biolmol. NMR 14: 105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 96: 13638-13643; Severinov and Muir, (1998) J. Biol. Chem. 273: 16205-16209; Shingledecker, et al. al., (1998) Gene 207: 187-195; Southworth, et al., (1998) EMBO J. 17: 918-926; Southworth, et al., (1999) Biotechniques 27: 110-120; Wood, et al. al., (1999) Nat. Biotechnol. 17: 889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95: 9226-9231; Wu, et al., (1998b) Biochim Biophys Acta 1387: 422-432; Xu, et al. al., (1999) Proc. Natl. Acad. Sci. USA 96: 388-393; Yamazaki, et al. al., (1998) J. Am. Chem. Soc., 120: 5591-5592 [Comb et al., U.S. Pat. No. 5,496,714; Comb et al., U.S. Pat. No. 5,834,247; Camarero and Muir, (1999), Journal of the American Chemical Society, 121: 5597-5598; Chong et al., (1997), Gene, 192: 271-281, Chong et al., (199 8), Nucleic Acids Research, 26: 5109-5115; Chong et al., (1998), Journal of Biological Chemistry, 273: 10567-10577; Cotton et al., (1999), Journal of the American Chemical Society, 121: 1100-1101; Evans et al., (1999), Journal of Biological Chemistry, 274: 18359-18363; Evans et al., (1999), Journal of Biological Chemistry, 274: 3923-3926;Evans et al., (1998), Protein Science, 7:2256-2264; Evans et al., (2000), Journal of Biological Chemistry, 275:9091-9094; Iwai and Pluckthun, (1999), FEBS News, 459:166-172; Mathys et al., (1999), Gene, 231:1-13; Mills et al., (1998), Proceedings of the National Academy of Sciences of the United States of America, 95:3543-3548; Muir et al., (1998), Proceedings of the National Academy of Sciences of the United States of America, 95:6705-6710; Otomo et al., (1999), Biochemistry, 38:16040-16044; Otomo et al., (1999), Journal of Biomolecular NMR, 14:105-114; Scott et al., (1999), Proceedings of the National Academy of Sciences of the United States of America, 96:13638-13643; Sever inov and Muir, (1998), Journal of Biological Chemistry, 273: 16205-16209; Shingledecker et al., (1998), Gene, 207: 187-195; Southworth et al., (1998), European Journal of Molecular Biology, 17: 918-926; Southworth et al., (1999), Biotechnology, 27: 110-120; Wood et al., (1999), ), Nature Biotechnology, 17: 889-892; Wu et al., (1998a), Proceedings of the National Academy of Sciences of the United States of America, 95: 9226-9231; Wu et al., (1998b), Biochimica et Biophysica Sinica, 1387: 422-432; Xu et al., (1999), Proceedings of the National Academy of Sciences of the United States of America, 96: 388-393; Yamazaki et al., (1998), Journal of the American Chemical Society, 120: 5591-5592]). For the application of intein in plant transgenesis, see Yang, et al., (Transgene Res 15: 583-593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol. 56: 375-392 (2005)) [Yang et al., (Transgenic Research, 15: 583-593 (2006)) and Evans et al., (Annu. Rev. Plant Biol. 56: 375-392 (2005))].

[0144] In another aspect, the insecticidal polypeptides disclosed herein can be encoded by two separate genes, wherein the inteins of the precursor protein are derived from two genes called split inteins, and the two parts of the precursor are connected by peptide bond formation. The formation of the peptide bond is achieved by intein-mediated trans-splicing. For this purpose, the first and second expression cassettes comprising two separate genes further encode inteins capable of mediating protein trans-splicing. Through trans-splicing, the proteins and polypeptides encoded by the first and second fragments can be connected by peptide bond formation. The trans-splicing inteins can be selected from the nucleolar and organellar genomes of different organisms including eukaryotes, archaea and true bacteria. Inteins that can be used are listed at neb.com / neb / inteins.html, which can be accessed on the World Wide Web using the "www" prefix. The nucleotide sequence encoding the intein can be split into 5' and 3' parts, which encode the 5' and 3' parts of the intein, respectively. It is possible to delete sequence portions (e.g., homing endonuclease domains) that are not required for intein splicing. The intein coding sequence is cleaved so that the 5' and 3' portions can undergo trans-splicing. To select an appropriate cleavage site for the intein coding sequence, the considerations disclosed by Southworth, et al., (1998) EMBO J. 17:918-926 can be followed. In constructing the first and second expression cassettes, the 5' intein coding sequence is linked to the 3' end of the first fragment encoding the N-terminal portion of the insecticidal polypeptide of the present disclosure, and the 3' intein coding sequence is linked to the 5' end of the second fragment encoding the C-terminal portion of the insecticidal polypeptide of the present disclosure.

[0145] In general, any split intein can be used to design a trans-splicing partner, including any naturally occurring or artificially split split intein. Several naturally occurring split inteins are known, for example, the split intein of the DnaE gene of Synechocystis sp. PCC6803 (see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 and Evans, et al., (2000) J Biol Chem. 275(13):9091-4 [Wu et al., (1998), Proc Natl Acad Sci USA. 95(16):9226-31 and Evans et al., (2000), J Biol Chem. 275(13):9091-4]) and the split intein of the DnaE gene from Nostoc punctata (see, Iwai, et al., (2006) FEBS Lett. 580(7): 1853-8 [Iwai et al., (2006), FEBS Bulletin, 580(7): 1853-8]). Non-split inteins are artificially split in the laboratory to produce new split inteins, for example: artificial split Ssp DnaB intein (see, Wu et al., (1998) Biochim Biophys Acta. 1387: 422-32 [Wu et al., (1998) Biochim Biophys Acta, 1387: 422-32]) and split Sce VMA intein (see, Brenzel et al., (2006) Biochemistry. 45 (6): 1571-8 [Brenzel et al., (2006) Biochemistry, 45 (6): 1571-8]) and artificially split fungal mini-intein (see, Elleuche et al., (2007) Biochem Biophys Res Commun. 355(3):830-4 [Elleuche et al., (2007), Biochimica et Biophysica Research Commun., 355(3):830-4]. There are also intein databases that catalog known inteins (see, for example, the online database available at bioinformatics.weizmann.ac.il / ~pietro / inteins / Inteinstable.html, which can be accessed on the World Wide Web using the "www" prefix).

[0146] Naturally occurring non-split inteins may have endonuclease activity or other enzymatic activity, which can generally be removed when designing an artificially split split intein. Such mini-inteins or minimized split inteins are well known in the art and are generally less than 200 amino acid residues long (see, Wu, et al., (1998) Biochim Biophys Acta. 1387: 422-32 [Wu et al., (1998), Biochim Biophys Acta, 1387: 422-32]). Suitable split inteins can have additional polypeptide elements added to their structure that make additional purification feasible, provided that these elements do not inhibit the splicing of the split intein, or are added in a manner that allows them to be removed prior to splicing. Protein splicing has been reported using proteins containing bacterial intein-like (BIL) domains (see, Amitai, et al., (2003) Mol Microbiol. 47: 61-73 [Amitai, et al., (2003) Mol Microbiol. 47: 61-73]) and hedgehog (Hog) autoprocessing domains (the latter combined with inteins when referred to as the Hog / intein superfamily or HINT family; see, Dassa, et al., (2004) J Biol Chem. 279: 32001-7 [Dassa et al., (2004), Journal of Biological Chemistry, 279: 32001-7])) and domains such as these can also be used to prepare artificially split inteins. In particular, non-splicing members of these families can be modified by molecular biology methods to introduce or restore splicing activity in these related species. Recent studies have shown that when an N-terminal split intein component is allowed to react with a C-terminal cleavage intein component not found in nature as its "partner", splicing can be observed; for example, splicing has been observed using partners with as little as 30% to 50% homology to the "natural" splicing partner (see, Dassa, et al., (2007) Biochemistry. 46(1):322-30). Other such mixtures of different split inteins have been shown to react with one another (see, Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it is within the capabilities of one skilled in the relevant art to determine whether a particular pair of polypeptides can bind to each other to provide a functional intein using routine methods and, if applicable, the exercise of inventive skill.

[0147] In another aspect, the insecticidal polypeptides disclosed herein are variants of circular arrangements. The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The method used to create new sequences is similar to that of naturally occurring protein pairs related by linear recombination of their amino acid sequences (Cunningham, et al., (1979) Proc. Natl. Acad. Sci. USA 76:3218-3222; Teather and Erfle, (1990) J. Bacteriol. 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem. 204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130; MacGregor, et al., (1996) FEBS Lett. 378: 263-266 [Cunningham et al., (1979), Proc. Natl. Acad. Sci. USA, 76: 3218-3222; Teather and Erfle, (1990), J. Bacteriol., 172: 3837-3841; Schimming et al., (1992), Eur. J. Biochem., 204: 13-19; Yamiuchi and Minamikawa, (1991), FES Bull., 260: 127-130; MacGregor et al., (1996), Eur. J. Biochem., 378: 263-266]. The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (J. Mol. Biol. 165: 407-413, 1983). In creating a variant of a circular arrangement, a new N-terminus is selected at an internal site (breakpoint) of the original sequence, and the new sequence has the same order of amino acids as the original sequence from the breakpoint until it reaches the original C-terminus or its nearby amino acids. At this point, the new sequence is directly or through another part of the sequence (joint) connected to the original N-terminus or its nearby amino acids, and the new sequence continues with the same sequence as the original sequence until it reaches the point at which the amino acid at the N-terminus of the breakpoint site of the original sequence is at or near the residue, which forms the new C-terminus of the chain. The length of the amino acid sequence of the joint can be selected empirically or with the guidance of structural information or by using a combination of two methods.When no structural information is available, the ability to design and take necessary steps without destroying the conformation of the pesticidal polypeptide (conformational flexibility; Karplus and Schulz, (1985) Naturwissenschaften 72:212-213) can be used to prepare a small series of linkers for testing, the lengths of which are designed to span from 0 to. The designed sequences vary in range and are chosen to be consistent with surface exposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. 20: 483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157: 105-132; solvent exposed surface area, Lee and Richards, (1971) J. Mol. Biol. 55: 379-400). It is assumed that each residue translates an average of 2.0 to This means that the lengths to be tested are between 0 and 30 residues, with 0 to 15 residues being the preferred range. An example of such an empirical series would be to construct a linker using a box sequence such as Gly-Gly-Gly-Ser repeated n times, where n is 1, 2, 3 or 4. One skilled in the art will recognize that there are many such sequences of varying length or composition that can be used as linkers, with the main consideration being that they are neither too long nor too short (cf., Sandhu, (1992) Critical Rev. Biotech. 12: 437-462 [cf., Sandhu, (1992), Biotech Review, 12: 437-462]); if they are too long, entropic effects may destabilize the three-dimensional folding and may also make folding kinetically impractical, and if they are too short, they may destabilize the molecule due to torsion or steric strain. One skilled in the analysis of protein structure information will recognize that the use of the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least limit the number of possibilities that must be tested in the empirical selection of linkers. They will also recognize that sometimes the position of the polypeptide chain terminus is ambiguous in structural models derived from X-ray diffraction or NMR spectroscopy data, and that when true, this situation needs to be taken into account in order to correctly estimate the length of the required linker. From those residues whose positions are well defined, two residues that are sequentially close to the chain termini are selected, and the distance between their c-alpha carbons is used to calculate the approximate length of the linker between them. Using the calculated lengths as a guide, residues with a range of numbers (2 to 100 for each residue) are then selected. Calculation) linkers. These linkers can consist of the original sequence, shortened or extended as necessary, and when extended, flexible and hydrophilic additional residues can be selected as described above; or optionally, the original sequence can be substituted with a series of linkers, an example of which is the Gly-Gly-Gly-Ser box method mentioned above; or optionally, a combination of the original sequence and the new sequence with an appropriate total length can be used. The sequence of the pesticidal polypeptide capable of folding into a biologically active state can be prepared by appropriate selection of the start (amino terminus) and end (carboxyl terminus) positions from the original polypeptide chain, while using the linker sequence as described above. The amino and carboxyl termini are selected from a common extension of the sequence called the breakpoint region using the method described below. Therefore, a novel amino acid sequence is generated by selecting the amino and carboxyl termini from within the same breakpoint region. In many cases, the selection of the new terminus will make the original position of the carboxyl terminus immediately adjacent to the position of the amino terminus. However, those skilled in the art will recognize that the selection of the terminus anywhere within the region may work, and these will effectively result in the deletion or addition of the amino or carboxyl portion of the new sequence. It is a core principle of molecular biology that the primary amino acid sequence of a protein determines the three-dimensional structure necessary for folding to express its biological function. Methods for obtaining and interpreting three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions are known to those skilled in the art. Examples of structural information relevant to the identification of breakpoint regions include the location and type of protein secondary structure (α and 3-10 helices, parallel and antiparallel β sheets, chain inversions and chain turns, and loops; Kabsch and Sander, (1983) Biopolymers 22: 2577-2637 [Kabsch and Sander, (1983), Biopolymers, 22: 2577-2637], the degree of solvent exposure of amino acid residues, the degree and type of residue interactions (Chothia, (1984), Annu. Rev. Biochim., 53: 537-572) and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol. 154: 511-533 [Alber and Mathews, (1987), Methods in Enzymology, 154: 511-533]). In some cases, additional information about the solvent exposure of the residues is known; one example is the site of post-translational attachment of a carbohydrate, which must be on the surface of the protein. When experimental structural information is not available or impossible to obtain, methods can also be used to analyze the primary amino acid sequence in order to predict protein tertiary and secondary structure, solvent accessibility, and the presence of turns and loops.When direct structural methods are not feasible, biochemical methods are sometimes applicable to empirically determine surface exposure; for example, identification of chain cleavage sites after limited proteolysis is used to infer surface exposure (Gentile and Salvatore, (1993) Eur. J. Biochem. 218: 603-621). Thus, using experimentally derived structural information or predictive methods (e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics 22: 81-99), the parental amino acid sequence is examined to classify regions according to whether they are essential for maintaining secondary and tertiary structure. The presence of sequences within regions known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and antiparallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence observed or predicted to have low solvent exposure are more likely to be part of the so-called hydrophobic core of the protein, and the amino and carboxyl termini should also be avoided. In contrast, those regions known or predicted to be surface turns or loops, and particularly those regions known not to be required for biological activity, are preferred sites for positioning the extremities of the polypeptide chain. Continuous stretches of amino acid sequence preferred based on the above criteria are referred to as breakpoint regions. A polynucleotide encoding a circularly arranged insecticidal polypeptide of the present disclosure having a new N-terminus / C-terminus, comprising a linker region separating the original C-terminus from the N-terminus, can be prepared essentially following the method described in Mullins, et al., (1994) J. Am. Chem. Soc. 116: 5529-5533 [Mullins et al., (1994), Journal of the American Chemical Society, 116: 5529-5533]. Multiple steps of polymerase chain reaction (PCR) amplification are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. Polynucleotides encoding the circularly arranged insecticidal polypeptides of the present disclosure having a new N-terminus / C-terminus, comprising a linker region separating the original C-terminus from the N-terminus, can be prepared based on the tandem duplication method described in Horlick, et al., (1992) Protein Eng. 5: 427-431 [Horlick et al., (1992), Protein Engineering, 5: 427-431]. Polymerase chain reaction (PCR) amplification of the new N-terminus / C-terminus gene is performed using the tandemly repeated template DNA.

[0148] In another aspect, a fusion protein is provided that comprises an amino acid sequence in its amino acid sequence that comprises an insecticidal polypeptide of the present disclosure. Methods for designing and constructing fusion proteins (and polynucleotides encoding them) are known to those skilled in the art. Polynucleotides encoding the insecticidal polypeptides of the present disclosure can be fused to signal sequences that will direct the localization of the insecticidal polypeptides of the present disclosure to the insecticidal polypeptides of the embodiments from prokaryotic or eukaryotic cells. For example, in E. coli, one may wish to direct protein expression to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the insecticidal polypeptides of the present disclosure can be fused in order to direct polypeptide expression to the bacterial periplasmic space include, but are not limited to: pelB signal sequence, maltose binding protein (MBP) signal sequence, MBP, ompA signal sequence, signal sequence of the periplasmic E. coli heat-labile enterotoxin B subunit, and signal sequence of alkaline phosphatase. Several vectors for constructing fusion proteins that will direct protein localization are commercially available, such as those available from New England Biolabs. (240 County Road, Ipswich, Massachusetts, 01938-2723) obtained from the pMAL series of vectors (particularly the pMAL-p series). In specific embodiments, the insecticidal polypeptides disclosed herein can be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of these polypeptides in Gram-negative bacteria (see, U.S. Patent Nos. 5,576,195 and 5,846,818). Plant plastid transit peptide / polypeptide fusions are well known in the art (see, U.S. Patent No. 7,193,133). Apoplast transit peptides such as rice or barley α-amylase secretion signals are also well known in the art. The plastid transit peptide is typically fused to the polypeptide to be targeted (e.g., fusion partner) at the N-terminus. In one embodiment, the fusion protein consists essentially of the plastid transit peptide of the disclosure to be targeted and the insecticidal polypeptide. In another embodiment, the fusion protein comprises the plastid transit peptide to be targeted and the polypeptide. In such an embodiment, the plastid transit peptide is preferably located at the N-terminus of the fusion protein. However, additional amino acid residues can be at the N-terminus of the plastid transit peptide, provided that the fusion protein is at least partially targeted to the plastid. In a specific embodiment, the plastid transit peptide is at the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein. When inserted into the plastid, most or all of the plastid transit peptides are usually cut from the fusion protein. Due to specific intercellular conditions or the specific combination of transit peptides / fusion partners used, the cutting position may vary slightly between plant species at different plant development stages. In one embodiment, the plastid transit peptide cutting is homogeneous, so that the cutting site is the same in the fusion protein population. In another embodiment, the plastid transit peptide is not uniform, so that the cutting site in the fusion protein population differs by 1-10 amino acids. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at the corresponding position of its C-terminus, and the same or compatible site can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminus. Care must be taken in designing these sites to ensure that the coding sequences for the transit peptide and the second protein remain "in frame" to allow synthesis of the desired fusion protein. In some cases, it is preferred to remove the initiator methionine codon for the second protein when introducing new restriction sites. The introduction of restriction endonuclease recognition sites on both parent molecules and their subsequent ligation by recombinant DNA techniques can result in the addition of one or more additional amino acids between the transit peptide and the second protein. This generally does not affect the targeting activity as long as the transit peptide cleavage site remains accessible and the function of the second protein is not altered by the addition of these additional amino acids at its N-terminus.Alternatively, one skilled in the art can use gene synthesis (Stemmer, et al., (1995) Gene 164:49-53) or similar methods to generate a precise cleavage site (with or without its initiator methionine) between the transit peptide and the second protein. In addition, the transit peptide fusion can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of the mature protein can affect the ability of the transit peptide to target the protein to the plastid and / or the efficiency of cleavage after protein entry. This may depend on the protein to be targeted. See, for example, Comai, et al., (1988) J. Biol. Chem. 263 (29): 15104-9.

[0149] In some embodiments, fusion proteins are provided that include an insecticidal polypeptide of the disclosure, and an insecticidal polypeptide connected by an amino acid linker.

[0150] In some embodiments, a fusion protein represented by a chemical formula selected from the group consisting of:

[0151] R 1 -LR 2 , R 2 -LR 1 , R 1 -R 2 or R 2 -R 1

[0152] Where R 1 is an insecticidal polypeptide disclosed herein. 1 The polypeptide is fused directly or via a linker (L) segment to the R 2 The term "directly" defines a fusion in which the polypeptides are linked without a peptide linker. Thus, "L" means R 1 and R 2 Chemically bonded or polypeptide segments fused in frame, most commonly, L is R 1 and R 2 R 1 The carboxyl terminus of L is connected to the amino terminus of L and the carboxyl terminus of L is connected to R 2 "In-frame fusion" means that R 1 and R 2There is no translation termination or interruption between the reading frames of the two proteins. The linker (L) is generally a polypeptide between 1 and 500 amino acids in length. The linker connecting the two molecules is preferably designed to (1) allow the two molecules to fold and function independently of each other, (2) have no tendency to develop ordered secondary structures that may interfere with the functional domains of the two proteins, (3) have minimal hydrophobic or charged characteristics that can interact with the functional protein domains, and (4) provide R 1 and R 2 The spatial separation of R 1 and R 2 Can interact with the corresponding receptor on single cell simultaneously.Usually, the surface amino acid in flexible protein region comprises Gly, Asn and Ser.It is expected that almost any arrangement of the amino acid sequence comprising Gly, Asn and Ser meets the standard of the above-mentioned joint sequence.Other neutral amino acids, such as Thr and Ala, can also be used in the joint sequence.Owing to adding unique restriction site in the joint sequence so that fusion is constructed, other amino acids can also be included in the joint.

[0153] In some embodiments, the linker comprises a sequence of a chemical formula selected from the group consisting of: (Gly 3 Ser) n , (Gly 4 Ser) n , (Gly 5 Ser) n , (Gly n Ser) n or (AlaGlySer) n, wherein n is an integer. An example of a highly flexible linker is a spacer (Schaller et al., 1975) rich in (GlySer) present in the pilli protein of a filamentous phage (e.g., phage M13 or fd). This region provides a long, flexible spacer region between two domains of pilli surface protein. Also included is a linker, in which an endopeptidase recognition sequence is included. Such a cleavage site may be valuable for separating the components of the fusion to determine whether they are properly folded and whether they have activity in vitro. Examples of various endopeptidases include, but are not limited to: plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, post-proline cleavage enzyme, V8 protease, thrombin and factor Xa. In some embodiments, the linker comprises the amino acids EEKKN (SEQ ID NO: 215) from a multigene expression vector (MGEV), which is cleaved by vacuolar proteases as disclosed in U.S. Patent Application Publication No. US 2007 / 0277263. In other embodiments, a peptide linker segment from the hinge region of a heavy chain immunoglobulin IgG, IgA, IgM, IgD or IgE provides an angular relationship between the attached polypeptides. Particularly useful are hinge regions in which cysteine ​​is replaced by serine. The linkers disclosed herein include sequences derived from the hinge region of mouse IgGγ2b in which cysteine ​​has been changed to serine. The fusion protein is not limited by the form, size or number of the linker sequences used, and the only requirement for the linker is that it does not interfere adversely with the folding and function of the individual molecules being fused.

[0154] In another aspect, a chimeric insecticidal polypeptide is provided, which is produced by connecting two or more parts of an insecticidal polypeptide gene of the present disclosure, which originally encodes a separate insecticidal protein to produce a chimeric gene. Translation of the chimeric gene results in a single chimeric insecticidal polypeptide having regions, motifs or domains derived from each original polypeptide.

[0155] It should be recognized that DNA sequences can be altered by various methods and that these changes may result in DNA sequences encoding proteins having amino acid sequences that differ from the amino acid sequences encoded by wild-type (or native) pesticidal proteins. In some embodiments, the insecticidal polypeptides of the present disclosure may be altered in various ways, including amino acid substitutions, deletions, truncations and insertions of one or more amino acids compared to any of SEQ ID NO: 1-SEQ ID NO: 107, and SEQ ID NO: 232-SEQ ID NO: 245, including 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 or more amino acid substitutions, deletions and / or insertions or combinations thereof. In some embodiments, the insecticidal polypeptides of the present disclosure comprise a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids from the N-terminus and / or C-terminus of the insecticidal polypeptides of the present disclosure.

[0156] Such methods of manipulation are generally known in the art. For example, amino acid sequence variants of the insecticidal polypeptides of the present disclosure can be prepared by DNA mutation. This can also be accomplished by one of several forms of mutagenesis and / or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will have the desired pesticidal activity. However, it should be understood that the ability of the insecticidal polypeptides of the present disclosure to confer pesticidal activity can be improved by using these techniques on the compositions of the present disclosure.

[0157] For example, conservative amino acid substitutions can be made at one or more, predicted, non-essential amino acid residues. A "non-essential" amino acid residue is a residue that can be changed from the wild-type sequence of the insecticidal polypeptide of the present disclosure without changing the biological activity. A "conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic acid, glutamine); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); amino acids with small aliphatic, non-polar or slightly polar residues (e.g., alanine, serine, threonine, proline); , glycine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cysteine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); amino acids with large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0158] Amino acid substitution can be carried out in the non-conservative region of retaining function.In general, this type of substitution is not carried out for conservative amino acid residues or for amino acid residues in a conservative motif, wherein this type of residue is necessary for protein activity.Examples of conservative and residues that may be necessary for protein activity include, for example, identical residues (for example, identical residues in homologues comparison) between all proteins included in the comparison of toxins similar to or related to the sequences of the embodiments.Examples of conservative but conservative amino acid substitutions and still retaining active residues include, for example, only conservatively substituted residues (for example, only conservatively substituted residues between all proteins included in the comparison of toxins similar to or related to the sequences of the embodiments).However, it will be appreciated by those skilled in the art that functional variants can have a small amount of conservative or non-conservative changes in conservative residues. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) [Dayhoff et al., (1978), Model of the Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington), which is incorporated herein by reference.

[0159] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1): 105-32). It is accepted that the relative hydropathic character of the amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0160] It is known in the art that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still produce a protein with similar biological activity, i.e., still obtain a biologically functionally equivalent protein. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, supra). 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). In making such changes, amino acid substitutions within the hydrophilicity index of +2 are preferred, within +1 are particularly preferred, and within +0.5 are even more particularly preferred.

[0161] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. US Patent No. 4,554,101 states that the greatest local average hydrophilicity of a protein (as governed by the hydrophilicity of its neighboring amino acids) correlates with the biological properties of the protein.

[0162] As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0.+0.1); glutamic acid (+3.0.+0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+0.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0163] Alternatively, the protein sequence of various proteins may be altered at the amino or carboxyl termini without substantially affecting activity. This may include insertions, deletions or changes introduced by modern molecular methods such as PCR, including PCR amplification, which alters or extends the protein coding sequence by including the amino acid coding sequence into the oligonucleotides used in the PCR amplification. Alternatively, the added protein sequence may include the entire protein coding sequence, such as those commonly used in the art to produce protein fusions. Such fusion proteins are often used to (1) increase expression of the protein of interest; (2) introduce binding domains, enzymatic activities or epitopes to facilitate protein purification, protein detection or other experimental uses known in the art; (3) target protein secretion or translation to subcellular organelles, such as the periplasmic space of Gram-negative bacteria, mitochondria or chloroplasts of plants or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

[0164] The variant nucleotide and amino acid sequences disclosed herein also encompass sequences derived from mutagenesis and procedures that cause recombination (such as DNA shuffling). In the case of such a method, one or more different insecticidal polypeptides of the coding region of the disclosure can be used to produce new insecticidal polypeptides of the disclosure having desired properties. In this way, a recombinant polynucleotide library is produced from a group of related sequence polynucleotides, which contain sequence regions with basic sequence identity and capable of homologous recombination in vitro or in vivo. For example, using this method, sequence motifs encoding a domain of interest can be shuffled between pesticidal genes and other known pesticidal genes to obtain new genes encoding improved properties of interest (such as increased insecticidal activity). The strategy of such DNA shuffling is known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer, (1994) Nature 370: 389-391; Crameri, et al., (1997) Nature Biotech. 15: 436-438; Moore, et al., (1997) J. Mol. Biol. 272: 336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri, et al., (1998) Nature 391:288-291 [Stemmer, (1994), Proc. Natl. Acad. Sci. USA, 91:10747-10751; Stemmer, (1994), Nature, 370:389-391; Crameri et al., (1997), Nature Biotechnology, 15:436-438; Moore et al., (1997), J. Molecular Biology, 272:336-347; Zhang et al., (1997), Proc. Natl. Acad. Sci. USA, 94:4504-4509; Crameri et al., (1998), Nature, 391:288-291]; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0165] Domain swapping or shuffling is another mechanism for generating altered insecticidal polypeptides of the disclosure.Domains can be swapped between insecticidal polypeptides of the disclosure, resulting in hybrid or chimeric toxins with improved insecticidal activity or target spectrum. Methods for producing recombinant proteins and testing them for pesticidal activity are well known in the art (see, e.g., Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930; Rang, et al., (1999) Appl. Environ. Microbiol. 65:2918-2925). Maagd et al., (1996), Applied and Environmental Microbiology, 62: 1537-1543; Ge et al., (1991), Journal of Biochemistry, 266: 17954-17958; Schnepf et al., (1990), Journal of Biochemistry, 265: 20923-20930; Rang et al., (1999), Applied and Environmental Microbiology, 65: 2918-2925]).

[0166] Alignment of homologs of insecticidal polypeptides (Figures 1, 2, 3, 4, 5, 6, 7 and 8) allowed the identification of residues that are highly conserved among homologs of these families.

[0167] Composition

[0168] Compositions comprising the insecticidal polypeptides of the disclosure are also contemplated. Compositions comprising a PIP-45-1 polypeptide of the disclosure and a PIP-45-2 polypeptide of the disclosure are contemplated. In some embodiments, the composition comprises an insecticidally effective concentration of a PIP-45-1 polypeptide of the disclosure and a PIP-45-2 polypeptide of the disclosure. Compositions comprising a PIP-64-1 polypeptide of the disclosure and a PIP-64-2 polypeptide of the disclosure are contemplated. In some embodiments, the composition comprises an insecticidally effective concentration of a PIP-64-1 polypeptide of the disclosure and a PIP-64-2 polypeptide of the disclosure. Compositions comprising a PIP-74-1 polypeptide of the disclosure and a PIP-74-2 polypeptide of the disclosure are contemplated. In some embodiments, the composition comprises an insecticidally effective concentration of a PIP-74-1 polypeptide of the disclosure and a PIP-74-2 polypeptide of the disclosure. Compositions comprising a PIP-75 polypeptide of the disclosure are contemplated. In some embodiments, the composition comprises an insecticidally effective concentration of a PIP-75 polypeptide of the disclosure. Compositions comprising a PIP-77 polypeptide of the disclosure are contemplated. In some embodiments, the composition comprises an insecticidally effective concentration of a PIP-77 polypeptide of the disclosure. In some embodiments, the composition further comprises an agriculturally acceptable carrier.

[0169] Antibody

[0170] Also contemplated are antibodies to the insecticidal polypeptides of the present disclosure of the embodiments or to variants or fragments thereof. Antibodies of the present disclosure include polyclonal and monoclonal antibodies and fragments thereof that retain their ability to bind to insecticidal proteins found in the insect gut. An antibody, monoclonal antibody, or fragment thereof is said to be capable of binding to a molecule if it is capable of reacting specifically with the molecule so as to bind the molecule to the antibody, monoclonal antibody, or fragment thereof. The term "antibody" (Ab) or "monoclonal antibody" (Mab) means a complete molecule that is capable of binding to a hapten as well as fragments or binding regions or domains thereof (e.g., Fab and F(ab).sub.2 fragments). Such fragments are typically produced by proteolytic cleavage, such as papain or pepsin. Alternatively, hapten-binding fragments may be produced by applying recombinant DNA technology or by synthetic chemistry. Methods for preparing antibodies of the present disclosure are generally known in the art. See, for example, Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, NY (1988) and references cited therein.Standard reference texts describing the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, NY (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, NY (1980) and Campbell, "Monoclonal Antibody Technology," In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al. al., (eds.), Elsevier, Amsterdam (1984) [Klein, Journal of Immunology: The Science of Cellular-Noncellular Differentiation, John & Wiley & Sons, New York, (1982); Dennett et al., Monoclonal Antibodies, Hybridomas: A New Dimension in Bioanalysis, Plainium Press, New York, (1980) and Campbell, "Monoclonal Antibody Technology", In: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon et al., (eds.), Elsevier, Amsterdam, (1984)]. See also, U.S. Patent Nos. 4,196,265, 4,609,893, 4,713,325, 4,714,681, 4,716,111, 4,716,117 and 4,720,459. Antibodies to the insecticidal polypeptides of the present disclosure or antigen-binding portions thereof can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256: 495. Other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can also be used. The animal system used to prepare hybridomas is the mouse system. Immunization protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known. Antibodies and monoclonal antibodies of the present disclosure can be prepared by using the insecticidal polypeptides of the present disclosure as antigens.

[0171] Kits are provided for detecting the presence of the insecticidal polypeptides of the present disclosure or detecting the presence of nucleotide sequences encoding the insecticidal polypeptides of the present disclosure in a sample. In one embodiment, the kit provides antibody-based reagents for detecting the presence of the insecticidal polypeptides of the present disclosure in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes for detecting the presence of one or more polynucleotides encoding the insecticidal polypeptides of the present disclosure. The kit is provided with appropriate reagents and controls for performing the detection method, as well as instructions for use of the kit.

[0172] Receptor identification and isolation

[0173] Also contemplated are receptors for the insecticidal polypeptides of the embodiments or variants or fragments thereof. Methods for identifying receptors are well known in the art (see, Hofmann, et al., (1988) Eur. J. Biochem. 173: 85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282). Brush border membrane vesicles from susceptible insects can be used to identify and isolate receptors that recognize the insecticidal polypeptides of the present disclosure. In addition to the radioactive labeling methods listed in the cited literature, the insecticidal polypeptides can be labeled with fluorescent dyes and other common labels such as streptavidin. Brush border membrane vesicles (BBMVs) of susceptible insects (such as soybean armyworm and stink bug) can be prepared according to the protocols listed in the references, separated on SDS-PAGE gels, and blotted on suitable membranes. The labeled insecticidal polypeptides of the present disclosure can be incubated with the blotted membranes of BBMVs, and the labeled insecticidal polypeptides of the present disclosure can be identified with labeled reporter genes. Identification of protein bands that interact with the insecticidal polypeptides of the present disclosure can be detected by N-terminal amino acid gas phase sequencing or mass spectrometry-based protein identification methods (Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc [Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc]). Once the protein is identified, the corresponding gene can be cloned from the genomic DNA or cDNA library of the susceptible insect, and the binding affinity can be measured directly with the insecticidal polypeptides of the present disclosure. The receptor function for the insecticidal activity of the insecticidal polypeptides of the present disclosure can be verified by RNAi-type gene knockout methods (Rajagopal, et al., (2002) J. Biol. Chem. 277: 46849-46851).

[0174] Nucleotide constructs, expression cassettes and vectors

[0175] The term "nucleotide construct" is used herein and is not intended to limit the embodiment to the nucleotide construct comprising DNA. It will be appreciated by those of ordinary skill in the art that nucleotide constructs, particularly polynucleotides and oligonucleotides consisting of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the method disclosed herein. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences. In addition, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules and sequences that can be used for the plant transformation method of the embodiments, including but not limited to those consisting of deoxyribonucleotides, ribonucleotides and combinations thereof. The deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc.

[0176] Another embodiment relates to a transformed organism, such as an organism selected from the group consisting of plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiment, an expression cassette comprising the DNA molecule, or a vector comprising the expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0177] The sequence of the embodiment is provided in a DNA construct for expression in an organism of interest. The construct will include 5' and 3' regulatory sequences that are effectively connected to the sequence of the embodiment. The term "effectively connected" as used herein refers to the functional connection between a promoter and a second sequence, wherein the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. Usually, effectively connecting means that the connected nucleotide sequence is continuous, and two protein coding regions are connected in the same reading frame when necessary. The construct may additionally include at least one other gene to be co-transformed into an organism. Alternatively, other genes may be provided on a multi-DNA construct.

[0178] This DNA construct has a plurality of restriction sites for inserting the insecticidal polypeptide gene sequence, which will be placed under the transcriptional regulation of the regulatory region. The DNA construct may additionally contain a selectable marker gene.

[0179] According to the transcription direction of 5' to 3', the DNA construct will generally include: transcription and translation initiation region (i.e., promoter), the DNA sequence of the embodiment, and transcription and translation termination region (i.e., termination region) with function in the organism used as the host. The transcription initiation region (i.e., promoter) can be natural, similar, foreign or heterologous to the host organism and / or sequence of the embodiment. In addition, the promoter can be a natural sequence or, alternatively, a synthetic sequence. The term "exogenous" as used herein means that the promoter is not found in the natural organism into which the promoter is introduced. In the case where the promoter is "exogenous" or "heterologous" for the sequence of the embodiment, it refers to that the promoter is not a natural or naturally occurring promoter for the sequence effectively connected to the embodiment. As used herein, the chimeric gene comprises a coding sequence effectively connected to a transcription initiation region heterologous to the coding sequence. When the promoter is a natural or natural sequence, the expression of the sequence effectively connected is transformed from wild-type expression, which results in a change in phenotype.

[0180] In some embodiments, the DNA construct may also include a transcription enhancer sequence. As used herein, the term "enhancer" refers to a DNA sequence that can stimulate promoter activity and may be an innate element or heterologous element of a promoter inserted to enhance the level or tissue specificity of a promoter. Various enhancers are known in the art, including, for example, introns having gene expression enhancing properties in plants (U.S. Patent Application Publication No. 2009 / 0144863), ubiquitin introns (i.e., maize ubiquitin intron 1 (see, e.g., NCBI sequence S94464; Christensen and Quail (1996) Transgenic Res. 5:213-218; Christensen et al. (1992) Plant Molecular Biology 18:675-689), ω enhancers or ω major enhancers (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al. (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256. al., (1987) Gene 60:217-25 [Gallie et al., (1989), Molecular Biology of RNA, ed.: Cech (Leeds, New York) 237-256 and Gallie et al., (1987), Gene, 60:217-25]), CaMy 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96 [Benfey et al., (1990), European Journal of Molecular Biology, 9:1685-96]), maize AdhI intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48; Kyozuka et al. (1990) Maydica 35:353-357 [Kyozuka et al., (1991), Molecular Genetics and Genomics, 228:40-48; Kyozuka et al., (1990), Maydica, 35:353-357]) and enhancers of U.S. Pat. No. 7,803,992, each of which is incorporated by reference, may also be used. The above list of transcription enhancers is not meant to be limiting. Any suitable transcription enhancer may be used in the embodiments.

[0181] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host, or any combination thereof).

[0182] Convenient termination regions are available from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acids Res. 18:7891-7903. Acid Res. 15: 9627-9639 [Guerineau et al., (1991), Molecular Genetics and Genomics, 262: 141-144; Proudfoot, (1991), Cell, 64: 671-674; Sanfacon et al., (1991), Genes and Development, 5: 141-149; Mogen et al., (1990), Plant Cell, 2: 1261-1272; Munroe et al., (1990), Gene, 91: 151-158; Ballas et al., (1989), Nucleic Acids Research, 17: 7891-7903 and Joshi et al., (1987), Nucleic Acids Research, 15: 9627-9639].

[0183] The nucleic acid can be optimized to increase expression in the host organism as appropriate. Thus, when the host organism is a plant, the synthetic nucleic acid can be synthesized using plant-preferred codons to improve expression. For a discussion of host-preferred codon usage, see, e.g., Campbell and Gowri, (1990) Plant Physiol. 92: 1-11. For example, although the nucleic acid sequences of the embodiments can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to account for codon preferences and GC content preferences specific to monocotyledonous or dicotyledonous plants, as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17: 477-498). Thus, the maize preferred codons of specific amino acids can be derived from known gene sequences of maize. The maize codons of 28 genes from maize plants are used and listed in Table 4 of Murray et al. (supra). Methods for synthesizing plant preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray, et al., (1989) Nucleic Acids Res.17:477-498, and Liu H et al.Mol Bio Rep 37:677-684, 2010 [Murray et al., (1989), Nucleic Acids Res., 17:477-498, and Liu H et al., Molecular Biology Reports, 37:677-684, 2010], which are incorporated herein by reference. The maize codon usage table can also be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=4577, which can be accessed using the www prefix. Table 2 shows the optimal codon analysis for maize (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).

[0184] Table 2

[0185]

[0186] Codon usage was compared using the Chi squared contingency test to identify optimal codons. Codons that appeared significantly more frequently (P\ 0.01) are indicated with an asterisk.

[0187] A soybean codon usage table is shown in Table 3 and can also be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=3847&aa=1&style=N, which can be accessed using the www prefix.

[0188] Table 3

[0189] TTT F 21.2 (10493) TCT S 18.4 (9107) TTC F 21.2 (10487) TCC S 12.9 (6409) TTA L 9.2 (4545) TCA S 15.6 (7712) TTG L 22.9 (11340) TCG S 4.8 (2397) CTT L 23.9 (11829) CCT P 18.9 (9358) CTC L 17.1 (8479) CCC P 10.1 (5010) CTA L 8.5 (4216) CCA P 19.1 (9461) CTG L 12.7 (6304) CCG P 4.7 (2312) ATT I 25.1 (12411) ACT T 17.1 (8490) ATC I 16.3 (8071) ACC T 14.3 (7100) ATA I 12.9 (6386) ACA T 14.9 (7391) ATG M 22.7 (11218) ACG T 4.3 (2147) GTT V 26.1 (12911) GCT A 26.7 (13201) GTC V 11.9 (5894) GCC A 16.2 (8026) GTA V 7.7 (3803) GCA A 21.4 (10577) GTG V 21.4 (10610) GCG A 6.3 (3123) TAT Y 15.7 (7779) TGT C 8.1 (3995) TAC Y 14.9 (7367) TGC C 8.0 (3980) TAA * 0.9 (463) TGA * 1.0 (480) TAG * 0.5 (263) TGG W 13.0 (6412) CAT H 14.0 (6930) CGT R 6.6 (3291) CAC H 11.6 (5759) CGC R 6.2 (3093) CAA Q 20.5 (10162) CGA R 4.1 (2018) CAG Q 16.2 (8038) CGG R 3.1 (1510) AAT N 22.4 (11088) AGT S 12.6 (6237) AAC N 22.8 (11284) AGC S 11.3 (5594) AAA K 26.9 (13334) AGA R 14.8 (7337) AAG K 35.9 (17797) AGG R 13.3 (6574) GAT D 32.4 (16040) GGT G 20.9 (10353) GAC D 20.4 (10097) GGC G 13.4 (6650) GAA E 33.2 (16438) GGA G 22.3 (11022) GAG E 33.2 (16426) GGG G 13.0 (6431)

[0190] In some embodiments, a recombinant nucleic acid molecule encoding an insecticidal polypeptide of the present disclosure has maize-optimized codons.

[0191] Other sequences are known to be modified to enhance gene expression in cellular hosts. These include clearing sequences that encode false polyadenylation signals, exon-intron splicing site signals, transposon-like repeats and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of a given cellular host, as calculated with reference to known genes expressed in the host cell. The term "host cell" as used herein refers to a cell that contains a vector and supports replication and / or expression of an expression vector. The host cell can be a prokaryotic cell such as Escherichia coli, or a eukaryotic cell such as yeast, insect, amphibian or mammalian cell or a monocotyledonous or dicotyledonous plant cell. The example of a monocotyledonous host cell is a maize host cell. When possible, the modified sequence is to avoid the occurrence of a predicted hairpin secondary mRNA structure.

[0192] The expression cassette may additionally contain a 5' leader sequence. These leader sequences may serve to enhance translation. Translation leader sequences are known in the art and include: picornavirus leader sequences, e.g., the EMCV leader (encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leader sequences, e.g., the TEV leader (tobacco etch virus) (Gallie, et al., (1995) Gene 165(2):233-238); MDMV leader (maize dwarf mosaic virus), human immunoglobulin heavy chain binding protein (BiP) (Macejak, et al., (1996) Gene 165(2):233-238); al., (1991) Nature 353:90-94 [Macejak et al., (1991), Nature, 353:90-94]); the untranslated leader sequence from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625 [Jobling et al., (1987), Nature, 325:622-625]); the tobacco mosaic virus leader sequence (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New New York, pp. 237-256 [Gallie et al., (1989), In: Molecular Biology of RNA, ed.: Cech (New York), pp. 237-256]); and the leader sequence of maize chlorotic mottle virus (MCMV) (Lommel, et al., (1991) Virology 81: 382-385 [Lommel et al., (1991), Virology, 81: 382-385]). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84: 965-968 [Della-Cioppa et al., (1987), Plant Physiol. 84: 965-968]. Such constructs may also contain a "signal sequence" or "leader sequence" to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures, such as chloroplasts (or other plastids), the endoplasmic reticulum, or the Golgi apparatus.

[0193] As used herein, "signal sequence" refers to a sequence known or suspected to cause co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Bacterial insecticidal toxins are often synthesized as protoxins, which are activated by proteolysis in the intestine of the target pest (Chang, (1987) Methods Enzymol. 153: 507-516 [Chang, (1987), Enzymology Methods, 153: 507-516]). In some embodiments, the signal sequence is located in the native sequence, or can be derived from the sequence of the embodiments. As used herein, the term "leader sequence" refers to any sequence that, when translated, produces an amino acid sequence sufficient to initiate co-translational transport of a peptide chain to a subcellular organelle. Thus, this includes leader sequences that target transport and / or glycosylation by entering the endoplasmic reticulum, entering the vacuole, plastids (including chloroplasts, mitochondria), etc. Nuclear-encoded proteins targeted to the chloroplast thylakoid lumen have a characteristic bipartite transit peptide consisting of a stromal targeting signal peptide and a lumen targeting signal peptide. The stromal targeting information is located in the amino-proximal part of the transit peptide. The lumen targeting signal peptide is located in the carboxyl-proximal part of the transit peptide and contains all the information for targeting to the lumen. Recent proteomic studies of higher plant chloroplasts have resulted in the identification of a number of nuclear-encoded lumenal proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001), whose lumenal targeting signal peptides may be used according to the present disclosure. Kieselbach et al., Photosynthesis Research, 78: 249-264, 2003 [Kieselbach et al., "Photosynthesis Research", 78: 249-264, 2003] reported about 80 proteins from Arabidopsis and homologous proteins from spinach and pea. In particular, Table 2 of the publication (which is incorporated herein by reference) discloses 85 proteins from the chloroplast cavity identified by their accession numbers (see also U.S. Patent Application Publication No. 2009 / 09044298).In addition, the recently published draft version of the rice genome (Goff et al, Science 296:92-100, 2002) is a suitable source for lumenal targeting signal peptides that can be used according to the present disclosure.

[0194] Suitable chloroplast transit peptides (CTPs) well known to those skilled in the art also include chimeric CTPs, including but not limited to: the N-terminal domain, central domain or C-terminal domain of CTPs from the following: rice 1-deoxy-D-xylulose-5-phosphate synthase, rice-superoxide dismutase, rice-soluble starch synthase, rice-NADP-dependent malic enzyme, rice-2-dehydro-3-deoxyheptanoic acid aldolase 2, rice-L-ascorbate peroxidase 5, rice-phosphoglucan water dikinase, maize ssRUBISCO, maize-β-glucosidase, maize-malate dehydrogenase, maize thioredoxin M-type (U.S. Patent Application Publication No. 2012 / 0304336). Chloroplast transit peptides of US Patent Publications US 20130205440A1, US 20130205441 A1 and US 20130210114 A1.

[0195] Insecticidal genes to be targeted to the chloroplast can be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and the organelle. In this way, nucleic acids of interest can be synthesized using chloroplast-preferred codons. See, e.g., U.S. Patent No. 5,380,831, which is incorporated herein by reference.

[0196] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or linkers may be used to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., conversion and transversion) may be involved.

[0197] Many promoters can be used to implement these embodiments. The promoter can be selected based on the desired results. The nucleic acid can be combined with a constitutive, tissue-preferred, inducible or other promoter for expression in a host organism. Suitable constitutive promoters for plant host cells include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999 / 43838 and U.S. Pat. No. 6,072,050; the core CaMV35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol.Biol.18:675-689 [Christensen et al., (1989), Plant Mol.Biol., 12:619-632 and Christensen et al., (1992), Plant Mol.Biol., 18:675-689]); pEMU (Last, et al., (1991) Theor.Appl.Genet.81:581-588 [Last et al., (1991), Theor.Appl.Genet., 81:581-588]); MAS (Velten, et al., (1984) EMBO J.3:2723-2730 [Velten et al., (1984), European Journal of Molecular Biology, 3:2723-2730]); ALS promoter (U.S. Pat. No. 5,659,026), etc. Other constitutive promoters include those discussed, for example, in U.S. Patent Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611.Suitable constitutive promoters also include promoters with strong expression in almost all tissues but low expression in pollen, including but not limited to: the banana streak virus (Acuminata Yunnan) promoter (BSV(AY)) disclosed in U.S. Patent No. 8,338,662, the banana streak virus (Acuminata Vietnam) promoter (BSV(AV)) disclosed in U.S. Patent No. 8,350,121, and the banana streak virus (Mysore) promoter (BSV(MYS)) disclosed in U.S. Patent No. 8,395,022.

[0198] Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for use in regulating expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters may respond to damage caused by insect feeding and include the potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. 28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1 and wun2 (U.S. Pat. No. 5,428,148); win1 and win2 (Stanford, et al., (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1989) Mol. Gen. Genet. 215:200-208); al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp, ​​et al., (1993) FEBS Letters 323:73-76); MPI gene (Corderok, et al., (1994) Plant Mol. Biol. 22:783-792; Eckelkamp, ​​et al., (1993) FEBS Letters 323:73-76); J. 6(2): 141-150 [Corderok et al., (1994), Plant Journal, 6(2): 141-150]) etc., which are incorporated herein by reference.

[0199] In addition, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced after being infected by pathogens; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and Van Loon, (1985) Plant Mol. Virol. 4: 111-116. See also WO 1999 / 43819, which is incorporated herein by reference.

[0200] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93: 14972-14977 [Marineau et al., (1987), Plant Molecular Biology, 9: 335-342; Matton et al., (1989), Molecular Plant-Microbe Interactions, 2: 325-331; Somsisch et al., (1986), Proceedings of the National Academy of Sciences of the United States of America, 83: 2427-2430; Somsisch et al., (1988), Molecular Genetics and Genomics, 2: 93-98 and Yang, (1996), Proceedings of the National Academy of Sciences of the United States of America, 93: 14972-14977]. See also, Chen, et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201; Siebertz, et al., (1989) Plant Cell 1:961-968 [Chen et al., (1996), Plant Journal, 10:955-966; Zhang et al., (1994), Proceedings of the National Academy of Sciences of the United States of America, 91:2507-2511; Warner et al., (1993), Plant Journal, 3:191-201; Siebertz et al., (1989), Plant Cell, 1:961-968]; U.S. Patent No. 5,750,386 (nematode-induced), and references cited therein. Of particular interest is the inducible promoter of the maize PRms gene, expression of which is induced by the pathogen Fusarium moniliforme (see, e.g., Cordero, et al., (1992) Physiol. Mol. Plant Path. 41: 189-200).

[0201] Chemically regulated promoters can be used to regulate gene expression in plants by using exogenous chemical regulators. Depending on the target, the promoter can be a chemical inducible promoter, wherein chemicals are used to induce gene expression, or a chemical repressible promoter, wherein chemicals are used to inhibit gene expression. Chemical inducible promoters are known in the art, and include but are not limited to: maize In2-2 promoter (activated by benzenesulfonamide herbicide safener), maize GST promoter (activated by hydrophobic electrophilic compounds used as pre-germination herbicides) and tobacco PR-1a promoter (activated by salicylic acid). Other chemically-regulated promoters of interest include steroid-responsive promoters (see, e.g., glucocorticoid-inducible promoters in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz, et al., (1998) Plant J. 14(2):247-257). al., (1991) Mol. Gen. Genet. 227: 229-237 [Gatz et al., (1991), Molecular Genetics and Genomics, 227: 229-237], and U.S. Patent Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0202] Tissue-preferred promoters can be used to target enhanced insecticidal polypeptide expression to specific plant tissues. Tissue-preferred promoters include those discussed in: Yamamoto, et al., (1997) Plant J. 12(2): 255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7): 792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3): 337-343; Russell, et al., (1997) Transgenic Res. 6(2): 157-168; Rinehart, et al., (1996) Plant Physiol. 112(3): 1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2): 525-535; Canevascini, et al., (1996) Plant Physiol. 112(2): 513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5): 773-778; Lam, (1994) Results Probl. Cell Differ. 20: 181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590 and Guevara-Garcia, et al., (1993) Plant J.4(3):495-505 [Yamamoto et al., (1997), Plant Journal, 12(2):255-265; Kawamata et al., (1997), Plant Cell Physiology, 38(7):792-803; Hansen et al., (1997), Molecular Genetics and Genomics, 254(3):337-343; Russell et al., (1997), Transgenic Research, 6(2):157-168; Rinehart et al., (1996), Plant Physiology, 112(3):1331-1341; Van Camp et al., (1996), Plant Physiology, 112(2):525-535; Canevascini et al., (1996), Plant Physiology, 112(2):513-524; Yamamoto et al., (1994), Plant Cell Physiology, 35(5):773-778; Lam, (1994), Consequences and Problems in Cell Differentiation, 20:181-196; Orozco et al., (1993), Plant Molecular Biology, 23(6):1129-1138; Matsuoka et al., (1993), Proceedings of the National Academy of Sciences of the United States of America, 90(20):9586-9590 and Guevara-Garcia et al., (1993), Plant Journal, 4(3):495-505]. If necessary, such promoters can be modified for weak expression.

[0203] Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2): 255-265; Kwon, et al., (1994) Plant Physiol. 105: 357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5): 773-778; Gotor, et al., (1993) Plant J. 3: 509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6): 1129-1138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590 [Yamamoto et al., (1997), Plant Journal, 12(2):255-265; Kwon et al., (1994), Textile Physiology, 105:357-67; Yamamoto et al., (1994), Plant Cell Physiology, 35(5):773-778; Gotor et al., (1993), Plant Cell, 3:509-18; Orozco et al., (1993), Plant Molecular Biology, 23(6):1129-1138 and Matsuoka et al., (1993), Proceedings of the National Academy of Sciences of the United States of America, 90(20):9586-9590].

[0204] Root-preferred or root-specific promoters are known and can be selected from many available from the literature or isolated de novo from different compatible species. See, e.g., Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990 ... Mol. Biol. 14(3):433-443 [Sanger et al., (1990), Plant Molecular Biology, 14(3):433-443] (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 [Miao et al., (1991), Plant Cell, 3(1):11-22] (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, which describes two root-specific promoters isolated from the hemoglobin gene from the nitrogen-fixing non-legume Parasponia andersonii and the related non-nitrogen-fixing non-legume Trematomentosa. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases root-specific promoter activity was retained. Leach and Aoyagi (1991) described their analysis of the promoters of the highly expressed roIC and roID root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1): 69-76). They concluded that enhancers and tissue-preferred DNA determinants were not associated in those promoters.Teeri et al. (1989) used gene fusions with lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is active especially in the epidermis of the root tip and that the TR2' gene is root-specific in intact plants and is stimulated by wounding in leaf tissue, a particularly desirable combination of features for use with insecticidal or larvicidal genes (see, EMBO J. 8(2): 343-350). The TR1' gene fused to nptII (neomycin phosphotransferase II) showed similar features. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772); and the rolB promoter (Capana, wt al., (1994) Plant Mol. Biol. 25(4):681-691). See also, U.S. Pat. Nos. 5,837,876, 5,750,386, 5,633,363, 5,459,252, 5,401,836, 5,110,732, and 5,023,179. The Arabidopsis root-preferred regulatory sequences are disclosed in US patent application US20130117883. The root-preferred Sorghum (bicolor) RCc3 promoter is disclosed in US patent application US20120210463.

[0205] "Seed preferred" promoters include "seed specific" promoters (those active during seed development such as promoters of seed storage proteins) as well as "seed germinating" promoters (those active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108, which is incorporated herein by reference. Such seed preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa maize protein); and milps (inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529, which is incorporated herein by reference). γ-zea protein and Glb-1 are endosperm specific promoters. For dicots, seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell 1: 1079-1093), bean β-phaseolin, napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, etc. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa maize, 22 kDa maize, 27 kDa maize, g-maize, wax, contractin 1, contractin 2, globulin 1, etc. See also, WO 2000 / 12733, which discloses seed-preferred promoters from end1 and end2 genes; which is incorporated herein by reference. In dicots, seed-specific promoters include, but are not limited to: the seed coat promoter from Arabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S. Pat. Nos. 7,294,760 and 7,847,153). Promoters with "preferred" expression in a particular tissue are expressed to a greater extent in that tissue than in at least one other plant tissue. Some tissue-preferred promoters are expressed almost exclusively in a particular tissue.

[0206] When low-level expression is desired, a weak promoter can be used. Generally, as used herein, the term "weak promoter" refers to a promoter that drives the expression of a coding sequence at a low level. Low-level expression is intended to be at a level of about 1 / 1000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts. Alternatively, it should be appreciated that the term "weak promoter" also encompasses promoters that drive expression only in a few cells but not in other cells to give a completely low expression level. When a promoter drives expression at an unacceptably high level, a portion of the promoter sequence can be deleted or modified to reduce the expression level.

[0207] Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 1999 / 43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, etc. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611, which are incorporated herein by reference.

[0208] The above list of promoters is not meant to be limiting.Any suitable promoter can be used in the embodiments.

[0209] Typically, the expression cassette will include a selectable marker gene for selecting transformed cells. Selectable marker genes are utilized to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2: 987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303: 209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16: 807-820); Estrella et al., (1983) Nature, 303: 209-213 and Meijer et al., (1991) Plant Mol. Biol., 16: 807-820]); streptomycin (Jones et al., (1987) Mol. Gen. Genet. 210: 86-91 [Jones et al., (1987) Mol. Genet., 210: 86-91]); streptomycin (Bretagne-Sagnard et al., (1996) Transgenic Res. 5: 131-137 [Bretagne-Sagnard et al., (1996) Transgenic Res., 5: 131-137]); bleomycin (Hille et al., (1990) Plant Mol.Biol.7:171-176 [Hille et al., (1990), Plant Mol.Biol., 7:171-176]); sulfonamides (Guerineau, et al., (1990) Plant Mol.Biol.15:127-136 [Guerineau et al., (1990), Plant Mol.Biol., 15:127-136]); bromoxynil (Stalker, et al., (1988) Science 242:419-423 [Stalker et al., (1988), Science, 242:419-423]); glyphosate (Shaw, et al., (1986) Science 233:478-481 [Shaw et al., (1986), Science, 233:478-481] and U.S. Patent Application Serial Nos. 10 / 004,357 and 10 / 427,692); glufosinate (DeBlock, et al., (1987) EMBO J. 6:2513-2518 [DeBlock et al., (1987), European Journal of Molecular Biology, 6:2513-2518]). See generally, Yarranton, (1992) Curr. Opin. Biotech. 3: 506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318; Yao, et al., (1992) Cell 71: 63-72; Reznikoff, (1992) Mol. Microbiol. 6: 2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48: 555-566; Brown, et al., (1987) Cell 49: 603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle, et al., (1990) Science 248: 480-483; Gossen, (1993) Ph. D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow, et al. al., (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti, et al. al., (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88: 5072-5076; Wyborski, et al., (1991) Nucleic Acids Res.19:4647-4653;Hillenand-Wissman,(1989)Topics Mol.Struc.Biol.10:143-162;Degenkolb,et al.,(1991)Antimicrob.Agents Chemother.35:1591-1595;Kleinschnidt,et al.,(1988)Biochemistry 27:1094-1104;Bonin,(1993)Ph.D.Thesis,University ofHeidelberg;Gossen,et al.,(1992)Proc.Natl.Acad.Sci.USA 89:5547-5551;Oliva,etal.,(1992)Antimicrob.Agents Chemother.36:913-919;Hlavka,et al.,(1985)Handbookof Experimental Pharmacology,Vol.78(Springer-Verlag,Berlin)and Gill,et al., (1988) Nature 334:721-724 [Yarranton, (1992), Current Opinion in Biotechnology, 3:506-511; Christopherson et al., (1992), Proceedings of the National Academy of Sciences of the United States of America, 89:6314-6318; Yao et al., (1992), Cell, 71:63-72; Reznikoff, (1992), Molecular Microbiology, 6:2419-2422; Barkley et al., (1980), In: Operon, pp. 177-220; Hu et al., (1987), Cell, 48:555-566 ; Brown et al., (1987), Cell, 49:603-612; Figge et al., (1988), Cell, 52:713-722; Deuschle et al., (1989), Proceedings of the National Academy of Sciences of the United States of America, 86:5400-5404; Fuerst et al., (1989), Proceedings of the National Academy of Sciences of the United States of America, 86:2549-2553; Deuschle et al., (1990), Science, 248:480-483; Gossen, (1993), PhD thesis, University of Heidelberg; Reines et al., (1993), Proceedings of the National Academy of Sciences of the United States of America, 90:191 7-1921; Labow et al., (1990), Molecular and Cellular Biology, 10:3343-3356; Zambretti et al., (1992), Proceedings of the National Academy of Sciences of the United States of America, 89:3952-3956; Baim et al., (1991), Proceedings of the National Academy of Sciences of the United States of America, 88:5072-5076; Wyborski et al., (1991), Nucleic Acids Research, 19:4647-4653; Hillenand-Wissman, (1989), Hot Spots Molecule Structural Biology, 10:143-162; Degenkolb et al., (1991), Antimicrobial Agents Chemotherapy, 35: 1591-1595; Kleinschnidt et al., (1988), Biochemistry, 27: 1094-1104; Bonin, (1993), PhD thesis, University of Heidelberg; Gossen et al., (1992), Proceedings of the National Academy of Sciences of the United States of America, 89: 5547-5551; Oliva et al., (1992), Antimicrobial Agents Chemotherapy, 36: 913-919; Hlavka et al., (1985), Handbook of Experimental Pharmacology, p. 78, (Springer-Verlag, Berlin) and Gill et al., (1988), Nature, 334: 721-724]. Such disclosures are incorporated herein by reference.

[0210] The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

[0211] DNA constructs

[0212] DNA constructs encoding polynucleotides of the insecticidal polypeptides of the present disclosure are contemplated. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide operably linked to a heterologous regulatory element. In some embodiments, the DNA construct comprises SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 111, SEQ ID NO: 119, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ The polynucleotide of ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 218, SEQ ID NO: 220 or SEQ ID NO: 222.In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 220 or SEQ ID NO: 222 encoding the PIP-45-1 polypeptide of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 and SEQ ID NO: 236, respectively. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-45-1 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 232, SEQ ID NO: 234 or SEQ ID NO: 236 and has insecticidal activity.In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-45-1 polypeptide.

[0213] In some embodiments, the DNA construct comprises a construct encoding the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 232, SEQ ID NO: 234, or SEQ ID NO: 235. : The invention also provides polynucleotides of a PIP-45-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to NO:236 and having insecticidal activity. In some embodiments, the DNA construct comprises a sequence encoding SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234, or SEQ ID NO: 46. : The invention also provides polynucleotides of a PIP-45-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to NO:236 and having insecticidal activity.

[0214] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 99.1% or greater sequence identity compared to SEQ ID NO: 1. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 99.4% or greater sequence identity compared to SEQ ID NO: 17. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 99.6% or greater sequence identity compared to SEQ ID NO: 19. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 87% or greater sequence identity compared to SEQ ID NO: 21. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 88% or greater sequence identity compared to SEQ ID NO: 23. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 99.1% or greater sequence identity compared to SEQ ID NO: 27. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 99.8% or more sequence identity compared to SEQ ID NO: 29. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 92.3% or more sequence identity compared to SEQ ID NO: 31. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 91.1% or more sequence identity compared to SEQ ID NO: 33. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 95.4% or more sequence identity compared to SEQ ID NO: 35. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 93% or more sequence identity compared to SEQ ID NO: 39. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 97.5% or more sequence identity compared to SEQ ID NO: 43. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO:45.

[0215] The present disclosure also encompasses transgenic plants or plant cells comprising a polynucleotide encoding a PIP-45-2 polypeptide. In some embodiments, the DNA construct comprises SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID The polynucleotide of NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 219, SEQ ID NO: 221 or SEQ ID NO: 223.In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 221 or SEQ ID NO: 223 encoding the PIP-45-2 polypeptide of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 and SEQ ID NO: 237, respectively. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-45-2 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 233, SEQ ID NO: 235 and SEQ ID NO: 237 and having insecticidal activity.In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-45-2 polypeptide.

[0216] In some embodiments, the DNA construct comprises a sequence encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:233, SEQ ID NO:235, or SEQ ID NO: 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a PIP-45-2 polypeptide having insecticidal activity. In some embodiments, the DNA construct comprises a sequence encoding SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235, or SEQ ID NO: 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a PIP-45-2 polypeptide having insecticidal activity.

[0217] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 99.2% or greater sequence identity compared to SEQ ID NO: 2. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 98.5% or greater sequence identity compared to SEQ ID NO: 18. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 96% or greater sequence identity compared to SEQ ID NO: 20. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 80% or greater sequence identity compared to SEQ ID NO: 22. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 81% or greater sequence identity compared to SEQ ID NO: 24. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 99.5% or greater sequence identity compared to SEQ ID NO: 28. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 98.5% or greater sequence identity compared to SEQ ID NO: 30. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 92% or greater sequence identity compared to SEQ ID NO: 32. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 91.5% or greater sequence identity compared to SEQ ID NO: 34. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 36. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 90% or greater sequence identity compared to SEQ ID NO: 40. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 94% or greater sequence identity compared to SEQ ID NO: 44. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-45-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO:46.

[0218] The present disclosure also encompasses a transgenic plant or plant cell comprising a polynucleotide encoding a PIP-64-1 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178 or SEQ ID NO: 224 encoding the PIP-64-1 polypeptide of SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 238, respectively. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 160, SEQ ID NO: 165 or SEQ ID NO: 224 encoding a PIP-64-1 polypeptide of SEQ ID NO: 53, SEQ ID NO: 58 and SEQ ID NO: 238. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-64-1 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide sufficiently homologous to the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 238 and having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 58, or SEQ ID NO: 238 and has insecticidal activity."Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of the PIP-64-1 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 238. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO: 238.

[0219] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 53. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide having at least 99.7% or greater sequence identity compared to SEQ ID NO: 58. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 238.

[0220] The present disclosure also encompasses a transgenic plant or plant cell comprising a polynucleotide encoding a PIP-64-2 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179 or SEQ ID NO: 225 encoding the PIP-64-2 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 239, respectively. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 166 or SEQ ID NO: 225 encoding a PIP-64-2 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 and SEQ ID NO: 239, respectively. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-64-2 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide sufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 or SEQ ID NO: 239 and having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and has insecticidal activity."Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of the PIP-64-2 polypeptide. In some embodiments, the DNA construct comprises a sequence encoding SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, or SEQ ID NO: 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a PIP-64-2 polypeptide having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and having insecticidal activity.

[0221] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 54. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 55. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide having at least 91% or greater sequence identity compared to SEQ ID NO: 59. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptide having at least 70% or greater sequence identity compared to SEQ ID NO: 239.

[0222] The present disclosure also encompasses a transgenic plant or plant cell comprising a polynucleotide encoding a PIP-74-1 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 180, SEQ ID NO: 182 or SEQ ID NO: 184 encoding the PIP-74-1 polypeptide of SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77, respectively. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-74-1 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-1 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of the PIP-74-1 polypeptide. In some embodiments, the polynucleotide encodes a PIP-74-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77.

[0223] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 73. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 75. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-1 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 77.

[0224] The present disclosure also encompasses transgenic plants or plant cells comprising a polynucleotide encoding a PIP-74-2 polypeptide. In some embodiments, the DNA construct comprises polynucleotides of SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185 encoding the PIP-74-2 polypeptides of SEQ ID NO: 74, SEQ ID NO: 76 and SEQ ID NO: 78, respectively. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-74-2 polypeptide. In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of the PIP-74-2 polypeptide. In some embodiments, the polynucleotide encodes a PIP-74-2 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78.

[0225] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-2 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 74. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-2 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 76. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-74-2 polypeptide having at least 75% or greater sequence identity compared to SEQ ID NO: 78.

[0226] The present disclosure also encompasses a transgenic plant or plant cell comprising a polynucleotide encoding a PIP-75 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 encoding a PIP-75 polypeptide of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 encoding a PIP-75 polypeptide of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In some embodiments, the DNA construct comprises a non-genomic nucleic acid molecule encoding a PIP-75 polypeptide. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide sufficiently homologous to the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and has insecticidal activity."Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-75 polypeptide. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and having insecticidal activity. In some embodiments, the polynucleotide encodes a PIP-75 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and having insecticidal activity.

[0227] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 75% or higher sequence identity compared to SEQ ID NO: 79. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 75% or higher sequence identity compared to SEQ ID NO: 80. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 86% or higher sequence identity compared to SEQ ID NO: 81. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 75% or higher sequence identity compared to SEQ ID NO: 84. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 75% or higher sequence identity compared to SEQ ID NO: 85. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 75% or higher sequence identity compared to SEQ ID NO: 86. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-75 polypeptide having at least 75% or higher sequence identity compared to SEQ ID NO: 87.

[0228] The present disclosure also encompasses DNA constructs comprising a polynucleotide encoding a PIP-77 polypeptide. In some embodiments, the DNA construct comprises SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, and SEQ ID NO: 215 encoding the PIP-45-2 polypeptide of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, and SEQ ID NO: 245, respectively. NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID The polynucleotide of NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230 or SEQ ID NO:231.In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 231 encoding a PIP-77 polypeptide of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 245, respectively. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245 and has insecticidal activity.In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and has insecticidal activity. "Sufficiently homologous" is used herein to refer to amino acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding homology of proteins taking into account amino acid similarity, etc. In some embodiments, the sequence homology is to the full-length sequence of a PIP-77 polypeptide.

[0229] In some embodiments, the DNA construct comprises a construct encoding the sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, or SEQ ID NO: 245. : The invention also provides polynucleotides of a PIP-77 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to NO:245 and having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least about 80% or more sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245 and having insecticidal activity.In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least about 90% or more sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245 and having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least about 95% or more sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245 and having insecticidal activity.

[0230] In some embodiments, the DNA construct comprises a sequence encoding SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 103. : The invention also provides polynucleotides of a PIP-77 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to NO:245 and having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least about 80% or more sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and having insecticidal activity. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least about 95% or greater sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and having insecticidal activity.

[0231] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 93% or more sequence identity compared to SEQ ID NO: 88. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 97% or more sequence identity compared to SEQ ID NO: 89. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 99% or more sequence identity compared to SEQ ID NO: 90. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 97% or more sequence identity compared to SEQ ID NO: 92. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 87% or more sequence identity compared to SEQ ID NO: 93. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 86% or more sequence identity compared to SEQ ID NO: 94. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 85% or more sequence identity compared to SEQ ID NO: 95. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 84% or more sequence identity compared to SEQ ID NO: 96. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 85% or more sequence identity compared to SEQ ID NO: 97. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 83% or more sequence identity compared to SEQ ID NO: 98. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 80% or more sequence identity compared to SEQ ID NO: 100. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 85% or more sequence identity compared to SEQ ID NO: 241. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 83% or more sequence identity compared to SEQ ID NO: 242. In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-77 polypeptide having at least 96% or greater sequence identity compared to SEQ ID NO: 245.

[0232] Plant transformation

[0233] The methods of these embodiments involve introducing a polypeptide or polynucleotide into a plant. "Introducing" as used herein means administering a polynucleotide or polypeptide to a plant in a manner that allows the sequence to access the interior of a plant cell. The methods of these embodiments do not rely on a specific method for introducing a polynucleotide or polypeptide into a plant, as long as the polynucleotide or polypeptide can access the interior of at least one cell of the plant. Methods for introducing a polynucleotide or polypeptide into a plant are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0234] As used herein, "stable transformation" means that the nucleotide construct introduced into the plant is integrated into the genome of the plant and can be inherited by its offspring. As used herein, "transient transformation" means that the polynucleotide is introduced into the plant but is not integrated into the genome of the plant, or the polypeptide is introduced into the plant. As used herein, "plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells and pollen).

[0235] Transformation protocols and protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocot or dicot). Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3: 2717-2722 [Paszkowski et al., (1984), European Journal of Molecular Biology, 3: 2717-2722]) and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050, 5,879,918, 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926 [Tomes et al., (1995), In: Plant Cell, Tissue and Organ Culture: Basic Methods, eds.: Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe et al., (1988), Biotechnology, 6:923-926]) and Lec1 transformation (WO 00 / 28058) For potato transformation see, Tu et al., (1998) Plant Molecular Biology 37:829-838 and Chong et al., (2000) Transgenic Research 9:71-78 [Tu et al., (1998), Plant Molecular Biology, 37:829-838 and Chong et al., (2000), Transgenic Research, 9:71-78]. Additional transformation procedures can be found in: Weissinger, et al., (1988) Ann. Rev. Genet.22:421-477;Sanford,et al.,(1987)Particulate Science andTechnology 5:27-37(onion);Christou,et al.,(1988)Plant Physiol.87:671-674(soybean);McCabe,et al.,(1988)Bio / Technology 6:923-926(soybean);Finer andMcMullen,(1991)In Vitro Cell Dev.Biol.27P:175-182(soybean);Singh,et al.,(1998)Theor.Appl.Genet.96:319-324(soybean);Datta,et al.,(1990)Biotechnology8:736-740(rice);Klein,et al.,(1988)Proc.Natl.Acad.Sci.USA 85:4305-4309(maize);Klein,et al.,(1988)Biotechnology 6:559-563(maize);US Patent Numbers5,240,855;5,322,783 and 5,324,646;Klein,et al.,(1988)Plant Physiol.91:440-444(maize);Fromm,et al.,(1990)Biotechnology 8:833-839(maize);Hooykaas-VanSlogteren,et al.,(1984)Nature(London)311:763-764;US Patent Number 5,736,369(cereals);Bytebier,et al.,(1987)Proc.Natl.Acad.Sci.USA 84:5345-5349(Liliaceae);De Wet,et al.,(1985)in The Experimental Manipulation of OvuleTissues,ed.Chapman,et al.,(Longman,New York),pp.197-209(pollen);Kaeppler,etal.,(1990)Plant Cell Reports 9:415-418 and Kaeppler,et al.,(1992)Theor.Appl.Genet.84:560-566(whisker-mediated transformation);D′Halluin,etal.,(1992)Plant Cell 4:1495-1505(electroporation);Li,et al.,(1993)Plant CellReports 12:250-255 and Christou and Ford,(1995)Annals of Botany 75:407-413(rice);Osjoda,et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens) [Weissinger et al., (1988), Annual Review of Genetics, 22:421-477; Sanford et al., (1987), Particle Science and Technology, 5:27-37 (onion); Christou et al., (1988), Plant Physiology, 87:671-674 (soybean); McCabe et al., (1988), Bio / Technology, 6:923-926 (soybean); Finer and McMullen, (1991), In Vitro Cell and Developmental Biology, 27P:175-182 (soybean); Singh et al., (1998), Theoretical and Applied Genetics, 96:31-32 (soybean)] 9-324 (soybean); Datta et al., (1990), Biotechnology, 8:736-740 (rice); Klein et al., (1988), Proc. Natl. Acad. Sci., 85:4305-4309 (maize); Klein et al., (1988), Biotechnology, 6:559-563 (maize); U.S. Pat. Nos. 5,240,855, 5,322,783 and 5,324,646; Klein et al., (1988), Plant Physiology, 91:440-444 (maize); Fromm et al., (1990), Biotechnology, 8:833-839 (maize); Hooykaas-Van Slogteren et al., (1984), Nature (London), 311:763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier et al., (1987), Proceedings of the National Academy of Sciences of the United States of America, 84:5345-5349 (Liliaceae); De Wet et al., (1985), In: Experimental Manipulation of Ovarian Tissue, ed.: Chapman et al., (Longman Publishers, New York), pp. 197-209 (pollen); Kaeppler et al., (1990), Plant Cell Reports, 9:415-418 and Kaeppler et al., (1992), Theoretical and Applied Genetics, 84:560-566 (whisker-mediated transformation); D'Halluin et al., (1993), Plant Cell Reports, 9:415-418 and Kaeppler et al., (1994), Theoretical and Applied Genetics, 9:415-418 and Kaeppler et al., (1995), Theoretical and Applied Genetics, 9:415-418 and Kaeppler et al., (1996), Theoretical and Applied Genetics, 9:415-418 and Kaeppler et al., (1997), Theoretical and Applied Genetics, 9:415-418 and Kaeppler et al., (1998), Theoretical and Applied Genetics, 9:415-418 and Kaeppler et al., (1999), Theoretical and Applied Genetics, 9:415-418 and Kaeppler et al., (1991), Theoretical and Applied Genetics, 9 92), Plant Cell, 4: 1495-1505 (electroporation); Li et al., (1993), Plant Cell Reports, 12: 250-255 and Christou and Ford, (1995), Annals of Botany, 75: 407-413 (rice); Osjoda et al., (1996), Nature Biotechnology, 14: 745-750 (maize via Agrobacterium tumefaciens)]; all of which are incorporated herein by reference.

[0236] In specific embodiments, various transient transformation methods can be used to provide the sequences of the embodiments to plants. Such transient transformation methods include, but are not limited to, directly introducing the insecticidal polypeptides of the present disclosure or variants or fragments thereof into plants or introducing the insecticidal polypeptides of the present disclosure transcripts into plants. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202: 179-185; Nomura, et al., (1986) Plant Sci. 44: 53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush, et al., (1994) The Journal of Cell Biology. Science 107: 775-784 [Crossway et al., (1986), Molecular Genetics and Genomics, 202: 179-185; Nomura et al., (1986), Plant Science, 44: 53-58; Hepler et al., (1994), Proceedings of the National Academy of Sciences of the United States of America, 91: 2176-2180 and Hush et al., (1994), Journal of Cell Science, 107: 775-784], all of which are incorporated herein by reference. Alternatively, the insecticidal polypeptides of the polynucleotides of the present disclosure may be transiently transformed into plants using techniques known in the art. Such techniques include viral vector systems and precipitation of the polynucleotides in a manner that precludes subsequent release of the DNA. Thus, transcription can be performed from the microparticle-bound DNA, but the frequency with which it is released for integration into the genome is greatly reduced. This method includes the use of particles coated with polyethyleneimine (PEI; Sigma #P3143).

[0237] The method for inserting polynucleotides at the specific position of plant genome is known in the art. In one embodiment, the site-specific recombination system is utilized to realize the insertion of polynucleotides at the desired genome position. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853, which are all incorporated herein by reference. In short, the polynucleotides of the present embodiment can be included in the transfer box with two different recombination sites flanking the flank. The transfer box is introduced into the plant, and the target site is stably incorporated into its genome by the plant, and two different recombination sites of the corresponding transfer box site of the target site flank are provided. Suitable recombinase is provided, and the transfer box is integrated into the target site. Thus, the polynucleotides of interest are integrated into the specific chromosome position in the plant genome.

[0238] Plant transformation vector can be made up of one or more DNA vectors needed for realizing plant transformation. For example, it is common practice in this area to use a plant transformation vector made up of more than one continuous DNA segment. In this area, these vectors are often referred to as "binary vectors". Binary vectors and vectors with helper plasmids are most commonly used for Agrobacterium-mediated transformation, wherein the size and complexity of the DNA segment required for realizing effective transformation are quite large, and it is advantageous to divide the function into the isolated DNA molecule. Binary vectors typically include plasmid vectors, which include the cis-acting sequences (such as, left and right borders) required for T-DNA transfer, the selectable markers that can be expressed in plant cells by engineering, and "genes of interest" (genes that can be expressed in plant cells by engineering, for this plant cell, it is desirable to produce transgenic plants). On this plasmid vector, there is also the sequence needed for bacterial replication. These cis-acting sequences are arranged in a way so as to allow effective transfer to and expression in plant cells. For example, this selectable marker gene and this pest-killing gene are located between this left and right borders. Typically the second plasmid vector contains trans-acting factors that mediate T-DNA transformation from Agrobacterium to plant cells. This plasmid often contains the toxic functions (Vir genes) that allow infection of plant cells by Agrobacterium and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5: 446-451). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transformation of these plants by other methods (e.g., microprojectile bombardment, microinjection, electroporation, polyethylene glycol, etc.).

[0239] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (for example, immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), and then using the appropriate selection of the maximum threshold level (depending on the selectable marker gene) to reclaim the transformed plant cells from a group of unconverted cell materials. After the heterologous exogenous DNA is integrated into the plant cell, then in the culture medium, using the appropriate selection of the maximum threshold level to kill these unconverted cells, and by regularly transferring to fresh culture medium to separate and propagate these assumed transformed cells that survive from this selection process. Attack by continuous passage and use appropriate selection, identify and propagate these cells transformed with this plasmid vector. Then, the existence of the interested heterologous gene that can be incorporated into the genome of this transgenic plant can be confirmed using molecular and biochemical methods.

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Claims

1. An insecticidal polypeptide, which consists of the amino acid sequence of SEQ ID NO:

79.

2. An insecticidal composition comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:

79.

3. A recombinant polynucleotide encoding an insecticidal polypeptide consisting of the amino acid sequence of SEQ ID NO:

79.

4. A DNA construct comprising a recombinant polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 79 and a heterologous regulatory sequence operably linked to the recombinant polynucleotide.

5. A method for producing a transgenic plant or plant cell comprising the DNA construct according to claim 4.

6. A method for inhibiting the growth of or killing corn rootworms, comprising contacting the corn rootworm with an insecticidal effective amount of the polypeptide of claim 1.

7. A method of controlling corn rootworm infestation in a transgenic plant, the method comprising expressing the polynucleotide of claim 3 or 4 in the plant.

8. Use of at least one insecticidal polypeptide according to claim 1 for inhibiting the growth of corn rootworms or their populations or killing corn rootworms or their populations.

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