Insecticidal proteins compositions and methods of use

Abstract

Disclosed herein are transformed plants, plant tissues, plant parts, plant cells, and plant seeds comprising a recombinant nucleic acid molecule encoding a polypeptide having pesticidal activity. Also disclosed herein are methods of protecting or treating a plant from infection by a plant pathogen or pest by transforming plants, plant tissues, plant parts, plant cells, and plant seeds with a recombinant nucleic acid molecule encoding a polypeptide having pesticidal activity.

Description

INSECTICIDAL PROTEINS COMPOSITIONS AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 517,808, 63 / 517,824, 63 / 517,831 , 63 / 517,840, 63 / 517,848, 63 / 517,853, and 63 / 517,855, filed on August 4, 2023, each of which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831 and PCT Rule 13 / er. The Sequence Listing XML file submitted in the USPTO Patent Center, “218903-0041-W001_Sequence Listing. xml,” was created on July 15, 2024, contains 20 sequences, has a file size of 36.0 Kbytes (36,864 bytes), and is incorporated by reference in its entirety into the specification.FIELD

[0003] This disclosure relates to the field of molecular biology, specifically, novel genes that encode pesticidal proteins useful for controlling pathogens and pests, particularly plant pests. These proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal compositions and in the production of transgenic pest-resistant plants. The disclosure also relates generally to compositions and methods for controlling pathogens and pests, particularly plant pests.INTRODUCTION

[0004] Across the world, crops are subjected to multiple threats e.g., pests, plant diseases, and weeds. Losses due to pests and diseases are greatly threatening global food supply, hence the necessity to develop solutions to avoid partial or complete destruction of cultures. The main solutions are chemicals, biocontrols or genetically modified organisms (GMO).

[0005] Current GMO strategies use genes expressing pesticidal proteins to produce transgenic crops. These pesticidal proteins are generally derived from Bacillus thuringiensis, a Gram-positive spore forming soil bacterium. The most prominent ones are called Cry (crystal protein) or VIP (Vegetative Insecticidal Protein). Others are named according to a recentlyrevisited Bacterial Pesticidal Protein Resource Center (BPPRC) nomenclature. Transgenic crops expressing insecticidal proteins are used to combat crop damage from insects.

[0006] The wide adoption of pesticidal protein-based technologies by farmers for controlling insects in the fields gave rise to resistance to these pesticidal proteins in some target pests in many parts of the world. One way of solving this problem is stacking pesticidal protein genes with different modes of action against insects in transformed plants. In order to find new pesticidal proteins with new modes of action, possible strategies involve discovering new pesticidal proteins from new sources or identifying pesticidal activity in known genes and redefining their coding for new pesticidal proteins. These new pesticidal proteins may be useful as alternatives to those from or derived from Bacillus thuringiensis for deployment in insect- and pest-resistant transformed plants, underlying the need for novel pesticidal proteins.SUMMARY

[0007] In one aspect, the disclosure relates to a method of protecting a plant from infection by a plant pathogen or pest, the method comprising: transforming the plant with a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , , 7, 10, 12, 15, or 18 to generate a transformed plant expressing the polypeptide, wherein said polypeptide has pesticidal activity against the plant pathogen or pest; and regenerating the transformed plant expressing the polypeptide. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the plant pathogen or pest is selected from the group consisting of fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), cotton boll worm (Helicoverpa armigera), black cutworm (Agrotis ipsilon), lesser cornstalk borer (Elasmopalpus lignosell us), Asian corn borer (Ostinia f urnacai is), southwestern corn borer (Diatraea grandiosella), sugarcane borer (Diatraea saccharalis), western bean cutworm (Striacosta albicosta), velvetbean caterpillar (Anticarsia gemmatalis), corn rootworm (Diabrotica virgifera), southern corn rootworm (Diabrotica undecimpunctata howardi), northern corn rootworm (Diabrotica barberi), soybean looper (Chrysodeixis includens), tobacco budworm (Chloridia virescens), beet armyworm (Spodoptera exigua), southern armyworm (Spodoptera eridania), and combinations thereof.

[0008] In a further aspect, the disclosure relates to a transformed plant, seed, or plant part comprising a recombinant nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18 stably incorporated into a genome of the transformed plant, seed, or plant part, wherein the transformed plant, seed, or plant part stably expresses the polypeptide, and wherein the polypeptide has pesticidal activity against a plant pathogen or pest. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the transformed plant, seed, or plant part is selected from the group consisting of rice, barley, sorghum, soybean, cotton, maize, rapeseed, sugar cane, tobacco, sunflower, and wheat.

[0009] Another aspect of the disclosure provides a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polynucleotide sequence encoding the polypeptide is operably linked to one or more promoter sequences.

[0010] Another aspect of the disclosure provides a vector comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

[0011] Another aspect of the disclosure provides a transformed host cell comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

[0012] Another aspect of the disclosure provides a method of treating a plant or plant part against a plant pathogen or pest, the method comprising: applying to the plant or plant part an effective amount of at least one polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against the plant pathogen or pest. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ I D NOs: 1 , 4, 7, 10, 12, 15, or 18.

[0013] Another aspect of the disclosure provides a composition having insecticidal activity against a plant pathogen or pest, the composition comprising an effective amount of at least one polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In an embodiment, the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

[0014] This disclosure provides for other aspects and embodiments that will be apparent considering the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a depiction of a sequence alignment between the GPA1073A (also termed “GEN05”) amino acid sequence (SEQ ID NO: 1) and a known Cry9Ga1 amino acid sequence (SEQ ID NO: 2).

[0016] FIG. 2 is an image of SDS-PAGE analysis of purified GPA1073A protein from a recombinant E.coli expression vector (~138 kDa).

[0017] FIGS. 3A-3B show graphs depicting the insecticidal activity of different concentrations of purified GPA1073A protein against fall armyworm, Spodoptera frugiperda (FAW) (FIG. 3A) and corn earworm, Helicoverpa zea (CEW) (FIG. 3B). Untreated control (UTC) was a negative control containing only the insect diet, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and 50 mM CAPS with 150 mlVI NaCI pH 11.0 was a negative control containing the buffer solution used for the purified protein assays.

[0018] FIG. 4 is a depiction of a sequence alignment between the GUN0345A (also termed “GEN06”) amino acid sequence (SEQ ID NO: 4) and a known Mpp51Aa3 amino acid sequence (SEQ ID NO: 5).

[0019] FIG. 5 is an image of SDS-PAGE analysis of purified GUN0345A protein from a recombinant E.coli expression vector (~31 kDa).

[0020] FIGS. 6A-6B show graphs depicting the insecticidal activity of different concentrations of purified GUN0345A protein against corn rootworm, Diabrotica virgifera (CRW) (FIG. 6A) and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) (FIG. 6B). Untreated control (UTC) was a negative control containing only the insect diet, Vip3A was a coleopteran negative control containing recombinant bacterial extract with lepidopteran controlling toxin Vip3Aa19, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and 20 mM sodium phosphate with 250 mM NaCI pH 8.0 was a negative control containing the buffer solution used for the purified protein assays.

[0021] FIG. 7 is a depiction of a sequence alignment between the GUN1183A (also termed “GEN07”) amino acid sequence (SEQ ID NO: 7) and a known Mpp46Ab1 amino acid sequence (SEQ ID NO: 8).

[0022] FIG. 8 is an image of SDS-PAGE analysis of purified GUN1183B protein from a recombinant E.coli expression vector (~38 kDa). GUN1183B is a truncated variant of GUN1183A, where a 35-amino acid signal peptide present on the N-temninus of GUN1183A is removed and absent in the truncated GUN1183B variant.

[0023] FIG. 9 shows a graph depicting the insecticidal activity of different concentrations of purified GUN1183B protein against corn earworm, Helicoverpa zea (CEW). Untreated control (UTC) was a negative control containing only the insect diet, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and 20 mM sodium phosphate with 250 mM NaCI pH 8.0 was a negative control containing the buffer solution used for the purified protein assays.

[0024] FIG. 10 is a depiction of a sequence alignment between the GUN0307A (also termed “GEN08”) amino acid sequence (SEQ ID NO: 10) and a known Mpp46Ab1 amino acid sequence (SEQ ID NO: 8).

[0025] FIG. 11 is an image of SDS-PAGE analysis of purified GUN0307A protein from a recombinant E.coli expression vector (~27 kDa).

[0026] FIGS. 12A-12B show graphs depicting the insecticidal activity of different concentrations of purified GUN0307A protein against corn rootworm, Diabrotica virgifera (CRW) (FIG. 12A) and corn earworm, Helicoverpa zea (CEW) (FIG. 12B). Untreated control (UTC) was a negative control containing only the insect diet, Vip3A was a coleopteran negative control containing recombinant bacterial extract with lepidopteran controlling toxin Vip3Aa19, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and PBS was a negative control containing the phosphate buffered saline buffer solution used for the purified protein assays.

[0027] FIG. 13 is a depiction of a sequence alignment between the GUN0527A (also termed “GEN09”) amino acid sequence (SEQ ID NO: 12) and a known Mpp46Aa1 amino acid sequence (SEQ ID NO: 13).

[0028] FIG. 14 is an image of SDS-PAGE analysis of purified GUN0527A protein from a recombinant E.coli expression vector (~36 kDa).

[0029] FIGS. 15A-15B show graphs depicting the insecticidal activity of different concentrations of purified GUN0527A protein against corn earworm, Helicoverpa zea (CEW) (FIG. 15A) and European corn borer, Ostrinia nubilalis (ECB) (FIG. 15B). Untreated control (UTC) was a negative control containing only the insect diet, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and 50 mM CAPS with 150 mM NaCI pH 11 and 10 mM sodium phosphate with 125 mM NaCI pH 8.0 were negative controls containing the buffer solutions used for the purified protein assays.

[0030] FIG. 16 is a depiction of a sequence alignment between the GUN0052A (also termed “GEN 10”) amino acid sequence (SEQ ID NO: 15) and a known Mpp3Aa8 amino acid sequence (SEQ ID NO: 16).

[0031] FIG. 17 is an image of SDS-PAGE analysis of purified GUN0052A protein from a recombinant E.coli expression vector (~32 kDa).

[0032] FIGS. 18A-18B show graphs depicting the insecticidal activity of different concentrations of purified GUN0052A protein against corn rootworm, Diabrotica virgifera (CRW)(FIG. 18A) and corn earworm, Helicoverpa zea (CEW) (FIG. 18B). Untreated control (UTC) was a negative control containing only the insect diet, Vip3A was a coleopteran negative control containing recombinant bacterial extract with lepidopteran controlling toxin Vip3Aa19, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and 10 mM sodium phosphate with 100 mM NaCI was a negative control containing the buffer solution used for the purified protein assays.

[0033] FIG. 19 is a depiction of a sequence alignment between the GPA1280A (also termed “GEN 11”) amino acid sequence (SEQ ID NO: 18) and a known Mpp3Aa8 amino acid sequence (SEQ ID NO: 16).

[0034] FIG. 20 is an image of SDS-PAGE analysis of purified GPA1280A protein from a recombinant E.coli expression vector (~37 kDa).

[0035] FIGS. 21 A-21 B show graphs depicting the insecticidal activity of different concentrations of purified GPA1280A protein against corn earworm, Helicoverpa zea (CEW) (FIG. 21 A) and fall armyworm, Spodoptera frugiperda (FAW) (FIG. 21 B). Untreated control (UTC) was a negative control containing only the insect diet, Gpp34 / Tpp35 was a lepidopteran negative control containing recombinant bacterial extract with coleopteran controlling binary toxin Gpp34Ab1 / Tpp35Ab1 , and 50 mM CAPS with 150 mM NaCI pH 11.0 was a negative control containing the buffer solution used for the purified protein assays.

[0036] Before any embodiments of this disclosure are explained in detail, it is to be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the accompanying figures. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.DETAILED DESCRIPTION

[0037] Described herein are compositions and methods comprising insecticidal proteins useful for conferring pesticidal activity. Disclosed compositions may include isolated, recombinant, and purified polypeptides having pesticidal activity. In some embodiments, the compositions and methods may comprise Cry9-, Mpp51-, Mpp46-, or Mpp3-like proteins having pesticidal activity. In other embodiments, the compositions and methods may comprise proteinshaving pesticidal activity that do not have any significant sequence identity with other known pesticidal proteins.

[0038] In some embodiments, recombinant nucleic acid molecules including DNA constructs and vectors that encode polypeptides having pesticidal activity are described herein. In some embodiments, nucleic acid molecules and polypeptides may be described as DNA constructs and expression cassettes for transforming plants, plant tissues, plant parts, plant cells, and plant seeds, as well as microorganisms. Polypeptides having pesticidal activity as described herein may provide useful alternatives to those currently deployed in commercial transgenic plants.

[0039] Unless otherwise defined herein, all technical and scientific terms used in connection with the present disclosure shall have the same meanings that are commonly understood by those of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0040] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0042] The term “about” or “approximately” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptableerror range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0043] “Amino acid” as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0044] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an organism to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0045] “Complement” or “complementary” as used herein can mean Watson-Crick (e.g., A- T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0046] The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” asused herein refers to a group of control organisms. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. The normal levels or ranges for a target or for a protein activity may be defined in accordance with standard practice. A control may be an organism or cell without a vector as detailed herein. A control may be an organism, or a sample therefrom, whose condition is known. The organism, or sample therefrom, may be healthy, exposed to a toxin, exposed to a toxin prior to treatment, exposed to a toxin during treatment, or exposed to a toxin after treatment, or a combination thereof.

[0047] “Functional” and “full-functional” as used herein describes protein that has biological activity. A “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional protein.

[0048] “Fusion protein” as used herein refers to a chimeric protein created through the joining of two or more genes or gene fragments that originally coded for separate polypeptides. The translation of the fusion gene results in a single polypeptide with functional properties derived from each of the original polypeptides.

[0049] “Genetic construct” or “construct” as used herein refers to the DNA or RNA nucleic acid molecules that comprise a polynucleotide that encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the organism to which the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the organism, the coding sequence will be expressed.

[0050] The term “heterologous” as used herein refers to nucleic acid comprising two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid that is recombinantly produced typically has two or more sequences from unrelated genes synthetically arranged to make a new functional nucleic acid, for example, a promoter from one source and a coding region from another source. The two nucleic acids are thus heterologous to each other in this context. When added to a cell, the recombinant nucleic acids would also be heterologous to the endogenous genes of the cell. Thus, in a chromosome, a heterologous nucleic acid would include a non-native (non-naturally occurring) nucleic acidthat has integrated into the chromosome, or a non-native (non-naturally occurring) extrachromosomal nucleic acid. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (for example, a “fusion protein,” where the two subsequences are encoded by a single nucleic acid sequence).

[0051] “Identical” or “identity” as used herein in the context of two or more polynucleotide or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of a single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Determining the percent sequence identity between any two or more nucleic acid or amino acid sequences can be accomplished using one or more mathematical algorithms. For example, identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0052] “Normal gene” as used herein refers to a gene that has not undergone a change, such as a loss, gain, or exchange of genetic material. The normal gene undergoes normal gene transmission and gene expression. For example, a normal gene may be a wild-type gene.

[0053] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single stranded or double stranded or may contain portionsof both double stranded and single stranded sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0054] “Open reading frame” refers to a stretch of codons that begins with a start codon and ends at a stop codon. In eukaryotic genes with multiple exons, introns are removed, and exons are then joined together after transcription to yield the final mRNA for protein translation. An open reading frame may be a continuous stretch of codons. In some embodiments, the open reading frame only applies to spliced mRNAs, not genomic DNA, for expression of a protein.

[0055] “Operably linked” as used herein means that expression of a gene is under the control of, or is influenced by, a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. Nucleic acid or amino acid sequences are “operably linked” (or “operatively linked”) when placed into a functional relationship with one another. For instance, a promoter or enhancer is operably linked to a coding sequence if it regulates, or contributes to the modulation of, the transcription of the coding sequence. Operably linked DNA sequences are typically contiguous, and operably linked amino acid sequences are typically contiguous and in the same reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous. Similarly, certain amino acid sequences that are noncontiguous in a primary polypeptide sequence may nonetheless be operably linked due to, for example folding of a polypeptide chain. With respect to fusion polypeptides, the terms “operatively linked” and “operably linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.

[0056] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins,receptors, and transport proteins. The terms “polypeptide”, “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, for example, enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. A domain may be comprised of a series of the same type of motif.

[0057] “Pest” as used herein includes, but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include, but are not limited to, insects selected from the orders of Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, and Trichoptera.

[0058] As used herein, “pesticidal activity,” “insecticidal,” “pesticidal,” or “insecticidal activity” means that the proteins, polypeptides, or toxins of the present disclosure, including proteins that have homology to such proteins, polypeptides, or toxins, are able to induce the stunting (sub- lethal effect) and / or killing (lethal effect) of insect pathogens or pests, including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera, and Coleoptera orders, or the Nematoda phylum.

[0059] As used herein, “plant cell” or “plant cells” means a cell obtained from or found in seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant cell also includes modified cells, such as protoplasts, obtained from the aforementioned tissues, as well as plant cell tissue cultures from which plants can be regenerated, plant calli and plant clumps. As used herein, “plant part” or “plant parts” means organs such as embryos, pollen, ovules, seeds, flowers, kernels, ears, cobs, leaves, husks, stalks, stems, roots, root tips, anthers, silk and the like. Asused herein, “plant” or “plants” means whole plants and their progeny. Progeny, variants and mutants of the regenerated plants are also included, provided that they comprise the introduced nucleic acid molecule as described herein.

[0060] “Promoter” as used herein means a synthetic or naturally derived molecule which is capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organelle in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens or pests, pesticides, metal ions, or inducing agents. Representative examples of promoters include the promoter of the 35S gene from the cauliflower mosaic virus, the promoter from the cassava vein mosaic virus, the promoter of the rice actinl gene, the promoter of the subterranean clover virus gene 4, the promoter region of the ubiquitin 4 gene, and the promoter region of the maize polyubiquitin 1 gene.

[0061] The term “recombinant” when used with reference to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (naturally occurring) form of the cell or express a second copy of a native gene that is otherwise normally or abnormally expressed, under expressed, or not expressed at all.

[0062] “Sample” or “test sample” as used herein can mean any sample in which the presence and / or level of a target is to be detected or determined or any sample comprising a vector as detailed herein. The sample may be a biological sample. Samples may include liquids, solutions, emulsions, or suspensions. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from an organism or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interferingcomponents, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0063] “Subject” and “organism” as used herein interchangeably refers to any plant, seed, plant part, or plant material including, but not limited to, a plant in need of the herein described compositions or methods. The plant may be, for example but not limited to, rice, barley, sorghum, soybean, cotton, maize, rapeseed, sugar cane, tobacco, sunflower, or wheat. The subject may be at any stage of development, such as, for example, seed, sprout, vegetative, budding, flowering, or ripening stages. The subject may be hermaphrodite or dioecious. In some embodiments, the subject may have a specific genetic marker. In some embodiments, the subject may be undergoing other forms of treatment.

[0064] “Substantially identical” can mean that a first and second amino acid or polynucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or greater amino acids or nucleotides, respectively.

[0065] As used herein, “introducing” means presenting to the plant cell, plant part, or plant, a nucleic acid molecule or construct in such a manner that it gains access to the interior of a cell of the plant. Methods of the present disclosure include introducing and expressing in a plant cell, plant part, or plant a nucleic acid sequence or construct as described herein. The methods described herein do not depend on the particular method for introducing the nucleic acid molecule or nucleic acid construct into the plant cell, plant part, or plant, only that it gains access to the interior of at least one cell of the plant or plant part. Methods of introducing nucleotide sequence, selecting transformation event, and regenerating whole plants, which may require routine modification in respect of a particular plant species, are known in the art. The methods may include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and sexual breeding. As such, the nucleic acid molecule or construct can be carried episomally or integrated into the genome of the host cell.

[0066] “Transformed plant cells” as used herein refer to plant cells that have been transformed that can be grown into plants by methods known in the art. These plants can thenbe grown, and either pollinated with the same transformed strain or different strains, where the resulting progeny have the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.

[0067] “Transformation event” means a product of organism or cell transformation with a heterologous DNA construct, the regeneration of a population of organisms resulting from the insertion of the recombinant DNA into the genome of the organism, and selection of a particular organism characterized by insertion of the gene construct into a particular genome location resulting in a transgenic cell of organism.

[0068] “Transformed organisms” or “transformed plants” refers to organisms or plants having integrated into their genome a genetic construct nucleic acid molecule. All cells of the transformed organisms or plants may have the genetic construct integrated into their genome. A transformed plant may be a fertile plant and more particularly a plant which agronomic properties (yield, grain quality, drought tolerance, etc.) are not impaired compared to the same plant not transformed. In some embodiments, organisms or plants are transformed using agrobacterium-mediated transformation. Other suitable transformation methods may include, for example, particle bombardment or silicon carbide whiskers, CRISPR, TALENs, or other genome modification techniques. Genome modification techniques may alter the genome of a plant through insertion or other alteration of the plant genome. In some embodiments, a modified plant comprising a nucleic acid encoding a polypeptide as disclosed herein is contemplated.

[0069] In some embodiments, the disclosed polynucleotides encoding a polypeptide may be introduced into the genome of a plant using genome editing technologies, or previously introduced polynucleotides in the genome of a plant may be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies including, but not limited to, TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. The disclosed polynucleotides may be introduced into a desired location in a plant genome using a CRISPR-Cas system for the purpose of site-specific insertion. The desired location in a plant genome may be any desired target site for insertion, such as a genomic region optimized for breeding, or may be a target site located in a genomic region with anexisting trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait.

[0070] In some embodiments, where the disclosed polynucleotide encoding the insecticidal polypeptide has previously been introduced into a genome, genome editing technologies may be used to alter or modify the introduced polynucleotide encoding the insecticidal polypeptide sequence. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position additional nucleotide sequences encoding additional insecticidally-active proteins in close proximity to the disclosed polynucleotide encoding the insecticidal polypeptide disclosed herein within the genome of a plant, in order to generate molecular stacks of insecticidally-active proteins.

[0071] “Transgene” as used herein refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. The introduction of a transgene has the potential to change the phenotype of an organism.

[0072] “Treatment” or “treating” or “treatment” when referring to protection of a subject from a toxin, means suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of damage or death due to exposure to a toxin, or completely eliminating a damage or death due to exposure to a toxin. A treatment may be either performed in an acute or chronic way. Preventing damage or death due to exposure to a toxin involves administering a composition of the present disclosure to a subject prior to exposure to a toxin. Suppressing damage or death due to exposure to a toxin involves administering a composition of the present disclosure to a subject exposure to a toxin but before the appearance of damage. Repressing or ameliorating damage or death due to exposure to a toxin involves administering a composition of the present disclosure to a subject after the appearance of damage.

[0073] “Variant,” with respect to a nucleotide or polynucleotide, means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referencednucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto.

[0074] “Variant,” with respect to a peptide, polypeptide, or protein, means differing in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retaining at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific polypeptide or to promote a specific response. Biological activity can also mean pesticidal or insecticidal activity. Variant can mean a functional fragment thereof, including functional truncated fragments and variants. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. A conservative substitution of an amino acid, for example, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. The hydrophilicity of amino acids may also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0075] In some embodiments, variant pesticidal proteins may be engineered by methods known in the art such that their sequence differs from a natural (i.e., native) or “wild-type” sequence. Protein engineering methods may be used to achieve, for example, improved pesticidal activities against specific pests (i.e., optimization) or altered target spectrum. As disclosed herein, suitable engineering methods for the generation of variant pesticidal proteins may include, but are not limited to, domain swapping, DNA shuffling, saturation mutagenesis,site-directed mutagenesis, oligonucleotide-mediated mutagenesis, cassette mutagenesis, and error-prone PCR techniques.

[0076] Variant nucleotide sequences and proteins disclosed herein encompass sequences and proteins derived from a mutagenic or recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create an engineered pesticidal protein possessing one or more desired properties. In this manner, libraries of recombinant polynucleotides can be generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, full- length coding sequences, sequence motifs encoding a domain of interest, or any fragment of a nucleotide sequence may be shuffled between nucleotide sequences encoding the pesticidal proteins described herein and other known pesticidal nucleotide sequences to obtain a new gene coding for an engineered protein having an improved property of interest, such as an increased insecticidal activity. Properties of interest may include, but are not limited to, pesticidal activity per unit of pesticidal protein, protein stability, and non-toxicity to non-target species, particularly humans, livestock, and plants and microbes that express the disclosed pesticidal proteins. DNA shuffling methods may involve only nucleotide sequences disclosed herein or may additionally involve shuffling of other nucleotide sequences known in the art. Strategies for such shuffling methods are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 :10747-10751 ; Stemmer (1994) Nature 370:389-391 ;Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al.(1998) Nature 391 :288-291 ; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0077] Domain swapping is another known technique for generating variant pesticidal proteins. Domains may be swapped between pesticidal polypeptides, resulting in hybrid or chimeric proteins having, for example, improved pesticidal activity or target spectrum. Methods for generating recombinant engineered proteins and testing them for pesticidal activity are known in the art. See, for example, Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328- 5330; de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930; and Rang, et al., 91999) Appl. Environ. Microbiol. 65:2918-2925.

[0078] Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of a nucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for the ability to confer pesticidal activity to identify mutants that retain activity or have improved activity. Following mutagenesis, the encoded pesticidal protein can be expressed recombinantly, and the activity of the variant protein can be determined using standard assay techniques known in the art.

[0079] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be a bacterial plasmid, viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, and may be a DNA plasmid. For example, the vector may encode a pesticidal protein.

[0080] Provided herein are nucleic acid molecules. A nucleic acid molecule may include a pesticidal gene polynucleotide such as that encoding any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, a selectable marker gene to allow transgenic plant selection, and / or a visual reporter marker such as GFP. The nucleic acid molecule may also comprise a nucleic acid that encodes a fusion protein.

[0081] Nucleic acid molecules described herein may include polynucleotides such as vectors and plasmids. The vector may be an expression vector or system to produce protein by routine techniques and readily available starting materials. The nucleic acid molecule may be recombinant. The nucleic acid molecule may comprise regulatory elements for gene expression of the coding sequences of the nucleic acid. Coding sequences in the nucleic acid molecule may be optimized for stability and high levels of expression. Regulatory elements may include a promoter, an enhancer, an initiation codon, a stop codon, and / or a polyadenylation signal.

[0082] In one aspect, the nucleic acid molecule may encode a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 75% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 85% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acidmolecule may encode a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 91% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 92% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 93% sequence identity to any one of SEQ I D NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 94% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 95% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 96% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 97% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 98% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99.2% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99.5% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99.8% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99.9% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity; or, the nucleic acid molecule may encode a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity.

[0083] In one embodiment, the present disclosure is directed to an isolated nucleic acid molecule encoding a polypeptide amino acid sequence having at least 70% or at least 90% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, and having pesticidal activity. The pesticidal polypeptides and nucleic acid molecules encoding the pesticidalpolypeptides of the present disclosure are particularly useful in agricultural crops for controlling and killing pests.

[0084] In one aspect, the present disclosure is directed to a method for producing a transgenic plant having pesticidal activity. The method may include transforming a plant cell with a nucleic acid molecule described herein, selecting a plant cell comprising the nucleic acid described herein, and regenerating a transgenic plant from the plant cell comprising the nucleic acid molecule described herein, wherein the transgenic plant expresses the nucleic acid molecule described herein and wherein the transgenic plant has pesticidal activity.

[0085] In one aspect, the present disclosure is directed to a method of protecting a plant from pest infestation related damage. The method may include introducing to the plant a nucleic acid molecule described herein, wherein the plant expresses the nucleic acid molecule and wherein the resulting polypeptide has pesticidal activity.

[0086] The plants or transgenic plants described herein may be protected from infection by plant pests including, but not limited to, fall armyworm (Spodoptera frugiperda) (FAW), corn earworm (Helicoverpa zea) (CEW), European corn borer (Ostrinia nubilalis), cotton boll worm (Helicoverpa armigera), black cutworm (Agrotis ipsilon), lesser cornstalk borer (Elasmopalpus lignosellus), Asian corn borer (Ostinia furnacalis), southwestern corn borer (Diatraea grandiose! la), sugarcane borer (Diatraea saccharalis), western bean cutworm (Striacosta albicosta), velvetbean caterpillar (Anticarsia gemmatalis), and combinations thereof.

[0087] In one aspect, the present disclosure is directed to a host cell comprising a nucleic acid molecule described herein. Suitable host cells may include prokaryote host cells and eukaryote host cells.

[0088] Particularly suitable prokaryote host cells may include archaea and bacteria. Particularly suitable eukaryote host cells may include plants and fungi. Suitable host cells may also include microbial cells such as Trichoderma, Aspergillus, Neurospora, Humicola, Penicillium, Fusarium, Thermomonospora, Bacillus, Pseudomonas, Escherichia, Clostridium, Cellulomonas, Streptomyces, Yarrowia, Pichia and Saccharomyces, and microalgal cells belonging to cyanobacterial species. Suitable plant host cells may include dicotyledons and monocotyledons. Suitable dicotyledons may include dicotyledons such as tobacco, cotton, soybean, sunflower, rapeseed, and monocotyledons such as wheat, rice, barley, sorghum, and maize.

[0089] In one aspect, the present disclosure is directed to a transgenic plant, a transgenic plant tissue, a transgenic plant cell, or a transgenic plant seed comprising a nucleic acid molecule described herein, and having pesticidal activity.

[0090] As described herein, the transformed plant cells, plant parts, or plants may have at least one nucleic acid molecule, nucleic acid construct, expression cassette or vector that encodes a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 75% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 80% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 85% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 91% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 92% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 93% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 94% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 95% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 96% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 97% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 98% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99.2% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 99.5% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 99.8% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99.9% sequence identity to any one of SEQ I D NOs: 1 , 4, 7, 10, 12, 15, or 18, or the at least one nucleic acid molecule, nucleic acid construct, expression cassette or vector may encode a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the transformed plant cells, transformed plant parts, or transformed plants have pesticidal activity.

[0091] The present disclosure also relates to homologs of the described insecticidal proteins, provided that the homologs retain insecticidal or pesticidal activity. Homolog sequences can be isolated from public or private collections and can also be prepared by various conventional methods, such as random mutagenesis, site-directed mutagenesis, gene synthesis or gene shuffling, based on all or a part of the peptide sequences presented in the present disclosure, or using all or part of their coding nucleotide sequences. Such homologs include, for example, deletions, insertions, or substitutions of one or more residues in the amino acid sequence of the protein, or a combination thereof. In some embodiments, a homolog mayinclude a protein having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least99.2% sequence identity, at least 99.5% sequence identity, at least 99.8% sequence identity, or at least 99.9% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

[0092] In addition to the full-length nucleotide sequence of a nucleic acid molecule encoding a polypeptide of any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, the nucleic acid molecule encoding any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18 may include a fragment or variant thereof that encodes a polypeptide capable of pesticidal activity. For nucleotide sequences, “fragment” as used herein means a portion of a nucleotide sequence of a nucleic acid molecule, for example, a portion of the nucleotide sequence encoding any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18. Fragments of a nucleotide sequence may retain the biological activity of the reference nucleic acid molecule. For example, a nucleic acid molecule encoding less than the entire amino acid sequence disclosed in any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18 may be used to encode a protein that retains its pesticidal activity. Alternatively, fragments of a nucleotide sequence can be used as hybridization probes or as an amplification primer. Fragments used as hybridization probes or primers generally do not need to retain biological activity. Thus, fragments of the nucleic acid molecules can be at least about 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 900 nucleotides, or up to the number of nucleotides present in a full-length nucleic acid molecule. A biologically active portion (fragment or variant) of the nucleic acid molecule can be prepared by isolating part of the sequence of the nucleic acid molecule, operably linking that fragment to a promoter, expressing the nucleotide sequence encoding the protein, and assessing the amount or activity of the protein.

[0093] In some embodiments, the nucleotide sequence or nucleic acid molecule encoding the polypeptide of any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18 can also be stacked with nucleotide sequences encoding for agronomic traits such as male sterility, stalk strength, flowering time or transformation technology traits such as cell cycle regulation or gene targeting. These stacked combinations can be created by any method including cross breeding plants by any conventional or TopCross™ methodology (DuPont Specialty Grains; Des Moines, Iowa), zinc finger nucleases (ZFNs), transcription activator- 1 ike effector nucleases (TALENs), clusteredregularly interspaced short palindromic repeats (CRISPR) and other genetic transformation. If the traits are stacked by genetically transforming the plants, the nucleotide sequences of interest can be combined at any time and in any order. For example, a transformed plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate expression cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters.

[0094] In one aspect, the present disclosure is directed to a vector that may comprise a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 75% sequence identity to any one of SEQ ID NOs: 1 , , 7, 10, 12, 15, or 18, at least 80% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 85% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 91 % sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 92% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 93% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 94% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 95% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 96% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 97% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 98% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99.2% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 99.5% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99.8% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, or at least 99.9% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

[0095] Suitable vectors are known in the art. Particularly suitable vectors include antibiotic resistance or thermostable antibiotic resistance, or coding for an enzyme that can complement an auxotrophy (natural, such as overcoming the absence of an indispensable amino acid, or engineered, such as URA3-deficient mutants where URA3 is necessary for uracil biosynthesis). Selectable markers include those conferring resistance to antibiotics such as kanamycin (nptllgene), hygromycin (aph IV) spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Selectable markers that allow a direct visual identification of transformation events can also be employed, for example, genes expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a betaglucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

[0096] In one aspect, the present disclosure is directed to a formulation that may include a recombinant polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 75% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 80% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 85% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 91% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 92% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 93% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 94% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 95% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 96% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 97% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 98% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, at least 99.2% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 99.5% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, at least 99.8% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, or at least 99.9% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, and having pesticidal activity. When applied to a plant, the recombinant polypeptide exhibits pesticidal activity.

[0097] Formulations of recombinant polypeptide comprising an acceptable carrier may be in the form of a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, encapsulations, or combinations thereof.

[0098] Formulations of recombinant polypeptide may include surface-active agents, inert carriers, preservatives, humectants, feeding stimulants, attractants, encapsulating agents,binders, emulsifiers, dyes, UV protectants, buffers, flow agents, fertilizers, solvents, dispersants, wetting agents, tackifiers, micronutrient donors, and combinations thereof.

[0099] In one aspect, the present disclosure is directed to a formulation that may include a transformed bacteria comprising a nucleic acid molecule as described herein, and having pesticidal activity. When applied to a plant, the transformed bacteria of the formulation express the nucleic acid molecule and the polypeptide exhibits pesticidal activity.[000100] Formulations of transformed bacteria comprising an acceptable carrier may be in the form of a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, encapsulations, or combinations thereof.[000101] Formulations of transformed bacteria may include surface-active agents, inert carriers, preservatives, humectants, feeding stimulants, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectants, buffers, flow agents, fertilizers, solvents, dispersants, wetting agents, tackifiers, micronutrient donors, and combinations thereof.[000102] Transformed bacteria comprising a nucleic acid molecule as described herein may be used in the same manner that Bacillus thuringiensis strains have previously been used as insecticidal sprays.[000103] The biological activity of interest of the formulations comprising recombinant polypeptide or transformed bacteria is the control of damage-causing plant pests. Such biological activity can be assayed by applying an effective amount of either formulation to a plant having a plant pest, or at risk of being infested by a plant pest, and determining whether the formulation controls the damage-causing plant pests.[000104] In one aspect, the present disclosure is directed to a method for protecting a plant from an insect pest. The method may include expressing in a plant, or a plant cell thereof, a nucleic acid molecule as described herein, wherein the nucleic acid molecule encoding the polypeptide is operably linked to a promoter capable of driving expression in the plant or plant cell thereof, and wherein the encoded polypeptide has pesticidal activity against the insect pest.EXAMPLESEXAMPLE 1[000105] In this example, the GPA1073A amino acid polypeptide sequence was determined.[000106] DNA sequence was isolated from sequenced DNA samples of Bacillus thuringiensis.[000107] Predicted protein sequences corresponding to coding regions in sequenced genomes were aligned using BLASTP against the known Cry9Ga1 protein sequence which has demonstrated insecticidal activity. Novel sequences with a query / subject coverage length of 50% amino acids or more, and an identity of 30% amino acids or more in the protein aligned region were considered as potential candidate-genes. GPA1073A (SEQ ID NO: 1) displayed an 88% sequence identity to Cry9Ga1 (SEQ ID NO: 2) using pairwise full-length protein alignment shown in FIG. 1.[000108] The specific amino acid polypeptide sequences of GPA1073A (SEQ ID NO: 1) and Cry9Ga1 (SEQ ID NO: 2) are provided below in Table 1.[000109] Table 1. Amino acid polypeptide sequences of GPA1073A (SEQ ID NO: 1) and Cry9Ga1 (SEQ ID NO: 2).EXAMPLE 2[000110] In this example, cloning and expression of GPA1073A were performed.[000111] To express GPA1073A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 2 (SEQ ID NO: 3). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing an N-terminal 6x-His TAG coding sequence to the GPA1073A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 2 shows an image of SDS-PAGE analysis of purified GPA1073A protein from the recombinant E.coli expression vector (~138 kDa). In some cases, the bacterial culture following induction was used for insect assays.[000112] Table 2. Optimized GPA1073A DNA coding sequence for GPA1073A expression in E. coli (SEQ ID NO: 3).EXAMPLE 3[000113] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GPA1073A protein to evaluate pesticidal efficacy against pestsincluding fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), and corn rootworm, Diabrotica virgifera (CRW).[000114] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion), were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000115] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion), were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °Cand 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000116] A transformant used as a negative control, as used in this example and in the following examples, was a whole cell bacterial culture of E. coli produced under the same conditions and from the same bacterial strain containing the same expression vector as the treatments containing the insecticidal proteins. The only difference was that the negative control expression vector contained a known gene encoding the CEW, FAW, ECB, SWCB, SCB, and VBC inactive protein Gpp34Ab1 / Tpp35Ab1 , which is active against CRW.[000117] Another transformant used as a negative control, as used in this example and in the following examples, was a whole cell bacterial culture of E. coli produced under the same conditions and from the same bacterial strain containing the same expression vector as the treatments containing the insecticidal proteins. The only difference was that the negative control expression vector contained a known gene encoding the CRW inactive protein Vip3Aa19, which is active against FAW and CEW.[000118] After five days, whole cell bacterial cultures containing GPA1073A protein exhibited fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), and sugarcane borer, Diatraea saccharalis (SCB) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 3). This was significant as none of the known Cry9-like pesticidal proteins have any documented insecticidal activity against these specific pests such as fall armyworm, Spodoptera frugiperda (FAW), and corn earworm, Helicoverpa zea (CEW).[000119] In contrast, the GPA1073A protein did not exhibit larval mortality or growth inhibition on corn rootworm, Diabrotica virgifera (CRW), as compared to the negative controls in bioassay (Table 3), demonstrating specificity of the insecticidal activity for the GPA1073A protein.[000120] Table 3. Measured insecticidal activity of whole recombinant E. coli culture expressing GPA1073A protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 4[000121] In this example, the insecticidal activity of GPA1073A purified protein (FIG. 2) against susceptible fall armyworm, Spodoptera frugiperda (FAW), and corn earworm, Helicoverpa zea (CEW) populations was determined in an artificial diet overlay bioassay.[000122] The same negative controls from Example 3 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000123] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR) and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000124] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000125] After five days, purified GPA1073A protein exhibited dose-dependent insecticidal activity against fall armyworm, Spodoptera frugiperda (FAW) and corn earworm, Helicoverpa zea (CEW), as larval mortality and growth inhibition (i.e., growth stunting) were found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIGS. 3A- 3B). This demonstrated that the mortality and growth inhibition of these specific pests were clearly linked to the presence and amount of GPA1073A protein content in the tested samples.EXAMPLE 5[000126] In this example, the potency of purified GPA1073A protein was evaluated and expressed as EC50, which represents the predicted protein concentration (pg / cm2) that impacts 50% of the infested insect larvae.[000127] The first step of the EC50 process was to determine the necessary protein concentration range to elicit growth inhibition or mortality responses of approximately 50% and100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of WCRMO-1 diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single neonate fall armyworm, Spodoptera frugiperda (FAW) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 25 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead) and growth inhibition.[000128] Once the appropriate protein concentration range was determined, five to nine different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a log-io transformation of the dose variable was used to analyze larval responses such as mortality (total dead larvae relative to total larvae tested) or growth inhibition per protein concentration.[000129] The predicted EC50 of GPA1073A was 2.89 pg / cm2with a 95% confidence interval of 2.00 - 4.14 pg / cm2for susceptible fall armyworm, Spodoptera frugiperda (FAW), while the EC50 for the control reference toxin Vip3Aa19 was 0.04 pg / cm2with a 95% confidence interval of 0.03 - 0.05 pg / cm2for susceptible FAW.EXAMPLE 6[000130] In this example, the GUN0345A amino acid polypeptide sequence was determined.[000131] Protein sequence from Microcystis aeruginosa was isolated from publicly available sequences.[000132] Protein sequence was aligned using BLASTP against known entomotoxins from the BPPRC data base (bpprc.org), identifying Mpp51Aa3 as the closest known protein sequence with demonstrated insecticidal activity to the GUN0345A sequence. GUN0345A (SEQ ID NO: 4) displayed only 15% sequence identity through pairwise full-length protein alignment with Mpp51Aa3 (SEQ ID NO: 5) (FIG. 4), demonstrating its sequence distance from any known insecticidal proteins.[000133] The specific amino acid polypeptide sequences of GUN0345A (SEQ ID NO: 4) andMpp51Aa3 (SEQ ID NO: 5) are provided below in Table 4.[000134] Table 4. Amino acid polypeptide sequences of GUN0345A (SEQ ID NO: 4) andMpp51Aa3 (SEQ ID NO: 5).EXAMPLE 7[000135] In this example, cloning and expression of GUN0345A were performed.[000136] To express GUN0345A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 5 (SEQ ID NO: 6). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing an N-terminal 6x-His TAG coding sequence to the GUN0345A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 5 shows an image of SDS-PAGE analysis of purified GUN0345A protein from the recombinant E.coli expression vector (~31 kDa). In some cases, the bacterial culture following induction was used for insect assays.[000137] Table 5. Optimized GUN0345A DNA coding sequence for GUN0345A expression in E. coli (SEQ ID NO: 6).EXAMPLE 8[000138] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GUN0345A protein to evaluate pesticidal efficacy against pests including corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), southern corn rootworm, Diabrotica undecimpunctata howardi (SCR), fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), and European corn borer, Ostrinia nubilalis (ECB).[000139] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000140] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm,Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000141] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000142] After five days, whole cell bacterial cultures containing GUN0345A protein exhibited corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 6). This was significant as none of the known Mpp51-like pesticidal proteins have any documented insecticidal activity against Diabrotica pests.[000143] In contrast, GUN0345A protein did not exhibit larval mortality or growth inhibition on the lepidopteran species, as compared to the negative controls in bioassay (Table 6), demonstrating specificity of the insecticidal activity for the GUN0345A protein.[000144] Table 6. Measured insecticidal activity of whole recombinant E. coli culture expressing GUN0345A protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 9[000145] In this example, the insecticidal activity of GUN0345A purified protein (FIG. 5) against susceptible corn rootworm, Diabrotica virgifera (CRW), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) populations was determined in an artificial diet overlay bioassay.[000146] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000147] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000148] After five days, purified GUN0345A protein exhibited dose-dependent insecticidal activity against corn rootworm, Diabrotica virgifera (CRW) and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR), as larval mortality and growth inhibition were found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIGS. 6A-6B). This demonstrated that the mortality and growth inhibition of these specific pests were clearly linked to the presence and amount of GUN0345A protein content in the tested samples.EXAMPLE 10[000149] In this example, the potency of purified GUN0345A protein was evaluated and expressed as LC50, which represents the predicted protein concentration (pg / cm2) that results in mortality of 50% of the infested insect larvae.[000150] The first step of the LC50 process was to determine the necessary protein concentration range to kill approximately 50% and 100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of WCRMO-1 diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single neonate corn rootworm, Diabrotica virgifera (CRW) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 24 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead).[000151] Once the appropriate protein concentration range was determined, five to nine different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a logw transformation of the dose variable was used to analyze larval mortality (total dead larvae relative to total larvae tested) per protein concentration.[000152] The predicted LC50 of GUN0345A was 5.34 pg / cm2with a 95% confidence interval of 4.00 - 7.25 pg / cm2for susceptible corn rootworm, Diabrotica virgifera (CRW), while the LC50for the reference binary toxin Gpp34Ab1 / Tpp35Ab1 was 12.42 pg / cm2with a 95% confidence interval of 7.17 - 26.39 g / cm2for susceptible CRW.EXAMPLE 11[000153] In this example, the GUN1183A amino acid polypeptide sequence was determined.[000154] Protein sequence from Melittangium boletus was isolated from publicly available sequences.[000155] Protein sequence was aligned using BLASTP against known entomotoxins from the BPPRC data base (bpprc.org), identifying Mpp46Ab1 as the closest known protein sequence with demonstrated insecticidal activity to the GUN1183A sequence. GUN1183A (SEQ ID NO: 7) displayed only 17% sequence identity through pairwise full-length protein alignment with Mpp46Ab1 (SEQ ID NO: 8) (FIG. 7), demonstrating its sequence distance to any known insecticidal protein.[000156] The specific amino acid polypeptide sequences of GUN1183A (SEQ ID NO: 7) and Mpp46Ab1 (SEQ ID NO: 8) are provided below in Table 7.[000157] Table 7. Amino acid polypeptide sequences of GUN1183A (SEQ ID NO: 7) and Mpp46Ab1 (SEQ ID NO: 8).EXAMPLE 12[000158] In this example, cloning and expression of GUN1183A were performed.[000159] To express GUN1183A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 8 (SEQ ID NO: 9). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing an N-terminal 6x-His TAG coding sequence to the GUN1183A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 8 shows an image of SDS-PAGE analysis of purified GUN1183B protein from a recombinant E.coli expression vector (~38 kDa). GUN1183B is a truncated variant of GUN1183A, where a 35- amino acid signal peptide present on the N-terminus of GUN1183A is removed and absent in the truncated GUN1183B variant. The specific amino acid polypeptide sequence of GUN1183B (SEQ ID NO: 20) is provided below in Table 9. In some cases, the bacterial culture following induction was used for insect assays.[000160] Table 8. Optimized GUN1183A DNA coding sequence for GUN1183A expression in E. coli (SEQ ID NO: 9).[000161] Table 9. Amino acid polypeptide sequence of the GUN1183B (SEQ ID NO: 20) variant.EXAMPLE 13[000162] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GUN1183B protein to evaluate pesticidal efficacy against pests including fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), soybean looper, Chrysodeixis includens (SBL), tobacco budworm, Chloridia virescens (TBW), and corn rootworm, Diabrotica virgifera (CRW).[000163] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000164] Corn rootworm, Diabrotica virgifera (CRW) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000165] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared bypartially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000166] After five days, whole cell bacterial cultures containing GUN1183B protein exhibited fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), soybean looper, Chrysodeixis includens (SBL), and tobacco budworm, Chloridia virescens (TBW) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 10). This was significant as none of the known Mpp46-like pesticidal proteins have any documented insecticidal activity against these specific lepidopteran pests such as fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), and European corn borer, Ostrinia nubilalis (ECB).[000167] In contrast, GUN1183B protein did not exhibit larval mortality or growth inhibition on CRW, as compared to the negative controls in bioassay (Table 10), demonstrating specificity of the insecticidal activity for the GUN1183B protein.[000168] Table 10. Measured insecticidal activity of whole recombinant E. coli culture expressing GUN1183B protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 14[000169] In this example, the insecticidal activity of GUN1183B purified protein (FIG. 8) against a susceptible corn earworm, Helicoverpa zea (CEW) population was determined in an artificial diet overlay bioassay.[000170] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000171] Corn earworm, Helicoverpa zea (CEW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semisolid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (dietoverlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000172] After five days, purified GUN1183B protein exhibited dose-dependent insecticidal activity against corn earworm, Helicoverpa zea (CEW), as larval mortality and growth inhibitionwere found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIG. 9). This demonstrated that the mortality and growth inhibition of this specific pest were clearly linked to the presence and amount of GUN1183B protein content in the tested samples.EXAMPLE 15[000173] In this example, the potency of purified GUN1183B protein was evaluated and expressed as EC50, which represents the predicted protein concentration (pg / cm2) that impacts 50% of the infested insect larvae.[000174] The first step of the EC50 process was to determine the necessary protein concentration range to elicit growth inhibition or mortality responses of approximately 50% and 100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of insect diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single neonate corn earworm, Helicoverpa zea (CEW) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 25 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead) and growth inhibition.[000175] Once the appropriate protein concentration range was determined, five to nine different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a logio transformation of the dose variable was used to analyze larval responses such as mortality (total dead larvae relative to total larvae tested) or growth inhibition per protein concentration.[000176] The predicted EC50 of GUN1183B was 5.54 pg / cm2with a 95% confidence interval of 3.28 - 7.93 pg / cm2for susceptible corn earworm, Helicoverpa zea (CEW), while the EC50 for the control reference toxin Vip3Aa19 was 0.11 pg / cm2with a 95% confidence interval of 0.09 - 0.13 pg / cm2for susceptible CEW.EXAMPLE 16[000177] In this example, the GUN0307A amino acid polypeptide sequence was determined.[000178] Protein sequence from Pseudoalteromonas luteoviolacea was isolated from publicly available sequences.[000179] Protein sequence was aligned using BLASTP against known entomotoxins from the BPPRC database (bpprc.org), identifying Mpp46Ab1 as the closest known protein sequence with demonstrated insecticidal activity to the GUN0307A sequence. GUN0307A (SEQ ID NO: 10) displayed only 37% sequence identity through pairwise full-length protein alignment with Mpp46Ab1 (SEQ ID NO: 8) (FIG. 10), demonstrating its sequence distance to any known insecticidal protein.[000180] The specific amino acid polypeptide sequences of GUN0307A (SEQ ID NO: 10) and Mpp46Ab1 (SEQ ID NO: 8) are provided below in Table 11.[000181] Table 11. Amino acid polypeptide sequences of GUN0307A (SEQ ID NO: 10) and Mpp46Ab1 (SEQ ID NO: 8).EXAMPLE 17[000182] In this example, cloning and expression of GUN0307A were performed.[000183] To express GUN0307A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 12 (SEQ ID NO: 11). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing an N-terminal 6x-His TAG coding sequence to the GUN0307A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHTEXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 11 shows an image of SDS-PAGE analysis of purified GUN0307A protein from the recombinant E.coli expression vector (~27 kDa). In some cases, the bacterial culture following induction was used for insect assays.[000184] Table 12. Optimized GUN0307A DNA coding sequence for GUN0307A expression in E. co / / (SEQ ID NO: 11).EXAMPLE 18[000185] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GUN0307A protein to evaluate pesticidal efficacy against pests including fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), corn rootworm, Diabrotica virgifera (CRW), and northern corn rootworm, Diabrotica barberi (NCR).[000186] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000187] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN).Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminarflow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000188] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000189] After five days, whole cell bacterial cultures containing GUN0307A protein exhibited corn earworm, Helicoverpa zea (CEW), corn rootworm, Diabrotica virgifera (CRW), and northern corn rootworm, Diabrotica barberi (NCR) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 13). This was significant as none of the known Mpp46-like pesticidal proteins have any documented insecticidal activity against these specific lepidopteran and coleopteran pests such as corn earworm, Helicoverpa zea (CEW), corn rootworm, Diabrotica virgifera (CRW), and northern corn rootworm, Diabrotica barberi (NCR).[000190] In contrast, GUN0307A protein did not exhibit larval mortality or growth inhibition on fall armyworm, Spodoptera frugiperda (FAW) or European corn borer, Ostrinia nubilalis (ECB), as compared to the negative controls in bioassay (Table 13), demonstrating specificity of the insecticidal activity for the GUN0307A protein.[000191] Table 13. Measured insecticidal activity of whole recombinant E. coli culture expressing GUN0307A protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 19[000192] In this example, the insecticidal activity of GUN0307A purified protein (FIG. 11) against susceptible corn earworm, Helicoverpa zea (CEW), and corn rootworm, Diabrotica virgifera (CRW) populations was determined in an artificial diet overlay bioassay.[000193] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000194] Corn rootworm, Diabrotica virgifera (CRW) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet. In a laminar flow hood, test samples were applied to the surface of the semisolid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paintbrushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000195] Corn earworm, Helicoverpa zea (CEW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, a single neonate larvae (less than 12 hours posteclosion) was introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000196] After five days, purified GUN0307A protein exhibited dose-dependent insecticidal activity against corn earworm, Helicoverpa zea (CEW) and corn rootworm, Diabrotica virgifera (CRW), as larval mortality and growth inhibition were found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIGS. 12A-12B). This demonstrated that the mortality and growth inhibition of these specific pests were clearly linked to the presence and amount of GUN0307A protein content in the tested samples.EXAMPLE 20[000197] In this example, the potency of purified GUN0307A protein was evaluated and expressed as EC50, which represents the predicted protein concentration (pg / cm2) that impacts 50% of the infested insect larvae.[000198] The first step of the EC50 process was to determine the necessary protein concentration range to elicit a growth inhibition or mortality response from approximately 50% and 100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of insect diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single neonate corn earworm, Helicoverpa zea (CEW) or corn rootworm, Diabrotica virgifera(CRW) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 26 °C or 24 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead) and growth inhibition.[000199] Once the appropriate protein concentration range was determined, five to nine different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a log transformation of the dose variable was used to analyze larval responses such as mortality (total dead larvae relative to total larvae tested) and growth inhibition per protein concentration.[000200] The predicted EC50 of GUN0307A was 22.86 pg / cm2with a 95% confidence interval of 15.71 - 33.74 pg / cm2for susceptible corn rootworm, Diabrotica virgifera (CRW), while the EC50 of the reference binary toxin Gpp34Ab1 / Tpp35Ab1 was 6.57 pg / cm2with a 95% confidence interval of 4.18 - 11.13 pg / cm2for susceptible CRW.[000201] The predicted EC50 of GUN0307A was 42.39 pg / cm2with a 95% confidence interval of 28.67 - 71.04 pg / cm2for susceptible corn earworm, Helicoverpa zea (CEW), while the EC50 of the control reference toxin Vip3Aa19 was 0.11 pg / cm2with a 95% confidence interval of 0.09 - 0.13 pg / cm2for susceptible CEW.EXAMPLE 21[000202] In this example, the GUN0527A amino acid polypeptide sequence was determined.[000203] Protein sequence from the bacteria Nocardiopsis alba was isolated from publicly available sequences. The GUN0527A protein sequence has the NCBI Reference Sequence ID of WP_238543783.1 and is listed as "follicular epithelium yolk protein subunit” through automated annotation.[000204] To relate GUN0527A to any known pesticidal protein, the protein sequence was aligned using BLASTP against known entomotoxins from the BPPRC database (bpprc.org), identifying Mpp46Aa1 as the closest known protein sequence with demonstrated insecticidal activity to the GUN0527A sequence. GUN0527A (SEQ ID NO: 12) displayed only 17%sequence identity through pairwise full-length protein alignment with Mpp46Aa1 (SEQ ID NO: 13) (FIG. 13), demonstrating its sequence distance to any known insecticidal protein.[000205] The specific amino acid polypeptide sequences of GUN0527A (SEQ ID NO: 12) and Mpp46Aa1 (SEQ ID NO: 13) are provided below in Table 14.[000206] Table 14. Amino acid polypeptide sequences of GUN0527A (SEQ ID NO: 12) and Mpp46Aa1 (SEQ ID NO: 13).EXAMPLE 22[000207] In this example, cloning and expression of GUN0527A were performed.[000208] To express GUN0527A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 15 (SEQ ID NO: 14). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing an N-terminal 6x-His TAG coding sequence to the GUN0527A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 14 shows an image of SDS-PAGE analysis of purified GUN0527A protein from the recombinant E.coli expression vector (~36 kDa). In some cases, the bacterial culture following induction was used for insect assays.[000209] Table 15. Optimized GUN0527A DNA coding sequence for GUN0527A expression in E. coli (SEQ ID NO: 14).EXAMPLE 23[000210] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GUN0527A protein to evaluate pesticidal efficacy against pests including fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), black cutworm, Agrotis ipsilon (BCW), and corn rootworm, Diabrotica virgifera (CRW).[000211] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000212] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN).Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000213] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000214] After five days, whole cell bacterial cultures containing GUN0527A protein exhibited fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 16). This was significant as none of the known Mpp46-like pesticidal proteins have any documented insecticidal activity against these specific lepidopteran pests such as fall armyworm, Spodopterafrugiperda (FAW), corn earworm, Helicoverpa zea (CEW), and European corn borer, Ostrinia nubilalis (ECB).[000215] In contrast, GUN0527A protein did not exhibit larval mortality or growth inhibition against CRW (Diabrotica virgifera), as compared to the negative controls in bioassay (Table 16), demonstrating specificity of the insecticidal activity for the GUN0527A protein.[000216] Table 16. Measured insecticidal activity of whole recombinant E. coli culture expressing GUN0527A protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 24[000217] In this example, the insecticidal activity of GUN0527A purified protein (FIG. 14) against susceptible corn earworm, Helicoverpa zea (CEW), and European corn borer, Ostrinia nubilalis (ECB) populations was determined in an artificial diet overlay bioassay.[000218] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000219] Corn earworm, Helicoverpa zea (CEW) and European corn borer, Ostrinia nubilalis (ECB) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000220] After five days, purified GUN0527A protein exhibited dose-dependent insecticidal activity against corn earworm, Helicoverpa zea (CEW) and European corn borer, Ostrinia nubilalis (ECB), as larval mortality and growth inhibition were found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIGS. 15A-15B). This demonstrated that the mortality and growth inhibition of these specific pests were clearly linked to the presence and amount of GUN0527A protein content in the tested samples.EXAMPLE 25[000221] In this example, the potency of purified GUN0527A protein was evaluated and expressed as EC50, which represents the predicted protein concentration (pg / cm2) that impacts 50% of the infested insect larvae.[000222] The first step of the EC50 process was to determine the necessary protein concentration range to elicit a growth inhibition or mortality response from approximately 50% and 100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of insect diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single neonate corn earworm, Helicoverpa zea (CEW) or European corn borer, Ostrinia nubilalis(ECB) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 26 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead) and growth inhibition.[000223] Once the appropriate protein concentration range was determined, five to nine different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a log transformation of the dose variable was used to analyze larval responses such as mortality (total dead larvae relative to total larvae tested) and growth inhibition per protein concentration.[000224] The predicted EC50 of GUN0527A was 11.22 pg / cm2with a 95% confidence interval of 8.53 - 14.65 pg / cm2for susceptible corn earworm, Helicoverpa zea (CEW), while the EC50 of the control reference toxin Vip3Aa19 was 0.11 pg / cm2with a 95% confidence interval of 0.09 - 0.13 pg / cm2for susceptible CEW.[000225] The predicted EC50 of GUN0527A was 3.33 pg / cm2with a 95% confidence interval of 1.49 - 5.39 pg / cm2for susceptible European corn borer, Ostrinia nubilalis (ECB), while the EC50 of the control reference toxin Cry1 F was 0.019 pg / cm2with a 95% confidence interval of 0.015 - 0.024 pg / cm2for susceptible ECB.EXAMPLE 26[000226] In this example, the GUN0052A amino acid polypeptide sequence was determined.[000227] Protein sequence from Vibrio sp. was isolated from the publicly available sequence reference WP_110166803.[000228] Protein sequence was aligned using BLASTP against known entomotoxins from the BPPRC database (bpprc.org), identifying Mpp3Aa8 as the closest known protein sequence with demonstrated insecticidal activity to the GUN0052A sequence. GUN0052A (SEQ ID NO: 15) displayed only 15% sequence identity through pairwise full-length protein alignment with Mpp3Aa8 (SEQ ID NO: 16) (FIG. 16), demonstrating its sequence distance to any known insecticidal protein.[000229] The specific amino acid polypeptide sequences of GUN0052A (SEQ ID NO: 15) andMpp3Aa8 (SEQ ID NO: 16) are provided below in Table 17.[000230] Table 17. Amino acid polypeptide sequences of GUN0052A (SEQ ID NO: 15) andMpp3Aa8 (SEQ ID NO: 16).EXAMPLE 27[000231] In this example, cloning and expression of GUN0052A were performed.[000232] To express GUN0052A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 18 (SEQ ID NO: 17). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing an N-terminal 6x-His TAG coding sequence to the GUN0052A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 17 shows an image of SDS-PAGE analysis of purified GUN0052A protein from the recombinant E.coli expression vector (~32 kDa). In some cases, the bacterial culture following induction was used for insect assays.[000233] Table 18. Optimized GUN0052A DNA coding sequence for GUN0052A expression in E. coli (SEQ ID NO: 17).EXAMPLE 28[000234] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GUN0052A protein to evaluate pesticidal efficacy against pests including fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR).[000235] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000236] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000237] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsiagemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000238] After five days, whole cell bacterial cultures containing GUN0052A protein exhibited corn earworm, Helicoverpa zea (CEW), corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 19). This was significant as none of the known Mpp3-like pesticidal proteins have any documented insecticidal activity against these specific lepidopteran and coleopteran pests such as corn earworm, Helicoverpa zea (CEW), corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR).[000239] In contrast, GUN0052A protein did not exhibit larval mortality or growth inhibition against fall armyworm, Spodoptera frugiperda (FAW) or European corn borer, Ostrinia nubilalis (ECB), as compared to the negative controls in bioassay (Table 19), demonstrating specificity of the insecticidal activity for the GUN0052A protein.[000240] Table 19. Measured insecticidal activity of whole recombinant E. coli culture expressing GUN0052A protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 29[000241] In this example, the insecticidal activity of GUN0052A purified protein (FIG. 17) against susceptible corn earworm, Helicoverpa zea (CEW), and corn rootworm, Diabrotica virgifera (CRW) populations was determined in an artificial diet overlay bioassay.[000242] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000243] Corn rootworm, Diabrotica virgifera (CRW) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect diets, WCRMO-1 diet. In a laminar flow hood, test samples were applied to the surface of the semisolid diet (diet-overlay) to soak-in and evaporate. Once dried, a single neonate larvae (less than 12 hours post-eclosion) was introduced into each well using a fine-tipped paint brush. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000244] Corn earworm, Helicoverpa zea (CEW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, a single neonate larva (less than 12 hours post-eclosion) was introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000245] After five days, purified GUN0052A protein exhibited dose-dependent insecticidal activity against corn earworm, Helicoverpa zea (CEW) and corn rootworm, Diabrotica virgifera (CRW), as larval mortality and growth inhibition were found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIGS. 18A-18B). This demonstrated that the mortality and growth inhibition of these specific pests were clearly linked to the presence and amount of GUN0052A protein content in the tested samples.EXAMPLE 30[000246] In this example, the potency of purified GUN0052A protein was evaluated and expressed as LC50 and EC50, which represent the predicted protein concentration (pg / cm2) that results in mortality of 50% of the infested insect larvae or impacts 50% of the infested insect larvae, respectively.[000247] The first step of the LC50 or EC50 process was to determine the necessary protein concentration range to kill or elicit a growth inhibition response from approximately 50% and 100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of insect diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single neonate corn earworm, Helicoverpa zea (CEW) or corn rootworm, Diabrotica virgifera (CRW) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 25 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead) and growth inhibition.[000248] Once the appropriate protein concentration range was determined, five different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a logio transformation of the dose variable was used to analyze larval mortality (total dead larvae relative to total larvae tested) or growth inhibition per protein concentration.[000249] The predicted EC50 of GUN0052A was 12.28 pg / cm2with a 95% confidence interval of 9.89 - 15.18 pg / cm2for susceptible corn earworm, Helicoverpa zea (CEW), while the EC50 of the control reference toxin Vip3Aa19 was 0.11 pg / cm2with a 95% confidence interval of 0.09 - 0.13 pg / cm2for susceptible CEW.[000250] The predicted LC50 of GUN0052A was 22.64 pg / cm2with a 95% confidence interval of 14.51 - 36.90 pg / cm2for susceptible corn rootworm, Diabrotica virgifera (CRW), while the LC50 of the reference binary toxin Gpp34Ab1 / Gpp35Ab1 was 12.42 pg / cm2with a 95% confidence interval of 7.17 - 26.39 pg / cm2for susceptible CRW.EXAMPLE 31[000251] In this example, GUN0052A was transiently expressed in Nicotiana benthamiana.[000252] Two nucleic acid experimental constructs were made for GUN0052A expression. For the first experimental construct, a DNA nucleotide sequence encoding GUN0052A was cloned between a constitutive promoter linked to a polyadenylation sequence present in a plant binary vector. For the second experimental construct, the DNA nucleotide sequence encoding GUN0052A was cloned between a constitutive promoter linked to a 5’ untranslated region (UTR) and a polyadenylation sequence present in a plant binary vector.[000253] The resulting binary plasmids were transferred into the agrobacterium strain GV3101 to generate a 516 strain and a 517 strain. A standard Nicotiana benthamiana agro-infiltration protocol (essentially as described in bio-protocol.org / bio101 / e95) was used to transiently transform leaf sectors with the agrobacterium 516 and 517 strains.[000254] Total proteins were extracted from the transformed N. benthamiana leaves, and GUN0052A expression was examined using western blot analyses and a monoclonal antibody specific to a 6xHis-Tag of the expressed recombinant protein as probe (Table 20).[000255] Table 20. GUN0052A protein expression in Nicotiana benthamiana tissue 3-4 days after infiltration.[000256] Leaf discs prepared from the agro-infiltrated portion of N. benthamiana leaves were then used in larval insect feeding assays using four different insect pests (Table 21). Strain 285 was used as a negative control in the experiment and did not express the GUN0052A protein. For the strain 285 control, a DNA nucleotide sequence encoding ZsGreen was cloned between a constitutive promoter linked to a polyadenylation sequence present in a plant binary vector.[000257] Table 21. Average leaf disc consumption score of Nicotiana benthamiana infiltrated leaf discs exposed for 5 days to pest larvae in wells of a 96-well plate. Leaf discs were scored on a scale from 3 (high feeding) to 1 (no feeding) based on the % leaf area remaining.EXAMPLE 32[000258] In this example, the GPA1280A amino acid polypeptide sequence was determined.[000259] DNA sequence was isolated from sequenced DNA samples of Bacillus thuringiensis.[000260] Predicted protein sequences corresponding to coding regions in sequenced genomes were aligned using BLASTP against the Mpp3Aa8 protein sequence, which has demonstrated insecticidal activity. Novel sequences with a query / subject coverage length of 50% amino acids or more and an identity of 30% amino acids or more in the protein aligned region were considered as potential candidate-genes. GPA1280A (SEQ ID NO: 18) displayed only 52% sequence identity through pairwise full-length protein alignment with Mpp3Aa8 (SEQ ID NO: 16) (FIG. 19), demonstrating its sequence distance to any known insecticidal protein.[000261] The specific amino acid polypeptide sequences of GPA1280A (SEQ ID NO: 18) and Mpp3Aa8 (SEQ ID NO: 16) are provided below in Table 22.[000262] Table 22. Amino acid polypeptide sequences of GPA1280A (SEQ ID NO: 18) and Mpp3Aa8 (SEQ ID NO: 16).EXAMPLE 33[000263] In this example, cloning and expression of GPA1280A were performed.[000264] To express GPA1280A, the DNA gene coding sequence was optimized for expression in E. coli, and is provided below in Table 23 (SEQ ID NO: 19). This sequence was cloned into the pHis Expression Vector (modified version of pRSF-1b (Novagen)), thus fusing anN-terminal 6x-His TAG coding sequence to the GPA1280A gene. The clone was transformed into E. coli strain BL21(DE3) and grown in an auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). Following induction, bacterial cells were harvested for recombinant protein purification prior to conducting insect larval activity assays. FIG. 20 shows an image of SDS-PAGE analysis of purified GPA1280A protein from the recombinant E.coli expression vector (~37 kDa). In some cases, the bacterial culture following induction was used for insect assays.[000265] Table 23. Optimized GPA1280A DNA coding sequence for GPA1280A expression in E. coli (SEQ ID NO: 19).EXAMPLE 34[000266] In this example, insecticidal toxicity bioassays were conducted with transformed bacterial whole cells expressing GPA1280A protein to evaluate pesticidal efficacy against pests including fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SOB), southern armyworm, Spodoptera eridania (SAW), and corn rootworm, Diabrotica virgifera (CRW).[000267] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000268] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000269] Fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW), European corn borer, Ostrinia nubilalis (ECB), velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), soybean looper, Chrysodeixis includens (SBL), beet armyworm, Spodoptera exigua (BAW), southern armyworm, Spodoptera eridania (SAW), tobacco budworm, Chloridia virescens (TBW), and black cutworm, Agrotis ipsilon (BCW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling either 24-well tissue culture plates, 96-well tissue culture plates (Costar®, Corning®) or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, single or several neonate larvae (less than 12 hours post-eclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5-7 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000270] After five days, whole cell bacterial cultures containing GPA1280A protein exhibited fall armyworm, Spodoptera frugiperda (FAW), corn earworm, Helicoverpa zea (CEW),velvetbean caterpillar, Anticarsia gemmatalis (VBC), southwestern corn borer, Diatraea grandiosella (SWCB), sugarcane borer, Diatraea saccharalis (SCB), and southern armyworm, Spodoptera eridania (SAW) larval mortality and growth inhibition greater than the negative controls in bioassay (Table 24). This was significant as none of the known Mpp3-like pesticidal proteins have any documented insecticidal activity against these specific lepidopteran pests such as fall armyworm, Spodoptera frugiperda (FAW) and corn earworm, Helicoverpa zea (CEW).[000271] In contrast, GPA1280A protein did not exhibit larval mortality or growth inhibition against corn rootworm, Diabrotica virgifera (CRW), as compared to the negative controls in bioassay (Table 24), demonstrating specificity of the insecticidal activity for the GPA1280A protein.[000272] Table 24. Measured insecticidal activity of whole recombinant E. coli culture expressing GPA1280A protein, Coleopteran-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidopteran-active Vip3Aa19 control.EXAMPLE 35[000273] In this example, the insecticidal activity of GPA1280A purified protein (FIG. 20) against susceptible fall armyworm, Spodoptera frugiperda (FAW), and corn earworm, Helicoverpa zea (CEW) populations was determined in an artificial diet overlay bioassay.[000274] The same negative controls from Examples 3 and 4 (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) were used in these assays.[000275] Corn rootworm, Diabrotica virgifera (CRW), northern corn rootworm, Diabrotica barberi (NCR), and southern corn rootworm, Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insectary (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling either 24-well tissue culture plates or 96- well tissue culture plates (Costar®, Corning®) with semi-solid insect diets: WCRMO-1 diet or Southern Corn Rootworm larval diet (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soakin and evaporate. Once dried, single or several neonate larvae (less than 12 hours posteclosion) were introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with membranes and ventilated with 000# pin holes. After 4-5 days of incubation at approximately 24 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000276] Fall armyworm, Spodoptera frugiperda (FAW) and corn earworm, Helicoverpa zea (CEW) eggs were obtained from a commercial insectary (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until eclosion. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect diets. Diets were either General Purpose Lepidoptera diet (Frontier Agricultural Sciences, Newark, DE) or multiple species diet (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, test samples were applied to the surface of the semi-solid diet (diet-overlay) to soak-in and evaporate. Once dried, a single neonate larva (less than 12 hours post-eclosion) was introduced into each well using fine-tipped paint brushes. Bioassay plates were sealed with perforated membranes or membranes ventilated with 000# pin holes. After 5 days incubation at approximately 26 °C and 50% relative humidity (RH), mortality, growth inhibition, and feeding inhibition were assessed.[000277] After five days, purified GPA1280A protein exhibited dose-dependent insecticidal activity against fall armyworm, Spodoptera frugiperda (FAW) and corn earworm, Helicoverpazea (CEW), as larval mortality and growth inhibition were found to be greater than the negative controls in bioassay, in a concentration-dependent manner (FIGS. 21A-21B). This demonstrated that the mortality and growth inhibition of these specific pests were clearly linked to the presence and amount of GPA1280A protein content in the tested samples.EXAMPLE 36[000278] In this example, the potency of purified GPA1280A protein was evaluated and expressed as EC50, which represents the predicted protein concentration (pg / cm2) that impacts 50% of the infested insect larvae.[000279] The first step of the EC50 process was to determine the necessary protein concentration range to elicit growth inhibition or mortality responses of approximately 50% and 100% of the insect larvae in bioassay. An aliquot (20 pL) of protein solution was applied to the top of the diet (approximately 200 pL of insect diet / well of a standard 96-well microtiter plate), and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dry, a single fall armyworm, Spodoptera frugiperda (FAW) larva was infested per well using a fine tipped watercolor brush. After larvae were infested, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a 26 °C dark growth chamber for 5 days. On day 5, each plate was removed from the growth chamber and larvae were assessed for mortality (alive or dead) and growth inhibition.[000280] Once the appropriate protein concentration range was determined, five to nine different concentrations of the purified protein were then tested in a dose-response artificial diet overlay bioassay. As described above, the assays were incubated for 5 days and assessed for lethal and sublethal effects. JMP® 16.2.0 software was used for statistical data analysis. Probit analysis using the Generalized Linear Model for a Binomial distribution with a logio transformation of the dose variable was used to analyze larval responses such as mortality (total dead larvae relative to total larvae tested) or growth inhibition per protein concentration.[000281] The predicted EC50 of GPA1280A was 4.98 pg / cm2with 95% confidence interval of 1.14 - 9.62 pg / cm2for susceptible fall armyworm, Spodoptera frugiperda (FAW), while the EC50 of the control reference toxin Vip3Aa19 was 0.04 pg / cm2with 95% confidence interval of 0.03 - 0.05 pg / cm2for susceptible FAW.EXAMPLE 37[000282] In this example, GPA1280A was transiently expressed in Nicotiana benthamiana.[000283] A nucleic acid experimental construct was made for GPA1280A expression. A DNA nucleotide sequence encoding GPA1280A plus a C-terminal HiBiT luciferase peptide tag was cloned between a constitutive promoter linked to a polyadenylation sequence present in a plant binary vector.[000284] The resulting binary plasmids were transferred into the agrobacterium strain GV3101 to generate a TWP152 strain. A standard Nicotiana benthamiana agro-infiltration protocol (essentially as described in bio-protocol.org / bio101 / e95) was used to transiently transform leaf sectors with the agrobacterium TWP152 strain.[000285] Total proteins were extracted from the transformed N. benthamiana leaves, and GPA1280A expression was examined using Promega’s Nano-Gio® HiBiT Lytic Detection Assay System (Table 25).[000286] Table 25. GPA1280A protein expression in Nicotiana benthamiana tissue 3-4 days after infiltration.[000287] Leaf discs prepared from the agro-infiltrated portion of N. benthamiana leaves were then used in larval insect feeding assays using three different insect pests (Table 26). Strain 285 was used as a negative control in the experiment and did not express the GPA1280A protein. For the strain 285 control, a DNA nucleotide sequence encoding ZsGreen was cloned between a constitutive promoter linked to a polyadenylation sequence present in a plant binary vector.[000288] Table 26. Average leaf disc consumption score of Nicotiana benthamiana infiltrated leaf discs exposed for 5 days to pest larvae in wells of a 96-well plate. Leaf discs were scored on a scale from 3 (high feeding) to 1 (no feeding) based on the % leaf area remaining.EXAMPLE 38[000289] Different plant binary nucleic acid experimental constructs are produced using various promoters, initiators, introns, enhancers, terminators, upstream regulatory constructs, downstream regulatory constructs, or other regulatory sequence elements that are operably linked to drive expression of a nucleotide sequence encoding any of the disclosed GPA1073A, GUN0345A, GUN1183A / B, GUN0307A, GUN0527A, GUN0052A, or GPA1280A proteins (SEQ ID NOs: 1 , 4, 7, 10, 12, 15, 18, or 20) in a target plant, such as maize cells.[000290] In some examples, these nucleic acid experimental constructs are operably linked to sequences encoding specific targeting peptides, such as a Zea mays chloroplast targeting signal peptide.[000291] Each of the experimental constructs are individually transformed into the maize inbred B104. A minimum of 10 individual, single copy transformation events with intact T-DNAs are produced for each construct. qRT-PCR and western blot analyses are performed on TO leaf material to select transgenic plants showing pesticidal protein expression.[000292] The selected transgenic plants and their progenies from the experimental constructs are grown in greenhouse conditions. Pesticidal activity and efficacy of the different transgenic plants are then evaluated against various pests.[000293] Fall armyworm, Spodoptera frugiperda (FAW) efficacy is tested in the greenhouse conditions using a method of artificial infestation of neonate (newly hatched larvae in the 1stlarval stage) FAW larvae onto the whorl leaves of the plant and then rating the leaves after the larvae have fed. FAW efficacy assays are deployed in a randomized complete block design of 4 replications of 3 infested plants. Negative (non-transgenic) and positive (transgenic plantsexpressing reference toxins) controls are utilized as comparators in the root damage assessment. Seeds are counted out and planted in 18 cell flats and placed in a greenhouse bay for germination. The greenhouse bays are set for corn growth with day temperature set to 26-29 °C at 50% RH, and night temperature set to 17-20 °C at 50% RH. The light to dark ratio is 16:8. The seedlings are transplanted into 1-gallon pots at V2 (approximately 14 days). The plants are allowed to grow to V5 / V6 growth stage and then each plant is infested with 30 neonate larvae. The neonate larvae are infested in the maize whorl using an inoculator that delivers a 1 mL aliquot of 2040 corn cob grits (used as a carrier) mixed with neonate larvae. Once infested, the larvae feed on the plants for 14 days. When the plants are deemed ready to rate, the Davis Scale for FAW damage is used to select efficacious plants. Analysis of variance (JMP) is run comparing the transgenic events to the appropriate controls.[000294] Corn earworm, Helicoverpa zea (CEW) efficacy is tested in the greenhouse conditions using a method of artificial infestation of neonate CEW (newly hatched larvae in the 1stlarval stage) at VT (a few days after each plant is hand pollinated) on the top of the ear in the pollinated silks. CEW efficacy assays are deployed in a randomized complete block design of 4 replications and 3 infested plants. Negative (non-transgenic) and positive (transgenic plants expressing reference toxins) controls are utilized as comparators in the ear damage assessment. Seeds are counted out and planted in 18 cell flats and placed in a greenhouse bay for germination. The greenhouse bays are set for corn growth with day temperature set to 26-29 °C at 50% RH, and night temperature set to 17-20 °C at 50% RH. The light to dark ratio is 16:8. The seedlings are transplanted into 3-gallon pots at V2 (approximately 14 days). After hand pollination, each ear is infested on the pollinated silks with 15 neonate larvae. Once infested, the larvae feed for 21 days. When the ears are deemed ready to rate, each ear is husked back and ear damage is measured in cm2per ear and efficacious plants are selected. Analysis of variance (JMP) is run comparing the transgenic events to the appropriate controls.[000295] Corn rootworm, Diabrotica virgifera (CRW) efficacy is tested in the greenhouse conditions using a method of artificial infestation of eggs into the plant and then rating the roots after the eggs have hatched and the larvae have fed. CRW efficacy assays are deployed in a randomized complete block design of 4 replicates of 3 infested plants. Negative (non- transgenic) and positive (transgenic plants expressing reference toxins) controls are utilized as comparators in the root damage assessment. Seeds are counted out and planted into 32 cell flats and placed in a greenhouse bay for germination. The greenhouse bays are set for corn growth with day temperature set to 26-29 °C at 50% RH, and night temperature set to 17-20 °Cat 50% RH. The light to dark ratio is 16:8. The seedlings are transplanted into 1 -gallon pots at V2 (approximately 14 days). The plants are allowed to acclimate for approximately 2 days and then are infested with CRW eggs. The eggs are delivered in a 0.16% agar solution at a rate of 500 eggs per mL. Each plant receives 2 ml_ of egg / agar solution. The solution is delivered in a 1 mL aliquot through a syringe or repeater pipette into each of 2 holes on either side of the plant, approximately 2 inches from the base of the plant and 2 inches deep. The eggs hatch after infestation in approximately 12 days. Once hatched, the larvae feed for approximately 17-21 days. Plants are checked throughout the feeding cycle to monitor feeding progress and proper time to rate. When the plants are determined to be ready, the plants are removed from the greenhouse and washed and rated in a root processing area of the greenhouse complex. The roots are rated using the Iowa State NIS corn injury scale. Analysis of variance (JMP) is run comparing the transgenic events to the appropriate controls.[000296] European corn borer, Ostrinia nubilalis (ECB) efficacy is tested in the greenhouse using a method of artificial infestation of neonate ECB (newly hatched larvae in the 1stlarval stage) at VT / R1 above primary ear and below the secondary ear and then rating the internal stalk and ear shank damage after the larvae have fed. ECB efficacy assays are deployed in a randomized complete block design of 4 replications of 3 infested plants. Negative (non- transgenic) and positive (transgenic plants expressing reference toxins) controls are utilized as comparators in stalk and ear shank damage assessment. Seeds are counted out and planted in 18 cell flats and placed in a greenhouse bay for germination. The greenhouse bays are set for corn growth with day temperature set to 26-29 °C at 50% RH, and night temperature set to IT- 20 °C at 50% RH. The light to dark ratio is 16:8. The seedlings are transplanted into 3-gallon pots at V2 (approximately 14 days). The plants are allowed to grow to VT / R1 growth stage and then each plant is infested one node above the primary ear and one node below the secondary ear with 50 neonate larvae (100 neonate larvae total). The neonate larvae are infested at the proper nodes where the leaf meets the stalk using an inoculator that delivers a 1 mL aliquot of 2040 corn cob grits (used as a carrier) mixed with neonate larvae. Once infested, the larvae feed for 45-60 days. When the plants are deemed ready to rate, each stalk and ear shank is split and the internal damage is measured in cm and efficacious plants are selected. Analysis of variance (JMP) is run comparing the transgenic events to the appropriate controls.[000297] In view of the above, it will be seen that several advantages of the disclosure are achieved, and other advantageous results attained. As various changes could be made in the above methods without departing from the scope of the disclosure, it is intended that all mattercontained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.***[000298] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure.Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.[000299] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.[000300] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes.[000301] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:[000302] Clause 1 . A method of protecting a plant from infection by a plant pathogen or pest, the method comprising: transforming the plant with a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18 to generate a transformed plant expressing the polypeptide, wherein said polypeptide has pesticidal activity against the plant pathogen or pest; and regenerating the transformed plant expressing the polypeptide.[000303] Clause 2. The method of clause 1, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000304] Clause 3. The method of clause 1 or 2, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000305] Clause 4. The method of any one of clauses 1-3, wherein the plant pathogen or pest is selected from the group consisting of fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), cotton boll worm (Helicoverpa armigera), black cutworm (Ag rotis ipsilon), lesser cornstalk borer (Elasmopalpus lignosellus), Asian corn borer (Ostinia furnacalis), southwestern corn borer (Diatraea grandiosella), sugarcane borer (Diatraea saccharalis), western bean cutworm (Striacosta albicosta), velvetbean caterpillar (Anticarsia gemmatalis), corn rootworm (Diabrotica virgifera), southern corn rootworm (Diabrotica undecimpunctata howardi), northern corn rootworm (Diabrotica barberi), soybean looper (Chrysodeixis includens), tobacco budworm (Chloridia virescens), beet armyworm (Spodoptera exigua), southern armyworm (Spodoptera eridania), and combinations thereof.[000306] Clause 5. A transformed plant, seed, or plant part comprising a recombinant nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18 stably incorporated into a genome of the transformed plant, seed, or plant part, wherein the transformed plant, seed, or plant part stably expresses the polypeptide, and wherein the polypeptide has pesticidal activity against a plant pathogen or pest.[000307] Clause 6. The transformed plant, seed, or plant part of clause 5, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000308] Clause 7. The transformed plant, seed, or plant part of clause 5 or 6, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000309] Clause 8. The transformed plant, seed, or plant part of any one of clauses 5-7, wherein the transformed plant, seed, or plant part is selected from the group consisting of rice, barley, sorghum, soybean, cotton, maize, rapeseed, sugar cane, tobacco, sunflower, and wheat.[000310] Clause 9. A recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ IDNOs: 1 , , 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest.[000311] Clause 10. The recombinant nucleic acid molecule of clause 9, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000312] Clause 11. The recombinant nucleic acid molecule of clause 9 or 10, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000313] Clause 12. The recombinant nucleic acid molecule of any one of clauses 9-11 , wherein the polynucleotide sequence encoding the polypeptide is operably linked to one or more promoter sequences.[000314] Clause 13. A vector comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest.[000315] Clause 14. The vector of clause 13, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18.[000316] Clause 15. The vector of clause 13 or 14, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000317] Clause 16. A transformed host cell comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest.[000318] Clause 17. The transformed host cell of clause 16, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000319] Clause 18. The transformed host cell of clause 16 or 17, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000320] Clause 19. A method of treating a plant or plant part against a plant pathogen or pest, the method comprising: applying to the plant or plant part an effective amount of at leastone polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against the plant pathogen or pest.[000321] Clause 20. The method of clause 19, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000322] Clause 21. The method of clause 19 or 20, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000323] Clause 22. A composition having insecticidal activity against a plant pathogen or pest, the composition comprising an effective amount of at least one polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.[000324] Clause 23. The composition of clause 22, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18.[000325] Clause 24. The composition of clause 22 or 23, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

Claims

CLAIMSWhat is claimed is:

1. A method of protecting a plant from infection by a plant pathogen or pest, the method comprising: transforming the plant with a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18 to generate a transformed plant expressing the polypeptide, wherein said polypeptide has pesticidal activity against the plant pathogen or pest; and regenerating the transformed plant expressing the polypeptide.

2. The method of claim 1 , wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

3. The method of claim 1 , wherein the polypeptide is any one of SEQ I D NOs: 1 , 4, 7, 10, 12, 15, or 18.

4. The method of claim 1 , wherein the plant pathogen or pest is selected from the group consisting of fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), cotton boll worm (Helicoverpa armigera), black cutworm (Agrotis ipsilori), lesser cornstalk borer (Elasmopalpus lignosellus), Asian corn borer (Ostinia furnacalis), southwestern corn borer (Diatraea grandiosella), sugarcane borer (Diatraea saccharalis), western bean cutworm (Striacosta al bicosta)., velvetbean caterpillar (Anticarsia gemmatalis), corn rootworm (Diabrotica virgifera), southern corn rootworm (Diabrotica undecimpunctata howardi), northern corn rootworm (Diabrotica barberi), soybean looper (Chrysodeixis includens), tobacco budworm (Chloridia virescens), beet armyworm (Spodoptera exigua), southern armyworm (Spodoptera eridania), and combinations thereof.

5. A transformed plant, seed, or plant part comprising a recombinant nucleic acid molecule encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18 stably incorporated into a genome of the transformed plant, seed, or plant part, wherein the transformed plant, seed, or plant part stably expresses the polypeptide, and wherein the polypeptide has pesticidal activity against a plant pathogen or pest.

6. The transformed plant, seed, or plant part of claim 5, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

7. The transformed plant, seed, or plant part of claim 5, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

8. The transformed plant, seed, or plant part of claim 5, wherein the transformed plant, seed, or plant part is selected from the group consisting of rice, barley, sorghum, soybean, cotton, maize, rapeseed, sugar cane, tobacco, sunflower, and wheat.

9. A recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest.

10. The recombinant nucleic acid molecule of claim 9, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

11. The recombinant nucleic acid molecule of claim 9, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

12. The recombinant nucleic acid molecule of claim 9, wherein the polynucleotide sequence encoding the polypeptide is operably linked to one or more promoter sequences.

13. A vector comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest.

14. The vector of claim 13, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

15. The vector of claim 13, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

16. A transformed host cell comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against a plant pathogen or pest.

17. The transformed host cell of claim 16, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18.

18. The transformed host cell of claim 16, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

19. A method of treating a plant or plant part against a plant pathogen or pest, the method comprising: applying to the plant or plant part an effective amount of at least one polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18, wherein the polypeptide has pesticidal activity against the plant pathogen or pest.

20. The method of claim 19, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

21. The method of claim 19, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

22. A composition having insecticidal activity against a plant pathogen or pest, the composition comprising an effective amount of at least one polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 12, 15, or 18.

23. The composition of claim 22, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

24. The composition of claim 22, wherein the polypeptide is any one of SEQ ID NOs: 1 , 4, 7, 10, 12, 15, or 18.

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