Insecticidal protein 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 a plant, plant tissue, plant part, plant cell, and plant seed with a recombinant nucleic acid molecule encoding a polypeptide having pesticidal activity.

Description

Cross-reference to related applications

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

[0002] Sequence List Reference This application is made in accordance with 37 CFR § 1.831 and PCT Rule 13 ter The sequence list XML was submitted in ST.26 XML format. The sequence list XML file “218903-0041-WO01_Sequence Listing.xml”, filed with the USPTO Patent Centre, was created on July 15, 2024, contains 20 sequences, has a file size of 36.0 kilobytes (36,864 bytes), and is incorporated in its entirety by reference in the specification. Technical Field

[0003] This disclosure relates to the field of molecular biology, and more specifically to novel genes encoding insecticidal proteins that can be used to control pathogens and pests, particularly plant pests. These proteins and the nucleic acid sequences encoding them can be used to prepare insecticidal compositions and produce transgenic pest-resistant plants. This disclosure also relates generally to compositions and methods for controlling pathogens and pests, particularly plant pests. Background Technology

[0004] Around the world, crops are threatened by a variety of factors, such as pests, plant diseases, and weeds. Because losses caused by pests and diseases pose a significant threat to global food supplies, it is essential to develop solutions to prevent partial or total destruction of crops. The main solutions are chemicals, biological control, or genetically modified organisms (GMOs).

[0005] Current GMO strategies use genes expressing insecticidal proteins to produce transgenic crops. These insecticidal proteins are typically derived from Bacillus thuringiensis (Bt). Bacillus thuringiensis (Cry) is a Gram-positive, spore-forming soil bacterium. Its most prominent insecticidal protein is called Cry (crystal protein) or VIP (vegetative-stage insecticidal protein). Other insecticidal proteins are named according to the recently revised Bacterial Insecticidal Protein Resource Center (BPPRC) nomenclature. Transgenic crops expressing insecticidal proteins are used to combat insect damage to crops.

[0006] Farmers widely employ insecticidal protein-based technologies to control insects in the field, leading to resistance to these proteins in some target pests in many parts of the world. One approach to address this problem is to superimpose insecticidal protein genes with different modes of insect resistance into transformative plants. Possible strategies for finding novel insecticidal proteins with new modes of action involve either discovering new insecticidal proteins from novel sources or identifying insecticidal activity in known genes and redefining their encoding for novel insecticidal proteins. These novel insecticidal proteins could serve as substitutes for insecticidal proteins derived from or derived from Bacillus thuringiensis in the development of transformative plants resistant to insects and pests, highlighting the need for new insecticidal proteins. Summary of the Invention

[0007] In one aspect, this disclosure relates to a method for protecting plants from infection by plant pathogens or pests, the method comprising: transforming the plant with a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 to produce a transformed plant expressing the polypeptide, wherein the polypeptide has insecticidal activity against the plant pathogen or pest; and regenerating the transformed plant expressing the polypeptide. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18. In yet another embodiment, the plant pathogen or pest is selected from the group consisting of: fall armyworm (… Spodoptera frugiperda ), corn ear worm ( Helicoverpa zea ), European corn borer ( Ostrinia nubilalis ), cotton bollworm ( Helicoverpa armigera ), Black Root Worm ( Agrotis ipsilon ), corn borer ( Elasmopalpus lignosellus Asian corn borer ( Ostinia furnacalis ), Southwest corn borer ( Diatraea grandiosella ), sugarcane borer ( Diatraea saccharalis ), Western bean rootworm ( Striacosta albicosta ), edamame noctuid moth ( Anticarsia gemmatalis ), corn rootworm ( Diabrotica virgifera Southern corn rootworm ( Diabrotica undecimpunctata howardi ), Northern corn rootworm ( Diabrotica barberi Soybean noctuid moth ( Chrysodeixis includens ), tobacco budworm ( Chloridia virescens ), cabbage brown moth ( Spodoptera exigua Southern armyworm ( Spodoptera eridania ) and their combinations.

[0008] In another aspect, this disclosure relates to a transformed plant, seed, or plant part comprising a recombinant nucleic acid molecule stably integrated into the genome of the transformed plant, seed, or plant part, the recombinant nucleic acid molecule encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, wherein the transformed plant, seed, or plant part stably expresses the polypeptide, and wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. In yet another embodiment, the transformed plant, seed, or plant part is selected from the group consisting of: rice, barley, sorghum, soybean, cotton, corn, rapeseed, sugarcane, tobacco, sunflower, and wheat.

[0009] Another aspect of this disclosure provides a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. In yet another embodiment, the polynucleotide sequence encoding the polypeptide is operatively linked to one or more promoter sequences.

[0010] Another aspect of this disclosure provides a vector comprising a recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0011] Another aspect of this 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 with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18.

[0012] Another aspect of this disclosure provides a method for 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 with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, wherein the polypeptide has insecticidal activity against the plant pathogen or pest. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0013] Another aspect of this disclosure provides a composition having insecticidal activity against plant pathogens or pests, the composition comprising an effective amount of at least one polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18. In one embodiment, the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18. In another embodiment, the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0014] This disclosure provides other aspects and implementations that will become apparent from the following specific embodiments and drawings. Attached Figure Description

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

[0016] Figure 2 It comes from recombinant Escherichia coli ( E. coliImages of SDS-PAGE analysis of purified GPA1073A protein in the expression vector (approximately 138 kDa).

[0017] Figures 3A to 3B The image depicts the effects of different concentrations of purified GPA1073A protein on fall armyworms (FAW). Figure 3A ) and corn earworm (CEW) Figure 3B The graph shows the insecticidal activity of the untreated control (UTC) and the lepidopteran control (Gpp34 / Tpp35). The untreated control (UTC) is a negative control containing only insect feed, the lepidopteran control (Gpp34Ab1 / Tpp35Ab1) is a negative control containing recombinant bacterial extract and the coleopteran control binary toxin Gpp34Ab1 / Tpp35Ab1, and the negative control (50 mM CAPS and 150 mM NaCl pH 11.0) is a negative control containing a buffer solution for protein purification assays.

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

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

[0020] Figures 6A to 6B The image depicts the effects of different concentrations of purified GUN0345A protein on the corn rootworm (CRW). Figure 6A ) and Southern Corn Rootworm (SCR) Figure 6B The graph shows the insecticidal activity of the untreated control (UTC) containing only insect feed. Vip3A is a Coleoptera negative control containing recombinant bacterial extract and the Lepidoptera control toxin Vip3Aa19. Gpp34 / Tpp35 is a Lepidoptera negative control containing recombinant bacterial extract and the Coleoptera control binary toxin Gpp34Ab1 / Tpp35Ab1. 20 mM sodium phosphate and 250 mM NaCl at pH 8.0 are negative controls containing a buffer solution used for protein purification assays.

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

[0022] Figure 8This is an image of SDS-PAGE analysis of purified GUN1183B protein from a recombinant E. coli expression vector (approximately 38 kDa). GUN1183B is a truncated variant of GUN1183A, in which the 35-amino acid signal peptide present at the N-terminus of GUN1183A is removed and is absent in the truncated GUN1183B variant.

[0023] Figure 9 A graph depicting the insecticidal activity of different concentrations of purified GUN1183B protein against the corn earworm (CEW) is shown. The untreated control (UTC) is a negative control containing only insect feed; Gpp34 / Tpp35 is a Lepidoptera negative control containing recombinant bacterial extract and the coleopteran control binary toxin Gpp34Ab1 / Tpp35Ab1; and 20 mM sodium phosphate and 250 mM NaCl at pH 8.0 is a negative control containing the buffer solution used for protein purification assays.

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

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

[0026] Figures 12A to 12B The image depicts the effects of different concentrations of purified GUN0307A protein on the corn rootworm (CRW). Figure 12A ) and corn earworm (CEW) Figure 12B The graph shows the insecticidal activity of the untreated control (UTC) containing only insect feed. Vip3A is a Coleoptera negative control containing recombinant bacterial extract and the lepidopteran control toxin Vip3Aa19. Gpp34 / Tpp35 is a Lepidopteran negative control containing recombinant bacterial extract and the Coleoptera control binary toxin Gpp34Ab1 / Tpp35Ab1. PBS is a negative control containing phosphate-buffered saline solution used for protein purification assays.

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

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

[0029] Figures 15A to 15B The image depicts the effects of different concentrations of purified GUN0527A protein on the corn earworm (CEW). Figure 15A ) and European corn borer (ECB) Figure 15B The graph shows the insecticidal activity of the untreated control (UTC) and the lepidopteran control (Gpp34 / Tpp35). The untreated control (UTC) is a negative control containing only insect feed. The lepidopteran control (Gpp34 / Tpp35) contains recombinant bacterial extract and the coleopteran control binary toxin Gpp34Ab1 / Tpp35Ab1. The negative controls (50 mM CAPS with 150 mM NaCl pH 11 and 10 mM sodium phosphate with 125 mM NaCl pH 8.0) contain buffer solutions for protein purification assays.

[0030] Figure 16 This is a depiction of the sequence alignment between the amino acid sequence of GUN0052A (also known as “GEN10”) (SEQ ID NO: 15) and the known amino acid sequence of Mpp3Aa8 (SEQ ID NO: 16).

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

[0032] Figures 18A to 18B The image depicts the effects of different concentrations of purified GUN0052A protein on the corn rootworm (CRW). Figure 18A ) and corn earworm (CEW) Figure 18B The graph shows the insecticidal activity of the untreated control (UTC) containing only insect feed. Vip3A is a Coleoptera negative control containing recombinant bacterial extract and the Lepidoptera control toxin Vip3Aa19. Gpp34 / Tpp35 is a Lepidoptera negative control containing recombinant bacterial extract and the Coleoptera control binary toxin Gpp34Ab1 / Tpp35Ab1. 10 mM sodium phosphate and 100 mM NaCl are negative controls containing a buffer solution used for protein purification assays.

[0033] Figure 19 This is a depiction of the sequence alignment between the amino acid sequence of GPA1280A (also known as “GEN11”) (SEQ ID NO: 18) and the known amino acid sequence of Mpp3Aa8 (SEQ ID NO: 16).

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

[0035] Figures 21A to 21B The image depicts the effects of different concentrations of purified GPA1280A protein on the corn earworm (CEW). Figure 21A ) and fall armyworm (FAW) Figure 21B The graph shows the insecticidal activity of the untreated control (UTC) and the lepidopteran control (Gpp34 / Tpp35). The untreated control (UTC) is a negative control containing only insect feed, the lepidopteran control (Gpp34Ab1 / Tpp35Ab1) is a negative control containing recombinant bacterial extract and the coleopteran control binary toxin Gpp34Ab1 / Tpp35Ab1, and the negative control (50 mM CAPS and 150 mM NaCl pH 11.0) is a negative control containing a buffer solution for protein purification assays.

[0036] Before explaining any embodiment of this disclosure in detail, it should be understood that the application of this disclosure is not limited to the construction details and component arrangements set forth in the following description or shown in the accompanying drawings. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive. Detailed Implementation

[0037] This document describes compositions and methods comprising insecticidal proteins that can be used to confer insecticidal activity. The disclosed compositions may include isolated, recombinant, and purified polypeptides having insecticidal activity. In some embodiments, the compositions and methods may comprise insecticidal Cry9-like, Mpp51-like, Mpp46-like, or Mpp3-like proteins. In other embodiments, the compositions and methods may comprise insecticidal proteins that do not share any significant sequence identity with other known insecticidal proteins.

[0038] In some embodiments, this document describes recombinant nucleic acid molecules comprising DNA constructs and vectors encoding peptides with insecticidal activity. In some embodiments, the nucleic acid molecules and peptides can be described as DNA constructs and expression cassettes for transforming plants, plant tissues, plant parts, plant cells, and plant seeds, as well as microorganisms. The insecticidal peptides described herein can provide useful alternatives to those currently used in commercial transgenic plants.

[0039] Unless otherwise defined herein, all technical and scientific terms used in conjunction with this disclosure shall have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, this document (including the definitions) shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials may be used in practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0040] As used herein, the terms “comprising,” “including,” “having,” “having,” “may,” “containing,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. Unless the context clearly indicates otherwise, the singular forms “a,” “and,” and “the” include plural references. This disclosure also considers other embodiments of the embodiments or elements “comprising,” “consisting of,” and “substantially consisting of,” as presented herein, whether or not explicitly stated.

[0041] In describing the numerical ranges in this article, each intermediate digit with the same precision is explicitly considered. For example, for the range of 6 to 9, the digits 7 and 8 are considered in addition to 6 and 9, and for the range of 6.0 to 7.0, the digits 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 considered.

[0042] As used herein, when applied to one or more target values, the term "about" or "approximately" means a value similar to the stated reference value, or a value within an acceptable margin of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, such as limitations of the measurement system. In some respects, the term "about" means a range of values ​​falling 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 or less than) of the stated reference value, unless otherwise stated or obvious from the context (unless such figures would exceed 100% of the possible value). Alternatively, according to practice in the art, "about" may mean within 3 or more standard deviations. Alternatively, for example, with respect to biological systems or processes, the term "about" may mean within the order of magnitude of the value, preferably within 5 times the value, and more preferably within 2 times the value.

[0043] As used herein, “amino acid” refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids may be represented herein by their commonly known three-letter symbols or by single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Amino acids include both side chains and the polypeptide backbone.

[0044] As used herein, "coding sequence" or "coding nucleic acid" means a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals, which direct expression in the cells of the organism administering the nucleic acid. The coding sequence may be codon-optimized.

[0045] As used in this article, “complementarity” or “complementarity” can refer to Watson-Crick base pairing (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs in a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences such that when they are aligned in reverse parallelism, the nucleotide bases at each position will be complementary.

[0046] The terms “control,” “reference level,” and “reference” are used interchangeably herein. A reference level can be a predetermined value or range used as a benchmark against which a measurement result is evaluated. As used herein, “control group” refers to a group of control organisms. A predetermined level can be a cutoff value from a control group. A predetermined level can be the mean value from a control group. A normal level or range of target or protein activity can be defined according to standard practice. A control can be an organism or cell that does not have a carrier as detailed herein. A control can be an organism or a sample thereof whose conditions are known. The organism or sample thereof can be healthy, exposed to a toxin, exposed to a toxin before treatment, exposed to a toxin during treatment, or exposed to a toxin after treatment, or a combination thereof.

[0047] As used in this article, "functional" and "fully functional" describe proteins that are biologically active. A "functional gene" is a gene that is transcribed into mRNA, which is then translated into a functional protein.

[0048] As used herein, a "fusion protein" refers to a chimeric protein created by linking two or more genes or gene segments that originally encode individual polypeptides. Translation of the fusion gene produces a single polypeptide that possesses the functional properties derived from each of the original polypeptides.

[0049] As used herein, a “genetic construct” or “construct” refers to a DNA or RNA nucleic acid molecule containing a polynucleotide encoding a protein. The coding sequence includes start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals, that direct expression in the cells of an organism administering the nucleic acid molecule. As used herein, the term “expressible form” refers to a gene construct containing the necessary regulatory elements operatively linked to a coding sequence encoding a protein such that the coding sequence will be expressed when the gene construct is present in the cells of an organism.

[0050] As used herein, the term "heterologous" refers to a nucleic acid containing two or more subsequences that are not identical to each other in nature. For example, recombinant nucleic acids typically have two or more sequences from unrelated genes that are synthesized and arranged to create new functional nucleic acids, such as a promoter from one source and a coding region from another. Therefore, in this context, the two nucleic acids are heterologous to each other. When recombinant nucleic acids are added to a cell, they also become heterologous to the cell's endogenous genes. Thus, in chromosomes, heterologous nucleic acids will include non-native (non-naturally occurring) nucleic acids or non-native (non-naturally occurring) extrachromosomal nucleic acids that have been integrated into the chromosome. Similarly, a heterologous protein indicates that the protein contains two or more subsequences that are not identical to each other in nature (e.g., a "fusion protein," where the two subsequences are encoded by a single nucleic acid sequence).

[0051] As used in the context of two or more polynucleotide or polypeptide sequences, “identical” or “identical” means that these sequences have a specified percentage of identical residues in a specified region. The percentage can be calculated as follows: First, align the two sequences. Then, compare the two sequences in a specified region. Determine the number of positions in both sequences where the same residues occur to obtain the number of matching positions. Divide the number of matching positions by the total number of positions in the specified region, and multiply the result by 100 to obtain the percentage of sequence identity. In cases where the two sequences have different lengths, or where the alignment produces one or more staggered ends, and the specified region being compared includes only a single sequence, the residues of that single sequence are counted in the denominator but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. The determination of the percentage of 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 can be determined manually or by using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0052] As used in this article, a "normal gene" is a gene that has not undergone alteration (such as loss, gain, or exchange of genetic material). Normal genes undergo normal gene transmission and expression. For example, a normal gene can be a wild-type gene.

[0053] As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide” refers to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, polynucleotide also encompasses the complementary strand of the depicted single strand. Many variants of polynucleotides can be used for the same purpose as a given polynucleotide. Therefore, polynucleotide also encompasses substantially the same polynucleotide and its complement. A single strand provides a probe that can hybridize to a target sequence under strict hybridization conditions. Therefore, polynucleotide also encompasses probes that hybridize under strict hybridization conditions. Polynucleotides can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Polynucleotides can be natural or synthetic nucleic acids, DNA, genomic DNA, cDNA, RNA, or hybrids, wherein the polynucleotide can contain combinations of deoxyribonucleotides and ribonucleotides, 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 or by recombinant methods.

[0054] An "open reading frame" (OPF) is a codon sequence that begins with a start codon and ends with a stop codon. In eukaryotic genes with multiple exons, introns are removed, and the exons are then joined together post-transcriptionally to produce the final mRNA for protein translation. An OPF can be a continuous sequence of codons. In some implementations, OPFs are applied only to spliced ​​mRNA, not to genomic DNA, to enable protein expression.

[0055] As used herein, “operably linked” means that gene expression is controlled or influenced by a promoter to which it is spatially linked. A promoter can be located at the 5' (upstream) or 3' (downstream) of a gene. The distance between the promoter and the gene can be approximately equal to the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be accommodated without loss of promoter function. Nucleic acid or amino acid sequences are “operably linked” (or “functionally linked”) when they are placed in a functional relationship with each other. For example, a promoter or enhancer is operably linked to a coding sequence if it regulates or helps regulate transcription of the coding sequence. Operatically linked DNA sequences are typically contiguous, and the amino acid sequences are typically contiguous and in the same reading frame. However, because enhancers often function up to several thousand bases or more apart from promoters, and intron sequences can have varying lengths, some polynucleotide elements can be operably linked but not adjacent. Similarly, certain amino acid sequences that are discontinuous in a primary polypeptide sequence can still be operably linked due to, for example, the folding of the polypeptide chain. Regarding fusion peptides, the terms "functional linkage" or "operable linkage" can refer to the fact that each component functions the same when linked to another component as it does when not linked.

[0056] A “peptide” or “polypeptide” is a linking sequence of two or more amino acids connected by peptide bonds. Polypeptides can be natural, synthetic, or a combination of natural and synthetic modifications. Peptides and polypeptides include proteins, such as binding proteins, receptors, and transport proteins. The terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein. A “primary structure” refers to the amino acid sequence of a particular peptide. A “secondary structure” refers to the locally ordered three-dimensional structure within a polypeptide. These structures are often referred to as domains, such as enzyme-catalyzing domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. A “domain” is a polypeptide portion that forms a compact unit of the polypeptide and is typically 15 to 350 amino acids long. Exemplary domains include those with enzymatic or ligand-binding activity. Typical domains consist of less organized portions, such as β-sheet segments and α-helical segments. A “tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. A “quaternary structure” refers to a three-dimensional structure formed by the non-covalent bonding of independent tertiary units. A “motif” is a portion of the polypeptide sequence and includes at least two amino acids. The length of the motif can be 2 to 20, 2 to 15, or 2 to 10 amino acids. The domain can consist of a series of motifs of the same type.

[0057] As used in this article, "pests" include, but are not limited to, insects, fungi, bacteria, nematodes, mites, ticks, etc. Insect pests include, but are not limited to, insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Trichophala, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermoptera, Isoptera, Pseudocoptera, Siphonaptera, and Trichoptera.

[0058] As used herein, “insectic activity,” “insectic,” “insecticidal,” or “insectic” means the protein, polypeptide, or toxin disclosed herein, including proteins homologous to such proteins, polypeptides, or toxins, capable of inducing developmental delay (sublethal effect) and / or killing insect pathogens or pests (lethal effect) in insect pathogens or pests (including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera, and Coleoptera or Nematoda).

[0059] As used herein, “plant cell” means a cell obtained or found from seeds, suspension cultures, plumules, meristematic regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen, and microspores. Plant cells also include modified cells (e.g., protoplasts) obtained from the above tissues, as well as plant cell tissue cultures, plant callus, and plant masses that can regenerate plants. As used herein, “plant part” means an organ, such as a plumule, pollen, ovule, seed, flower, grain, spike, rachis, leaf, husk, culm, stem, root, root tip, anther, and fimbriae. As used herein, “plant” means a whole plant and its offspring. This also includes the offspring, variants, and mutants of regenerated plants, provided they contain nucleic acid molecules introduced as described herein.

[0060] As used herein, a "promoter" refers to a synthetic or naturally derived molecule that endows, activates, or enhances nucleic acid expression in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. Promoters may also contain distal enhancer or repressor elements, which can be located up to thousands of base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively regulate the expression of genomic elements, or differentially regulate the expression of genomic elements relative to the cell, tissue, or organelle in which expression occurs, or relative to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens or pests, pesticides, metal ions, or inducers. Representative examples of promoters include the promoter of the 35S gene from cauliflower mosaic virus, the promoter of cassava vein mosaic virus, the promoter of rice actin 1 gene, the promoter of underground clover virus gene 4, the promoter region of ubiquitin 4 gene, and the promoter region of maize polyubiquitin 1 gene.

[0061] When the term "recombinant" is used to refer to, for example, cells, nucleic acids, proteins, or vectors, it means that the cell, nucleic acid, protein, or vector has been modified by introducing a heterologous nucleic acid or protein or by altering the native nucleic acid or protein, or that the cell is derived from a cell that has been so modified. Thus, for example, a recombinant cell expresses a gene not present in the cell's native (naturally occurring) form, or expresses a second copy of a native gene that is otherwise normally or abnormally expressed, underexpressed, or not expressed at all.

[0062] As used herein, "sample" or "test sample" can mean any sample in which the presence and / or level of a target is to be detected or determined, or any sample containing a carrier as detailed herein. A sample can be a biological sample. A sample can include a liquid, solution, emulsion, or suspension. A sample can be obtained by any means known in the art. A sample can be used directly as obtained from a living organism, or it can be pretreated, for example by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to alter the characteristics of the sample in ways discussed herein or known in the art.

[0063] The terms "subject" and "organism," used interchangeably herein, refer to any plant, seed, plant part, or plant material, including but not limited to plants requiring the compositions or methods described herein. Plants may be, for example, but not limited to, rice, barley, sorghum, soybean, cotton, corn, rapeseed, sugarcane, tobacco, sunflower, or wheat. The subject may be at any stage of development, such as the seed stage, germination stage, vegetative growth stage, sprouting stage, flowering stage, or maturity stage. The subject may be hermaphroditic or dioecious. In some embodiments, the subject may possess specific genetic markers. In some embodiments, the subject may undergo other forms of treatment.

[0064] "Substantially identical" can mean that the first and second amino acid sequences or polynucleotide sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in the region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 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 more amino acids or nucleotides.

[0065] As used herein, “introduction” means the presentation of a nucleic acid molecule or construct to a plant cell, plant part, or plant in a manner that allows the nucleic acid molecule or construct to enter the interior of a plant cell. The methods disclosed herein include the introduction and expression of nucleic acid sequences or constructs as described herein in plant cells, plant parts, or plants. The methods described herein do not rely on a specific method for introducing a nucleic acid molecule or construct into a plant cell, plant part, or plant, as long as it allows entry into at least one cell of the plant or plant part. Methods for introducing nucleotide sequences, selecting transformation events, and regenerating intact plants (which may require routine modifications for a particular plant species) are known in the art. These methods may include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and sexual breeding. Thus, nucleic acid molecules or constructs can be carried or integrated into the genome of a host cell in the form of episomes.

[0066] As used herein, "transformed plant cell" refers to a plant cell that has been transformed and can be grown into a plant by methods known in the art. These plants can then be grown and pollinated with the same or different transforming strains, wherein the resulting offspring possess the identified desired phenotypic trait. Two or more generations can be grown to ensure the stable maintenance and heritability of the expression of the desired phenotypic trait, and then the seeds are harvested to ensure that the expression of the desired phenotypic trait has been achieved.

[0067] "Transformation event" refers to the product of transforming an organism or cell with a heterologous DNA construct, regenerating a population of organisms by inserting recombinant DNA into the genome of an organism, and selecting a specific organism characterized by inserting a gene construct into a specific genomic location to produce transgenic cells.

[0068] A “transformed organism” or “transformed plant” is an organism or plant in which a genetic construct nucleic acid molecule has been integrated into its genome. All cells of a transformed organism or plant may have a genetic construct integrated into its genome. A transformed plant may be a fertile plant, and more specifically, a plant whose agronomic characteristics (yield, grain quality, drought tolerance, etc.) are not impaired compared to the same untransformed plant. In some embodiments, Agrobacterium-mediated transformation is used to transform the organism or plant. Other suitable transformation methods may include, for example, particle bombardment or silicon carbide whiskers, CRISPR, TALEN, or other genome modification technologies. Genome modification technologies can alter the plant genome through insertion or other changes to the plant genome. In some embodiments, modified plants comprising nucleic acids encoding polypeptides as disclosed herein are considered.

[0069] In some implementations, genome editing technologies can be used to introduce a disclosed polynucleotide encoding a polypeptide into the plant genome, or to edit previously introduced polynucleotides in the plant genome. For example, a disclosed polynucleotide can be introduced into a desired location in the plant genome using double-strand break techniques, including but not limited to TALENs, large-scale nucleases, zinc finger nucleases, and CRISPR-Cas. For site-specific insertion, a CRISPR-Cas system can be used to introduce the disclosed polynucleotide into a desired location in the plant genome. The desired location in the plant genome can be any desired target site for insertion, such as a genomic region optimized for breeding, or a target site located in a genomic region with an existing target trait. The existing target trait can be an endogenous trait or a previously introduced trait.

[0070] In some embodiments, when the disclosed polynucleotide encoding an insecticidal polypeptide has been previously introduced into the genome, genome editing techniques can be used to alter or modify the introduced polynucleotide encoding the insecticidal polypeptide sequence. Alternatively, double-strand break techniques can be used to add additional nucleotide sequences to the introduced polynucleotide. These additional sequences include additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing techniques can be used to localize additional nucleotide sequences encoding additional insecticidal active proteins within the plant genome adjacent to the disclosed polynucleotide encoding the insecticidal polypeptide disclosed herein, in order to produce a molecular stack of insecticidal active proteins.

[0071] As used in this article, "transgenic" refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into a different organism. This non-native DNA fragment can retain the transgenic organism's ability to produce RNA or protein, or it can alter the normal function of the transgenic organism's genetic code. The introduction of transgenics has the potential to alter the phenotype of an organism.

[0072] When "treatment" refers to protecting a subject from a toxin, it means curbing, inhibiting, reversing, mitigating, improving, or preventing the progression of damage or death caused by exposure to a toxin, or completely eliminating damage or death caused by exposure to a toxin. Treatment can be carried out in an acute or chronic manner. Prevention of damage or death caused by exposure to a toxin involves administering the disclosed composition to the subject prior to exposure to the toxin. Curbing damage or death caused by exposure to a toxin involves administering the disclosed composition to the subject exposed to the toxin prior to the occurrence of damage. Inhibiting or improving damage or death caused by exposure to a toxin involves administering the disclosed composition to the subject after the occurrence of damage.

[0073] Regarding nucleotides or polynucleotides, "variant" means (i) a portion or fragment of the reference nucleotide sequence; (ii) a complementary sequence of the reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to the reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to the reference nucleic acid, its complementary sequence, or a substantially identical sequence under stringent conditions.

[0074] The term "variant" in relation to a peptide, polypeptide, or protein means a protein that differs in its amino acid sequence through the insertion, deletion, or conserved substitution of amino acids, but retains at least one biological activity. A variant can also mean a protein having a substantially identical amino acid sequence to a reference protein that retains 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 insecticidal or insecticidal activity. A variant can mean a functional fragment, including functionally truncated fragments and variants. A variant can also mean multiple copies of a polypeptide. Multiple copies can be tandem or separated by linkers. Conserved substitution of amino acids, for example, replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor alterations. As understood in the art, these minor alterations can be identified in part by considering the hydrophilicity index of the amino acid. The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices can be substituted and still retain protein function. The hydrophilicity of amino acids can also be used to reveal substitutions that allow a protein to retain its biological function. In the case of peptides, considering the hydrophilicity of amino acids allows for the calculation of the peptide's maximum local average hydrophilicity. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, biologically compatible amino acid substitutions should be understood as depending on the relative similarity of the amino acids (especially the side chains of those amino acids), as revealed by properties such as hydrophobicity, hydrophilicity, charge, size, and others.

[0075] In some embodiments, variant insecticidal proteins can be engineered using methods known in the art to make their sequences different from natural (i.e., native) or "wild-type" sequences. Protein engineering methods can be used to achieve, for example, improved insecticidal activity (i.e., optimization) or altered target profiles against specific pests. Suitable engineering methods for generating variant insecticidal proteins, as disclosed herein, may include, but are not limited to, domain exchange, DNA shuffling, saturation mutagenesis, site-directed mutagenesis, oligonucleotide-mediated mutagenesis, cassette mutagenesis, and error-prone PCR techniques.

[0076] The variant nucleotide sequences and proteins disclosed herein encompass sequences and proteins derived from mutagenesis or recombination processes, such as DNA shuffling. Using such processes, one or more distinct coding sequences can be manipulated to produce engineered insecticidal proteins with one or more desired properties. In this way, recombinant polynucleotide libraries containing sequence regions with fundamental sequence identity can be generated from a population of related sequence polynucleotides, which can undergo homologous recombination in vitro or in vivo. For example, using this method, any fragment of a full-length coding sequence, a sequence motif encoding a target domain, or a nucleotide sequence can be shuffled between the nucleotide sequence encoding the insecticidal protein described herein and other known insecticidal nucleotide sequences to obtain a new gene encoding an engineered protein with improved target properties (e.g., enhanced insecticidal activity). Target properties may include, but are not limited to, insecticidal activity per unit of insecticidal protein, protein stability, and non-toxicity to non-target species (particularly humans, livestock, and plants and microorganisms expressing the disclosed insecticidal protein). DNA shuffling methods may involve only the nucleotide sequences disclosed herein, or may additionally involve the 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. Patent Nos. 5,605,793 and 5,837,458.

[0077] Domain exchange is another known technique for generating variant insecticidal proteins. Domains can be exchanged between insecticidal peptides, resulting in hybrid or chimeric proteins with, for example, improved insecticidal activity or target spectrum. Methods for generating recombinant engineered proteins and testing their insecticidal 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 prepared by randomly introducing mutations along all or part of the nucleotide coding sequence (e.g., through saturation mutagenesis), and the resulting mutants can be screened for their ability to confer insecticidal activity to identify mutants that retain or improve activity. After mutagenesis, the encoded insecticidal protein can be recombinantly expressed, and the activity of the variant protein can be determined using standard assays known in the art.

[0079] As used herein, "vector" refers to a nucleic acid sequence containing an origin of replication. Vectors can be bacterial plasmids, viral vectors, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA vectors or RNA vectors. Vectors can be self-replicating extrachromosomal vectors and can be DNA plasmids. For example, a vector can encode an insecticidal protein.

[0080] This document provides information on nucleic acid molecules. Nucleic acid molecules may include, for example, insecticidal gene polynucleotides encoding any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, selective marker genes allowing selection in transgenic plants, and / or visual reporter markers (e.g., GFP). Nucleic acid molecules may also contain nucleic acids encoding fusion proteins.

[0081] The nucleic acid molecules described herein may include polynucleotides, such as vectors and plasmids. Vectors may be expression vectors or systems for producing proteins using conventional techniques and readily available starting materials. Nucleic acid molecules may be recombinant. Nucleic acid molecules may contain regulatory elements for gene expression of the coding sequence of the nucleic acid. The coding sequence in the nucleic acid molecule may be optimized to achieve stability and high expression levels. Regulatory elements may include promoters, enhancers, start codons, stop codons, and / or polyadenylation signals.

[0082] In one aspect, the nucleic acid molecule can encode a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity; the nucleic acid molecule can encode a polypeptide having at least 75% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity; the nucleic acid molecule can encode a polypeptide having at least 80% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity; the nucleic acid molecule can encode a polypeptide having at least 85% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity; the nucleic acid molecule can encode a polypeptide having at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity; the nucleic acid molecule can encode a polypeptide having at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity; the nucleic acid molecule can encode a polypeptide having at least 75 ... The nucleic acid molecule may encode a polypeptide having at least 91% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 92% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 93% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 94% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 95 ...2% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 93% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 94% sequence identity with any of SEQ ID NO: 1, 4, 7, 10 The nucleic acid molecule may encode a polypeptide having at least 96% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 97% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 98% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 and having insecticidal activity; the nucleic acid molecule may encode a polypeptide having at least 99% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18.A polypeptide with 2% sequence identity and insecticidal activity; the nucleic acid molecule may encode a polypeptide with at least 99.5% sequence identity and insecticidal activity to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18; the nucleic acid molecule may encode a polypeptide with at least 99.8% sequence identity and insecticidal activity to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18; the nucleic acid molecule may encode a polypeptide with at least 99.9% sequence identity and insecticidal activity to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18; or the nucleic acid molecule may encode a polypeptide having the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 and having insecticidal activity.

[0083] In one embodiment, this disclosure relates to an isolated nucleic acid molecule encoding a polypeptide amino acid sequence having at least 70% or at least 90% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, and having insecticidal activity. The insecticidal polypeptides of this disclosure and the nucleic acid molecules encoding these insecticidal polypeptides are particularly useful in crops for the control and killing of pests.

[0084] In one aspect, this disclosure relates to a method for producing transgenic plants with insecticidal activity. The method may include transforming plant cells with the nucleic acid molecules described herein, selecting plant cells containing the nucleic acids described herein, and regenerating the transgenic plant from the plant cells containing the nucleic acid molecules described herein, wherein the transgenic plant expresses the nucleic acid molecules described herein, and wherein the transgenic plant has insecticidal activity.

[0085] In one aspect, this disclosure relates to a method for protecting plants from damage related to pest infestation. The method may include introducing a nucleic acid molecule described herein into a plant, wherein the plant expresses the nucleic acid molecule, and wherein the resulting polypeptide has insecticidal activity.

[0086] The plants or genetically modified plants described in this article can be protected from plant pests, including but not limited to fall armyworm (…). Spodoptera frugiperda (FAW), corn earworm ( Helicoverpa zea (CEW), European corn borer ( Ostrinia nubilalis ), cotton bollworm ( Helicoverpa armigera ), Black Root Worm ( Agrotis ipsilon ), corn borer ( Elasmopalpus lignosellus Asian corn borer ( Ostinia furnacalis ), Southwest corn borer ( Diatraea grandiosella ), sugarcane borer ( Diatraea saccharalis), Western bean rootworm ( Striacosta albicosta ), edamame noctuid moth ( Anticarsia gemmatalis ) and their combinations.

[0087] In one aspect, this disclosure relates to a host cell comprising the nucleic acid molecules described herein. Suitable host cells may include prokaryotic host cells and eukaryotic host cells.

[0088] Particularly suitable prokaryotic host cells can include archaea and bacteria. Particularly suitable eukaryotic host cells can include plants and fungi. Suitable host cells can also include microbial cells, such as Trichoderma ( ). Trichoderma Aspergillus ( ) Aspergillus Neurospora ( Neurospora ), genus *Pythium* Humicola ), Penicillium ( Penicillium Fusarium ( ) Fusarium Thermomonas genus ( Thermomonospora ), Bacillus spp. ( Bacillus ), Pseudomonas spp. Pseudomonas ), Escherichia coli ( Escherichia Clostridium ( Clostridium ), Fibromospora ( Cellulomonas Streptomyces ( Streptomyces ), Yersinia ( Yarrow ), Pichia pastoris ( Peach ) and yeast ( Saccharomyces ), as well as microalgal cells belonging to cyanobacteria species. Suitable plant host cells can include dicotyledons and monocotyledons. Suitable dicotyledons can include dicotyledons (e.g., tobacco, cotton, soybean, sunflower, rapeseed) and monocotyledons (e.g., wheat, rice, barley, sorghum, and corn).

[0089] In one respect, this disclosure relates to a transgenic plant, transgenic plant tissue, transgenic plant cell, or transgenic plant seed that contains the nucleic acid molecules described herein and has insecticidal 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 encoding a polypeptide, wherein the polypeptide has at least 70% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 75% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 80% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 85% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 90% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 91% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; and at least 91% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. The sequence number NO: 1, 4, 7, 10, 12, 15, or 18 has at least 92% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 93% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 94% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 95% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 96% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 97% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; or at least 98% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; or at least 98% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; or at least 98% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; or at least 97% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; or at least 98% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; or at least 98% sequence identity with any of The sequence name is at least 99% identical to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, at least 99.2% identical to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, at least 99.5% identical to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, at least 99.8% identical to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, or at least 99% identical to any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.9% sequence identity, or the at least one nucleic acid molecule, nucleic acid construct, expression cassette, or vector can encode a polypeptide having an amino acid sequence having any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, wherein the transformed plant cells, transformed plant parts, or transformed plant have insecticidal activity.

[0091] This disclosure also relates to homologs of the insecticidal protein, provided that the homolog retains insecticidal or insecticidal activity. Homologous sequences can be isolated from public or private gene banks, or prepared using all or part of the peptide sequences provided in this disclosure, or using all or part of their encoding nucleotide sequences, by various conventional methods, such as random mutagenesis, site-directed mutagenesis, gene synthesis, or gene shuffling. Such homologs include, for example, deletions, insertions, or substitutions of one or more residues in the amino acid sequence of a protein, or combinations thereof. In some embodiments, homologs may include proteins 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 least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.2% sequence identity, at least 99.5% sequence identity, at least 99.8% sequence identity, or at least 99.9% sequence identity with any of the proteins in SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18.

[0092] In addition to the full-length nucleotide sequence of the nucleic acid molecule encoding the polypeptide of any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, the nucleic acid molecule encoding any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 may include a fragment or variant thereof encoding a polypeptide capable of insecticidal activity. For nucleotide sequences, as used herein, “fragment” means a portion of the nucleotide sequence of a nucleic acid molecule, for example, a portion of the nucleotide sequence encoding any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. A fragment of the nucleotide sequence may retain the biological activity of the reference nucleic acid molecule. For example, a nucleic acid molecule encoding a shorter than the complete amino acid sequence disclosed in any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 may be used to encode a protein that retains its insecticidal activity. Alternatively, a fragment of the nucleotide sequence may be used as a hybridization probe or as an amplification primer. Fragments used as hybridization probes or primers generally do not need to retain biological activity. Therefore, a fragment of a nucleic acid molecule 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 at most the number of nucleotides present in a full-length nucleic acid molecule. The biologically active portion (fragment or variant) of the nucleic acid molecule can be prepared by the following steps: isolating a portion of the nucleic acid molecule's sequence, operatively linking the 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 a polypeptide of any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18 may also be superimposed with a nucleotide sequence encoding an agronomic trait (e.g., male sterility, stem strength, flowering time) or a transformational trait (e.g., cell cycle regulation or gene targeting). These superimposed combinations can be generated by any method, including hybridization of plants using any conventional or TopCross™ method (DuPont Specialty Grains; DesMoines, Iowa), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly spaced short palindromic repeats (CRISPR), and other genetic transformations. If traits are superimposed through genetically transformed plants, target nucleotide sequences can be combined at any time and in any order. For example, a transformed plant containing one or more desired traits can be used as a target to introduce other traits through subsequent transformations. Traits can be introduced simultaneously with target polynucleotides provided by any combination of transformation cassettes in a co-transformation scheme. For example, if two sequences are to be introduced, they can be contained in separate expression boxes (trans) or in the same transformation box (cis). The expression of the sequences can be driven by the same promoter or different promoters.

[0094] In one aspect, this disclosure relates to a vector that can contain a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 75% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 80% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 85% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 90% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 91% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; and at least 91% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. The sequence shares at least 92% identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 93% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 94% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 95% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 96% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 97% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; and at least 98% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. The sequence of any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 has at least 99% sequence identity, at least 99.2% sequence identity, at least 99.5% sequence identity, at least 99.8% sequence identity, or at least 99.9% sequence identity.

[0095] Suitable vectors are known in the art. Particularly suitable vectors include those for antibiotic resistance or heat-stable antibiotic resistance, or those encoding enzymes that can supplement auxotrophs (natural auxotrophs, such as those overcoming the lack of essential amino acids; or engineered auxotrophs, such as URA3-deficient mutants, where URA3 is essential for uracil biosynthesis). Selective markers include those conferring antibiotic resistance, such as resistance to kanamycin (nptll gene), hygromycin (aph IV), spectinomycin (aadA), and gentamicin (aac3 and aacC4); or those conferring herbicide resistance, such as resistance to glufosinate (bar or pat), dicamba (DMO), and glyphosate (aroA or EPSPS). Selective markers that allow direct visual identification of transformation events may also be used, such as genes expressing colored or fluorescent proteins (e.g., luciferase or green fluorescent protein (GFP)), or genes expressing β-glucuronidase or the uidA gene (GUS), for which a variety of chromogenic substrates are known to be available.

[0096] In one aspect, this disclosure relates to a formulation comprising a recombinant polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 75% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 80% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 85% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 91% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; and at least 91% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. The sequence shares at least 92% identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 93% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 94% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 95% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 96% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 97% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; at least 98% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18; and with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18. The recombinant polypeptide has at least 99% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, at least 99.2% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, at least 99.5% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, at least 99.8% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, or at least 99.9% sequence identity with any of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, and exhibits insecticidal activity. When applied to plants, the recombinant polypeptide exhibits insecticidal activity.

[0097] Formulations containing recombinant peptides with acceptable carriers may be in the form of suspensions, solutions, emulsions, powders, dispersible granules, wettable powders, emulsifiable concentrates, aerosols, impregnated granules, adjuvants, coating pastes, encapsulating agents, or combinations thereof.

[0098] Formulations of recombinant peptides may include surfactants, inert carriers, preservatives, humectants, feeding stimulants, attractants, encapsulating agents, adhesives, emulsifiers, dyes, UV protectants, buffers, flow agents, fertilizers, solvents, dispersants, wetting agents, thickeners, micronutrient donors, and combinations thereof.

[0099] In one aspect, this disclosure relates to a formulation that may comprise transforming bacteria containing nucleic acid molecules as described herein and exhibiting insecticidal activity. When applied to plants, the transforming bacteria of the formulation express nucleic acid molecules, and the peptides exhibit insecticidal activity.

[0100] Formulations containing transforming bacteria with an acceptable carrier may be in the form of suspensions, solutions, emulsions, powders, dispersible granules, wettable powders, emulsifiable concentrates, aerosols, impregnated granules, adjuvants, coating pastes, encapsulating agents, or combinations thereof.

[0101] Preparations for transforming bacteria may include surfactants, inert carriers, preservatives, humectants, feeding stimulants, attractants, encapsulating agents, adhesives, emulsifiers, dyes, UV protectants, buffers, flow agents, fertilizers, solvents, dispersants, wetting agents, thickeners, micronutrient donors, and combinations thereof.

[0102] Transforming bacteria containing nucleic acid molecules as described herein can be used in the same manner as Bacillus thuringiensis strains previously used as insecticide sprays.

[0103] The target bioactivity of formulations containing recombinant peptides or transformed bacteria is the control of damaging plant pests. Such bioactivity can be determined by applying an effective amount of either formulation to plants infested with or at risk of infestation by plant pests and determining whether the formulation controls the damaging plant pests.

[0104] In one aspect, this disclosure relates to a method for protecting plants from insect pests. The method may include expressing a nucleic acid molecule as described herein in a plant or its plant cells, wherein the nucleic acid molecule encoding a polypeptide is operatively linked to a promoter capable of driving expression in the plant or its plant cells, and wherein the encoded polypeptide has insecticidal activity against insect pests.

[0105] Example Example 1 In this embodiment, the amino acid polypeptide sequence of GPA1073A was determined.

[0106] DNA sequences were isolated from sequencing DNA samples of Bacillus thuringiensis.

[0107] BLASTP was used to align predicted protein sequences corresponding to coding regions in the sequenced genome with Cry9Ga1 protein sequences known to exhibit insecticidal activity. Within the aligned protein region, a new sequence was considered a potential candidate gene if its query / object coverage length was 50% or more and it shared 30% or more amino acid identity. Figure 1 The paired full-length protein alignment shown shows that GPA1073A (SEQ ID NO: 1) has 88% sequence identity with Cry9Ga1 (SEQ ID NO: 2).

[0108] The specific amino acid polypeptide sequences of GPA1073A (SEQ ID NO: 1) and Cry9Ga1 (SEQ ID NO: 2) are provided in Table 1 below.

[0109] Table 1. Amino acid polypeptide sequences of GPA1073A (SEQ ID NO: 1) and Cry9Ga1 (SEQ ID NO: 2).

[0110]

[0111] Example 2 In this embodiment, GPA1073A is cloned and expressed.

[0112] To express GPA1073A, the DNA gene coding sequence was optimized in Escherichia coli (E. coli). E. coli The sequence was expressed in a vector (SEQ ID NO: 3) and is provided in Table 2 below. This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GPA1073A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMDMillipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 2 Images of SDS-PAGE analysis of purified GPA1073A protein from a recombinant E. coli expression vector (approximately 138 kDa) are shown. In some cases, induced bacterial cultures are used for insect assays.

[0113] Table 2. Optimized GPA1073A DNA coding sequence for GPA1073A expression in Escherichia coli (SEQ ID NO:3).

[0114]

[0115] Example 3 In this embodiment, insecticidal toxicity bioassays were performed using whole cells of transformed bacteria expressing the GPA1073A protein to evaluate insecticidal efficacy against pests including fall armyworm (G. spp.). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Clouded oyster (ECB)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB)), sugarcane borer ( Diatraea saccharalis (SCB) and corn rootworm ( Diabrotica virgifera (CRW)).

[0116] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpuncatata Howard (SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0117] Fall armyworm ( Spodoptera frugiperda (FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Clouded oyster (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diarrhea sugary (SCB) eggs, soybean noctuid moth ( Including Chrysodeixis (SBL)) eggs, cabbage brown moth ( Spodoptera small (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon (BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0118] As used in this and the following examples, the transformant used as the negative control was a whole-cell culture of *E. coli* produced under the same conditions by the same bacterial strain containing the same expression vector as the treatment group containing the insecticidal protein. The only difference was that the negative control expression vector contained a known gene encoding the inactive proteins Gpp34Ab1 / Tpp35Ab1 of CEW, FAW, ECB, SWCB, SCB, and VBC, which is active against CRW.

[0119] As used in this and the following examples, another transformant used as a negative control was a whole-cell culture of *E. coli* produced under the same conditions by the same bacterial strain containing the same expression vector as the treatment group containing the insecticidal protein. The only difference was that the negative control expression vector contained a known gene encoding the CRW inactive protein Vip3Aa19, which is active against both FAW and CEW.

[0120] Five days later, in bioassays, whole-cell bacterial cultures containing the GPA1073A protein showed positive results for fall armyworm ( ). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Clouded oyster (ECB)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB) and sugarcane borer ( Diatraea saccharalis (SCB) larval mortality and growth inhibition were greater than in the negative control (Table 3). This is important because known Cry9-like insecticidal proteins are effective against these specific pests (e.g., fall armyworm). Spodoptera frugiperda (FAW) and corn earworm ( Helicoverpa zea (CEW) does not have any documented insecticidal activity.

[0121] In contrast, in bioassays, GPA1073A protein showed significantly higher efficacy against maize rootworms (compared to the negative control). Diabrotica virgifera (CRW) did not show larval mortality or growth inhibition (Table 3), demonstrating the specificity of the insecticidal activity of the GPA1073A protein.

[0122] Table 3. Insecticidal activity of whole recombinant E. coli cultures expressing GPA1073A protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control.

[0123]

[0124] Example 4 In this embodiment, the purified protein of GPA1073A was determined in an artificial feed cover bioassay. Figure 2 For susceptible fall armyworms ( Spodoptera frugiperda (FAW) groups and corn earworms ( Helicoverpa zea (CEW) colony insecticidal activity.

[0125] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Example 3 were used in these assays.

[0126] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpuncatata Howard(SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0127] Fall armyworm ( Spodoptera frugiperda (FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Clouded oyster (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diarrhea sugary (SCB) eggs, soybean noctuid moth ( Including Chrysodeixis (SBL)) eggs, cabbage brown moth ( Spodoptera small (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon(BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0128] Five days later, the purified GPA1073A protein was effective against fall armyworm (…). Spodoptera frugiperda (FAW) and corn earworm ( Helicoverpa zea (CEW) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition (i.e., growth retardation) were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figures 3A to 3B This demonstrates that the death and growth inhibition of these specific pests are significantly correlated with the presence and amount of GPA1073A protein in the test samples.

[0129] Example 5 In this embodiment, the potency of the purified GPA1073A protein was evaluated and denoted as EC50, which represents the predicted protein concentration (μg / cm³) affecting 50% of infected insect larvae. 2 ).

[0130] The first step of the EC50 method is to determine the range of protein concentrations required in the bioassay to induce growth inhibition or mortality in approximately 50% and 100% of insect larvae. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL of WCRMO-1 feed / wells of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single newly emerged fall armyworm is infected into each well using a fine-tipped water color brush. Spodoptera frugiperda(FAW) larvae. After larval infection, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 25°C for 5 days. On day 5, each plate was removed from the growth chamber and the mortality (survival or death) and growth inhibition of the larvae were assessed.

[0131] Once the appropriate protein concentration range was determined, five to nine different concentrations of purified protein were tested in a dose-response artificial feed cover bioassay. As described above, the analytes were incubated for 5 days and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformations are used to analyze larval responses at each protein concentration, such as mortality (total larval deaths relative to total test larvae) or growth inhibition.

[0132] For those susceptible to fall armyworm ( Spodoptera frugiperda (FAW)), the predicted EC50 of GPA1073A is 2.89 μg / cm. 2 The 95% confidence interval is 2.00 µg / cm³. 2 Up to 4.14 µg / cm 2 For susceptible FAW, the EC50 of the control reference toxin Vip3Aa19 was 0.04 µg / cm³. 2 The 95% confidence interval is 0.03 μg / cm³. 2 Up to 0.05 μg / cm 2 .

[0133] Example 6 In this embodiment, the amino acid polypeptide sequence of GUN0345A was determined.

[0134] Isolate from Microcystis aeruginosa (Microcystis aeruginosa) using publicly available sequences. Microcystis aeruginosa The protein sequence of ).

[0135] Using BLASTP, the protein sequence was compared with known insect toxins in the BPPRC database (bpprc.org). Mpp51Aa3 was identified as the known protein sequence most closely related to GUN0345A and possessing proven insecticidal activity. Pairwise full-length protein alignment of GUN0345A (SEQ ID NO: 4) with Mpp51Aa3 (SEQ ID NO: 5) showed only 15% sequence identity. Figure 4 This demonstrates that it has a sequence distance from any known insecticidal protein.

[0136] The specific amino acid polypeptide sequences of GUN0345A (SEQ ID NO: 4) and Mpp51Aa3 (SEQ ID NO: 5) are provided in Table 4 below.

[0137] Table 4. Amino acid sequences of GUN0345A (SEQ ID NO: 4) and Mpp51Aa3 (SEQ ID NO: 5).

[0138]

[0139] Example 7 In this embodiment, GUN0345A is cloned and expressed.

[0140] To express GUN0345A, the DNA gene coding sequence was optimized for expression in *E. coli* and is provided in Table 5 below (SEQ ID NO: 6). This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GUN0345A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 5 Images of SDS-PAGE analysis of purified GUN0345A protein from a recombinant E. coli expression vector (approximately 31 kDa) are shown. In some cases, induced bacterial cultures are used for insect assays.

[0141] Table 5. Optimized GUN0345A DNA coding sequence for GUN0345A expression in Escherichia coli (SEQ ID NO: 6).

[0142]

[0143] Example 8 In this embodiment, whole cells of transformed bacteria expressing the GUN0345A protein were used for insecticidal bioassays to evaluate the insecticidal efficacy against pests, including corn rootworms. Diabrotica virgifera (CRW)), Northern Corn Root Worm ( Diabrotica barberi (NCR)), Southern maize rootworm ( Diabrotica undecimpunctata howardi (SCR)), Fall armyworm ( Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW) and European corn borer ( Ostrinia nubilalis(ECB)).

[0144] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0145] Fall armyworm ( Spodoptera frugiperda (FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Ostrinia nubilalis (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diatraea saccharalis (SCB) eggs, soybean noctuid moth ( Chrysodeixis includens (SBL)) eggs, cabbage brown moth ( Spodoptera exigua (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon(BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0146] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0147] Five days later, in bioassays, whole-cell bacterial cultures containing the GUN0345A protein showed positive results for corn rootworms ( Diabrotica virgifera (CRW)), Northern Corn Root Worm ( Diabrotica barberi (NCR) and Southern Corn Rootworm ( Diabrotica undecimpunctata howardi (SCR) larval mortality and growth inhibition were greater in the SCR control than in the negative control (Table 6). This is important because the known Mpp51-like insecticidal protein is effective against root leaf beetles (SCR). Diabrotica None of the pests possess any documented insecticidal activity.

[0148] In contrast, in bioassays, GUN0345A protein did not show larval mortality or growth inhibition in Lepidoptera species compared to the negative control (Table 6), demonstrating the specificity of the insecticidal activity of GUN0345A protein.

[0149] Table 6. Insecticidal activity of whole recombinant E. coli cultures expressing GUN0345A protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control measured.

[0150]

[0151] Example 9 In this embodiment, the purified protein GUN0345A was determined in an artificial feed cover bioassay. Figure 5 ) for susceptible corn rootworms ( Diabrotica virgifera (CRW) group and southern maize rootworm ( Diabrotica undecimpunctata howardi (SCR) colony insecticidal activity.

[0152] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0153] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0154] Five days later, the purified GUN0345A protein showed efficacy against corn rootworm ( Diabrotica virgifera (CRW) and Southern Corn Rootworm ( Diabrotica undecimpunctata howardi (SCR) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figures 6A to 6B This demonstrates that the death and growth inhibition of these specific pests are significantly correlated with the presence and amount of GUN0345A protein in the test samples.

[0155] Example 10 In this embodiment, the potency of the purified GUN0345A protein was evaluated and expressed as LC50, which represents the predicted protein concentration (μg / cm³) that causes death in 50% of infected insect larvae. 2 ).

[0156] The first step of the LC50 method is to determine the range of protein concentrations required to kill approximately 50% and 100% of insect larvae in a bioassay. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL of WCRMO-1 feed / wells of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single newly emerged corn rootworm is infected in each well using a fine-tipped water color brush. Diabrotica virgifera (CRW) larvae. After larval infection, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 24°C for 5 days. On day 5, each plate was removed from the growth chamber and the mortality status of the larvae (survival or death) was assessed.

[0157] Once the appropriate protein concentration range was determined, five to nine different concentrations of purified protein were tested in a dose-response artificial feed cover bioassay. As described above, the analytes were incubated for 5 days and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformation used to analyze larval mortality at each protein concentration (total dead larvae relative to total test larvae).

[0158] For susceptible corn rootworms ( Diabrotica virgifera (CRW)), the predicted LC50 of GUN0345A is 5.34 μg / cm³. 2 The 95% confidence interval is 4.00 µg / cm³. 2 Up to 7.25 µg / cm 2 For susceptible CRW, the LC50 of the binary toxin Gpp34Ab1 / Tpp35Ab1 is 12.42 μg / cm³. 2 The 95% confidence interval is 7.17 μg / cm³. 2 Up to 26.39 μg / cm 2 .

[0159] Example 11 In this embodiment, the amino acid polypeptide sequence of GUN1183A was determined.

[0160] Isolation from Boletus cellulosum (a publicly available sequence) Melittangium boletus The protein sequence of ).

[0161] Using BLASTP, the protein sequence was compared with known insect toxins in the BPPRC database (bpprc.org). Mpp46Ab1 was identified as the known protein sequence most closely related to GUN1183A and possessing proven insecticidal activity. Pairwise full-length protein alignment of GUN1183A (SEQ ID NO: 7) with Mpp46Ab1 (SEQ ID NO: 8) showed only 17% sequence identity. Figure 7 This demonstrates that it has a sequence distance from any known insecticidal protein.

[0162] The specific amino acid polypeptide sequences of GUN1183A (SEQ ID NO: 7) and Mpp46Ab1 (SEQ ID NO: 8) are provided in Table 7 below.

[0163] Table 7. Amino acid sequences of GUN1183A (SEQ ID NO: 7) and Mpp46Ab1 (SEQ ID NO: 8).

[0164]

[0165] Example 12 In this embodiment, GUN1183A is cloned and expressed.

[0166] To express GUN1183A, the DNA gene coding sequence was optimized for expression in *E. coli* and is provided in Table 8 below (SEQ ID NO: 9). This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GUN1183A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMD Millipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 8 Images of the purified GUN1183B protein from a recombinant *E. coli* expression vector (approximately 38 kDa) using SDS-PAGE analysis are shown. GUN1183B is a truncated variant of GUN1183A, in which the 35-amino acid signal peptide present at the N-terminus of GUN1183A is removed and is absent in the truncated GUN1183B variant. Table 9 below provides the specific amino acid polypeptide sequence of GUN1183B (SEQ ID NO: 20). In some cases, induced bacterial cultures are used for insect assays.

[0167] Table 8. Optimized GUN1183A DNA coding sequence for GUN1183A expression in Escherichia coli (SEQ ID NO: 9).

[0168]

[0169] Table 9. Amino acid polypeptide sequences of the GUN1183B (SEQ ID NO: 20) variant.

[0170]

[0171] Example 13 In this embodiment, whole cells of transformed bacteria expressing the GUN1183B protein were used for insecticidal bioassays to evaluate insecticidal efficacy against pests, including fall armyworm (GUN1183B). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Ostrinia nubilalis (ECB)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB)), soybean cutworm ( Including Chrysodeixis (SBL)), tobacco budworm ( Chloridia virescens (TBW) and corn rootworm ( Diabrotica virgifera (CRW)).

[0172] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0173] Corn rootworm ( Diabrotica virgifera (CRW) eggs were obtained from a commercial insect bank (CropCharacteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0174] Fall armyworm ( Spodoptera frugiperda(FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Clouded oyster (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diarrhea sugary (SCB) eggs, soybean noctuid moth ( Including Chrysodeixis (SBL)) eggs, cabbage brown moth ( Spodoptera small (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon (BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0175] Five days later, in bioassays, whole-cell bacterial cultures containing the GUN1183B protein showed positive results for fall armyworm ( ). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Clouded oyster (ECB)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB)), soybean cutworm ( Including Chrysodeixis (SBL)) and tobacco budworm ( Chloridia virescens(TBW) larval mortality and growth inhibition were greater than in the negative control (Table 10). This is important because the known Mpp46-like insecticidal protein is effective against these specific lepidopteran pests (e.g., fall armyworm). Spodoptera fruitless (FAW)), corn ear worm ( Helicoverpa zea (CEW) and European corn borer ( Ostrinia cloudy (ECB) does not have any documented insecticidal activity.

[0176] In contrast, in bioassays, GUN1183B protein did not show larval mortality or growth inhibition against CRW compared to the negative control (Table 10), demonstrating the specificity of the insecticidal activity of GUN1183B protein.

[0177] Table 10. Insecticidal activity of whole recombinant E. coli cultures expressing GUN1183B protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control as measured.

[0178]

[0179] Example 14 In this embodiment, the purified protein of GUN1183B was determined in an artificial feed cover bioassay. Figure 8 ) for susceptible corn ear borers ( Helicoverpa zea (CEW) colony insecticidal activity.

[0180] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0181] Corn ear worm ( Helicoverpa zea(CEW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with commercially available semi-solid insect feed. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0182] Five days later, the purified GUN1183B protein showed efficacy against corn earworm ( Helicoverpa zea (CEW) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figure 9 This demonstrates that the death and growth inhibition of this specific pest are significantly correlated with the presence and amount of GUN1183B protein in the test samples.

[0183] Example 15 In this embodiment, the potency of the purified GUN1183B protein was evaluated and denoted as EC50, which represents the predicted protein concentration (μg / cm³) affecting 50% of infected insect larvae. 2 ).

[0184] The first step of the EC50 method is to determine the range of protein concentrations required in the bioassay to induce growth inhibition or mortality in approximately 50% and 100% of insect larvae. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL insect feed / well of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single newly emerged corn ear borer is infected in each well using a fine-tipped water color brush. Helicoverpa zea(CEW) larvae. After larval infection, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 25°C for 5 days. On day 5, each plate was removed from the growth chamber and the larval mortality (survival or death) and growth inhibition were assessed.

[0185] Once the appropriate protein concentration range was determined, five to nine different concentrations of purified protein were tested in a dose-response artificial feed cover bioassay. As described above, the analytes were incubated for 5 days and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformations are used to analyze larval responses at each protein concentration, such as mortality (total larval deaths relative to total test larvae) or growth inhibition.

[0186] For susceptible corn earworm ( Helicoverpa zea (CEW)), the predicted EC50 of GUN1183B is 5.54 μg / cm³. 2 The 95% confidence interval is 3.28 µg / cm³. 2 Up to 7.93 µg / cm 2 For susceptible CEW, the EC50 of the reference toxin Vip3Aa19 was 0.11 μg / cm³. 2 The 95% confidence interval is 0.09 μg / cm³. 2 Up to 0.13 μg / cm 2 .

[0187] Example 16 In this embodiment, the amino acid polypeptide sequence of GUN0307A was determined.

[0188] Isolation from *Pseudomonas sarcodactylis* (a publicly available sequence) Pseudoalteromonas yellow-violet The protein sequence of ).

[0189] Using BLASTP, the protein sequence was compared with known insect toxins in the BPPRC database (bpprc.org). Mpp46Ab1 was identified as the known protein sequence most closely related to GUN0307A and possessing proven insecticidal activity. GUN0307A (SEQ ID NO: 10) showed only 37% sequence identity with Mpp46Ab1 (SEQ ID NO: 8) through paired full-length protein alignment. Figure 10 This demonstrates that it has a sequence distance from any known insecticidal protein.

[0190] The specific amino acid polypeptide sequences of GUN0307A (SEQ ID NO: 10) and Mpp46Ab1 (SEQ ID NO: 8) are provided in Table 11 below.

[0191] Table 11. Amino acid sequences of GUN0307A (SEQ ID NO: 10) and Mpp46Ab1 (SEQ ID NO: 8).

[0192]

[0193] Example 17 In this embodiment, GUN0307A is cloned and expressed.

[0194] To express GUN0307A, the DNA gene coding sequence was optimized for expression in *E. coli* and is provided in Table 12 below (SEQ ID NO: 11). This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GUN0307A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMDMillipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 11 Images of SDS-PAGE analysis of purified GUN0307A protein from a recombinant E. coli expression vector (approximately 27 kDa) are shown. In some cases, induced bacterial cultures are used for insect assays.

[0195] Table 12. Optimized GUN0307A DNA coding sequence for GUN0307A expression in Escherichia coli (SEQ ID NO: 11).

[0196]

[0197] Example 18 In this embodiment, insecticidal toxicity bioassays were performed using whole cells of transformed bacteria expressing the GUN0307A protein to evaluate insecticidal efficacy against pests, including fall armyworm (GUN0307A). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Clouded oyster (ECB)), corn rootworm ( Diabrotica virgifera (CRW) and Northern Corn Rootworm ( Diabrotica barberi (NCR)).

[0198] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0199] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpuncatata Howard (SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0200] Fall armyworm ( Spodoptera frugiperda (FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Clouded oyster (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diarrhea sugary (SCB) eggs, soybean noctuid moth ( Including Chrysodeixis (SBL)) eggs, cabbage brown moth ( Spodoptera small (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon(BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0201] Five days later, in bioassays, whole-cell bacterial cultures containing the GUN0307A protein showed positive results for corn ear borers (GUN0307A). Helicoverpa zea (CEW)), corn rootworm ( Diabrotica virgifera (CRW) and Northern Corn Rootworm ( Diabrotica barberi (NCR) larval mortality and growth inhibition were greater than in the negative control (Table 13). This is important because the known Mpp46-like insecticidal protein is effective against these specific lepidopteran and coleopteran pests (e.g., corn earworm). Helicoverpa zea (CEW)), corn rootworm ( Diabrotica virgifera (CRW)), Northern Corn Root Worm ( Diabrotica barberi (NCR) does not have any documented insecticidal activity.

[0202] In contrast, in bioassays, the GUN0307A protein was significantly more effective against fall armyworm (GUN0307A) than the negative control. Spodoptera frugiperda (FAW) or European corn borer ( Ostrinia nubilalis (ECB) did not show larval death or growth inhibition (Table 13), demonstrating the specificity of the insecticidal activity of the GUN0307A protein.

[0203] Table 13. Insecticidal activity of whole recombinant E. coli cultures expressing GUN0307A protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control measured.

[0204]

[0205] Example 19 In this embodiment, the purified protein GUN0307A was determined in an artificial feed cover bioassay. Figure 11 ) for susceptible corn ear borers ( Helicoverpa zea (CEW) population and corn rootworm ( Diabrotica virgifera (CRW) colony insecticidal activity.

[0206] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0207] Corn rootworm ( Diabrotica virgifera (CRW) eggs were obtained from a commercial insect bank (CropCharacteristics, Inc., Farmington, MN). Bioassay chambers were prepared by filling 96-well tissue culture plates (Costar®, Corning®) with a portion of the semi-solid insect feed WCRMO-1. In a laminar flow hood, test samples were applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0208] Corn ear worm ( Helicoverpa zea(CEW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect feed. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, a single newly emerged larva (less than 12 hours after emergence) was introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0209] Five days later, the purified GUN0307A protein showed efficacy against corn earworm ( Helicoverpa zea (CEW) and corn rootworm ( Diabrotica virgifera (CRW) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figures 12A to 12B This demonstrates that the death and growth inhibition of these specific pests are significantly correlated with the presence and amount of GUN0307A protein in the test samples.

[0210] Example 20 In this embodiment, the potency of the purified GUN0307A protein was evaluated and denoted as EC50, which represents the predicted protein concentration (μg / cm³) affecting 50% of infected insect larvae. 2 ).

[0211] The first step of the EC50 method is to determine the range of protein concentrations required in the bioassay to induce growth inhibition or mortality in approximately 50% and 100% of insect larvae. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL insect feed / well of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single newly emerged corn ear borer is infected in each well using a fine-tipped water color brush. Helicoverpa zea (CEW) larvae or corn rootworms ( Diabrotica virgifera(CRW) larvae. After larval infection, each plate was covered with a sealing film (ExcelScientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 26°C or 24°C for 5 days. On day 5, each plate was removed from the growth chamber, and larval mortality (survival or death) and growth inhibition were assessed.

[0212] Once the appropriate protein concentration range was determined, five to nine different concentrations of purified protein were tested in a dose-response artificial feed cover bioassay. As described above, the analytes were incubated for 5 days and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformations were used to analyze larval responses at each protein concentration, such as mortality (total dead larvae relative to total test larvae) and growth inhibition.

[0213] For susceptible corn rootworms ( Diabrotica virgifera (CRW)), the predicted EC50 of GUN0307A is 22.86 μg / cm³. 2 The 95% confidence interval is 15.71 µg / cm³. 2 Up to 33.74 µg / cm 2 For susceptible CRW, the reference EC50 for the binary toxins Gpp34Ab1 / Tpp35Ab1 is 6.57 μg / cm³. 2 The 95% confidence interval is 4.18 μg / cm³. 2 Up to 11.13 μg / cm 2 .

[0214] For susceptible corn earworm ( Helicoverpa zea (CEW)), the predicted EC50 for GUN0307A is 42.39 μg / cm³. 2 The 95% confidence interval is 28.67 µg / cm³. 2 Up to 71.04 µg / cm 2 For susceptible CEW, the EC50 of the reference toxin Vip3Aa19 was 0.11 μg / cm³. 2 The 95% confidence interval is 0.09 μg / cm³. 2 Up to 0.13 μg / cm 2 .

[0215] Example 21 In this embodiment, the amino acid polypeptide sequence of GUN0527A was determined.

[0216] Isolate from the bacterium Nocardia leucocephala (Noctiluca spp.) using publicly available sequences. Nocardiopsis alba The protein sequence of GUN0527A is shown. The NCBI reference sequence ID for the protein sequence is WP_238543783.1, and it is automatically annotated as "follicular epithelial vitellin subunit".

[0217] To associate GUN0527A with any known insecticidal protein, the protein sequence was compared with known insect toxins in the BPPRC database (bpprc.org) using BLASTP. Mpp46Aa1 was identified as the known protein sequence most closely related to GUN0527A and possessing proven insecticidal activity. A pairwise full-length protein comparison of GUN0527A (SEQ ID NO: 12) with Mpp46Aa1 (SEQ ID NO: 13) showed only 17% sequence identity. Figure 13 This demonstrates that it has a sequence distance from any known insecticidal protein.

[0218] The specific amino acid polypeptide sequences of GUN0527A (SEQ ID NO: 12) and Mpp46Aa1 (SEQ ID NO: 13) are provided in Table 14 below.

[0219] Table 14. Amino acid sequences of GUN0527A (SEQ ID NO: 12) and Mpp46Aa1 (SEQ ID NO: 13).

[0220]

[0221] Example 22 In this embodiment, GUN0527A is cloned and expressed.

[0222] To express GUN0527A, the DNA gene coding sequence was optimized for expression in *E. coli* and is provided in Table 15 below (SEQ ID NO: 14). This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GUN0527A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMDMillipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 14 Images of SDS-PAGE analysis of purified GUN0527A protein from a recombinant E. coli expression vector (approximately 36 kDa) are shown. In some cases, induced bacterial cultures are used for insect assays.

[0223] Table 15. Optimized GUN0527A DNA coding sequence for GUN0527A expression in Escherichia coli (SEQ ID NO: 14).

[0224]

[0225] Example 23 In this embodiment, whole cells of transformed bacteria expressing the GUN0527A protein were used for insecticidal bioassays to evaluate insecticidal efficacy against pests, including fall armyworm (GUN0527A). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Ostrinia nubilalis (ECB)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB)), sugarcane borer ( Diatraea saccharalis (SCB)), soybean noctuid moth ( Chrysodeixis includens (SBL)), Cabbage Brown Noctuid Moth ( Spodoptera exigua (BAW)), Southern Armyworm ( Spodoptera eridania (SAW)), tobacco budworm ( Chloridia virescens (TBW)), Black Root Worm ( Agrotis ipsilon (BCW) and corn rootworm ( Diabrotica virgifera (CRW)).

[0226] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0227] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpunctata howardi(SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0228] Fall armyworm ( Spodoptera frugiperda (FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Ostrinia nubilalis (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diatraea saccharalis (SCB) eggs, soybean noctuid moth ( Chrysodeixis includens (SBL)) eggs, cabbage brown moth ( Spodoptera exigua (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon(BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0229] Five days later, in bioassays, whole-cell bacterial cultures containing the GUN0527A protein showed positive results for fall armyworm (GUN0527A). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), European corn borer ( Ostrinia nubilalis (ECB)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), sugarcane borer ( Diatraea saccharalis (SCB)), soybean noctuid moth ( Chrysodeixis includens (SBL)), Cabbage Brown Noctuid Moth ( Spodoptera exigua (BAW)), Southern Armyworm ( Spodoptera eridania (SAW)), tobacco budworm ( Chloridia virescens (TBW) and black-cutting rootworm ( Black cutworm (BCW) larval mortality and growth inhibition were greater than in the negative control (Table 16). This is important because the known Mpp46-like insecticidal protein is effective against these specific lepidopteran pests (e.g., fall armyworm). Fall armyworm (FAW)), corn ear worm ( Corn earworm (CEW) and European corn borer ( European corn borer (ECB) does not have any documented insecticidal activity.

[0230] In contrast, in bioassays, GUN0527A protein significantly reduced CRW ( ) compared to the negative control. Diabrotica virgiferaNo larval mortality or growth inhibition was observed (Table 16), demonstrating the specificity of the insecticidal activity of the GUN0527A protein.

[0231] Table 16. Insecticidal activity of whole recombinant E. coli cultures expressing GUN0527A protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control measured.

[0232]

[0233] Example 24 In this embodiment, the purified protein GUN0527A was determined in an artificial feed cover bioassay. Figure 14 ) for susceptible corn ear borers ( Corn earworm (CEW) group and European corn borer ( European corn borer (ECB) colony insecticidal activity.

[0234] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0235] Corn ear worm ( Corn earworm (CEW) eggs and European corn borer ( European corn borer (ECB) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect feed. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0236] Five days later, the purified GUN0527A protein showed efficacy against corn earworm ( Corn earworm (CEW) and European corn borer ( European corn borer(ECB) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figures 15A to 15B This demonstrates that the death and growth inhibition of these specific pests are significantly correlated with the presence and amount of GUN0527A protein in the test samples.

[0237] Example 25 In this embodiment, the potency of the purified GUN0527A protein was evaluated and denoted as EC50, which represents the predicted protein concentration (μg / cm³) affecting 50% of infected insect larvae. 2 ).

[0238] The first step of the EC50 method is to determine the range of protein concentrations required in the bioassay to induce growth inhibition or mortality in approximately 50% and 100% of insect larvae. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL insect feed / well of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single newly emerged corn ear borer is infected in each well using a fine-tipped water color brush. Corn earworm (CEW) larvae or European corn borer ( European corn borer (ECB) larvae. After larval infection, each plate was covered with a sealing film (ExcelScientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 26°C for 5 days. On day 5, each plate was removed from the growth chamber and the larval mortality (survival or death) and growth inhibition were assessed.

[0239] Once the appropriate protein concentration range was determined, five to nine different concentrations of purified protein were tested in a dose-response artificial feed cover bioassay. As described above, the analytes were incubated for 5 days and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformations were used to analyze larval responses at each protein concentration, such as mortality (total dead larvae relative to total test larvae) and growth inhibition.

[0240] For susceptible corn earworm ( Corn earworm (CEW)), the predicted EC50 for GUN0527A is 11.22 μg / cm³. 2 The 95% confidence interval is 8.53 µg / cm³. 2 Up to 14.65 µg / cm 2For susceptible CEW, the EC50 of the reference toxin Vip3Aa19 was 0.11 μg / cm³. 2 The 95% confidence interval is 0.09 μg / cm³. 2 Up to 0.13 μg / cm 2 .

[0241] For those susceptible to the European corn borer ( European corn borer (ECB)), the predicted EC50 for GUN0527A is 3.33 μg / cm³. 2 The 95% confidence interval is 1.49 µg / cm³. 2 Up to 5.39 µg / cm 2 For susceptible ECB, the EC50 of the reference toxin Cry1F was 0.019 μg / cm³. 2 The 95% confidence interval is 0.015 μg / cm³. 2 Up to 0.024 μg / cm 2 .

[0242] Example 26 In this embodiment, the amino acid polypeptide sequence of GUN0052A was determined.

[0243] Isolation from Vibrio spp. was performed using publicly available sequence reference WP_110166803. Vibrio sp. The protein sequence of ).

[0244] Using BLASTP, the protein sequence was compared with known insect toxins in the BPPRC database (bpprc.org). Mpp3Aa8 was identified as the known protein sequence most closely related to GUN0052A and possessing proven insecticidal activity. Pairwise full-length protein alignment of GUN0052A (SEQ ID NO: 15) with Mpp3Aa8 (SEQ ID NO: 16) showed only 15% sequence identity. Figure 16 This demonstrates that it has a sequence distance from any known insecticidal protein.

[0245] The specific amino acid polypeptide sequences of GUN0052A (SEQ ID NO: 15) and Mpp3Aa8 (SEQ ID NO: 16) are provided in Table 17 below.

[0246] Table 17. Amino acid sequences of GUN0052A (SEQ ID NO: 15) and Mpp3Aa8 (SEQ ID NO: 16).

[0247]

[0248] Example 27 In this embodiment, GUN0052A is cloned and expressed.

[0249] To express GUN0052A, the DNA gene coding sequence was optimized for expression in *E. coli* and is provided in Table 18 below (SEQ ID NO: 17). This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GUN0052A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMDMillipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 17 Images of SDS-PAGE analysis of purified GUN0052A protein from a recombinant E. coli expression vector (approximately 32 kDa) are shown. In some cases, induced bacterial cultures are used for insect assays.

[0250] Table 18. Optimized GUN0052A DNA coding sequence for GUN0052A expression in Escherichia coli (SEQ ID NO: 17).

[0251]

[0252] Example 28 In this embodiment, insecticidal toxicity bioassays were performed using whole cells of transformed bacteria expressing the GUN0052A protein to evaluate insecticidal efficacy against pests, including fall armyworm (GUN0052A). Fall armyworm (FAW)), corn ear worm ( Corn earworm (CEW)), European corn borer ( European corn borer (ECB)), corn rootworm ( Western corn rootworm (CRW)), Northern Corn Root Worm ( Northern corn rootworm (NCR) and Southern Corn Rootworm ( Southern corn rootworm (SCR)).

[0253] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0254] Corn rootworm ( Western corn rootworm (CRW) eggs, northern corn rootworm ( Northern corn rootworm (NCR) eggs and southern maize rootworm ( Southern corn rootworm(SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0255] Fall armyworm ( Fall armyworm (FAW) eggs, corn ear borer ( Corn earworm (CEW) eggs, European corn borer ( European corn borer (ECB) eggs, velvety bean borer (Anticarsia gemmatalis (VBC)) eggs, Southwestern corn borer ( Southwestern corn borer (SWCB)) eggs, sugarcane borer ( Diatraea saccharalis (SCB) eggs, soybean noctuid moth ( Beet armyworm (SBL)) eggs, cabbage brown moth ( Beet armyworm (BAW) eggs, Southern armyworm ( Southern beetworm (SAW) eggs, tobacco budworms ( Green cloverworm (TBW)) eggs and black-cutting rootworm ( Black cutworm(BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0256] Five days later, in bioassays, whole-cell bacterial cultures containing the GUN0052A protein showed positive results for corn ear borers (…). Corn earworm (CEW)), corn rootworm ( Western corn rootworm (CRW)), Northern Corn Root Worm ( Northern corn rootworm (NCR) and Southern Corn Rootworm ( Southern corn rootworm (SCR) larval mortality and growth inhibition were greater in the SCR than in the negative control (Table 19). This is important because known Mpp3-like insecticidal proteins are effective against these specific lepidopteran and coleopteran pests (e.g., corn ear borer). Corn earworm (CEW)), corn rootworm ( Western corn rootworm (CRW)), Northern Corn Root Worm ( Northern corn rootworm (NCR) and Southern Corn Rootworm ( Southern corn rootworm (SCR) does not have any documented insecticidal activity.

[0257] In contrast, in bioassays, the GUN0052A protein was significantly more effective against fall armyworm ( ) compared to the negative control. Spodoptera frugiperda (FAW) or European corn borer ( European corn borer (ECB) did not show larval mortality or growth inhibition (Table 19), demonstrating the specificity of the insecticidal activity of the GUN0052A protein.

[0258] Table 19. Insecticidal activity of whole recombinant E. coli cultures expressing GUN0052A protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control measured.

[0259]

[0260] Example 29 In this embodiment, the purified protein GUN0052A was determined in an artificial feed cover bioassay. Figure 17 ) for susceptible corn ear borers ( Corn earworm (CEW) population and corn rootworm ( Western corn rootworm (CRW) colony insecticidal activity.

[0261] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0262] Corn rootworm ( Western corn rootworm (CRW) eggs were obtained from a commercial insect bank (CropCharacteristics, Inc., Farmington, MN). Bioassay chambers were prepared by filling 96-well tissue culture plates (Costar®, Corning®) with a portion of the semi-solid insect feed WCRMO-1. In a laminar flow hood, test samples were applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, single newly emerged larvae (less than 12 hours post-emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0263] Corn ear worm ( Corn earworm(CEW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect feed. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, a single newly emerged larva (less than 12 hours after emergence) was introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0264] Five days later, the purified GUN0052A protein showed efficacy against corn earworm ( Corn earworm (CEW) and corn rootworm ( Western corn rootworm (CRW) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figures 18A to 18B This demonstrates that the death and growth inhibition of these specific pests are significantly correlated with the presence and amount of GUN0052A protein in the test samples.

[0265] Example 30 In this embodiment, the potency of the purified GUN0052A protein was evaluated and denoted as LC50 and EC50, which represent the predicted protein concentration (μg / cm³) that caused death in 50% of infected insect larvae or affected 50% of infected insect larvae, respectively. 2 ).

[0266] The first step in the LC50 or EC50 method is to determine the range of protein concentrations required in the bioassay to kill or induce a growth-inhibiting response of approximately 50% and 100% of insect larvae. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL of insect feed per well of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single newly emerged corn ear borer is infected in each well using a fine-tipped water-color brush. Helicoverpa zea (CEW) larvae or corn rootworms ( Diabrotica virgifera(CRW) larvae. After larval infection, each plate was covered with a sealing film (ExcelScientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 25°C for 5 days. On day 5, each plate was removed from the growth chamber and the larval mortality (survival or death) and growth inhibition were assessed.

[0267] Once the appropriate protein concentration range was determined, five different concentrations of purified protein were tested in a dose-response artificial feed-covered bioassay. As described above, the analytes were incubated for 5 days, and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformation was used to analyze larval mortality (total dead larvae relative to total test larvae) or growth inhibition at each protein concentration.

[0268] For susceptible corn earworm ( Helicoverpa zea (CEW)), the predicted EC50 for GUN0052A is 12.28 μg / cm³. 2 The 95% confidence interval is 9.89 µg / cm³. 2 Up to 15.18 µg / cm 2 For susceptible CEW, the EC50 of the reference toxin Vip3Aa19 was 0.11 μg / cm³. 2 The 95% confidence interval is 0.09 μg / cm³. 2 Up to 0.13 μg / cm 2 .

[0269] For susceptible corn rootworms ( Diabrotica virgifera (CRW)), the predicted LC50 of GUN0052A is 22.64 μg / cm³. 2 The 95% confidence interval is 14.51 µg / cm³. 2 Up to 36.90 µg / cm 2 For susceptible CRW, the LC50 of the reference binary toxins Gpp34Ab1 / Gpp35Ab1 is 12.42 μg / cm³. 2 The 95% confidence interval is 7.17 μg / cm³. 2 Up to 26.39 μg / cm 2 .

[0270] Example 31 In this embodiment, GUN0052A is made from Tobacco Benedict (Tobacco Benedict). Nicotiana benthamiana (Instantaneous expression in )

[0271] Two nucleic acid constructs for GUN0052A expression were prepared. For the first construct, the DNA nucleotide sequence encoding GUN0052A was cloned between a constitutive promoter linked to a polyadenylated sequence present in the plant binary vector. For the second construct, the DNA nucleotide sequence encoding GUN0052A was cloned between a constitutive promoter linked to the 5' untranslated region (UTR) and a polyadenylated sequence present in the plant binary vector.

[0272] The resulting binary plasmid was transferred into Agrobacterium strain GV3101 to produce strains 516 and 517. Leaf portions were transiently transformed with Agrobacterium 516 and 517 using the standard Agrobacterium benthamiana infiltration protocol (essentially as described in bio-protocol.org / bio101 / e95).

[0273] Total protein was extracted from transformed tobacco leaves and GUN0052A expression was examined using Western blot analysis and a monoclonal antibody specific to the 6xHis tag of the expressed recombinant protein as a probe (Table 20).

[0274] Table 20. Expression of GUN0052A protein in *Nicotiana benthamiana* tissue 3 to 4 days after infiltration.

[0275]

[0276] Leaf discs prepared from Agrobacterium-infiltrated portions of tobacco leaves were then used for insect feeding assays using larvae of four different insect pests (Table 21). Strain 285 was used as a negative control in the experiments and did not express the GUN0052A protein. For the strain 285 control, the DNA nucleotide sequence encoding ZsGreen was cloned between constitutive promoters linked to polyadenylated sequences present in the plant binary vector.

[0277] Table 21. Average leaf disc consumption score of leaf discs in *Nicotiana benthamiana* exposed to pest larvae for 5 days in the wells of a 96-well plate. Leaf discs were scored on a scale of 3 (high feeding) to 1 (no feeding) based on the percentage of remaining leaf area.

[0278]

[0279] Example 32 In this embodiment, the amino acid polypeptide sequence of GPA1280A was determined.

[0280] DNA sequences were isolated from sequencing DNA samples of Bacillus thuringiensis.

[0281] BLASTP was used to align predicted protein sequences corresponding to coding regions in the sequenced genome with the Mpp3Aa8 protein sequence, which exhibits insecticidal activity. Within the protein alignment region, a potential candidate gene was considered when the query / object coverage length of the new sequence was 50% or more and it had 30% or more amino acid identity. GPA1280A (SEQ ID NO: 18) showed only 52% sequence identity with Mpp3Aa8 (SEQ ID NO: 16) through paired full-length protein alignment. Figure 19 This demonstrates that it has a sequence distance from any known insecticidal protein.

[0282] The specific amino acid polypeptide sequences of GPA1280A (SEQ ID NO: 18) and Mpp3Aa8 (SEQ ID NO: 16) are provided in Table 22 below.

[0283] Table 22. Amino acid sequences of GPA1280A (SEQ ID NO: 18) and Mpp3Aa8 (SEQ ID NO: 16).

[0284]

[0285] Example 33 In this embodiment, GPA1280A is cloned and expressed.

[0286] To express GPA1280A, the DNA gene coding sequence was optimized for expression in *E. coli* and is provided in Table 23 below (SEQ ID NO: 19). This sequence was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)) to fuse the N-terminal 6x-His tag coding sequence with the GPA1280A gene. This clone was transformed into *E. coli* strain BL21(DE3) and grown in auto-induction medium (OVERNIGHT EXPRESS™ LB medium, EMDMillipore). After induction, bacterial cells were harvested for recombinant protein purification and then for insect larval activity assays. Figure 20 Images of SDS-PAGE analysis of purified GPA1280A protein from a recombinant E. coli expression vector (approximately 37 kDa) are shown. In some cases, induced bacterial cultures are used for insect assays.

[0287] Table 23. Optimized GPA1280A DNA coding sequence for GPA1280A expression in Escherichia coli (SEQ ID NO: 19).

[0288]

[0289] Example 34 In this embodiment, insecticidal toxicity bioassays were performed using whole cells of transformed bacteria expressing the GPA1280A protein to evaluate insecticidal efficacy against pests including fall armyworm (G. spp.). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB)), sugarcane borer ( Diatraea saccharalis (SCB)), Southern armyworm ( Spodoptera eridania (SAW) and corn rootworm ( Diabrotica virgifera (CRW)).

[0290] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0291] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0292] Fall armyworm ( Spodoptera frugiperda (FAW) eggs, corn ear borer ( Helicoverpa zea (CEW) eggs, European corn borer ( Ostrinia nubilalis (ECB) eggs, edamame noctuid moth ( Anticarsia gemmatalis (VBC) eggs, Southwest corn borer ( Diatraea grandiosella (SWCB)) eggs, sugarcane borer ( Diatraea saccharalis (SCB) eggs, soybean noctuid moth ( Chrysodeixis includens (SBL)) eggs, cabbage brown moth (Spodoptera exigua (BAW) eggs, Southern armyworm ( Spodoptera eridania (SAW) eggs, tobacco budworms ( Chloridia virescens (TBW)) eggs and black-cutting rootworm ( Agrotis ipsilon (BCW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by filling 24-well, 96-well (Costar®, Corning®), or 128-cell bioassay trays (Frontier Agricultural Sciences, Newark, DE) with a commercially available semi-solid insect feed portion. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method), allowing it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 to 7 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0293] Five days later, in bioassays, whole-cell bacterial cultures containing the GPA1280A protein showed positive results for fall armyworm ( ). Spodoptera frugiperda (FAW)), corn ear worm ( Helicoverpa zea (CEW)), edamame noctuid moth ( Anticarsia gemmatalis (VBC)), Southwest Corn Borer ( Diatraea grandiosella (SWCB)), sugarcane borer ( Diatraea saccharalis (SCB) and Southern Armyworm ( Spodoptera eridania (SAW) larval mortality and growth inhibition were greater than in the negative control (Table 24). This is important because known Mpp3-like insecticidal proteins are effective against these specific lepidopteran pests (e.g., fall armyworm). Spodoptera frugiperda (FAW) and corn earworm ( Helicoverpa zea (CEW) does not have any documented insecticidal activity.

[0294] In contrast, in bioassays, GPA1280A protein showed significantly higher efficacy against maize rootworms (compared to the negative control). Diabrotica virgifera (CRW) did not show larval mortality or growth inhibition (Table 24), demonstrating the specificity of the insecticidal activity of the GPA1280A protein.

[0295] Table 24. Insecticidal activity of whole recombinant E. coli cultures expressing GPA1280A protein, Coleoptera-specific Gpp34Ab1 / Tpp35Ab1 control, or Lepidoptera-active Vip3Aa19 control measured.

[0296]

[0297] Example 35 In this embodiment, the purified protein of GPA1280A was determined in an artificial feed cover bioassay. Figure 20 For susceptible fall armyworms ( Spodoptera frugiperda (FAW) groups and corn earworms ( Helicoverpa zea (CEW) colony insecticidal activity.

[0298] The same negative controls (Gpp34Ab1 / Tpp35Ab1 and Vip3Aa19) from Examples 3 and 4 were used in these assays.

[0299] Corn rootworm ( Diabrotica virgifera (CRW) eggs, northern corn rootworm ( Diabrotica barberi (NCR) eggs and southern maize rootworm ( Diabrotica undecimpunctata howardi (SCR) eggs were obtained from a commercial insect bank (Crop Characteristics, Inc., Farmington, MN). Bioassay chambers were prepared by partially filling 24-well or 96-well tissue culture plates (Costar®, Corning®) with semi-solid insect feed: WCRMO-1 feed or Southern Corn Rootworm larval feed (Frontier Agricultural Sciences, Newark, DE). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, one or several newly emerged larvae (less than 12 hours after emergence) were introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a membrane and ventilated with 000# pinholes. After incubation at approximately 24°C and 50% relative humidity (RH) for 4 to 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0300] Fall armyworm ( Spodoptera frugiperda (FAW) eggs and corn earworm ( Helicoverpa zea(CEW) Eggs were obtained from a commercial insect bank (Benzon Research Inc., Carlisle, PA). Eggs were incubated under controlled temperature and humidity until emergence. Bioassay chambers were prepared by partially filling 96-well tissue culture plates (Costar®, Corning®) with commercially available semi-solid insect feed. The feed was either a general lepidopteran feed (Frontier Agricultural Sciences, Newark, DE) or a multi-species feed (Southland Products Incorporated, Lake Village, AR). In a laminar flow hood, the test sample was applied to the surface of the semi-solid feed (feed covering method) to allow it to penetrate and evaporate. Once dry, a single newly emerged larva (less than 12 hours after emergence) was introduced into each well using a fine-tipped paintbrush. The bioassay plate was sealed with a perforated membrane or a membrane vented with 000# pinholes. After incubation at approximately 26°C and 50% relative humidity (RH) for 5 days, mortality, growth inhibition, and feeding inhibition were assessed.

[0301] Five days later, the purified GPA1280A protein was effective against fall armyworm (…). Spodoptera frugiperda (FAW) and corn earworm ( Helicoverpa zea (CEW) exhibited dose-dependent insecticidal activity because larval mortality and growth inhibition were found in a concentration-dependent manner to be greater than in the negative control in bioassays. Figures 21A to 21B This demonstrates that the death and growth inhibition of these specific pests are significantly correlated with the presence and amount of GPA1280A protein in the test samples.

[0302] Example 36 In this embodiment, the potency of the purified GPA1280A protein was evaluated and denoted as EC50, which represents the predicted protein concentration (μg / cm³) affecting 50% of infected insect larvae. 2 ).

[0303] The first step of the EC50 method is to determine the range of protein concentrations required in the bioassay to induce growth inhibition or mortality in approximately 50% and 100% of insect larvae. Aliquots (20 μL) of the protein solution are applied to the top of the feed (approximately 200 μL insect feed / well of a standard 96-well microtiter plate) and subsequently dried in a laminar flow hood for approximately 30 minutes. Once dried, a single fall armyworm is infected into each well using a fine-tipped water color brush. Spodoptera frugiperda(FAW) larvae. After larval infection, each plate was covered with a sealing film (Excel Scientific, Inc., Thermalseal RTSTM, TSS-RTQ-100) and placed in a dark growth chamber at 26°C for 5 days. On day 5, each plate was removed from the growth chamber and the mortality (survival or death) and growth inhibition of the larvae were assessed.

[0304] Once the appropriate protein concentration range was determined, five to nine different concentrations of purified protein were tested in a dose-response artificial feed cover bioassay. As described above, the analytes were incubated for 5 days and lethal and sublethal effects were assessed. Statistical analysis was performed using JMP® 16.2.0 software. Probit analysis of the binomial distribution was performed using a generalized linear model, with the dose variable logarithmic. 10 Transformations are used to analyze larval responses at each protein concentration, such as mortality (total larval deaths relative to total test larvae) or growth inhibition.

[0305] For those susceptible to fall armyworm ( Spodoptera frugiperda (FAW) The predicted EC50 of GPA1280A is 4.98 μg / cm³. 2 The 95% confidence interval is 1.14 µg / cm³. 2 Up to 9.62 µg / cm 2 For susceptible FAW, the EC50 of the control reference toxin Vip3Aa19 was 0.04 μg / cm³. 2 The 95% confidence interval is 0.03 µg / cm³. 2 Up to 0.05 µg / cm 2 .

[0306] Example 37 In this embodiment, GPA1280A is made from Tobacco Benzoinus (Tobacco Benzoinus). Nicotiana benthamiana (Instantaneous expression in )

[0307] Nucleic acid constructs for GPA1280A expression were prepared. The DNA nucleotide sequence encoding GPA1280A with a C-terminal HiBiT luciferase peptide tag was cloned between constitutive promoters linked to a polyadenylated sequence present in the plant binary vector.

[0308] The resulting binary plasmid was transferred into Agrobacterium strain GV3101 to produce strain TWP152. Leaf portions were transiently transformed with Agrobacterium tumefaciens strain TWP152 using the standard Agrobacterium tumefaciens infiltration protocol (essentially as described in bio-protocol.org / bio101 / e95).

[0309] Total protein was extracted from transformed tobacco leaves, and GPA1280A expression was examined using Promega’s Nano-Glo® HiBiT lysozyme assay system (Table 25).

[0310] Table 25. Expression of GPA1280A protein in *Nicotiana benthamiana* tissue 3 to 4 days after infiltration.

[0311]

[0312] Leaf discs prepared from Agrobacterium-infiltrated portions of tobacco leaves were then used for insect feeding assays using larvae of three different insect pests (Table 26). Strain 285 was used as a negative control in the experiments and did not express the GPA1280A protein. For the strain 285 control, the DNA nucleotide sequence encoding ZsGreen was cloned between constitutive promoters linked to polyadenylated sequences present in the plant binary vector.

[0313] Table 26. Average leaf disc consumption score of leaf discs in *Nicotiana benthamiana* exposed to pest larvae for 5 days in the wells of a 96-well plate. Leaf discs were scored on a scale of 3 (high feeding) to 1 (no feeding) based on the percentage of remaining leaf area.

[0314]

[0315] Example 38 Various plant binary nucleic acid experimental constructs are generated using a variety of promoters, initiators, introns, enhancers, terminators, upstream regulatory constructs, downstream regulatory constructs, or other regulatory sequence elements. These elements are operatively linked to drive the expression of nucleotide sequences encoding any of the disclosed proteins GPA1073A, GUN0345A, GUN1183A / B, GUN0307A, GUN0527A, GUN0052A, or GPA1280A (SEQ ID NO: 1, 4, 7, 10, 12, 15, 18, or 20) in target plants (e.g., maize cells).

[0316] In some instances, these nucleic acid experimental constructs are coupled with those encoding specific target peptides (e.g., maize). Zea mays The sequence of the chloroplast targeting signal peptide is operatively linked.

[0317] Each experimental construct was individually transformed into the maize inbred line B104. For each construct, at least 10 single-copy transformation events with intact T-DNA were generated. qRT-PCR and Western blot analysis were performed on T0 leaf materials to select transgenic plants exhibiting insecticidal protein expression.

[0318] Selected transgenic plants from the experimental constructs and their progeny were grown under greenhouse conditions. The insecticidal activity and efficacy of different transgenic plants against various pests were then evaluated.

[0319] Under greenhouse conditions, newly hatched (first larval stage) fall armyworms were artificially infected onto the heart leaves of plants. Spodoptera frugiperda The efficacy of FAW (Fecal Oil Witch) was tested by larvae feeding on the leaves and then scoring the results. The FAW efficacy assay employed a randomized complete block design with four replicates and three infected plants per block. Negative (non-GMO) and positive (GMO plants expressing the reference toxin) controls were used as comparisons in the root damage assessment. Seeds were counted and planted in 18-cell seedling trays and germinated in greenhouse compartments. The greenhouse compartments were configured for maize growth with daytime temperatures of 26°C to 29°C and 50% RH, and nighttime temperatures of 17°C to 20°C and 50% RH. The light-to-dark ratio was 16:8. At V2 (approximately 14 days), seedlings were transplanted into 1-gallon pots. Plants were allowed to grow to the V5 / V6 growth stage, and then each plant was infected with 30 newly infected larvae. Newly infected larvae were inoculated into the whorl of maize plants using an inoculator that delivered 1 mL aliquots of coarsely ground 2040 maize cob powder (used as a carrier) mixed with the newborn larvae. Once infected, the larvae fed on the plants for 14 days. Effective plants were selected using the Davis scale for FAW damage when plants were deemed suitable for scoring. Analysis of variance (JMP) was performed to compare transgenic events with appropriate controls.

[0320] Under greenhouse conditions, newly hatched (first larval stage) corn ear borers were artificially infected at the top of the pollinated spike silks using VT (several days after artificial pollination of each plant). Helicoverpa zea The efficacy of CEW was tested using the method described in the CEW efficacy assay. The CEW efficacy assay employed a randomized complete block design with four replicates and three infected plants per block. Negative (non-transgenic) and positive (transgenic plants expressing the reference toxin) controls were used as comparisons in ear damage assessment. Seeds were counted and planted in 18-cell seedling trays and germinated in greenhouse compartments. The greenhouse compartments were configured for maize growth with daytime temperatures of 26°C to 29°C and 50% RH, and nighttime temperatures of 17°C to 20°C and 50% RH. The light-to-dark ratio was 16:8. At V2 (approximately 14 days), seedlings were transplanted into 3-gallon pots. Following artificial pollination, each ear was infected with 15 newly hatched larvae on the pollinating silks. Once infected, the larvae were allowed to feed for 21 days. When the ear was deemed ready for scoring, each ear was dehulled and measured in cm. 2 Ear damage was measured per ear, and effective plants were selected. Analysis of variance (JMP) was performed to compare transgenic events with appropriate controls.

[0321] Under greenhouse conditions, a method was used to test for corn rootworms by artificially infecting plants with eggs, followed by scoring of the roots after egg hatching and larval feeding. Diabrotica virgifera The efficacy of CRW (Reference Toxin-Exposed Wheat) was assessed. A randomized complete block design was used for CRW efficacy determination, with four replicates and three infected plants per block. Negative (non-transgenic) and positive (transgenic plants expressing the reference toxin) controls were used as comparisons in root damage assessment. Seeds were counted and planted in 32-cell seedling trays and placed in greenhouse compartments for germination. The greenhouse compartments were configured for maize growth with daytime temperatures of 26°C to 29°C and 50% RH, and nighttime temperatures of 17°C to 20°C and 50% RH. The light-to-dark ratio was 16:8. At V2 (approximately 14 days), seedlings were transplanted into 1-gallon pots. Plants were allowed to acclimatize for approximately 2 days before being infected with CRW eggs. Eggs were delivered in 0.16% agar solution at a concentration of 500 eggs per mL. Each plant received 2 mL of egg / agar solution. The solution was delivered in 1 mL aliquots to each of two wells on either side of the plant using a syringe or repeat pipette, approximately 2 inches from the base of the plant and 2 inches deep. Eggs hatched approximately 12 days after infection. Once hatched, the larvae were allowed to feed for approximately 17 to 21 days. The plants were examined throughout the feeding cycle to monitor feeding progress and appropriate scoring times. When the plants were deemed ready, they were removed from the greenhouse and washed and scored in the root processing area of ​​the greenhouse complex. Roots were scored using the Iowa State University NIS Maize Damage Scale. Analysis of variance (JMP) was performed to compare transgenic events with appropriate controls.

[0322] In greenhouses, artificial inoculation of newly hatched (first larval stage) European corn borers was performed using VT / R1 larvae above the primary ear and below the secondary ear. Clouded oysterThe efficacy of ECB was tested by scoring damage to the internal stem and pedicel after larval feeding. The ECB efficacy assay employed a randomized complete block design with four replicates and three infected plants per block. Negative (non-transgenic) and positive (transgenic plants expressing the reference toxin) controls were used as comparisons in assessing stem and pedicel damage. Seeds were counted and planted in 18-cell seedling trays and germinated in greenhouse compartments. The greenhouse compartments were configured for maize growth with daytime temperatures of 26°C to 29°C and 50% RH, and nighttime temperatures of 17°C to 20°C and 50% RH. The light-to-dark ratio was 16:8. At V2 (approximately 14 days), seedlings were transplanted into 3-gallon pots. Plants were allowed to grow to the VT / R1 growth stage, and then 50 newborn larvae (out of a total of 100 newborn larvae) were used to infect one node above the primary ear and one node below the secondary ear per plant. Newly infected larvae were inoculated at appropriate nodes where leaves meet stems using an inoculator that delivered 1 mL aliquots of coarsely ground 2040 corn cob powder (used as a carrier) mixed with the newborn larvae. Once infected, the larvae were allowed to feed for 45 to 60 days. When plants were deemed ready for scoring, each stem and peduncle was dissected, and internal damage was measured in cm. Effective plants were selected. Analysis of variance (JMP) was performed to compare transgenic events with appropriate controls.

[0323] In summary, it can be seen that this disclosure achieves several advantages and obtains other beneficial results. Since various changes can be made to the methods described above without departing from the scope of this disclosure, all content contained in the above description and shown in the accompanying drawings is intended to be illustrative rather than restrictive.

[0324]

[0325] The foregoing description of specific aspects will so fully reveal the general nature of the invention that others can readily modify and / or adapt these specific aspects for various applications by applying knowledge within the art, without departing from the general conception of this disclosure, without excessive experimentation. Therefore, based on the teachings and guidance presented herein, such modifications and adjustments are intended to fall within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is for descriptive rather than limiting purposes, and that the terminology or terminology of this specification should be interpreted by those skilled in the art based on the teachings and guidance.

[0326] The breadth and scope of this disclosure should not be limited by any of the foregoing exemplary aspects, but should be defined solely by the appended claims and their equivalents.

[0327] All publications, patents, patent applications and / or other documents cited in this application are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication, patent, patent application and / or other document is individually indicated as incorporated herein by reference for all purposes.

[0328] For the sake of completeness, various aspects of the invention are set forth in the following numbered clauses: Clause 1. A method for 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 with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 to produce a transformed plant expressing the polypeptide, wherein the polypeptide has insecticidal activity against the plant pathogen or pest; and regenerating the transformed plant expressing the polypeptide.

[0329] Clause 2. The method according to Clause 1, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0330] Clause 3. The method according to Clause 1 or 2, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0331] Clause 4. The method according to any one of Clauses 1 to 3, wherein the plant pathogen or pest is selected from the group consisting of: fall armyworm ( Spodoptera frugiperda ), corn ear worm ( Helicoverpa zea ), European corn borer ( Clouded oyster ), cotton bollworm ( Helicoverpa armigera ), Black Root Worm ( Agrotis ipsilon ), corn borer ( Elasmopalpus lignosellus Asian corn borer ( Ostinia furnacalis ), Southwest corn borer ( Diatraea grandiosella ), sugarcane borer ( Diatraea saccharalis ), Western bean rootworm ( White-striped wrasse ), edamame noctuid moth ( Anticarsia gemmatalis ), corn rootworm ( Diabrotica virgin-bearing Southern corn rootworm ( Diabrotica undecimpuncatata Howard ), Northern corn rootworm ( Diabrotica barberi Soybean noctuid moth ( Including Chrysodeixis ), tobacco budworm ( Chloride greening ), cabbage brown moth ( Spodoptera small Southern armyworm ( Spodoptera eridania ) and their combinations.

[0332] Clause 5. A transformed plant, seed, or plant part comprising a recombinant nucleic acid molecule stably integrated into the genome of said transformed plant, seed, or plant part, said recombinant nucleic acid molecule encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, wherein said transformed plant, seed, or plant part stably expresses said polypeptide, and wherein said polypeptide has insecticidal activity against plant pathogens or pests.

[0333] Clause 6. The transformed plant, seed or plant part according to Clause 5, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0334] Clause 7. The transformed plant, seed or plant part as described in Clause 5 or 6, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0335] Clause 8. The transformed plant, seed or plant part according to any one of Clauses 5 to 7, wherein said transformed plant, seed or plant part is selected from the group consisting of: rice, barley, sorghum, soybean, cotton, corn, rapeseed, sugarcane, tobacco, sunflower and wheat.

[0336] Clause 9. A recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, wherein said polypeptide has insecticidal activity against plant pathogens or pests.

[0337] Clause 10. The recombinant nucleic acid molecule according to Clause 9, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0338] Clause 11. The recombinant nucleic acid molecule according to Clause 9 or 10, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0339] Clause 12. A recombinant nucleic acid molecule according to any one of Clauses 9 to 11, wherein the polynucleotide sequence encoding the polypeptide is operatively linked to one or more promoter sequences.

[0340] Clause 13. A vector comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, said polypeptide having insecticidal activity against plant pathogens or pests.

[0341] Clause 14. The vector according to Clause 13, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0342] Clause 15. The vector according to Clause 13 or 14, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0343] Clause 16. A transformed host cell comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, said polypeptide having insecticidal activity against plant pathogens or pests.

[0344] Clause 17. The transformed host cell according to Clause 16, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0345] Clause 18. The transformed host cell according to Clause 16 or 17, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0346] Clause 19. A method for 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 with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, wherein the polypeptide has insecticidal activity against the plant pathogen or pest.

[0347] Clause 20. The method according to Clause 19, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0348] Clause 21. The method according to Clause 19 or 20, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0349] Clause 22. A composition having insecticidal activity against plant pathogens or pests, said composition comprising an effective amount of at least one polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0350] Clause 23. The composition according to Clause 22, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

[0351] Clause 24. The composition according to Clause 22 or 23, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

Claims

1. A method for protecting plants from infection by plant pathogens or pests, the method comprising: The plant is transformed with a nucleic acid molecule encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 to produce a transformed plant expressing the polypeptide, wherein the polypeptide has insecticidal activity against the plant pathogen or pest; and The transformed plant that regenerates and expresses the polypeptide.

2. The method of claim 1, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

3. The method according to claim 1, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

4. The method according to claim 1, wherein the plant pathogen or pest is selected from the group consisting of: fall armyworm (… Spodoptera frugiperda ), corn ear worm ( Helicoverpa zea ), European corn borer ( Ostrinia nubilalis ), cotton bollworm ( Helicoverpa armigera ), Black Root Worm ( Agrotis ipsilon ), corn borer ( Elasmopalpus lignosellus Asian corn borer ( Ostinia furnacalis ), Southwest corn borer ( Diatraea grandiosella ), sugarcane borer ( Diatraea saccharalis ), Western bean rootworm ( Striacosta albicosta ), edamame noctuid moth ( Anticarsia gemmatalis ), corn rootworm ( Diabrotica virgifera Southern corn rootworm ( Diabrotica undecimpunctata howardi ), Northern corn rootworm ( Diabrotica barberi Soybean noctuid moth ( Chrysodeixis includens ), tobacco budworm ( Chloridia virescens ), cabbage brown moth ( Spodoptera exigua Southern armyworm ( Spodoptera eridania ) and their combinations.

5. A transformed plant, seed, or plant part comprising a recombinant nucleic acid molecule stably integrated into the genome of said transformed plant, seed, or plant part, said recombinant nucleic acid molecule encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18, wherein said transformed plant, seed, or plant part stably expresses said polypeptide, and wherein said polypeptide has insecticidal activity against plant pathogens or pests.

6. The transformed plant, seed, or plant part according to claim 5, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18.

7. The transformed plant, seed, or plant part according to claim 5, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15, or 18.

8. The transformed plant, seed, or plant part according to claim 5, wherein the transformed plant, seed, or plant part is selected from the group consisting of: rice, barley, sorghum, soybean, cotton, corn, rapeseed, sugarcane, tobacco, sunflower, and wheat.

9. A recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, wherein the polypeptide has insecticidal activity against plant pathogens or pests.

10. The recombinant nucleic acid molecule according to claim 9, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

11. The recombinant nucleic acid molecule according to claim 9, wherein the polypeptide is any one of SEQ ID NO: 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 operatively linked to one or more promoter sequences.

13. A vector comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, said polypeptide having insecticidal activity against plant pathogens or pests.

14. The vector according to claim 13, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

15. The carrier according to claim 13, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

16. A transformed host cell comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18, said polypeptide having insecticidal activity against plant pathogens or pests.

17. The transformed host cell of claim 16, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

18. The transformed host cell according to claim 16, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

19. A method for treating a plant or part of a plant against a plant pathogen or pest, the method comprising: Apply an effective amount of at least one polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18 to the plant or plant part, wherein the polypeptide has insecticidal activity against the plant pathogen or pest.

20. The method of claim 19, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

21. The method of claim 19, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

22. A composition having insecticidal activity against plant pathogens or pests, said composition comprising an effective amount of at least one polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

23. The composition of claim 22, wherein the polypeptide has at least 90% sequence identity with any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

24. The composition of claim 22, wherein the polypeptide is any one of SEQ ID NO: 1, 4, 7, 10, 12, 15 or 18.

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