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 Application Nos. 63 / 565,152 and 63 / 565,195, filed March 14, 2024, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence List Reference This application is made in accordance with 37 CFR § 1.831 and PCT Rule 13 three times The sequence list XML was submitted in ST.26 XML format. The sequence list XML file "218903-0051-WO01_GEN00013WOPCT_SequenceListing.xml" filed with the USPTO Patent Center was created on February 21, 2025, contains 114 sequences, has a file size of 284 kilobytes (290,816 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 engineered genes encoding insecticidal proteins that can be used to control pathogens and pests (particularly plant pests). These engineered 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 strategies use genes that express insecticidal proteins to produce transgenic crops. These insecticidal proteins are typically derived from Bacillus thuringiensis (Bt). Bacillus thuringiensis Bt ("Bt") is a Gram-positive, spore-forming soil bacterium. Currently available commercial insecticidal proteins include Bt Cry (crystal protein) or VIP (vegetative stage insecticidal protein). 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 fields, 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 action into transformative plants. To find novel insecticidal proteins with new modes of action, possible strategies involve discovering new insecticidal proteins from novel sources or through protein engineering. Therefore, novel insecticidal proteins are needed for the control of plant pests. Summary of the Invention

[0007] In one aspect, this disclosure relates to compositions and methods for protecting plants from infection by plant pathogens or pests. In some embodiments, these methods include transforming a plant with a nucleic acid molecule encoding a polypeptide having at least 97% or 98% sequence identity with any of SEQ ID NO: 1-60 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 is any of SEQ ID NO: 1-60. In 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 ( Clouded oyster ), cotton bollworm ( Helicoverpa armigera ), Black Root Worm ( Agrotis Y ), 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 ) 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 97% or 98% sequence identity with any of SEQ ID NO: 1-60, 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 is any of SEQ ID NO: 1-60. In 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 97% or 98% sequence identity with any of SEQ ID NO: 1-60, wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide is any of SEQ ID NO: 1-60. In 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 97% or 98% sequence identity with any of SEQ ID NO: 1-60, wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide is any of SEQ ID NO: 1-60.

[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 97% or 98% sequence identity with any of SEQ ID NO: 1-60, wherein the polypeptide has insecticidal activity against plant pathogens or pests. In one embodiment, the polypeptide is any of SEQ ID NO: 1-60.

[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 97% or 98% sequence identity with any of SEQ ID NO: 1-60, wherein the polypeptide has insecticidal activity against the plant pathogen or pest. In one embodiment, the polypeptide is any of SEQ ID NO: 1-60.

[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 97% or 98% sequence identity with any of SEQ ID NO: 1-60. In one embodiment, the polypeptide is any of SEQ ID NO: 1-60.

[0014] Another aspect of this disclosure provides a composition having insecticidal activity against plant pathogens or pests, the composition comprising a polynucleotide encoding at least one polypeptide disclosed herein, wherein the polynucleotide has at least 95% sequence identity with any of SEQ ID NO: 61-113. In one embodiment, the polypeptide is any of SEQ ID NO: 1-60.

[0015] Another aspect of this disclosure provides a polynucleotide comprising a polynucleotide sequence encoding any engineered insecticidal polypeptide described herein, the polynucleotide being operatively linked to a heterologous regulatory element.

[0016] Another aspect of this disclosure provides cells comprising any of the polynucleotides described herein. In one embodiment, the cell is a plant cell or a bacterial cell.

[0017] Another aspect of this disclosure provides plants containing any of the polynucleotide or cellular modifications described herein.

[0018] This disclosure provides other aspects and implementations that will become apparent from the following specific embodiments and drawings. Attached Figure Description

[0019] Figure 1 This is a table describing SEQ ID NO: 1-114.

[0020] Figure 2 This is a depiction of sequence alignments between SEQ ID NO: 50, 51 and 114, wherein each of the engineered variants 188A (SEQ ID NO: 50) and 401A (SEQ ID NO: 51) is an engineered variant of the native Cry1B-like toxin sequence of SEQ ID NO: 114.

[0021] Figure 3 This is the homology table for SEQ ID NO: 50, 51, 114.

[0022] Figure 4A and Figure 4B This is a description of the sequence alignment between SEQ ID NO: 1-49.

[0023] Figure 5A and Figure 5B This is the homology table for SEQ ID NO: 1-49.

[0024] Figure 6 This is a model illustration showing the tertiary structure (including domain I, domain II and domain III) characteristic of the classic 3-domain crystal protein (Cry) of Bacillus thuringiensis.

[0025] 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

[0026] This document describes compositions and methods that can include native and engineered Cry-like insecticidal proteins, which can be used to confer insecticidal activity. The disclosed compositions may include isolated, recombinant, and purified peptides having insecticidal activity. In some embodiments, the engineered insecticidal peptides include engineered Cry1B-like insecticidal peptides. In some embodiments, the engineered insecticidal peptides described herein have at least 95%, 97%, or 98% sequence identity with any of SEQ ID NO: 1-60. In some embodiments, this document describes recombinant polynucleotides comprising DNA constructs and expression vectors encoding engineered peptides having insecticidal activity. In some embodiments, nucleic acid molecules and peptides may be described as DNA constructs and expression cassettes for transforming plants, plant tissues, plant parts, plant cells, and plant seeds, as well as microorganisms. The insecticidal peptides described herein can provide useful alternatives to those currently used in commercial transgenic plants.

[0027] 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.

[0028] 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 “comprising,” “composed of,” and “substantially composed of” the embodiments or elements presented herein, whether or not explicitly stated.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] As used in this article, “derived from” and “originating from” refer to a DNA or amino acid sequence, or a portion thereof, that has a portion or all of a sequence found in a native gene or protein.

[0036] 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.

[0037] As used herein, a "fusion protein" refers to a chimeric protein resulting from the fusion of two or more genes or gene segments that originally encode a single polypeptide. Translation of the fusion gene produces a single polypeptide that possesses functional properties derived from each of the original polypeptides. As used herein, a "chimeric protein" refers to a polypeptide that comprises at least one polypeptide segment derived from two heterologous genes or two heterologous polypeptides.

[0038] 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.

[0039] 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 a new functional nucleic acid, 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 will also be 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). Heterologous polynucleotides can be produced using any gene-editing or molecular biology technique. As used herein, a "heterologous domain" refers to a protein domain region that combines with one or more naturally occurring domain regions to form a non-native (i.e., non-naturally occurring) engineered fusion protein, wherein the heterologous domain and one or more naturally occurring domain regions do not exhibit the same interactions found in nature. For example, in some non-limiting exemplary embodiments described herein, domain III from a first Cry protein may be combined and linked with domains I and II from a second Cry protein, wherein domain III from the first Cry protein is a "heterologous domain" relative to the naturally occurring domains I and II from the second Cry protein.

[0040] 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.

[0041] As used in this article, a "natural gene" is a gene that has not undergone alteration (such as loss, gain, or exchange of genetic material). Natural genes undergo normal gene transmission and expression. For example, a natural gene can be a wild-type gene.

[0042] 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.

[0043] 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.

[0044] As used herein, “operably linked” means that gene expression is controlled or influenced by a regulatory element (e.g., a promoter) spatially linked to it. The regulatory element can be located at the 5' (upstream) or 3' (downstream) of the gene. The distance between the regulatory element and the gene can be approximately equal to the distance between the regulatory element and the gene it controls in the gene from which it originates. Variations in this distance can be accommodated without loss of regulatory 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, if a regulatory element regulates or helps regulate the transcription of a coding sequence, then the regulatory element is operably linked to the coding sequence. Operatically linked DNA sequences are typically contiguous, and the amino acid sequences are typically contiguous and within the same reading frame. However, because enhancers often function up to several thousand bases or more separate 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.

[0045] 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.

[0046] 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 (i.e., optimized) or altered insecticidal activity against specific pests. Suitable engineering methods for producing 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.

[0047] The variant nucleotide sequences and proteins disclosed herein encompass sequences derived from mutagenesis or recombination processes (e.g., 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.

[0048] Domain exchange is another known engineering mechanism for generating variant insecticidal proteins, such as modified Cry toxin peptides. For example, domains can be exchanged between different Cry family toxin insecticidal peptides (e.g., Cry1B, Cry1N, Cry1F, Cry1C, Cry1D, etc.) to generate hybrid or chimeric fusion protein toxins with altered 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.

[0049] 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.

[0050] The proteins disclosed herein can be engineered to produce different physical properties, such as enhanced resistance to insects or insecticidal activity, altered insecticidal or resistance spectra, or reduced phytotoxicity to plants. The engineered proteins can be variants, mutants, fragments, or chimeric proteins derived from the starting polypeptide sequence.

[0051] "Pests" include, but are not limited to, insects, fungi, bacteria, nematodes, mites, ticks, etc. Insect pests can include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Trichophala, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermoptera, Isoptera, Pseudocoptera, Silicaria, and Trichoptera.

[0052] In some embodiments described herein, insect pests may include larvae and adults of the order Coleoptera, including weevils from the families Anthribidae, Bruchidae, and Curculionidae, including but not limited to: the cotton boll weevil (… Anthonomus grandis Boheman); Sunflower stem weevil ( Cylindrocopterus sprinkled LeConte); Citrus root weevil ( Diaprepes abbreviatus Linnaeus; Alfalfa leaf weevil ( Hyper dotted Fabricius); Rice water weevil ( Lissorhoptrus oryzophilus Kuschel); West Indian sugarcane weevil ( Metamasius hemipterus hemipterus Linnaeus; Silky Sugarcane Weevil ( M. hemipterus silky Olivier); Valley Elephant ( Sitophilus granarius Linnaeus; Rice elephant ( S. oryzae Linnaeus; Red sunflower seed weevil ( Yellow-tailed eagle LeConte); Gray sunflower seed weevil ( S. sordidus LeConte); Maize cryptid ( Sphenophorus maidis Chittenden; New Guinea sugarcane weevil ( Rhabdoscelus obscure Boisduval; flea beetles, cucumber beetles, root leaf beetles, leaf beetles, potato beetles, and leaf miners of the Chrysomelidae family, including but not limited to: wasteland corn flea beetles ( Chaetocnema ectypa Horn); Corn Flea Beetle ( C. flea market Melsheimer); Grape leaf beetle ( Brown sedge Fabricius); Northern corn root leaf beetle ( Diabrotica barberi Smith & Lawrence); Southern Corn Root Leaf Beetle ( D. undecimpunctata Howard Barber); Western corn root leaf beetle ( D. virgifera virgifera LeConte); Colorado potato beetle ( Leptinotarsa ​​ten-lineata Say); Black-horned mud worm ( Oulema melanopus Linnaeus); Yellow-striped flea beetle ( Phyllotreta crucifera Goeze (corn flea beetle); sunflower leaf beetle ( Zygogram exclamation Fabricius; beetles of the family Coccinellidae, including but not limited to: Mexican bean beetle ( Epilachna varivesti Mulsant; scarab beetles and other beetles of the Scarabaeidae family, including but not limited to: the cane grub of the Childes ( Small Antitrogus Britton); Northern Covered-face Golden Tortoise ( Cyclocephalus northern Arrow (white grub); Southern-faced golden beetle ( C. immaculata Olivier (white grub); Grey-backed sugarcane beetle ( Dermolepida albohirtum Waterhouse); Sugarcane Beetle ( Euetheola humilis ruddyLeConte); French sugarcane beetle ( Lepidiota frenchi Blackburn); Carrot Beetle ( Humpbacked Tomarus DeGeer); Sugarcane grub ( T. subtropical Blatchley); white grub ( Phyllophaga hairy Burmeister); June beetle ( P. latifrons LeConte); Japanese beetle ( Japanese popillia Newman); European golden beetle ( Rhizotrogus majalis Razoumowsky; carpet beetles (dermatids) of the family Dermestidae; wireworms of the family Elateridae, including the genera *Eleodes* and *Melanotus*, which include wireworms ( Common M. Gyllenhal; *Acer spp.* Conoderus spp.); Wireworm (Limonius spp.); Click beetle ( Agriots spp.); genus *Smilodon* ( Ctenicera spp.); *Aeolus* ( ) Aeolus spp.); bark beetles of the family Scolytidae; beetles of the family Tenebrionidae; beetles of the family Cerambycidae, such as, but not limited to, the longhorn beetle ( Almond fryanus Westwood; and beetles of the family Buprestidae, including but not limited to leaf miners (Westwood); Aphanisticus cochinchina seed Obenberger).

[0053] In other embodiments, insect pests may include immature stages and adults of the order Diptera, including leaf miners, corn leaf miners (… Agromyza smallcornis Loew); gall midges, including but not limited to: sorghum gall midge ( Contarina sorghum Coquillett, wheat gall midge ( Mayetiola destructor Say (Black Forest Fly), Sunflower Seed Gall Mosquito ( Neolasioptera murtfeldtiana Felt), wheat wax moth ( Sitodiplosis mosellana Géhin; fruit flies of the family Tephritidae, Swedish straw fly ( Oscinella frit Linnaeus); fly larvae, including but not limited to: genus *Linnaeus* (…). Delia spp.), including gray ground fly ( Delia's flat Meigen (seed fly), wheat root fly ( D. narrowedFallen (wheat seed fly); summer toilet fly ( Fannia canicularis Linnaeus, spotted toilet fly ( F. femoral Stein (small dung fly); American wheat straw fly ( Meromyza americana Fitch); housefly ( Fly domestic Linnaeus; stable fly ( Stomoxys calcitrans Linnaeus); genus *Linnaeus*, including *Flelidae*, *Hornflies*, *Blowflies*, and *Goldflies*. Chrysomya spp.), genus *Fly* ( Phormia spp.) and other fly pests; horseflies ( Horsefly spp.); stomach flies ( Gastrophilus spp.); sheep fly ( Estrus spp.); cowfly ( Hypodermis spp.); deer fly ( Chrysops spp.); sheep lice ( Sheep's hawk Linnaeus (sheep lice fly); and other suborders of Brachycera ( Brachycera Insects; mosquitoes, including those of the genus Aedes ( House spp.), Anopheles ( Anopheles spp.), Culex genus ( Mosquito spp.); blackflies, including the genus Protoceratops ( Proximum spp.) and genus *Nematophores* ( Simulacrum spp.); midges, sandflies, fungus gnats and other longhornoids ( Nematocera )insect.

[0054] Lepidoptera insects can include, but are not limited to, armyworms, root-cutting insects, inchworms, and cutworms of the family Noctuidae: cutworms ( Agrotis ipsilon Hufnagel (black rootworm); western cutworm ( A. orthogonality Morrison (Western rootworm); Yellow cutworm ( A. grain Denis & Schiffermüller); Gracilaria granulosa (A. underground Fabricius); cotton leafworm ( Alabama clay Hubner; Bean moth ( Anticarsia gemmatalis Hubner (Eriocheir aegypti); Cutworm (Agrostis spp.) Athetis mindara Barnesand McDunnough (rough-skinned root-cutting insect); Emerald-patterned diamond ( Island Earias Boisduval; patina diamond ( E. vittella Fabricius; Citrus cutworm ( Turn around ( Xylomyces ) courtierGrote; Dark Tiger ( The harvester Harris (dark root-cutting worm); American cotton bollworm ( Helicoverpa armigera Hubner); Corn ear worm ( H. zea Boddie (cotton bollworm); tobacco shoot moth ( Heliothis virescens Fabricius (Tobacco budworm); Alfalfa moth ( Hypena scabra Fabricius); scapulae ( Mammestra configuratus Walker); Cabbage moth ( M. brassicae Linnaeus; Zebra Noctuid ( Painted lady Harris); Armyworm ( Pseudaletia single-pointed Haworth); Soybean borer ( Pseudoplusia including Walker; Western bean moth ( Rich white-skinned Smith); Fall armyworm ( Spodoptera frugiperda JE Smith (fall armyworm); beet armyworm ( S. small Hubner (Brown cabbage moth); Spodoptera litura (… S. litura Fabricius; Powdered Noctuid moth ( Trichoplusia if Hubner; borers, nest moths, web moths, fruit moths, and spotted moths from the families Pyralidae and Crombidae, such as the small wax moth ( Achroia grisella Fabricius); Navel Orange Moth ( Amyelois transitella Walker); Mediterranean mealybug ( Anagasta kuehniella Zeller); Almond borer ( Caution frame Walker); Spotted stem borer ( Kilo part Swinhoe; Rice stem borer ( C. suppressalis Walker); sugarcane stalk borer ( C. earthly Pagenstecher); Rice moth ( Corcyra cephalonica Stainton); Corn root borer ( Crab little fog Clemens); annual grass webworm ( C. teterrellus Zincken); Rice leaf roller ( Cnaphalocrocis medinalis Guenee); Grape leafroller ( Funeral Desmia Hubner); Melon moth ( Hyalinous diaphania Linnaeus; pickle worms ( D. nitidalis Stoll); Southwest corn borer ( Diarrhea grandiloquent Dyar); Sugarcane borer ( D. saccharalisFabricius); small corn stalk borer ( Elasmopalpus wooden stool Zeller); Mexican rice borer ( Eoreuma loftini Dyar); Tobacco powder moth ( Ephesus small Hubner); Galleria mellonella (Mellonella mellonella) Galleria mellonella Linnaeus); Sugarcane leaf roller ( Hedyleptus accepted Butler); Grassland webworm ( Herpetogramma licarsisalis Walker); sunflower borer ( Homoeosome small gift Hulst); yellow lawn borer ( Loxosthege stycticalis Linnaeus); Bean pod borer ( Maruca testicle Geyer); Tea leaf roller (Orthaga thyrisalis Walker); European corn borer ( Ostrinia cloudy Hubner); Indian meal moth ( Plodia interpunctella Hubner); Rice stem borer ( Grasshopper uncertain Walker); Celery leaf roller ( Red-bellied woodpecker Guenee; and leafrollers, bud borers, seed borers, and fruit borers of the Tortricidae family: Western Blackhead Bud Roller ( Acleris gloverana Walsingham); Eastern Blackhead Bud Roller ( A. variana Fernald); Summer fruit roller ( Adoxophyes orana Fischer von Rösslerstamm); Apple leafroller ( ) Archippus spp.), including fruit tree leafrollers ( A. argyrospila Walker) and European leafroller ( A. rose Linnaeus); *Linnaeus* (Silver-striped Roller) Argyrotaenia spp.); Brazilian apple leafroller ( Good-for-nothing healthy Meyrick); genus *Meyrick* (… Choristoneura spp.); spotted sunflower moth ( Shellfish guest Walsingham; Hazelnut borers ( Cydia latiferra Walsingham; codling moth ( C. apple tree Linnaeus; Grape berry moth ( Endopiza viteana Clemens); Grape leafroller ( Eupoecilia ambiguous Hubner); Oriental fruit moth ( Grapholite pest Busck); European grape moth ( Lobesia girl Denis & Schiffermüller); Variegated Leaf Roller ( Platynota flavedana Clemens); Omnivorous leafroller ( P. stultana Walsingham); Eye-spotted bud roller ( Spilonota ocellana Denis & Schiffermüller); and sunflower bud moth ( Suleima helianthana Riley).

[0055] Other lepidopteran agricultural pests may include, but are not limited to: the autumn geometrid moth ( Alsophila orchard Harris); Peach-leaf moth ( Anarsia lineatella Zeller); Orange-spotted oak beetle ( Aniseed senatorial office JE Smith); Tussah silkworm (Antheraea pernyi Guérin-Méneville); Domestic silkworm ( Silkworm to die Linnaeus); cotton leaf-piercing moth ( Bucculatrix thurberiella Busck); Alfalfa White Butterfly ( Necks Eurythema Boisduval); Walnut boat moth (Datana most complete Grote & Robinson; Siberian pine caterpillar ( Siberian Dendrolimus Tschetwerikov); Elm looper ( Ennomos' signatures Hübner; Linden inchworm ( Erannis tiliaria Harris); Sugarcane bud moth ( Erechthias flavistriata Walsingham; Brown-tailed tussock moth ( Euproctis chrysorrhoea Linnaeus); Grape leaf spot moth ( American Harrisia Guérin-Méneville; *Cephalopoda* ( Heliothis subflexa Guenée; Pasture moth ( Hemileuca olives Cockrell); American white moth ( Hyphantria wedge Drury); Tomato leaf miner ( Keiferia small tomato Walsingham); Eastern Hemlock Geometrid Moth ( Lambdina fiscellaria boxwood Hulst); Western Hemlock Geometrid Moth ( L. fiscellaria lugubrosa Hulst); Willow Moth ( Leucoma willows Linnaeus; Gypsy moth ( Lymantria dispar Linnaeus; Genus *Linnaeus* (tent caterpillars) Malacosome spp.); Five-spotted hawk moth ( Five-spotted Manduca Haworth); Tobacco hawk moth ( M. sixth Haworth); Winter geometrid moth ( Operophtera brumata Linnaeus; genus *Linnaeus* (… Orgy spp.); Spring geometrid moth ( Paleacrite spring Peck); Citrus Swallowtail ( Butterfly cresphontes Cramer); California oak leafminer ( Phrygianidae Californian Packard); Citrus leafminer ( Phyllocnistis citrella Stainton); Apple leafminer ( Phyllonorycter blancardella Fabricius); Giant cabbage white butterfly ( Cabbage Pieris Linnaeus); Cabbage white butterfly ( P. rapae Linnaeus); Green-veined cabbage white butterfly ( P. napi Linnaeus; Artichoke moth ( Platyptilia carduidactyl Riley); Diamondback moth ( Plutella xylostella Linnaeus); Bollworm ( ) Pectinophora gossypiella Saunders; Southern cabbage white butterfly ( Pontia protodice Boisduval & Leconte); Omnivorous inchworm ( Sick sandflies Guenee); Red Camelback Caterpillar ( Sitotroga cerealella JE Smith); Wheat moth ( Sitotroga cerealella Olivier); Pine-leaved moth ( Thaumetopoea pityocampa Schiffermüller); clothes moth ( Tineola bisselliella Hummel); Tomato leaf miner ( Absolutely safe Meyrick); Nesting moth ( Yponomeuta pan Linnaeus).

[0056] In some implementations, insect pests may include species of the order Hemiptera, including but not limited to the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae, Delphacidae, Diaspididae, and Eriococcidae. The family includes: Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae, Membracidae, Miridae, Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae, and Tingidae.

[0057] Non-limiting examples of insect pests of significant agronomic importance in the order Hemiptera include: the green bug (… Acrosternum cheerful Say); Pea aphid ( Acyrthisiphon pea Harris); Coccidioides ( ) Adelges (spp.) (ball aphid); alfalfa brown mirid bug ( Adelphocoris rapidus Say); Pumpkin-shaped stink bug ( Sad duck De Geer); Bean aphid ( Aphid ravenous Koch); Black bean aphid ( A. beans Scopoli); cotton aphid ( A. gossypii Glover (melon aphid); corn root aphid ( A. maidaridicis Forbes); Apple aphid ( A. apple De Geer); Spiraea aphid ( A. small hole Patch); Sugarcane scale ( Aulacaspis tegalensis Zehntner); Eggplant Aphid (a type of aphid without webs) Nightshade Kaltenbach; whitefly ( Bemisia tabaci Gennadius (sweet potato whitefly); silver leaf whitefly ( B. argentifoliiBellows & Perring; Wheat bug ( Blissus leucopterus white-winged hawk Say); Family: Viperidae ( Blostomatidae spp.); cabbage aphid ( Brevicoryne cabbages Linnaeus); pear psyllid ( Cacopsylla pyricola Foerster; potato mirid bug ( Calocorus Norwegian Gmelin); Strawberry aphid ( Chaetosiphon fragaefolii Cockerell; Cimicidae spp.; Coreidae spp.; Lace bug ( Corythuca gossypii Fabricius); Tomato stink bug ( Cyrtopeltis modesta Distant); Tobacco mirid bug ( C. notatus Distant (smoke-bearing bug); foaming cicada ( God yellow-painted Stål); Citrus whitefly ( Citrus citronella Ashmead; soapberry mirid bug ( Diaphnocorus chlorine Say); Wheat double-tailed aphid ( Diuraphis noxious Kurdjumov / Mordvilko (Russian wheat aphid); Two-spined scale ( Duplachionaspis divergens Green); Rose apple aphid ( Dysaphis plantain Passerini; Cotton Red Bug ( Dysdercus suturellus Herrich-Schaffer); sugarcane mealybug ( Dysmicoccus boninsis Kuwana; potato leafhopper ( Bean sprouts Harris); Apple woolly aphid ( Eriosoma woolly Hausmann); Grape leafhopper ( ) Erythroneura spp.); Island sugarcane planthopper ( Eumetopina flavipes Muir; *Muir* genus ( Eurygaster spp.); Brown bug ( Euschistus servant Say); a little brown bug ( E. variolarius Palisot de Beauvois; genus *Palaeotropium* (red-glanded long bug) Graptostethus spp. (fruit bug group); peach mealybug ( Hyalopterus plumi Geoffroy); Pittosporum tobira ( Icerya purchasi Maskell); Onion bug ( Allium labopodicola Knight); Gray flyfly ( Laodelphax striped Fallén; Pine nut periwinkle ( Leptoglossus corculus Say); Sugarcane lace bug ( Leptodictya lukewarm Herrich-Schaeffer); Pseudo-cabbage aphid ( Lipaphis erysimi Kaltenbach; Common green mirid bug ( Lygocoris pabulinus Linnaeus; pasture mirid bug ( Lygus lineolaris Palisot de Beauvois); Western pasture mirid bug (L. Hesperus Knight); Common Lygus ( L. pratensis Linnaeus; European pasture mirid bug ( L. rugulipennis Poppius; Potato aphid ( Euphorbia macrosiphum Thomas); Aster leafhopper ( Macrosteles quadrilineatus Forbes); Seventeen-year cicada ( Seventeen Magicicada Linnaeus; sugarcane grasshopper ( Mahanarva fimbriolata Stål); sugarcane black aphid ( Sugar ants Zehntner; Melanaspis clusters Green); Wheat webless long-tubed aphid ( Metopolophium dirhodum Walker); Peach aphid ( Myzus persica Sulzer (green peach aphid); lettuce aphid ( Nasonovia blackcurrant Mosley); Black-tailed Leafhopper ( Nephotettix cinquefoil Uhler); Black-spotted leafhopper ( N. nigropictus Stål); Southern Green Bug ( Nezara greenish Linnaeus); Brown planthopper ( Nilaparvata is mourning. Stål); Small long bug ( Nyssa heather Schilling); False wheat bug ( Nysius radish Howard); Rice Green Bug ( Oebalus combative Fabricius); Milkweed bug ( Oncopeltus fasciatus Dallas); Wilderness Mischievous Bug ( Orthopedic plain Linnaeus; *Pemphigus* spp. (root aphids and gall aphids); corn planthopper ( Pilgrim maid Ashmead; sugarcane planthopper ( Perkinsiella saccharicida Kirkaldy); Walnut phylloxera ( Phylloxera devastating Pergande); citrus mealybug ( Planococcus citri Risso); Apple mirid bug ( Plesiocoris rugicolfis Fallen); Four-lined mirid bug ( Poecilocapsus lineatus Fabricius; cotton bollworm ( Pseudomoscelis seriatusReuter); Mealybug genus ( Pseudococci spp. (other mealybug complex); Bombyx mori mealybug ( Elongated cushion Newstead); sugarcane leafhopper ( Pyrilla perpusilla Walker); Red bug family ( Pyrrhocoridae spp.); Pear-shaped shield scale ( Quadraspidiotus pernicious Comstock); Assassinidae ( Reduviidae spp.); corn aphid ( Rhopalosiphum maidis Fitch); Gramineae constrictor aphid ( R. paddy Linnaeus (bird cherry-oat aphid); pink sugarcane mealybug ( Saccharococcus sacchari Cockerell); wheat aphid ( Schizaphis grass Rondani); Yellow sugarcane aphid ( Yellow siskin Forbes); Wheat aphid ( Sitobion oat Fabricius); White-backed planthopper ( Sogatella furcifera Horváth); rice planthopper ( Sogatodes oryzicola Muir); White-spotted mirid bug ( Spanagonicus albofasciatus Reuter); Alfalfa spotted aphid ( Therioaphis maculata Buckton); Gramineae family ( Tinidae spp.); Orange black aphid ( Toxoptera orange Boyer de Fonscolombe); orange-brown aphid ( T. citricida Kirkaldy); Hemp whitefly ( Trialeurodes abutiloneus ) and greenhouse whiteflies ( T. vaporariorum Westwood); persimmon psyllid ( Trioza persimmon Ashmead; Apple leafhopper ( Typhlocyba pomaria McAtee.

[0058] In other embodiments, insect pests may also include adult and larval mites of the order Acari (mites), including but not limited to: wheat leafroller ( Aceraceae Keifer; Elm mite ( Panonychus elm Koch (European red mite); Wheat mite ( Petrobia latentis Müller (brown wheat mite); Benedict's leaf mite ( Steneotarsonemus Bancroft Michael (cane stem mite); spider mites and red mites of the family Tetranychidae, African sugarcane leaf mite ( Oligonychus grypus Baker & Pritchard, Indian small claw mite ( O. indicus Hirst (cane leaf mite), Banksy mite ( O. pratensisBanks (Grassland Spider Mite), Sugarcane Spider Mite ( O. stickneyi McGregor); Two-spotted spider mite ( Spider mite Koch); Mayermites ( T. mcdanieli McGregor); Carmine spider mite ( T. cinnabar Boisduval (red leaf mite); Turkestan leaf mite ( T. turkestani Ugarov & Nikolski (Strawberry Spider Mite); Flat Mite of the Tenuipalpidae family, Liu's Short-haired Mite ( Brevipalpus lewisi McGregor (citrus flat mite); rust mites and bud mites of the family Eriophyidae, as well as other leaf-feeding mites and mites important to human and animal health, namely dust mites of the family Epidermoptidae, hair follicle mites of the family Demodicidae, grain mites of the family Glycyphagidae, and ticks of the family Ixodidae. *Ixodactylum scapulae* ( Ixodes scapularis Say (deer tick); Holocircly hard tick ( I. whole cycle Neumann (Australasian paralyzed tick); Mutant leather tick ( Dermacentor variabilis Say (American canine tick); American flower tick ( Amblyomma American Linnaeus (Lone Star Tick); and scabies mites and itch mites from the families Psoroptidae, Pyemotidae, and Sarcoptidae.

[0059] In addition, insect pests can also include species of the order Thysanura, such as silverfish ( Sugar silverfish Linnaeus and small silverfish ( Thermobia domestica Packard (spotted wrasse).

[0060] Insect pests can also include species of the order Isoptera, including species of the family Termitidae, such as, but not limited to, *Noscheretella nobilis* (termites). Cylindrotermes nordenskioeldi Holmgren and aggressive spiny termites (Holmgren) and aggressive spiny termites Pseudocanthotermes military Hagen (sugarcane termite).

[0061] Insect pests can also include species of the order Thysanoptera, including but not limited to thrips, such as the needleless thrips ( Stenchaetothrips minutus van Deventer (cane thrips).

[0062] In other embodiments, arthropod pests may include: spiders of the order Araneae, such as the brown-skinned flower spider (… Loxosceles reclusaGertsch & Mulaik (brown hermit spider); and red-spotted spider (Gertsch & Mulaik) Robber slaughterer Fabricius (black widow spider); and centipedes of the order Scutigeromorpha, such as the house centipede (Scutigeromorpha var. spp.). Scutigera coleoptrata Linnaeus).

[0063] 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).

[0064] In some embodiments described herein, the composition, plant, cell, and method may further comprise at least one additional insecticidal protein as an insecticidal synergist, for example, contributing to reducing the likelihood of resistance development or broadening the insect inhibition spectrum. These additional insecticidal proteins may be isolated from organisms including, for example, *Bacillus* sp., *Pseudomonas* sp., *Photorhabdus* sp., *Xenorhabdus* sp., *Clostridium bifermentans*, and *Paenibacillus popilliae*. In some embodiments, transgenic or modified plants expressing insecticidal proteins as described herein may also be hybridized with transgenic events expressing other insecticidal proteins and / or other transgenic traits (e.g., other insect control traits, herbicide tolerance genes, genes conferring yield or stress tolerance traits, etc.), or such traits may be combined in a single vector, thereby linking all traits.

[0065] Other insecticidal proteins may include, but are not limited to: insecticidal proteins from the genus *Pseudomonas*, such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7: 1-13); insecticidal proteins from *Pseudomonas protegens* strains CHAO and Pf-5 (formerly *Pseudomonas fluorescens*) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank accession number EU400157); insecticidal proteins from *Pseudomonas taiwanensis* (Liu et al., (2010) J. Agric. Food Chem., 58: 12343-12349); and insecticidal proteins from *Pseudomonas pseudoalcaligenes* (Zhang et al., (2009) Annals of Microbiology). 59:45-50 and Li et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168); Insecticidal proteins from the genera *Bacillus* and *Bacillus* (Hinchliffe et al., (2010) The Open Toxicology Journal, 3: 101-118 and Morgan et al., (2001) Applied and Envir. Micro.67:2062-2069); US Patent Nos. 6,048,838 and 6,379,946; PIP-1 peptide of US Patent No. 9,688,730; AflP-1A and / or AflP-1B peptides of US 9,475,847; PIP-47 peptide of US Publication No. US20160186204; PCT Publication No. WO The following peptides are listed: IPD045, IPD064, IPD074, IPD075, and IPD077 (serial number 2016 / 114973); IPD080 (serial number PCT / US17 / 56517); IPD078, IPD084, IPD085, IPD086, IPD087, IPD088, and IPD089 (serial number PCT / US17 / 54160); and PIP-72 (serial number US20160366891). The following peptides are listed: PtlP-50 and PtlP-65 (US Publication No. US20170166921); IPD098, IPD059, IPD108, and IPD109 (US Serial No. 62 / 521084); IPD110, IPD113, and PtlP-83 (US Publication No. US20160347799); PtlP-96 (US Publication No. US20170233440); IPD079 (PCT Publication No. WO2017 / 23486); and PCT Publication No. WO2017 / 23486. IPD082 peptide (serial number PCT / US 17 / 30602); IPD090 peptide (serial number PCT / US 17 / 30602); IPD093 peptide (serial number 62 / 434020); IPD103 peptide (serial number PCT / US 17 / 39376); IPD101 peptide (serial number 62 / 438179); US serial number...IPD121 peptide 62 / 508,514; and δ-endotoxins, including but not limited to Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, C Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71 and Cry72 d-endotoxin peptides, as well as the cytolytic cyt1 and cyt2 genes of Bacillus thuringiensis. These are members of the insecticidal protein class of Bacillus thuringiensis (see Crickmore et al., “Bacillus thuringiensis toxin nomenclature” (2011), bpprc.org).

[0066] Examples of d-endotoxins include, but are not limited to: the Cry1A protein of U.S. Patent Nos. 5,880,275, 7,858,849, and 8,878,007; the Cry1Ac variant of U.S. Patent Nos. 9,512,187; and the DIG of U.S. Patent Nos. 8,304,604, 8,304,605, and 8,476,226. 3 or DIG 11. Toxins (N-terminal deletion variants of α-helix 1 and / or α-helix 2 of cry proteins (e.g., Cry1A, Cry3A); Cry1B of U.S. Patent Application Serial No. 10 / 525,318, U.S. Patent Application Publication No. US20160194364, and U.S. Patent Nos. 9,404,121 and 8,772,577; Cry1B of PCT Publication No. WO2016 / 61197 and Serial No. PCT / US17 / 27160) Variant 1B; Cry1C (US Patent No. 6,033,874); Cry1D (US Patent No. 20170233759); Cry1E (PCT Sequence No. PCT / US17 / 53178); Cry1F (US Patent Nos. 5,188,960 and 6,218,188); Cry1A / F chimeras (US Patent Nos. 7,070,982, 6,962,705 and 6,713,063); PCT Publication No. WO Cry1L protein of 2017 / 0233759; engineered Cry1G as described in WO2018111553A1; CryU variant published in US20170240603; Cry2 proteins, such as Cry2Ab protein of US Patent No. 7,064,249 and Cry2A.127 protein of US Patent No. 7,208,474; Cry3A proteins, including but not limited to engineered hybrid insecticidal proteins (eHIP) produced by fusing a unique combination of variable and conserved segments of at least two different Cry proteins (US Patent Application Publication No. 2010 / 0233759). 017914); Cry4 protein; Cry5 protein; Cry6 protein; Cry8 protein of U.S. Patent Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,339,092, 7,378,499, 7,462,760 and 9,593,345; Cry9 proteins, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families, including the Cry9 protein of U.S. Patent Nos. 9,000,261 and 8,802,933, and WO 2017 / 132188; Naimov et al. (2008) Applied and Environmental Microbiology, 74: 7145 Cry15 protein of US Patent No. 7151; Cry14 protein of US Patent No. 8,933,299; Cry22; Cry34Ab1 protein of US Patent Nos. 6,127,180, 6,624,145 and 6,340,593; truncated Cry34 protein of US Patent No. 8,816,157; ​​CryET33 and cryET34 proteins of US Patent Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; US Patent Publication Nos. 2006 / 0191034, 2012 / 0278954 and PCT Publication No. WO The following proteins are listed: CryET33 and CryET34 homologs (US Patent No. 2012 / 139004); Cry35Ab1 protein (US Patent Nos. 6,083,499, 6,548,291, and 6,340,593); Cry46 protein (US Patent No. 9,403,881); Cry51 protein; Cry binary toxin; TIC901 or related toxins; TIC807 (US Patent Application Publication No. 2008 / 0295207); TIC853 (US Patent No. 8,513,493); ET29, ET37, TIC809, TIC810, TIC812, TIC127, and TIC128 (US Patent No. 2006 / 033867); engineered hemipteran toxic protein (US Patent Application Publication No. 20160150795); and AXMI (US Patent No. 8,236,757). 027, AXMI 036 and AXMI 038; AXMI of US Patent No. 7,923,602 031, AXMI 039, AXMI 040, AXMI 049; WO 2006 / 083891 of AXMI 018, AXMI 020 and AXMI 021; AXMI of WO 2005 / 038032 010; AXMI of WO2005 / 021585 003; AXMI of U.S. Patent Application Publication No. 2004 / 0250311 008; AXMI of U.S. Patent Application Publication No. 2004 / 0216186 006; AXMI of U.S. Patent Application Publication No. 2004 / 0210965 007; AXMI of U.S. Patent Application No. 2004 / 0210964 009; AXMI of U.S. Patent Application Publication No. 2004 / 0197917 014; AXMI of U.S. Patent Application Publication No. 2004 / 0197916 004; AXMI of WO 2006 / 119457 028 and AXMI 029; AXMI of WO 2004 / 074462 007, AXMI 008, AXMI 0080rf2, AXMI 009, AXMI 014 and AXMI 004; AXMI of U.S. Patent No. 8,084,416 150; AXMI of U.S. Patent Application Publication No. 2011 / 0023184 205; AXMI in U.S. Patent Application Publication No. 2011 / 0263488 011, AXMI 012, AXMI 013, AXMI 015, AXMI 019, AXMI 044, AXMI 037, AXMI 043, AXMI 033, AXMI 034, AXMI 022, AXMI 023, AXMI 041, AXMI 063 and AXMI 064; AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI099 of U.S. Patent Nos. 8,461,421 and 8,461,422 8. AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132, AXMI137, AXMI138; AXMI of U.S. Patent Application Publication No. 2010 / 0197592 R1 and related proteins; AXMI221Z, AXMI222z, AXMI223z, AXMI224z, and AXMI225Z of WO 2011 / 103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 of WO 2011 / 103247; and AXMI21 of U.S. Patent No. 8,334,431. 115. AXMI 113. AXMI 005, AXMI 163 and AXMI 184; AXMI of U.S. Patent Application Publication No. 2010 / 0298211 001, AXMI 002, AXMI 030, AXMI 035 and AXMI 045; AXMI of U.S. Patent Application Publication No. 2009 / 0144852 066 and AXMI 076; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169 of U.S. Patent No. 8,318,900 AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189; US Patent No. 8461421, AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097 , AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMI111, AXMI112, AX MI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI12 9. AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137; AXMI192 of U.S. Patent No. 8,461,415; AXMI281 of U.S. Patent Application Publication No. US20160177332; AXMI422 of U.S. Patent No. 8,252,872; Cry proteins (e.g., Cry1A and Cry3A) with modified proteolytic sites in U.S. Patent No. 8,319,019; Cry1Ac, Cry2Aa, and Cry1Ca toxin proteins from Bacillus thuringiensis strain VBTS 2528 in U.S. Patent Application Publication No. 2011 / 0064710;And the Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S, MP214S, MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253, MP259S, and MP287S from US Serial No. 62 / 607372. , MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, MP312S, MP314S, MP319S, MP325S, MP326S, MP327S, MP328S, MP334S, MP337S, MP342 S, MP349S, MP356S, MP359S, MP360S, MP437S, MP451S, MP452S, MP466S, MP468S, MP476S, MP482S, MP522S, MP529S, MP548S, MP552S, MP562S, MP564S, MP56 6S, MP567S, MP569S, MP573S, MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S, MP629S, MP630 S, MP631S, MP632S, MP633S, MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S, MP652S, MP653S, MP661S, MP666S, MP672S, MP696S, MP704S, MP72 4S, MP729S, MP739S, MP755S, MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S, MP815S, MP828S, MP831S, MP844S, MP852, MP865S, MP87 9S, MP887S, MP891S, MP896S, MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206, MP1233 and MP1311. ;

[0067] Other exemplary additional insecticidal proteins for controlling lepidopteran pests may include insect inhibitory proteins, such as, but not limited to, Cry1A (US Patent No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (US Patent Publication Serial No. 10 / 525,318), Cry1C (US Patent No. 6,033,874), Cry1D, Cry1E, Cry1F, and Cry1A / F chimeras (US Patent Nos. 7,070,982; 6,962,705); And 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab (US Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI 001, AXMI 002, AXMI 030, AXMI 035, AXMI 045 (US Patent Publication 2013) 0117884 A1), AXMI 52. AXMI 58. AXMI 88. AXMI 97. AXMI 102. AXMI 112. AXMI 117. AXMI 100 (US Patent Publication 2013) 0310543 A1), AXMI 115. AXMI 113. AXMI 005 (US Patent Publication 2013) 0104259 A1), AXMI 134 (US Patent Publication 2013-0167264 A1), AXMI-150 (US Patent Publication 2010-0160231 A1), AXMI-184 (US Patent Publication 2010) 0004176 A1), AXMI 196. AXMI 204, AXMI 207, AXMI 209 (US Patent Publication 2011) 0030096 A1), AXMI 218, AXMI 220 (US Patent Publication 2014) 0245491 A1), AXMI 221z, AXMI 222z, AXMI 223z, AXMI 224z, AXMI 225z (US Patent Publication 2014) 0196175 A1), AXMI 238 (US Patent Publication 2014) 0033363 A1), AXMI 270 (US Patent Publication 2014) 0223598 A1), AXMI 345 (US Patent Publication 2014) 0373195 A1), DIG 3 (US Patent Publication 2013) 0219570 A1), DIG 5 (US Patent Publication 2010) 0317569 A1), DIG 11 (US Patent Publication 2010) 0319093A1), AfIP 1A and its derivatives (US Patent Publication 2014) 0033361 A1), AfIP 1B and its derivatives (US Patent Publication 2014) 0033361 A1), PIP 1APIP 1B (US Patent Publication 2014) 0007292 A1), PSEEN3174 (US Patent Publication 2014-0007292 A1), AECFG-592740 (US Patent Publication 2014-0007292 A1), Pput_1063 (US Patent Publication 2014-0007292 A1), Pput_1064 (US Patent Publication 2014) 0007292 A1), GS 135 and its derivatives (US Patent Publication 2012) 0233726 A1), GS153 and its derivatives (US Patent Publication 2012) 0192310A1), GS154 and its derivatives (US Patent Publication 2012) 0192310 A1), GS155 and its derivatives (US Patent Publication 2012) 0192310 A1), US Patent Publication 2012 SEQ ID NO: 2 and its derivatives as described in 0167259 A1, and US Patent Publication 2012 The SEQ ID NO: 2 and its derivatives described in 0047606 A1, and US Patent Publication 2011 SEQ ID NO: 2 and its derivatives as described in 0154536 A1, US Patent Publication 2011 SEQ ID NO: 2 and its derivatives as described in 0112013 A1, and US Patent Publication 2010 SEQ ID NO: 2 and 4 and their derivatives as described in 0192256 A1, US Patent Publication 2010 The SEQ ID NO: 2 and its derivatives described in 0077507 A1, and US Patent Publication 2010 The SEQ ID NO: 2 and its derivatives described in 0077508 A1, and US Patent Publication 2009 SEQ ID NO: 2 and its derivatives as described in 0313721 A1, and US Patent Publication 2010 SEQ ID NO: 2 or 4 and its derivatives as described in 0269221 A1, SEQ ID NO: 2 and its derivatives as described in US Patent No. 7,772,465 B2, CF161_0085 and its derivatives as described in WO2014 / 008054 A2, and US Patent Publication 2008 0172762 A1, 2011 0055968A1 and 2012 Lepidoptera toxic proteins and their derivatives as described in 0117690 A1; SEQ ID NO: 2 and its derivatives as described in US Patent No. 7,510,878 B2; SEQ ID NO: 2 and its derivatives as described in US Patent No. 7,812,129 B1; and so on.

[0068] In other embodiments, exemplary additional insecticidal proteins for controlling coleopteran pests may include insect inhibitory proteins, such as, but not limited to, Cry3Bb (US Patent No. 6,501,009), Cry1C variant, Cry3A variant, Cry3, Cry3B, Cry34 / 35, 5307, and AXMI. 134 (US Patent Publication 2013) 0167264 A1), AXMI 184 (US Patent Publication 2010-0004176 A1), AXMI-205 (US Patent Publication 2014-0298538 A1), AXMI-207 (US Patent Publication 2013) 0303440 A1), AXMI 218, AXMI 220 (US Patent Publication 20140245491A1), AXMI 221z, AXMI 223z (US Patent Publication 2014-0196175 A1), AXMI-279 (US Patent Publication 2014) 0223599 A1), AXMI R1 and its variants (US Patent Publication 2010) 0197592 A1), TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG 10 (US Patent Publication 2010) 0319092 A1), eHIP (US Patent Publication 2010 / 0017914), IP3 and its variants (US Patent Publication 2012) 0210462 A1) and ω hexapeptide toxin Hv1a (US Patent Publication 2014) 0366227 A1).

[0069] In other embodiments, exemplary additional insecticidal proteins for controlling hemipteran pests may include hemipteran active proteins, such as, but not limited to, TIC1415 (US Patent Publication 2013-0097735 A1), TIC807 (US Patent No. 8,609,936), TIC834 (US Patent Publication 2013-0269060 A1), AXMI-036 (US Patent Publication 2010-0137216 A1), and AXMI-171 (US Patent Publication 2013-0055469 A1).

[0070] Other insecticidal proteins used to control pests in the Coleoptera, Lepidoptera, and Hemiptera orders can be found on the Bacillus thuringiensis toxin nomenclature website (btnomenclature.info).

[0071] As used herein, “plant cell” means any cell obtained or found from seeds, suspension cultures, plumules, meristematic regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen, protoplasts, and microspores. Plant cells also include modified cells (e.g., protoplasts) obtained from the aforementioned 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.

[0072] 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 the rice actin 1 gene, the promoter of the underground clover virus gene 4, the promoter region of the ubiquitin 4 gene, and the promoter region of the maize polyubiquitin 1 gene. A "regulatory element" is a polynucleotide sequence that affects gene transcription. Regulatory elements can include, but are not limited to, promoters, enhancers, terminators, or other sequences that affect gene transcription.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] "Substantially identical" can mean that the first and second amino acid sequences or polynucleotide sequences are identical at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 30, respectively. The region of 0, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids or nucleotides is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0077] 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.

[0078] "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.

[0079] A “transformed organism” or “transformed plant” is an organism or plant in which a heterologous nucleic acid molecule has been integrated into its genome. All cells of the transformed organism or plant may have genetic constructs integrated into its genome. A transformed plant may be a fertile plant, and more specifically, a plant whose agronomical characteristics (yield, grain quality, drought tolerance, etc.) are not impaired compared to the untransformed identical 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 by inserting or changing the plant genome. In some embodiments, modified plants comprising nucleic acids encoding polypeptides as disclosed herein are considered.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] When "treatment" refers to protecting a subject from toxins, it means curbing, inhibiting, reversing, mitigating, improving, or preventing the progression of damage or death caused by exposure to toxins, or completely eliminating damage or death caused by exposure to toxins. Treatment can be carried out in an acute or chronic manner. Prevention of damage or death caused by exposure to toxins involves administering the composition of the present invention to the subject prior to exposure to the toxin. Curbing damage or death caused by exposure to toxins involves administering the composition of the present invention to the subject exposed to the toxin prior to the occurrence of damage. Inhibiting or improving damage or death caused by exposure to toxins involves administering the composition of the present invention to the subject after the occurrence of damage. Treatment can originate from the expression of a transgene or from the topical application of the disclosed polypeptide to a plant.

[0084] Regarding 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.

[0085] The term "variant" in relation to a peptide or polypeptide means a protein that differs in its amino acid sequence by 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 with at least one biological activity. Representative examples of "biological activity" include the ability to bind to a particular polypeptide or to promote a particular response (e.g., insecticide resistance or activity). A variant can mean a functional fragment of the polypeptide. A variant can also mean multiple copies of the polypeptide. Multiple copies can be tandem or separated by linkers. Conservative 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.

[0086] As used herein, "vector" refers to a nucleic acid sequence containing the origin of replication. Vectors can be bacterial plasmids, viral vectors, bacteriophages, bacterial artificial chromosomes, plant expression vectors, animal expression vectors, archaea vectors, 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.

[0087] This document provides information on nucleic acid molecules. Nucleic acid molecules may include, for example, insecticidal gene polynucleotides encoding any of SEQ ID NO: 1-60, 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.

[0088] 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. Polynucleotides may be recombinant. Polynucleotides may contain regulatory elements for gene expression of the coding sequence of the nucleic acid. The coding sequence in the polynucleotide may be optimized for stability and high expression levels. Regulatory elements may include promoters, enhancers, start codons, stop codons, and / or polyadenylation signals.

[0089] On one hand, the polynucleotide can encode a polypeptide having at least 70% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 75% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 80% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 85% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 90% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 91% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 70% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 75% sequence identity with any of SEQ ID NO: 1-60 and having insecticidal activity; a polynucleotide can encode a polypeptide having at least 70 ... The following are examples of polypeptides: 1-60, each having at least 92% sequence identity and insecticidal activity; 1-60, each having at least 93% sequence identity and insecticidal activity; 1-60, each having at least 94% sequence identity and insecticidal activity; 1-60, each having at least 95% sequence identity and insecticidal activity; 1-60, each having at least 96% sequence identity and insecticidal activity; 1-60, each having at least 97% sequence identity and insecticidal activity; 1-60, each having at least 98% sequence identity and insecticidal activity; 1-60, each having at least 98% sequence identity and insecticidal activity. The following are examples of polypeptides: SEQ ID NO: 1-60, having at least 99% sequence identity and insecticidal activity; SEQ ID NO: 1-60, having at least 99.2% sequence identity and insecticidal activity; SEQ ID NO: 1-60, having at least 99.5% sequence identity and insecticidal activity; SEQ ID NO: 1-60, having at least 99.8% sequence identity and insecticidal activity; SEQ ID NO: 1-60, having at least 99.2% sequence identity and insecticidal activity.A polypeptide with 9% sequence identity and insecticidal activity; or, a polynucleotide encoding an amino acid sequence having any of SEQ ID NO: 1-60 and having insecticidal activity.

[0090] In one embodiment, this disclosure relates to an isolated polynucleotide encoding a polypeptide amino acid sequence having at least 90% sequence identity with any of SEQ ID NO: 1-60 and possessing insecticidal activity. The insecticidal polypeptides of this disclosure and the polynucleotides encoding these insecticidal polypeptides are particularly useful in crops for the control and killing of pests.

[0091] In one aspect, this disclosure relates to a method for producing transgenic plants with insecticidal activity. The method may include transforming or editing plant cells with the polynucleotides described herein, selecting plant cells containing the polynucleotides described herein, and regenerating the transgenic plant from plant cells containing the nucleic acid molecules described herein, wherein the transgenic plant expresses the polynucleotides described herein, and wherein the transgenic plant has insecticidal activity.

[0092] In one aspect, this disclosure relates to a method for protecting plants from pest-related damage. The method may include introducing a polynucleotide described herein into a plant, wherein the plant expresses the polynucleotide, and wherein the resulting polypeptide has insecticidal activity.

[0093] 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 ( Clouded oyster ), cotton bollworm ( Helicoverpa armigera ), Black Root Worm ( Agrotis Y ), 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 ), Western corn rootworm ( Diabrotica virgifera virgifera Coleoptera species, Lepidoptera species, Hemiptera species, and combinations thereof.

[0094] In one aspect, this disclosure relates to a host cell comprising the polynucleotides described herein. Suitable host cells may include prokaryotic host cells and eukaryotic host cells.

[0095] Particularly suitable prokaryotic host cells can include archaea and bacterial cells. 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).

[0096] In one respect, this disclosure relates to a transgenic plant, transgenic plant tissue, transgenic plant cell, or transgenic plant seed that contains the polynucleotides described herein and has insecticidal activity.

[0097] As described herein, the transformed plant cells, plant parts, or plants may have at least one polynucleotide, nucleic acid construct, expression cassette, or vector encoding a polypeptide that has at least 70% sequence identity with any of SEQ ID NO: 1-60, at least 75% sequence identity with any of SEQ ID NO: 1-60, at least 80% sequence identity with any of SEQ ID NO: 1-60, at least 85% sequence identity with any of SEQ ID NO: 1-60, at least 90% sequence identity with any of SEQ ID NO: 1-60, at least 91% sequence identity with any of SEQ ID NO: 1-60, at least 92% sequence identity with any of SEQ ID NO: 1-60, at least 93% sequence identity with any of SEQ ID NO: 1-60, at least 94% sequence identity with any of SEQ ID NO: 1-60, and at least 95% sequence identity with any of SEQ ID NO: 1-60. Any one of SEQ ID NO: 1-60 has 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% 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, or the at least one polynucleotide, nucleic acid construct, expression cassette, or vector can encode a sequence containing SEQ ID NO: A polypeptide of any of the amino acid sequences 1-60, wherein the transformed plant cell, transformed plant part, or transformed plant has insecticidal activity.

[0098] This disclosure also relates to homologs of any of SEQ ID NO: 1-60, 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, genetic engineering, gene editing, 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: 1-60.

[0099] In some aspects of this disclosure, polynucleotide sequences encoding shuffled insecticidal peptides are disclosed, the shuffled insecticidal peptides comprising amino acid substitutions, deletions, insertions, and fragments thereof. Insecticidal toxins may have one or more of exchanged or shuffled domains to alter the physical properties of the toxin, such as improving efficacy, changing the spectrum, or reducing phytotoxicity. In one embodiment, a Cry family insecticidal peptide is engineered to have at least one shuffled Cry domain to alter insecticidal activity.

[0100] Insecticidal proteins can be derived from Bacillus thuringiensis (“Bt”), a Gram-positive, spore-forming soil bacterium. Currently commercially available insecticidal proteins include Bt Cry (crystal protein), as well as many other active insecticidal proteins lacking commercial efficacy, spectrum, or stability. The disclosed implementation addresses the Bt-derived insecticidal protein Cry1B family (Cry1B toxins, proteins; see Crickmore et al., “Bacillus thuringiensis toxin nomenclature” (2011) and Crickmore et al., “A structure-based nomenclature for…”). Bacillus thuringiensis Some efficacy, spectrum and / or stability issues in "and other bacteria-derived pesticidal proteins" (2021), see bpprc.org.

[0101] Members of the three-domain (3d) Cry family are globular molecules composed of three domains linked by a single linker (Bravo et al., 2007).

[0102] These proteins are synthesized as prototoxins and stored within bacteria as crystalline inclusion bodies. When ingested by insects, these prototoxins are processed by midgut proteases into active toxins. These active toxins then bind to specific receptors in the insect cell membrane, leading to cell destruction and insect death (Palma et al., 2014).

[0103] A characteristic feature of the 3d-Cry family members is the presence of two different protoxin lengths: 135 kDa and 65 kDa. The 135 kDa protein is the protoxin, with the actual toxin located at the N-terminus. The C-terminal half has no toxic function but is responsible for protein crystallization. The 65 kDa protein consists only of the toxin portion and lacks the C-terminal portion. Many of these naturally truncated toxins can still crystallize with the help of "helper" proteins and other factors (Adalat et al., 2017).

[0104] Another common feature of these proteins is that they contain up to five conserved amino acid segments in the toxic core of the protoxin, and three additional conserved amino acid segments outside the toxic core and toward the C-terminus of the protoxin (Palma et al., 2014).

[0105] Classic 3d-Cry family members all possess very similar and conservative three-domain structures, such as... Figure 6 As shown.

[0106] Domain I (N-terminal domain) comprises seven α-helices, with the central α5 helix being hydrophobic and contained within six other amphiphilic helices. Domain I is responsible for membrane insertion and pore formation. Domain II comprises three antiparallel β-sheets with exposed loop regions. At one end of these β-sheets, or the tip of the wedge-shaped toxin, there is an aggregation of six loops. This “loop cluster” contains one of the most variable regions of the protein and may be involved in receptor recognition and receptor-mediated cytotoxicity during insecticidal activity, making it one of the key elements determining insecticidal specificity and activity. Domain III (C-terminal domain) consists of two antiparallel β-sheets and is also involved in receptor binding. The boundary / connector sequences that connect or link Domains I to II and Domains II to III are generally highly conserved. As used herein, “Boundary I region” refers to the boundary / connector region that connects Domain I to Domain II, and “Boundary II region” refers to the boundary / connector region that connects Domain II to Domain III.

[0107] In some embodiments, the engineered insecticidal protein or peptide comprises domain I and heterologous domain II and / or domain III from Cry1B family Bt insecticidal toxins. In some embodiments, the engineered insecticidal protein comprises domain II and heterologous domain I and / or domain III from Cry1B family Bt insecticidal toxins. In some embodiments, the engineered insecticidal protein comprises domains I and II and heterologous domain III from Cry1B family Bt insecticidal toxins.

[0108] In addition to the full-length nucleotide sequence of the polynucleotide encoding the polypeptide of any of SEQ ID NO: 1-60, the polynucleotide encoding any of SEQ ID NO: 1-60 may include fragments or variants thereof encoding a polypeptide having insecticidal activity. For nucleotide sequences, as used herein, “fragment” means a portion of the nucleotide sequence of a polynucleotide, for example, a portion of the nucleotide sequence encoding any of SEQ ID NO: 1-60. Fragments of nucleotide sequences may retain the biological activity of the reference polynucleotide. For example, a polynucleotide encoding a shorter complete amino acid sequence than that disclosed in any of SEQ ID NO: 1-60 may be used to encode a protein that retains its insecticidal activity. In other embodiments, fragments of any of SEQ ID NO: 1-60 may be used to alter the biological activity of another insecticidal or non-insectic polypeptide sequence by adding, exchanging, or mutating the fragment of any of SEQ ID NO: 1-60 to another insecticidal or non-insectic polypeptide sequence. Alternatively, fragments of nucleotide sequences may be used as hybridization probes or as amplification primers. 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 this polynucleotide can be prepared by the following steps: isolating a portion of the nucleic acid molecule, operatively linking the fragment to a promoter, expressing the nucleotide sequence encoding the protein, and assessing the amount or activity of the protein.

[0109] In some embodiments, the nucleotide sequence or nucleic acid molecule encoding the polypeptide of any of SEQ ID NO: 1-60 may also be superimposed with a nucleotide sequence encoding an agronomic trait (e.g., male sterility, stem strength, flowering time, other insecticidal proteins, RNA interference transgenes) 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, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly spaced short palindromic repeats (CRISPR), and other genetic transformations or edits. If traits are superimposed through genetically transformed plants, the 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, these two sequences can be contained in separate expression cassettes (trans) or in the same transformation cassette (cis). The expression of a sequence can be driven by the same promoter or different promoters.

[0110] In one aspect, this disclosure relates to a vector that may comprise a polynucleotide encoding a polypeptide having at least 70% sequence identity with any of SEQ ID NO: 1-60, at least 75% sequence identity with any of SEQ ID NO: 1-60, at least 80% sequence identity with any of SEQ ID NO: 1-60, at least 85% sequence identity with any of SEQ ID NO: 1-60, at least 90% sequence identity with any of SEQ ID NO: 1-60, at least 91% sequence identity with any of SEQ ID NO: 1-60, at least 92% sequence identity with any of SEQ ID NO: 1-60, at least 93% sequence identity with any of SEQ ID NO: 1-60, at least 94% sequence identity with any of SEQ ID NO: 1-60, and at least 95% sequence identity with any of SEQ ID NO: 1-60. Any one of SEQ ID NO: 1-60 has 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% 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.

[0111] In one aspect, this disclosure relates to a formulation comprising a recombinant polypeptide having at least 70% sequence identity with any of SEQ ID NO: 1-60, at least 75% sequence identity with any of SEQ ID NO: 1-60, at least 80% sequence identity with any of SEQ ID NO: 1-60, at least 85% sequence identity with any of SEQ ID NO: 1-60, at least 90% sequence identity with any of SEQ ID NO: 1-60, at least 91% sequence identity with any of SEQ ID NO: 1-60, at least 92% sequence identity with any of SEQ ID NO: 1-60, at least 93% sequence identity with any of SEQ ID NO: 1-60, at least 94% sequence identity with any of SEQ ID NO: 1-60, at least 95% sequence identity with any of SEQ ID NO: 1-60, and at least 95% sequence identity with any of SEQ ID NO: 1-60. Any one of SEQ ID NO: 1-60 has at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% 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, and exhibits insecticidal activity. When applied to plants, the recombinant polypeptide exhibits insecticidal activity.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] Some aspects described herein also cover antibodies that specifically bind to the chimeric insecticidal proteins of this disclosure. The antibodies may optionally be monoclonal antibodies or polyclonal antisera. In some embodiments, the antibodies are selective for the chimeric protein and do not bind to one or more parental molecules, and can be used to distinguish the chimeric protein from the parental protein. Such antibodies can be generated using standard immunological techniques for generating polyclonal antisera, and, if desired, the antibody-generating cells of the immunized host can be immortalized for use as a source for monoclonal antibody production. This disclosure also covers an insecticidal protein that cross-reacts with antibodies (particularly monoclonal antibodies) generated against one or more chimeric insecticidal proteins disclosed herein.

[0121] The following experimental examples are provided by way of illustration rather than limitation.

[0122] Example Example 1 In this embodiment, engineered peptide sequences were designed in a computer. Using previously identified native Cry1B-like toxins (originally discovered in Bacillus thuringiensis species, PCT / US2022 / 028078; as disclosed herein, SEQ ID NO: 114), novel insecticidal engineered variants (SEQ ID NO: 1-60) with enhanced insecticidal activity, altered insecticidal activity profiles, or enhanced stability were generated using strategic engineering methods.

[0123] Figure 2 A depiction of sequence alignments between SEQ ID NO: 50, 51 and 114 is shown, wherein each of the exemplary engineered variants of engineered variant 188A (SEQ ID NO: 50) and engineered variant 401A (SEQ ID NO: 51) is an engineered variant of the native Cry1B-like toxin sequence of SEQ ID NO: 114. Figure 3 The amino acid identity / homology table for SEQ ID NO: 50, 51 and 114 is shown.

[0124] Figure 4A and Figure 4B A further depiction of sequence alignments between engineered variants of SEQ ID NO: 1-49 is shown, wherein each of the exemplary engineered variants is an engineered variant of the native Cry1B-like toxin sequence of SEQ ID NO: 114. Figure 5A and Figure 5B An amino acid homology table for engineered variants of SEQ ID NO: 1-49 is shown.

[0125] Example 2 To express engineered insecticidal peptide variants, optimized for use in Escherichia coli (E. coli) was synthesized. E. coli The DNA gene coding sequence expressed in the vector was cloned into the pHis expression vector (a modified form of pRSF-1b (Novagen)), thereby fusing the N-terminal 6x-His tag coding sequence with the engineered 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, followed by insect larval activity assays. In some cases, the induced bacterial culture was used for insect assays.

[0126] Example 3 Insecticidal bioassays were performed on whole-cell transformed bacteria expressing engineered proteins to assess insecticidal efficacy against pests including armyworms and cutworms. Spodoptera spp. ), corn ear borer, bollworm ( ) Helicoverpa spp. ), corn borer, genus Corn borer ( Ostrinia spp. ), genus *Zygota* ( Diatraea spp. ), genus *Leptochloa* ( Anticarsia spp. ) and root firefly beetle ( Diabrotica spp. ).

[0127] 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). Insecticidal efficacy was assessed using bioassays similar to those described by Huynh et al. (2017) and Ludwick et al. (2018). Mortality, growth inhibition, and feeding inhibition were evaluated after incubation (Table 1).

[0128] 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. Insecticidal efficacy was assessed using bioassays similar to those described by Wang et al. (2019). Mortality, growth inhibition, and feeding inhibition were evaluated after 5 days of incubation (Table 1).

[0129] Table 1. Measurement of insecticidal activity of whole recombinant Escherichia coli cultures expressing engineered proteins.

[0130]

[0131] (+): Indicates that insecticidal activity has been measured. (-): Indicates that no insecticidal activity was measured. NT: Indicates an untested condition. Example 4 Use of Benedict's tobacco ( Nicotiana benthamiana Transient expression of Agrobacterium-mediated gene in plants was used to identify insecticidal activity in plants (Kapila et al., 1997; Schob et al., 1997). Leaf discs constitutively expressing this gene were measured to assess their reduction in feeding damage from the following pests: Cry1Fa-rFAW, Vip3A-rFAW, sFAW, CEW, ECB, SBL, BCW, SAW, TBW, BAW, and the white spot moth (Gnaphalium affineum). Trichoplusia ni (CL) (Table 2).

[0132] Table 2. Measured insecticidal activity of leaf discs expressing engineered proteins.

[0133]

[0134] (+): Indicates that insecticidal activity has been measured. (-): Indicates that no insecticidal activity was measured. NT: Indicates an untested condition. Example 5 In this embodiment, 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 engineered insecticidal proteins disclosed herein in target plants (e.g., maize cells).

[0135] In some instances, these nucleic acid experimental constructs are coupled with those encoding specific target peptides (e.g., maize). Corn The sequence of the chloroplast targeting signal peptide is operatively linked.

[0136] 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.

[0137] 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.

[0138] Under greenhouse conditions, newly hatched (first larval stage) fall armyworms were artificially infected onto the heart leaves of plants. Spodoptera frugiperda The insecticidal efficacy against FAW (fecal microorganisms) was tested by evaluating leaves after the larvae had fed on them. 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.

[0139] 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 insecticidal efficacy against CEW was tested using the method described in the study. The CEW 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 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 scored 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.

[0140] Under greenhouse conditions, a method was used to test the efficacy of corn rootworms by artificially infecting plants with eggs, followed by root scoring after egg hatching and larval feeding. Diabrotica virgifera The insecticidal efficacy of CRW (Reference Toxin) was assessed. CRW efficacy was determined using 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 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 at 50% RH and nighttime temperatures of 17°C to 20°C at 50% RH. The light-to-dark ratio was 16:8. Seedlings were transplanted into 1-gallon pots at V2 (approximately 14 days). 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.

[0141] 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 insecticidal efficacy of ECB was tested by scoring damage to the internal stem and pedicel after larval feeding. ECB efficacy was determined using 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 set 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 met 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 suitable 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] For the sake of completeness, various aspects of the invention described herein are set forth in the following numbered clauses: Clause 1. A method for protecting plants from infection by plant pathogens or pests, said method comprising: The plant is transformed with a nucleic acid molecule encoding a polypeptide having at least 97% sequence identity with any of SEQ ID NO: 1-60 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.

[0147] Clause 2. The method according to Clause 1, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

[0148] Clause 3. The method described in Clause 1 or 2, 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 ) and their combinations.

[0149] Clause 4. 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 97% sequence identity with any of SEQ ID NO: 1-60, wherein said transformed plant, seed, or plant part stably expresses said polypeptide, and wherein said polypeptide has insecticidal activity against plant pathogens or pests.

[0150] Clause 5. The transformed plant, seed or plant part according to Clause 4, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

[0151] Clause 6. The transformed plant, seed, or plant part as described in Clause 4 or 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.

[0152] Clause 7. A recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 97% sequence identity with any of SEQ ID NO: 1-60, wherein the polypeptide has insecticidal activity against plant pathogens or pests.

[0153] Clause 8. The recombinant nucleic acid molecule according to Clause 7, wherein the polypeptide is any one of SEQ ID NO: 1-60.

[0154] Clause 9. A recombinant nucleic acid molecule according to Clause 7 or 8, wherein the polynucleotide sequence encoding the polypeptide is operatively linked to one or more promoter sequences.

[0155] Clause 10. A vector comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 97% sequence identity with any of SEQ ID NO: 1-60, said polypeptide having insecticidal activity against plant pathogens or pests.

[0156] Clause 11. The vector according to Clause 10, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

[0157] Clause 12. 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 97% sequence identity with any one of SEQ ID NO: 1-60, said polypeptide having insecticidal activity against plant pathogens or pests.

[0158] Clause 13. The transformed host cell according to Clause 12, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

[0159] Clause 14. 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 97% sequence identity with any one of SEQ ID NO: 1-60 to the plant or plant part, wherein the polypeptide has insecticidal activity against the plant pathogen or pest.

[0160] Clause 15. The method according to Clause 14, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

[0161] Clause 16. A composition having insecticidal activity against plant pathogens or pests, said composition comprising an effective amount of at least one polypeptide having at least 97% sequence identity with any one of SEQ ID NO: 1-60.

[0162] Clause 17. The composition according to Clause 16, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

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 97% sequence identity with any of SEQ ID NO: 1-60 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 according to claim 1, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

3. 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 ) and their combinations.

4. 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 97% sequence identity with any of SEQ ID NO: 1-60, wherein the transformed plant, seed, or plant part stably expresses the polypeptide, and wherein the polypeptide has insecticidal activity against plant pathogens or pests.

5. The transformed plant, seed, or plant part according to claim 4, wherein the polypeptide has at least 98% sequence identity with any one of SEQ ID NO:1-60.

6. The transformed plant, seed, or plant part according to claim 4, 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.

7. A recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 97% sequence identity with any of SEQ ID NO: 1-60, wherein the polypeptide has insecticidal activity against plant pathogens or pests.

8. The recombinant nucleic acid molecule according to claim 7, wherein the polypeptide is any one of SEQ ID NO: 1-60.

9. The recombinant nucleic acid molecule of claim 7, wherein the polynucleotide sequence encoding the polypeptide is operatively linked to one or more promoter sequences.

10. A vector comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide having at least 97% sequence identity with any one of SEQ ID NO: 1-60, said polypeptide having insecticidal activity against plant pathogens or pests.

11. The vector according to claim 10, wherein the polypeptide has at least 98% sequence identity with any one of SEQ ID NO: 1-60.

12. 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 97% sequence identity with any one of SEQ ID NO: 1-60, said polypeptide having insecticidal activity against plant pathogens or pests.

13. The transformed host cell according to claim 12, wherein the polypeptide has at least 98% sequence identity with any one of SEQ ID NO: 1-60.

14. 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 97% sequence identity with any one of SEQ ID NO: 1-60 to the plant or plant part, wherein the polypeptide has insecticidal activity against the plant pathogen or pest.

15. The method of claim 14, wherein the polypeptide has at least 98% sequence identity with any of SEQ ID NO: 1-60.

16. A composition having insecticidal activity against plant pathogens or pests, said composition comprising an effective amount of at least one polypeptide having at least 97% sequence identity with any one of SEQ ID NO: 1-60.

17. The composition of claim 16, wherein the polypeptide has at least 98% sequence identity with any one of SEQ ID NO: 1-60.

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