Insecticidal nematode proteins and methods of use

Abstract

As disclosed herein, insect-parasitic nematodes are considered, "beneficial nematodes" since they kill insect pests, produce and release insecticidal proteins into their hosts. Those insecticidal proteins have oral toxicity to some insect pests. The present disclosure provides methods to identify those proteins and provides examples of proteins that have insecticidal activity, such as oral toxicity to a variety of pest insects.

Description

[0001]UC2023-989-2-PCT / / 3868.091WO1 INSECTICIDAL NEMATODE PROTEINS AND METHODS OF USE PRIORITY This patent application claims the benefit of priority to U.S. Provisional Application Serial No.63 / 530,831, filed August 4, 2023, which is incorporated by reference herein in its entirety. STATEMENT OF GOVERNMENT RIGHTS This invention was made with government support under grant 2020- 67013-31855 awarded by the United States Department of Agriculture. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on July 29, 2024, is name 3868091WO1.xml and is 6,284 bytes in size. BACKGROUND Food security is one of the most important global challenges of our age and insect pests continue to be a problem in agricultural systems, contributing to between 36%-42% losses in some staple crop systems. Reducing crop damage by pest insects is one strategy to increase food production and security. Toxins from the insect pathogen Bacillus thuringiensis (Bt) are widely used in biological control. Bt toxins, which are specifically toxic to insects and not mammals, are applied (sprayed) on crops and produced by transgenic crops to control insect pests in agriculture. However, few of the transgenic Bt technologies currently available for cultivation can prevent infestation by sap-sucking pests, and resistance to these toxins in some populations of insect pests is a problem, making Bt toxins less effective. One way to combat resistance to Bt toxins is to use a variety of toxins with different modes of action, including the development of novel toxins. SUMMARY As disclosed herein, insect-parasitic nematodes, which are considered "beneficial nematodes" since they kill insect pests, produce and release insecticidal proteins into their hosts. Those insecticidal proteins have oral toxicity to some insect pests. The present disclosure provides methods to identify those UC2023-989-2-PCT / / 3868.091WO1 proteins and provides three examples of proteins from Steinenema that have insecticidal activity. In particular, one of the proteins has significant oral toxicity to a variety of pest insects. Specifically, a purification method for insecticidal activity guided protein purification of insect-parasitic nematode venom was established and one target insecticidal protein was further characterized, a chymotrypsin-like protease (CLP). The recombinant CLP was found to be efficacious against chewing and sucking insect pests of plants. In one embodiment, the CLP encoding gene is from S. carpocapsae (L596_023060). That gene can be employed to produce recombinant protein or introduced to a plant, e.g., to prepare a transgenic plant such as a transgenic tomato plant. In one embodiment, the insecticidal protein is from Steinernema. In one embodiment, the insecticidal protein is from S. carpocapsae. In one embodiment, the insecticidal protein comprises SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 97%, 98%, or 99%, amino acid sequence identity thereto. In one embodiment, an orthologous protein is present in Steinernema scapterisci (L892_g23626), with 72% amino acid similarity to (L596_023060) in S. carpocapsae. In one embodiment, the insecticidal protein is expressed in a transgenic plant. Such expression can lead to decreased crop loss, e.g., from insect herbivory and can serve as an alternative to or is used in combination with other insecticides, such as Bt toxin, to prevent or decrease emerging resistance to other insecticides, e.g., resistance to Bt. In one embodiment, the insecticidal protein may be expressed in a heterologous cell, e.g., a microbial cell such as bacterium, algae or yeast, and that recombinant protein sprayed onto plants. Topical application may be accomplished through liquid sprayers where the enzyme is suspended in water or some other mixture, e.g., one that includes a carrier, to improve application or the length of time that the enzyme would be active and viable. In one embodiment, a composition is provided comprising an insecticidal amount of an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at UC2023-989-2-PCT / / 3868.091WO1 least 90% amino acid sequence identity thereto. In one embodiment, the composition further comprises other insecticidal compounds, e.g., other insecticidal proteins from S. carpocapsae, S. feltiae, or S. hermaphroditum. In one embodiment, the composition further comprises a carrier. In one embodiment, the carrier is a water soluble, biocompatible liquid. In one embodiment, the carrier provides for sustained release. In one embodiment, the composition can be aerosolized. In one embodiment, the polypeptide in the composition comprises SEQ ID NO:1, an isolated polypeptide comprising SEQ ID NO:1 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto. In one embodiment, the polypeptide in the composition comprises SEQ ID NO:2, an isolated polypeptide comprising SEQ ID NO:2 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto. In one embodiment, the polypeptide in the composition comprises SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto. Also provided is a non-nematode recombinant host cell that expresses a polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto. In one embodiment, the host cell is a bacterial cell. In one embodiment, the host cell is a plant cell. In one embodiment, the host cell is a crop plant cell. In one embodiment, the host cell is a vegetable cell. In one embodiment, the host cell is a yeast cell. In one embodiment, the host cell is an insect cell. Further provided is a method of preparing an insect-resistant transgenic plant, comprising: introducing to plant cells isolated nucleic acid encoding a polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto. In one embodiment, the plant is a crop plant. In one embodiment, the plant is a tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rape seed, pulses, or sunflower. UC2023-989-2-PCT / / 3868.091WO1 A transgenic plant, the genome of which comprises nucleic acid encoding a polypeptide comprising, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto, is provided. In one embodiment, the plant is a crop plant. In one embodiment, the plant is a tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rape seed, pulses, or sunflower. BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Collection of excreted / secreted proteins from parasitic nematode IJs. Insect homogenate is soaked into small pieces of sponge. The nematodes are exposed to the homogenate-soaked sponge for a set number of hours; usually 12 or 18 for S. carpocapsae. This exposure activates them, and they are then washed and transferred to clean PBS buffer where ESPs are collected for subsequent use. FIG. 2. Activated S. carpocapsae and S. feltiae IJs release proteins. A silver-stained protein gel of venom proteins from naive nematodes or nematodes that have been activated for 0 to 18 hours in host tissue; proteins collected from S. carpocapsae and S. feltiae. Each lane contains 1% of the total protein volume collected. The 0 hr sample was collected from non-activated IJs. L, protein ladder. Modified from Chang et al. 2019. FIGS. 3A-3B. ESPs released by activated S. carpocapsae IJs are toxic to insects. A) A survival curve of flies injected with 20ng of S. carpocapsae ESPs, shown as survival in hours post injection. ESPs collected from IJs exposed to hosts for different amounts of time, 6-42 hours, but the ESPs collected and tested were all only 3 hours old, as the activated IJs are washed after exposure and proteins are only collected for 3 hours. B) A survival curve of flies injected with 20ng of S. carpocapsae ESPs, shown as survival in days post injection. Modified from Chang et al.2019. FIGS. 4A-4B. Purification of insecticidal protein / s on Anion exchange chromatography: (A) Anion exchange (Mono-Q) chromatogram showing separation of proteins, red box depicts the peak which was most toxic to fruit fly when injected. (B) Silver stained SDS-PAGE gel of total ESPs collected from S. carpocapsae. (C) Silver stained SDS-PAGE gel of Mono-q column eluted fractions, red box depicted the most toxic fraction when injected to fruit flies. (D) UC2023-989-2-PCT / / 3868.091WO1 Toxicity of the tested fractions eluted from Mono-q column. Fraction 37 showed highest toxicity within seven days post injection, this fraction was further fractionated. FIGS. 5A-5C. Purification of fraction 37 on size exclusion chromatography: (A) Chromatograph showing separation of proteins on size exclusion chromatography. (B) Silver stained SDS-PAGE gel of size exclusion column eluted fractions, red box depicted the most toxic fraction when injected to fruit flies. (C) Toxicity of fractions eluted from size exclusion column. Fraction 56 from size exclusion showed highest toxicity within seven days. Little protein was in this fraction by this point, and no clear bands were visible on the gel. FIG. 6. Protease activity of the recombinant CLP: The recombinant CLP was assayed for protease activity along with two control proteins. CLP has measurable proteolytic activity. FIGS. 7A-7C. Feeding assays with CLP: The recombinant CLP was fed orally using café assay to Drosophila melanogaster and killed 96 % of flies within 72 hours. CLP did not kill Manduca sexta, but it did cause severe weight loss in larvae. It killed 85% of whiteflies within 72 hours when fed orally. It was also toxic to Plutella xylostella and Spodoptera frugiperda (data not shown). FIGS. 8A-8B. Feeding assays with CLP (referred to as NIP1 in this figure): It was toxic to Plutella xylostella and Spodoptera frugiperda in oral feeding assays using the concentrations described. DETAILED DESCRIPTION Definitions A "vector" or “delivery” vehicle refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide or polypeptide, and which can be used to mediate delivery of the polynucleotide or polypeptide to a cell or intercellular space, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, nanoparticles, or microparticles and other delivery vehicles. In one embodiment, a polynucleotide to be delivered, sometimes referred to as a "target polynucleotide" or "transgene," may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest and / or a selectable or detectable marker. UC2023-989-2-PCT / / 3868.091WO1 "Transduction," "transfection," "transformation" or "transducing" as used herein, are terms referring to a process for the introduction of an exogenous polynucleotide into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell. Transduction, transfection or transformation of a polynucleotide in a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays. Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non- viral gene delivery techniques. The introduced polynucleotide may be stably or transiently maintained in the host cell. "Gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression. "Gene transfer" refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene. "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification. The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. UC2023-989-2-PCT / / 3868.091WO1 A "transcriptional regulatory sequence" refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use in the present disclosure generally include at least one transcriptional promoter and may also include one or more enhancers and / or terminators of transcription. "Operably linked" refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. By way of illustration, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence. An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it. "Heterologous" means derived from a genotypically distinct entity from the entity to which it is compared. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a transcriptional regulatory element such as a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element. A "terminator" refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator). The degree to which transcription is disrupted is typically a function of the base sequence and / or the length of the terminator sequence. In particular, as is well known in numerous molecular biological systems, particular DNA sequences, generally referred to as "transcriptional termination sequences" are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and / or disengage from the DNA being transcribed. Typical example of such sequence- specific terminators includes polyadenylation ("polyA") sequences, e.g., SV40 polyA. In addition to or in place of such sequence-specific terminators, insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there UC2023-989-2-PCT / / 3868.091WO1 is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated. Terminators may thus prevent transcription from only one direction ("uni-directional" terminators) or from both directions ("bi-directional" terminators) and may be comprised of sequence- specific termination sequences or sequence-non-specific terminators or both. A variety of such terminator sequences are known in the art; and illustrative uses of such sequences within the context of the present disclosure are provided below. "Host cells," "cell lines," "cell cultures," "packaging cell line" and other such terms denote eukaryotic cells, such as plant cells, or microbial cells such as bacterial or yeast cells, useful in the present disclosure, e.g., to produce recombinant polypeptide. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell. "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct. A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) UC2023-989-2-PCT / / 3868.091WO1 from the promoter. Promoters include AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous promoters. An "expression vector" is a vector comprising a region which encodes a gene product of interest and is used for effecting the expression of the gene product in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art. The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component. An "isolated" polynucleotide, e.g., plasmid, virus, polypeptide or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Isolated nucleic acid, peptide or polypeptide is present in a form or setting that is different from that in which it is found in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. The isolated nucleic acid molecule may be present in single-stranded or double- stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single-stranded), but may contain both the sense and anti-sense strands (i.e., the molecule may be double-stranded). Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source UC2023-989-2-PCT / / 3868.091WO1 mixture. For example, a 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or a 1000-fold enrichment. The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell. By way of a non-limiting example, an exogenous nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene. "Transformed" or "transgenic" is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells of the present disclosure may be produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector. The term "sequence homology" means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less, or 2 bases or less. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%). Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or 2 or less. Alternatively, two UC2023-989-2-PCT / / 3868.091WO1 protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. The two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. The term "corresponds to" is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97% or more, e.g., 99% or 100%, sequence identity. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA". The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, at least 90 to 95 percent sequence identity, or at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide UC2023-989-2-PCT / / 3868.091WO1 sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. As used herein, "substantially pure" or "purified" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), for instance, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, or more than about 85%, about 90%, about 95%, and about 99%. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. As used herein, the term "plant" is used in its broadest sense. It includes, but is not limited to, any species of grass (e.g., forage, grain-producing, turf grass species), ornamental or decorative, crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, herb plant, woody plant, flower plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g., microalga) and a plurality of plant cells that are largely differentiated into a colony (e.g., volvox) or a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit, et cetera. The term "transgenic" when used in reference to a plant or leaf or fruit or seed or plant biomass, for example a "transgenic plant," transgenic leaf," "transgenic fruit," "transgenic fruit," "transgenic seed," "transgenic biomass," or a "transgenic host cell" refers to a plant or leaf or fruit or seed or biomass that contains at least one heterologous or foreign gene in one or more of its cells. The term "transgenic plant material" refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells. The term "transgene" refers to a foreign gene that is placed into an organism (e.g., a plant) or host cell by the process of transfection. The term "foreign gene" or heterologous gene refers to any nucleic acid (e.g., gene UC2023-989-2-PCT / / 3868.091WO1 sequence) that is introduced into the genome of an organism or tissue of an organism or a host cell by experimental manipulations, such as those described herein, and may include gene sequences found in that organism so long as the introduced gene does not reside in the same location, as does the naturally occurring gene. As used herein, the term “heterologous” when used in reference to a nucleic acid or protein refers to a nucleic acid or protein that has been manipulated in some way. For example, a heterologous nucleic acid includes a nucleic acid from one species introduced into another species. A heterologous nucleic acid also includes a nucleic acid that is native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, present in a locus within the genome, expressed from an autonomously replicating vector, linked to a non-native promoter, linked to a mutated promoter, or linked to an enhancer sequence, etc.). Heterologous nucleic acids may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). In some cases, heterologous nucleic acids are distinguished from endogenous plant genes in that the heterologous nucleic acids can be joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the nucleic acid. In another example, the heterologous nucleic acids are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed). As used herein, a “native” nucleic acid or polypeptide means a DNA, RNA or amino acid sequence or segment that has not been manipulated in vitro, i.e., has not been isolated, purified, and / or amplified. As used herein, the term "wild type" when made in reference to a gene refers to a functional gene common throughout an outbred population. As used herein, the term "wild- type" when made in reference to a gene product refers to a functional gene product common throughout an outbred population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild- type" form of the gene. As used herein, the term "wild type" when made in reference to a plant refers to the plant type common throughout an outbred population that has not been genetically UC2023-989-2-PCT / / 3868.091WO1 manipulated to contain an expression cassette, e.g., any of the expression cassettes described herein. Preparation of Expression Cassettes to Express Protein To prepare expression cassettes encoding one or more of the insecticidal proteins disclosed herein or truncated forms thereof, a peptide thereof, or a fusion thereof, for transformation, the recombinant DNA sequence or segment may be circular or linear, double-stranded or single-stranded. A DNA sequence which encodes an RNA sequence that is substantially complementary to a mRNA sequence encoding a gene product of interest is typically a "sense" DNA sequence cloned into a cassette in the opposite orientation (i.e., 3' to 5' rather than 5' to 3'). Generally, the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA in a cell. As used herein, "chimeric" means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species. Aside from DNA sequences that serve as transcription units, or portions thereof, a portion of the DNA may be untranscribed, serving a regulatory or a structural function. For example, the DNA may itself comprise a promoter that is active in eukaryotic cells, prokaryotic cells, or in certain cell types, or may utilize a promoter already present in the genome that is the transformation target. In one embodiment, the pEarlyGate 103 plant expression vector with the CaMV promoter could be used for expression of the SEQ ID NO:1 protein in plants. In one embodiment, expression is inducible. In one embodiment, a tissue-specific promoter (or enhancer) is employed. In inducible embodiments, tissue-specific plant promotors such as AP3 (petals), chi (pollen and anthers), Pht1;1 (roots), SynR2 (roots), pCL (tuber), and Hahb-4 LPF (roots and leaves), among others, could be used. Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA. Such elements may or may not be necessary for the function of the DNA but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell. UC2023-989-2-PCT / / 3868.091WO1 The recombinant DNA to be introduced into the cells may contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA, puro, hyg, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956). Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Exemplary reporter genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E. coli, the green, red, or blue fluorescent protein gene, and the luciferase gene. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein. The recombinant DNA can be readily introduced into the host cells, e.g., plant, bacterial, yeast, or prokaryotic cells, by transfection with an expression vector comprising the recombinant DNA by any procedure useful for the introduction into a particular cell, e.g., physical or biological methods, to yield a transformed (transgenic) cell having the recombinant DNA so that the DNA sequence of interest is expressed by the host cell. In one embodiment, the recombinant DNA is stably integrated into the genome of the cell. Physical methods to introduce a recombinant DNA into a host cell include calcium-mediated methods, lipofection, particle bombardment, microinjection, UC2023-989-2-PCT / / 3868.091WO1 electroporation, and the like. Biological methods to introduce the DNA of interest into a host cell include the use of viruses. To confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular gene product, e.g., by immunological means (ELISAs and Western blots) or by other molecular assays. To detect and quantitate RNA produced from introduced recombinant DNA segments, RT-PCR may be employed. In this application of PCR, it is first important to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique demonstrates the presence of an RNA species and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and only demonstrate the presence or absence of an RNA species. While Southern blotting and PCR may be used to detect the recombinant DNA segment in question, they do not provide information as to whether the recombinant DNA segment is being expressed. Expression may be evaluated by specifically identifying the peptide products of the introduced DNA sequences or evaluating the phenotypic changes brought about by the expression of the introduced DNA segment in the host cell. Vectors for Delivery of Transgenes Delivery vectors include, for example, plasmids, viral vectors, microparticles, nanoparticles, liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a gene to a host cell, e.g., to provide for recombinant expression of a polypeptide encoded by the gene. Vectors can also comprise other components or functionalities that further modulate gene delivery and / or gene expression, or that otherwise provide beneficial properties. Such other components include, for example, components UC2023-989-2-PCT / / 3868.091WO1 that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector by the cell; components that influence localization of the transferred gene within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the gene. Such components also might include markers, such as detectable and / or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. A variety of such marker genes have been described, including bifunctional (i.e., positive / negative) markers (see, e.g., WO 92 / 08796; and WO 94 / 28143). Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts. A large variety of such vectors are known in the art and are generally available. Vectors for gene delivery within the scope of the disclosure include, but are not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, and viral vectors, including viral and non-viral vectors which are present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA / DOPE, DOGS / DOPE or DMRIE / DOPE liposomes, and / or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA / DOPE) complexes. Exemplary gene viral vectors are described below. Vectors may be administered via any route including, but not limited to, electroporation and / or iontophoresis. In one embodiment, vectors are locally administered. In one embodiment, an isolated polynucleotide or vector having that polynucleotide comprises nucleic acid encoding a polypeptide or fusion protein that has substantial identity, e.g., at least 80% or more, e.g., 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% and up to 100%, amino acid sequence identity to, for example, SEQ ID NO:1. UC2023-989-2-PCT / / 3868.091WO1 Peptides, Polypeptides and Fusion Proteins The peptide or fusion proteins of the disclosure can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant DNA approaches (see above). The solid phase peptide synthetic method is an established and widely used method. These polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion- exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography. Once isolated and characterized, chemically modified derivatives of a given peptide or fusion thereof, can be readily prepared. For example, amides of the peptide or fusion thereof of the present disclosure may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. One method for amide formation at the C-terminal carboxyl group is to cleave the peptide or fusion thereof from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine. Salts of carboxyl groups of a peptide or fusion thereof may be prepared in the usual manner by contacting the peptide, polypeptide, or fusion thereof with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like. N-acyl derivatives of an amino group of the peptide or fusion thereof may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide, polypeptide, or fusion thereof. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy polypeptide or polypeptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired. Formyl-methionine, pyroglutamine and trimethyl-alanine may be substituted at the N-terminal residue of the polypeptide. Other amino-terminal modifications include aminooxypentane modifications. UC2023-989-2-PCT / / 3868.091WO1 In one embodiment, a peptide or fusion protein has substantial identity, e.g., at least 80% or more, e.g., 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% and up to 100%, amino acid sequence identity to, for example, SEQ ID NOS: 1, 2 or 3. Substitutions may include substitutions which utilize the D rather than L form, as well as other well-known amino acid analogs, e.g., unnatural amino acids VXFK^DV^Į^^Į-disubstituted amino acids, N-alkyl amino acids, lactic acid, and the like. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2- carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, SHQLFLOODPLQH^^RUQLWKLQH^^FLWUXOLQH^^Į-methyl-alanine, para-benzoyl-phenylalanine, SKHQ\OJO\FLQH^^ SURSDUJ\OJO\FLQH^^ VDUFRVLQH^^ İ-N,N,N-WULPHWK\OO\VLQH^^ İ-N- acetyllysine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- K\GUR[\O\VLQH^^ ^-N-methylarginine, and other similar amino acids and imino acids and tert-butylglycine. Conservative amino acid substitutions may be employed--that is, for example, aspartic-glutamic as acidic amino acids; lysine / arginine / histidine as polar basic amino acids; leucine / isoleucine / methionine / valine / alanine / proline / glycine non-polar or hydrophobic amino acids; serine / threonine as polar or hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting peptide, polypeptide or fusion polypeptide. Whether an amino acid change results in a functional peptide, polypeptide or fusion polypeptide can readily be determined by assaying the specific activity of the peptide, polypeptide or fusion polypeptide. UC2023-989-2-PCT / / 3868.091WO1 Amino acid substitutions falling within the scope of the disclosure, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic; trp, tyr, phe. The disclosure also envisions a peptide, polypeptide or fusion polypeptide with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another. Acid addition salts of the peptide, polypeptide or fusion polypeptide or of amino residues of the peptide, polypeptide or fusion polypeptide may be prepared by contacting the polypeptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art. Methods to Administer Recombinant Protein to Plants In one embodiment, the isolated protein, peptide or fusion polypeptide may be sprayed onto plants. Non-limiting effective amounts of the isolated protein, peptide or fusion polypeptide include but are not limited to a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of the isolated protein, peptide or fusion polypeptide in a liquid form sprayed on a plant, or from about 10 milligrams per acre to about 100 grams per acre of the isolated protein, peptide or fusion polypeptide applied to a field of plants, or from about 0.001 to about 0.1 microgram per milliliter of the isolated protein, peptide or fusion polypeptide. Where compositions as described herein are topically applied to a plant, the concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, UC2023-989-2-PCT / / 3868.091WO1 stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants is about 1 nanomole (nmol) of the isolated protein, peptide or fusion polypeptide per plant, for example, from about 0.05 to 1 nmol the isolated protein, peptide or fusion polypeptide per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of the isolated protein, peptide or fusion polypeptide per plant. In certain embodiments, about 40 to about 50 nmol of the isolated protein, peptide or fusion polypeptide is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of the isolated protein, peptide or fusion polypeptide is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of the isolated protein, peptide or fusion polypeptide is applied. In certain embodiments, a composition of about 0.5 to about 1.5 milligrams per milliliter of the isolated protein, peptide or fusion polypeptide is applied. In certain embodiments, about 1 nmol to about 5 nmol of the isolated protein, peptide or fusion polypeptide is applied to a plant. In certain embodiments, the isolated protein, peptide or fusion polypeptide composition as topically applied to the plant contains at least one type of isolated protein, peptide or fusion polypeptide at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines can require correspondingly larger amounts of the isolated protein, peptide or fusion polypeptide. Embodiments of such compositions include those where the composition includes a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment. In many embodiments, the composition is formulated in a form that is ingested by the insect. In embodiments, the composition further includes one or more components selected from a carrier agent, a surfactant, an organosilicone, an herbicidal molecule, a pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, or an insect growth regulator, or any combination thereof. The composition can include or be in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, UC2023-989-2-PCT / / 3868.091WO1 matrix, soil drench, or seed treatment. In some embodiments, the contacting includes providing the isolated protein, peptide or fusion polypeptide in a composition that further includes one or more components selected from a carrier agent, a surfactant, an organosilicone, an herbicidal molecule, a pesticide, a fungicide, a safener, an insect attractant, or an insect growth regulator, or any combination thereof. In some embodiments, the insecticidal composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment. In some embodiments, the insecticidal composition is provided in a form that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. The insecticidal compositions can further include one or more components selected from a carrier agent, a surfactant, an organosilicone, an herbicidal molecule, a pesticide, a fungicide, a safener, an insect attractant, or an insect growth regulator, or any combination thereof. The insecticidal compositions can further include at least one pesticidal agent selected from a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae. In some embodiments, the combination of the isolated protein, peptide or fusion polypeptide and the pesticidal agent provides a level of insect control that is greater than the sum of the effects of the isolated protein, peptide or fusion polypeptide and the pesticidal agent components if tested separately. The form of a spray treatment (applied prior to, contemporaneously with, or following application of the composition including the isolated protein, peptide or fusion polypeptide) of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, UC2023-989-2-PCT / / 3868.091WO1 2.0, 2.1, 2.2, 2.3, or 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of the isolated protein, peptide or fusion polypeptide into plant cells from a topical application on the surface Transgenic Plants The nucleic acids disclosed herein provide a means for production of healthy plants with insect resistance. The related nucleic acids can be isolated and identified by mutation of nucleic acid sequences encoding SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and / or by hybridization to DNA and / or RNA isolated from other species using segments of these nucleic acids as probes. In one embodiment, the nucleic acids described herein can include any nucleic acid that can selectively hybridize to nucleic acid encoding SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a polypeptide with at least 88%, 90% or more amino acid sequence identity thereto. The term “selectively hybridize’’ includes hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences. Such selective hybridization substantially excludes non target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or 60-99% sequence identity, or 70-99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 97-99% sequence identity, or 100% sequence identity (or complementarity) with each other. In some embodiments, a selectively hybridizing sequence has about at least about 80% sequence identity or complementarity with nucleic acid encoding SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. Thus, the nucleic acids of the disclosure include those with about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more of the same nucleotides as those encoding SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The identical UC2023-989-2-PCT / / 3868.091WO1 nucleotides or amino acids can be distributed throughout the nucleic acid or the protein and need not be contiguous. Note that if a value of a variable that is necessarily an integer, e.g., the number of nucleotides or amino acids in a nucleic acid or protein, is described as a range, e.g., 90-99% sequence identity what is meant is that the value can be any integer between 90 and 99 inclusive, i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, or any range between 90 and 99 inclusive, e.g., 91-99%, 91-98%, 92-99%, etc. The terms “stringent conditions’’ or “stringent hybridization conditions’’ include conditions under which a probe will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are somewhat sequence-dependent and can vary in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences can be identified with up to 100% complementarity to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of sequence similarity are detected (heterologous probing). The probe can be approximately 20-500 nucleotides in length but can vary greatly in length from about 18 nucleotides to equal to the entire length of the target sequence. In some embodiments, the probe is about 10-50 nucleotides in length, or about 18-25 nucleotides in length, or about 18-50 nucleotides in length, or about 18-100 nucleotides in length. Typically, stringent conditions will be those where the salt concentration is less than about 1.5 M Na+ion (or other salts), typically about 0.01 to 1.0 M Na+ion concentration (or other salts), at pH 7.0 to 8.3 and the temperature is at least about 30 °C for shorter probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for longer probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt’ s solution. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C, and a wash in 1 x SSC to 2 x SSC (where 20 x SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55 °C Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1M NaCl, 1 % SDS at 37 °C, and a wash in 0.5 x SSC to 1 x SSC at 55 to 60 °C. Exemplary UC2023-989-2-PCT / / 3868.091WO1 high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C, and a wash in 0.1 x SSC at 60 to 65 °C. Specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution. Thus, high stringency conditions can include a wash that includes 0.1 x SSC at 60 to 65 °C. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem. 138:267-84 (1984)): Tm= 81.5 °C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L where M is the molarity of monovalent cations; % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. The Tm is reduced by about 1 °C for each 1 % of mismatching. Thus, the Tm, hybridization and / or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with greater than or equal to 90% sequence identity are sought, the Tm can be decreased 10 °C. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can include hybridization and / or a wash at 1, 2, 3 or 4° C lower than the thermal melting point (Tm). Moderately stringent conditions can include hybridization and / or a wash at 6, 7, 8, 9 or 10 °C lower than the thermal melting point (Tm). Low stringency conditions can include hybridization and / or a wash at 11, 12, 13, 14, 15 or 20 °C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and a desired Tm, those of ordinary skill can identify and isolate nucleic acids with sequences related to nucleic acid encoding SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Those of skill in the art also understand how to vary the hybridization and / or wash solutions to isolate desirable nucleic acids. For example, if the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32 UC2023-989-2-PCT / / 3868.091WO1 °C (formamide solution), it may be desirable to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY -HYBRIDIZATION WITH NUCLEIC ACID PROBES, part 1, chapter 2,“Overview of principles of hybridization and the strategy of nucleic acid probe assays,’’ Elsevier, N.Y. (1993); and in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, chapter 2, Ausubel, et ah, eds, Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise stated, in the present application high stringency is defined as hybridization in 4 x SSC, 5 x Denhardt’s (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg / ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65 °C, and a wash in 0.1 x SSC, 0.1% SDS at 65 °C. However, because specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution, the high stringency conditions can more simply be expressed as including a wash in 0.1 x SSC at 60 to 65 °C. The following terms are used to describe the sequence relationships between two or more nucleic acids or polypeptides: (a) “reference sequence,’’ (b) “comparison window,’’ (c) “sequence identity,’’ (d) “percentage of sequence identity’’ and (e) “substantial identity.’’ As used herein, “reference sequence’’ is a defined sequence used as a basis for sequence comparison. The reference sequence can be a nucleic acid sequence or an amino acid sequence. A reference sequence may be a subset or the entirety of a specified sequence. For example, a reference sequence may be a segment of a full-length cDNA or of a genomic DNA sequence, or the complete cDNA or complete genomic DNA sequence, or a domain of a polypeptide sequence. As used herein, "comparison window" refers to a contiguous and specified segment of a nucleic acid or an amino acid sequence, wherein the nucleic acid / amino acid sequence can be compared to a reference sequence and wherein the portion of the nucleic acid / amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment UC2023-989-2-PCT / / 3868.091WO1 of the two sequences. The comparison window can vary for nucleic acid and polypeptide sequences. Generally, for nucleic acids, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or more nucleotides. For amino acid sequences, the comparison window is at least about 10 amino acids, and can optionally be 15, 20, 30, 40, 50, 100 or more amino acids. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the nucleic acid or amino acid sequence, a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, may permit optimal alignment of compared sequences; by the homology alignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53; by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software. Package, Version 8 (available from Genetics Computer Group (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp, (1989) CABIOS 5: 151-3; Corpet, et al., (1988) Nucleic Acids Res.16: 10881-90; Huang, et al., (1992) Computer Applications in the Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. An example of a good program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60, which is similar to the method described by Higgins and Sharp, (1989) CABIOS 5:151-53 (and is hereby incorporated by reference). The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences UC2023-989-2-PCT / / 3868.091WO1 against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., eds., Greene Publishing and Wiley- Interscience, New York (1995). GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP makes a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more. GAP presents one member of the family of best alignments. There may be many members of this family. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). For example, sequence identity / similarity values provided herein can refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et ah, (1997) Nucleic Acids Res. 25:3389-402). As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real UC2023-989-2-PCT / / 3868.091WO1 proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low- complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Ci-ayerie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination. The terms “substantial identity’’ indicates that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, or at least 60%, or at least 70%, or at least 80%, or at least 90% or at least 95% sequence identity, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or any percentage value within the range of 55-100% sequence identity relative to the reference sequence over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra. Alternatively, substantial identity is present when a second polypeptide is immunologically reactive with antibodies raised against the first polypeptide (e.g., a polypeptide with SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3). Thus, a polypeptide is substantially identical to a first polypeptide, for example, where the two polypeptides differ only by a conservative substitution. In addition, a polypeptide can be substantially identical to a first polypeptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical. Polypeptides that are "substantially similar" share sequences as noted above except that some residue positions, which are not identical, may differ by conservative amino acid changes. The polypeptide can include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 N-terminal amino acid residues of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. Alternatively, the polypeptides may include the last 21, 22, 23, 24, UC2023-989-2-PCT / / 3868.091WO1 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 C-terminal amino acid residues of the SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. There is a signal peptide that includes the first 16 amino acids of SEQ ID NO:1, which may be omitted or substituted with a different signal peptide. The protease domain includes residues from amino acid position 56 through 292. There are several amino acids likely for active function, including H100, D148, and S243. Plants Modified to Express or Contain Insecticidal Proteins To engineer healthy plants with increased tolerance to insects, one of skill in the art can introduce an insecticidal protein such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an insecticidal fragment thereof, or a polypeptide with at least 90% amino acid sequence identity thereto, or nucleic acids encoding such polypeptides into the plants. Introduction of an insecticidal protein such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an insecticidal fragment thereof, or a polypeptide with at least 90% amino acid sequence identity thereto, or expression of increased levels of an insecticidal protein such as SEQ ID NO:1, an insecticidal fragment thereof, or a polypeptide with at least 88%, 89%, 90% or more amino acid sequence identity thereto, can enhance a plant’s resistance to one or more species of insect. For example, introduction of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or expression of increased levels of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, in a plant can increase the plant’s resistance to insect infestation by at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 33% compared to a wild-type plant of the same species that does not comprise SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, e.g., does not comprise an expression cassette encoding an insecticidal protein such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an insecticidal fragment thereof, or a polypeptide with at least 88%, 89%, 90% or more amino acid sequence identity thereto. For example, one of skill in the art can generate genetically modified plants that contain nucleic acids encoding an insecticidal protein such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an insecticidal fragment thereof, or a polypeptide with at least 88%, 89%, 90% or more amino acid sequence identity thereto within UC2023-989-2-PCT / / 3868.091WO1 their somatic and / or germ cells. Such genetic modification can be accomplished by various procedures. Non-limiting examples of methods of introducing a modification into the genome of a plant cell can include microinjection, viral delivery, recombinase technologies, homologous recombination, TALENS, CRISPR, and / or ZFN, see, e.g., Clark and Whitelaw Nature Reviews Genetics 4:825-833 (2003); which is incorporated by reference herein in its entirety. For example, nucleases such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and / or meganucleases can be employed with guide nucleic acid that allows the nuclease to target the genomic site(s). In some cases of the various aspects described herein, a targeting vector can be used to introduce a transcriptional element, promotor, deletion, or modification of the genomic chromosomal sites. A "targeting vector" is a vector generally has a 5' flanking region and a 3' flanking region homologous to segments of the gene of interest. The 5' flanking region and a 3' flanking region can surround a DNA sequence comprising a modification and / or a foreign DNA sequence to be inserted into the gene. For example, foreign DNA to be inserted may encode a non-native promotor (as described below) and / or a selectable marker, such as an antibiotics resistance gene. Examples for suitable selectable markers include chloramphenicol resistance, gentamycin resistance, kanamycin resistance, spectinomycin UHVLVWDQFH^ ^6SHF5^^^ QHRP\FLQ^ UHVLVWDQFH^ JHQH^ ^1(2^^ DQG^ K\JURP\FLQ^ ȕ- phosphotransferase markers (genes). The 5' flanking region and the 3' flanking region can be homologous to regions within the gene, or such flanking regions can flank the coding region of gene to be deleted, mutated, or replaced with the unrelated DNA sequence. In some cases, the targeting vector does not comprise a selectable marker. DNA comprising the targeting vector and the native gene of interest are contacted under conditions that favor homologous recombination (e.g., by transforming plant cell(s) with the targeting vector). A typical targeting vector contains nucleic acid fragments of not less than about 0.1 kb nor more than about 10.0 kb from both the 5' and the 3' ends of the genomic locus which encodes the gene to be modified (e.g., the genomic site(s)). These two fragments can be separated by an intervening fragment of nucleic acid that includes the modification to be introduced. When the resulting construct UC2023-989-2-PCT / / 3868.091WO1 recombines homologously with the chromosome at this locus, it results in the introduction of the modification, e.g., an insertion, substitution, or a deletion of a portion of the genomic site(s). In some cases, a Cas9 / CRISPR system can be used to create a modification in genomic site(s). Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems are useful for, e.g., RNA- programmable genome editing (see e.g., Marraffini & Sontheimer. Nature Reviews Genetics 11: 181-190 (2010); Sorek et al. Nature Reviews Microbiology 20086: 181-6; Karginov and Hannon. Mol Cell 20101 :7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012337:815-820; Bikard and Marraffini Curr Opin Immunol 201224:15-20; Bikard et al. Cell Host & Microbe 201212: 177-186; all of which are incorporated by reference herein in their entireties). A CRISPR guide RNA can be used that can target a Cas enzyme to the desired location in the genome, where it generates a double strand break. This technique is available in the art and described, e.g., at Mali et al. Science 2013339:823-6; which is incorporated by reference herein in its entirety and kits for the design and use of CRISPR-mediated genome editing are commercially available, e.g. the PRECISION X CAS9 SMART NUCLEASE™ System (Cat No. CAS900A-1) from System Biosciences, Mountain View, CA. In another example, one of skill in the art can prepare an expression cassette or expression vector that can express one or more insecticidal proteins such as SEQ ID NO:1, an insecticidal fragment thereof, or a polypeptide with at least 90% amino acid sequence identity thereto. Plant cells can be transformed by the expression cassette or expression vector, and whole plants (and their seeds) can be generated from the plant cells that were successfully transformed with nucleic acids encoding an insecticidal protein such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an insecticidal fragment thereof, or a polypeptide with at least 88%, 89%, 90% or more amino acid sequence identity thereto. Some procedures for making such genetically modified plants and their seeds are described below. Promoters: The nucleic acids described herein can be operably linked to a promoter, which provides for expression of mRNA from the nucleic acids. The promoter can be a promoter functional in plants and / or seeds and can be a promoter functional during plant growth and development. In some cases, the UC2023-989-2-PCT / / 3868.091WO1 promotor can be a non-native promotor that is not found in the plant cell, plant seed, or plant the nucleic acid is expressed in. A nucleic acid is operably linked to the promoter when it is located downstream from the promoter, to thereby form an expression cassette. Most endogenous genes have regions of DNA that are known as promoters, which regulate gene expression. Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA. Promoter sequences are also known to be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that allows gene expression to be turned on and off in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the Ptac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous DNAs is advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired. Expression cassettes generally include, but are not limited to, a plant promoter such as the CaMV 35S promoter (Odell et ah, Nature. 313:810-812 (1985)), or others such as CaMV 19S (Lawton et ah, Plant Molecular Biology. 9:315-324 (1987)), nos (Ebert et ah, Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), Adhl (Walker et ah, Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et ah, Proc. Natl. Acad. Sci. USA.87:4144-4148 (1990)), a-tubulin, ubiquitin, actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et ah, Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., Plant Molecular Biology.12:579-589 (1989)) or those associated with the R gene UC2023-989-2-PCT / / 3868.091WO1 complex (Chandler et al., The Plant Cell. 1: 1175-1183 (1989)). Further suitable promoters include the poplar xylem-specific secondary cell wall specific cellulose synthase 8 promoter, cauliflower mosaic virus promoter, the Z10 promoter from a gene encoding a 10 kDa zein protein, a Z27 promoter from a gene encoding a 27 kDa zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene (Coruzzi et al., EMBO J. 3: 1671 (1971)) and the actin promoter from rice (McElroy et al., The Plant Cell.2: 163-171 (1990)). Seed specific promoters, such as the phaseolin promoter from beans, may also be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324 (1985). The promotor can include a drought-regulated promoter. For example, the drought-regulated promotor can include a 7DA2 promoter as disclosed in U.S. Patent No. 10,106,813, issued October 23, 2018 to Han, K.H., et. al., and incorporated herein by reference. The promotor can include a cold-stress-responsive promoter for delayed flowering, such as to avoid flowering during cold temperatures. For example, the cold-stress-responsive promotor can be a Cor15A promotor. (Baker, S. et. al., The ^ƍ-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold- , drought- and ABA-regulated gene expression, Plant Mol. Biol. 24: 701-713 (1994), incorporated herein by reference). Alternatively, tissue specific promoter sequences may be employed. cDNA clones from a particular tissue can be isolated and those clones which are expressed specifically in that tissue are identified, for example, using Northern blotting. In one embodiment, the gene isolated is not present in a high copy number but is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones can then be localized using techniques well known to those of skill in the art. A nucleic acid can be combined with the promoter by standard methods to yield an expression cassette, for example, as described in Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL. Second Edition (Cold Spring Harbor, NY: Cold Spring Harbor Press (1989); MOLECULAR CLONING: A LABORATORY MANUAL. Third Edition (Cold Spring Harbor, NY: Cold Spring Harbor Press (2000)). Briefly, a plasmid containing a promoter such as the 35 S CaMV promoter can be constructed as described in Jefferson (Plant Molecular Biology Reporter 5:387-405 (1987)) or obtained from Clontech UC2023-989-2-PCT / / 3868.091WO1 Lab in Palo Alto, California (e.g., pBI121 or pBI221). Typically, these plasmids are constructed to have multiple cloning sites having specificity for different restriction enzymes downstream from the promoter. The nucleic acids can be subcloned downstream from the promoter using restriction enzymes and positioned to ensure that the DNA is inserted in proper orientation with respect to the promoter so that the DNA can be expressed as sense RNA. Once the nucleic acid sequence is operably linked to a promoter, the expression cassette so formed can be subcloned into a plasmid or other vector (e.g., an expression vector). In some embodiments, a cDNA clone encoding a protein is isolated from plant tissue, for example, a root, stem, leaf, seed, or flower tissue. For example, cDNA clones from selected species (that encode a protein with homology to any of those described herein) are made from isolated mRNA from selected plant tissues. In another example, a nucleic acid encoding a mutant or modified insecticidal protein can be prepared by available methods or as described herein. For example, the nucleic acid encoding a mutant or modified protein can be any nucleic acid with a coding region that hybridizes to a segment of a nucleic acid encoding SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Using restriction endonucleases, the entire coding sequence for the modified protein is subcloned downstream of the promoter in a 5’ to 3’ sense orientation. Targeting Sequences: Additionally, expression cassettes can be constructed and employed to target the proteins to an intracellular compartment within plant cells, into a membrane, or to direct an encoded protein to the extracellular environment. This can generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of the nucleic acid. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and can then be posttranslational removed. Transit peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. By facilitating transport of the protein into compartments inside or outside the cell, these sequences can increase the accumulation of a particular gene product in a particular location. For example, see U.S. Patent No. 5,258,300. UC2023-989-2-PCT / / 3868.091WO1 3' Sequences: When the expression cassette is to be introduced into a plant cell, the expression cassette can also optionally include 3' nontranslated plant regulatory DNA sequences that act as a signal to terminate transcription and allow for the polyadenylation of the resultant mRNA. The 3' nontranslated regulatory DNA sequence may include from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequences. For example, 3' elements that can be used include those derived from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et ah, Nucleic Acid Research. 11:369-385 (1983)), or the terminator sequences for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and / or the 3' end of the protease inhibitor I or II genes from potato or tomato. Other 3' elements known to those of skill in the art can also be employed. These 3' nontranslated regulatory sequences can be obtained as described in Methods in Enzymology. 153:292 (1987). Many such 3' nontranslated regulatory sequences are already present in plasmids available from commercial sources such as Clontech, Palo Alto, California. The 3’ nontranslated regulatory sequences can be operably linked to the 3’ terminus of the nucleic acids by standard methods. Selectable and Screenable Marker Sequences: To improve identification of transformants, a selectable or screenable marker gene can be employed with the expressible nucleic acids. "Marker genes" are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can ‘select’ for by chemical means, e.g., by use of a selective agent (e.g., an herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by ‘screening’ (e.g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the disclosure. Included within the terms selectable or screenable marker genes are also genes which encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins UC2023-989-2-PCT / / 3868.091WO1 detectable, e.g., by ELISA; and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S). With regard to selectable secretable markers, the use of a gene that encodes a polypeptide that becomes sequestered in the cell wall, where the polypeptide includes a unique epitope may be advantageous. Such a secreted antigen marker can employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that imparts efficient expression and targeting across the plasma membrane and can produce protein that is bound in the cell wall and yet is accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy such requirements. Examples of proteins suitable for modification in this manner include extensin or hydroxyproline rich glycoprotein (HPRG). For example, the maize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is well characterized in terms of molecular biology, expression, and protein structure and therefore can readily be employed. However, any one of a variety of extensins and / or glycine- rich wall proteins (Keller et ah, EMBO J. 8:1309-1314 (1989)) could be modified by the addition of an antigenic site to create a screenable marker. Numerous other possible selectable and / or screenable marker genes will be apparent to those of skill in the art in addition to those forth herein below. Therefore, it will be understood that the discussion herein is exemplary rather than exhaustive. In light of the techniques disclosed herein and the general recombinant techniques that are known in the art, the present disclosure readily allows the introduction of any gene, including marker genes, into a recipient cell to generate a transformed plant cell, e.g., a monocot cell or dicot cell. Possible selectable markers for use in connection with the present disclosure include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199: 183-188 (1985)) which codes for kanamycin resistance and can be selected for using kanamycin, G418, and the like; a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein (Hinchee et al., Bio / Technology. 6:915-922 (1988)) thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., Science. 242:419-423 (1988)); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, UC2023-989-2-PCT / / 3868.091WO1 sulfonylurea or other ALS-inhibiting chemicals (European Patent Application 154,204 (1985)); a methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase gene that confers resistance to 5 -methyl tryptophan. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0218571 (1987)). An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the gene that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (Ei.S. Patent No. 5,550,318). The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twell et al., Plant Physiol.91:1270-1274 (1989)) causing rapid accumulation of ammonia and cell death. The success in using this selective system in conjunction with monocots was surprising because of the major difficulties that have been reported in transformation of cereals (Potrykus, Trends Biotech. 7:269-273 (1989)). Screenable markers that may be employed include, but are not limited to, a b-glucuronidase or u id A gene (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R- locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts , 18* Stadler Genetics Symposium, J.P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282 (1988)); a b-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA.75:3737-3741 (1978)), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA. 80:1101 (1983)) which encodes a catechol dioxygenase that can convert chromogenic catechols; an a-amylase gene (Ikuta et al., Bio / technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable of oxidizing tyrosine to DOPA and UC2023-989-2-PCT / / 3868.091WO1 dopaquinone which in turn condenses to form the easily detectable compound melanin; a b-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science. 234:856- 859.1986), which allows for bioluminescence detection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm. 126: 1259-1268 (1985)), which may be employed in calcium-sensitive bioluminescence detection, or a green or yellow fluorescent protein gene (Niedz et al., Plant Cell Reports. 14:403 (1995)). For example, genes from the maize R gene complex can be used as screenable markers. The R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue. Maize strains can have one, or as many as four, R alleles that combine to regulate pigmentation in a developmental and tissue specific manner. A gene from the R gene complex does not harm the transformed cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector. If a maize line carries dominant alleles for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, Al, A2, Bzl and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation. Exemplary lines include Wisconsin 22 that contains the rg-Stadler allele and TR112, a K55 derivative that is r-g, b, PI. Alternatively, any genotype of maize can be utilized if the Cl and R alleles are introduced together. The R gene regulatory regions may be employed in chimeric constructs to provide mechanisms for controlling the expression of chimeric genes. More diversity of phenotypic expression is known at the R locus than at any other locus (Coe et al., in Corn and Corn Improvement, eds. Sprague, G.F. & Dudley, J.W. (Am. Soc. Agron., Madison, WI), pp. 81-258 (1988)). It is contemplated that regulatory regions obtained from regions 5' to the structural R gene can be useful in directing the expression of genes, e.g., insect resistance, drought resistance, herbicide tolerance or other protein coding regions. For the purposes of the present disclosure, it is believed that any of the various R gene family members may be successfully employed (e.g., P, S, Fc, etc.). However, one that can be used is Sn (particularly Sn:bol3). Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific UC2023-989-2-PCT / / 3868.091WO1 deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R. A further screenable marker contemplated for use is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for population screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening. Other Optional Sequences: An expression cassette can also further comprise plasmid DNA. Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. The additional DNA sequences include origins of replication to provide for autonomous replication of the vector, additional selectable marker genes, e.g., encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette and sequences that enhance transformation of prokaryotic and eukaryotic cells. Another vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoort et ah, U.S. Patent No. 4,940,838) as exemplified by vector pGA582. This binary Ti plasmid vector has been previously characterized by An (Methods in Enzymology. 153:292 (1987)) and is available from Dr. An. This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as rice cells. The binary Ti vectors may include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the co / El replication of origin and a wide host range replicon. The binary Ti vectors carrying an expression cassette of the disclosure can be used UC2023-989-2-PCT / / 3868.091WO1 to transform both prokaryotic and eukaryotic cells but may be used to transform dicot plant cells. In Vitro Screening of Expression Cassettes: Once the expression cassette is constructed and subcloned into a suitable plasmid, it can be screened for the ability to substantially inhibit the translation of an mRNA coding for a seed storage protein by standard methods such as hybrid arrested translation. For example, for hybrid selection or arrested translation, a preselected antisense DNA sequence is subcloned into an SP6 / T7 containing plasmids (as supplied by ProMega Corp.). For transformation of plants cells, suitable vectors include plasmids such as described herein. Typically, hybrid arrest translation is an in vitro assay that measures the inhibition of translation of an mRNA encoding a particular seed storage protein. This screening method can also be used to select and identify preselected antisense DNA sequences that inhibit translation of a family or subfamily of zein protein genes. As a control, the corresponding sense expression cassette is introduced into plants and the phenotype assayed. DNA Delivery of the DNA Molecules into Host Cells: The present disclosure generally includes steps directed to introducing nucleic acids, such as a preselected cDNA encoding the insecticidal protein, into a recipient cell to create a transformed cell. In some instances, the frequency of occurrence of cells taking up exogenous (foreign) DNA may be low. Moreover, it is most likely that not all recipient cells receiving DNA segments or sequences will result in a transformed cell wherein the DNA is stably integrated into the plant genome and / or expressed. Some may show only initial and transient gene expression. However, certain cells from virtually any dicot or monocot species may be stably transformed, and these cells regenerated into transgenic plants, through the application of the techniques disclosed herein. Another aspect of the disclosure is a plant with increased drought tolerance and / or delayed flowering and increased biomass, wherein the plant has an introduced nucleic acid. The plant can be a monocotyledon or a dicotyledon. Another aspect of the disclosure includes plant cells (e.g., embryonic cells or other cell lines) that can regenerate fertile transgenic plants and / or seeds. The cells can be derived from either monocotyledons or dicotyledons. Suitable examples of plant species include grasses, softwoods, hardwoods, wheat, rice, maize, barley, rye, Brachypodium, Arabidopsis, alfalfa, oats, sorghum, millet, miscanthus, UC2023-989-2-PCT / / 3868.091WO1 switchgrass, poplar, eucalyptus, sugarcane, bamboo, tobacco, cucumber, tomato, soybean, and the like. In some embodiments, the plant or cell is a monocotyledon plant or cell. For example, the plant or cell can be a grass plant or cell. In some embodiments, the plant or cell is a dicotyledon plant or cell. For example, the plant or cell can be a hardwood plant or cell. The cell(s) may be in a suspension cell culture or may be in an intact plant part, such as an immature embryo, or in a specialized plant tissue, such as callus, such as Type I or Type II callus. Transformation of the cells of the plant tissue source can be conducted by any one of a number of methods known to those of skill in the art. Examples are: Transformation by direct DNA transfer into plant cells by electroporation (U.S. Patent No. 5,384,253 and U.S. Patent No. 5,472,869, Dekeyser et ah, The Plant Cell. 2:591-602 (1990)); direct DNA transfer to plant cells by PEG precipitation (Hayashimoto et al, Plant Physiol. 93:857-863 (1990)); direct DNA transfer to plant cells by microprojectile bombardment (McCabe et ah, Bio / Technology. 6:923-926 (1988); Gordon-Kamm et ah, The Plant Cell. 2:603- 618 (1990); U.S. Patent No.5,489,520; U.S. Patent No.5,538,877; and U.S. Patent No.5,538,880) and DNA transfer to plant cells via infection with Agrobacterium. Methods such as microprojectile bombardment or electroporation can be carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction. One method for dicot transformation, for example, involves infection of plant cells with Agrobacterium tumefaciens using the leaf-disk protocol (Horsch et ak, Science 227:1229-1231 (1985). Monocots such as Zea mays can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase-containing enzyme (U.S. Patent No. 5,384,253; and U.S. Patent No.5,472,869). For example, embryogenic cell lines derived from immature Zea mays embryos can be transformed by accelerated particle treatment as described by Gordon-Kamm et al. (The Plant Cell. 2:603-618 (1990)) or U.S. Patent No. 5,489,520; U.S. Patent No. 5,538,877 and U.S. Patent No. 5,538,880, cited above. Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as UC2023-989-2-PCT / / 3868.091WO1 described in U.S. application Serial No. 08 / 112,245 and PCT publication WO 95 / 06128. Furthermore, methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0604662, 1994) and Saito et al. (European Patent 0672752, 1995). Methods such as microprojectile bombardment or electroporation are carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction. The choice of plant tissue source for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells. Type I or Type II embryonic maize callus and immature embryos are desirable Zea mays tissue sources. Similar tissues can be transformed for softwood or hardwood species. Selection of tissue sources for transformation of monocots is described in detail in U.S. Application Serial No. 08 / 112,245 and PCT publication WO 95 / 06128. The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA or RNA carrying the nucleic acids for an effective period of time. This may range from a less than one second pulse of electricity for electroporation to a 2-3 days co-cultivation in the presence of plasmid-bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed. Electroporation: Where one wishes to introduce DNA by means of electroporation, it is contemplated that the method of Krzyzek et al. (U.S. Patent No. 5,384,253) may be advantageous. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target UC2023-989-2-PCT / / 3868.091WO1 recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells can be made more susceptible to transformation, by mechanical wounding. To effect transformation by electroporation, one may employ either friable tissues such as a suspension cell cultures, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. The cell walls of the preselected cells or organs can be partially degraded by exposing them to pectin-degrading enzymes (pectinases or pectolyases) or mechanically wounding them in a controlled manner. Such cells would then be receptive to DNA uptake by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA. Microprojectile Bombardment: A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, microparticles may be coated with DNA and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. In an illustrative embodiment, non-embryogenic BMS cells were bombarded with intact cells of the bacteria E. coli or Agrobacterium tumefaciens containing plasmids with either the b-glucoronidase or bar gene engineered for expression in maize. Bacteria were inactivated by ethanol dehydration prior to bombardment. A low level of transient expression of the b-glucoronidase gene was observed 24-48 hours following DNA delivery. In addition, stable transformants containing the bar gene were recovered following bombardment with either E. coli or Agrobacterium tumefaciens cells. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that particles may increase the level of DNA delivery but are not, in and of themselves, necessary to introduce DNA into plant cells. The microprojectile bombardment is an effective means of reproducibly stably transforming monocots that avoids the need to prepare and isolate protoplasts (Christou et al., PNAS.84:3962-3966 (1987)), avoids the formation of partially degraded cells, and the susceptibility to Agrobacterium infection is not UC2023-989-2-PCT / / 3868.091WO1 required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension (Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990)). The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregate and may contribute to a higher frequency of transformation, by reducing damage inflicted on the recipient cells by an aggregated projectile. For bombardment, cells in suspension may be concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Using techniques set forth herein, one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from about 1 to 10 and average about 1 to 3. In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment can influence transformation frequency. Physical factors are those that involve manipulating the DNA / microprojectile precipitate or those that affect the path and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmid DNA. One may wish to adjust various bombardment parameters in small scale studies to fully optimize the conditions and / or to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells, and which may therefore UC2023-989-2-PCT / / 3868.091WO1 influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. Execution of such routine adjustments will be known to those of skill in the art. An Example of Production and Characterization of Stable Transgenic Plants: After effecting delivery of a nucleic acid to recipient cells by any of the methods discussed above, the transformed cells can be identified for further culturing and plant regeneration. As mentioned above, to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the expressible nucleic acids. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait. Selection: An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive cells will not be amenable to further culturing. To use the har-bialaphos or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg / 1 bialaphos or about 1-3 mM glyphosate, as appropriate. While ranges of about 1-3 mg / 1 bialaphos or about 1-3 mM glyphosate can be employed, it is proposed that ranges of at least about 0.1-50 mg / 1 bialaphos or at least about 0.1-50 mM glyphosate will find utility in the practice of the disclosure. Tissue can be placed on any porous, inert, solid or semi solid support for bombardment, including but not limited to filters and solid culture medium. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this disclosure is not limited to them. An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are UC2023-989-2-PCT / / 3868.091WO1 pigmented. These cells may be cultured further, either in suspension or on solid media. The R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the Cl and B genes will result in pigmented cells and / or tissues. The enzyme luciferase is also useful as a screenable marker in the context of the present disclosure. In the presence of the substrate luciferin, cells expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time. It is further contemplated that combinations of screenable and selectable markers may be useful for identification of transformed cells. For example, selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those providing 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone. In an illustrative embodiment embryogenic Type II callus of Zea mays L. can be selected with sub-lethal levels of bialaphos. Slowly growing tissue was subsequently screened for expression of the luciferase gene and transformants can be identified. Regeneration and Seed Production: Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants. One example of a growth regulator that can be used for such purposes is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA + 2,4-D or perhaps even picloram. Media improvement in these and like ways can facilitate the growth of cells at specific developmental stages. Tissue can be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least two weeks, then transferred to media conducive to maturation of embryoids. Cultures are typically UC2023-989-2-PCT / / 3868.091WO1 transferred every two weeks on this medium. Shoot development signals the time to transfer to medium lacking growth regulators. The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO2, and at about 25-250 microeinsteins / sec-m of light. Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Con™. Regenerating plants can be grown at about 19 °C to 28 °C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Mature plants are then obtained from cell lines that are known to express the trait. In some embodiments, the regenerated plants are self-pollinated. In addition, pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants. The trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture are of interest if the traits are to be commercially useful. Regenerated plants can be repeatedly crossed to inbred plants to introgress the nucleic acids into the genome of the inbred plants. This process is referred to as backcross conversion. When a sufficient number of crosses to the recurrent inbred parent have been completed to produce a product of the backcross conversion process that is substantially isogenic with the recurrent inbred parent except for the presence of the introduced nucleic acids, the plant is self-pollinated at least once to produce a homozygous backcross converted inbred containing the nucleic acids. Progeny of these plants are true breeding. Alternatively, seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants. UC2023-989-2-PCT / / 3868.091WO1 Seed from the fertile transgenic plants can then be evaluated for the presence and / or expression of the nucleic acids (or insecticidal proteins). Transgenic plant and / or seed tissue can be analyzed for expression using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a product of insecticidal activity. Once a transgenic seed expressing the insecticidal sequence in the plant is identified, the seed can be used to develop true breeding plants. The true breeding plants are used to develop a line of plants with an increase in the percent of the insecticidal protein and growth of the plant while still maintaining other desirable functional agronomic traits. Adding the trait of increased resistance to insects and growth and normal to improved growth of the plant can be accomplished by back- crossing with this trait and with plants that do not exhibit this trait and studying the pattern of inheritance in segregating generations. Those plants expressing the target trait in a dominant fashion may be selected. Back-crossing is carried out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of an increased resistance to insects, and / or normal to improved growth. The resulting progeny are then crossed back to the parent that expresses the increased trait. The progeny from this cross will also segregate so that some of the progeny carry the trait and some do not. This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the trait and normal to improved growth of the plant. Such expression of the increased insect resistanceand / or normal to improved growth of plant can be expressed in a dominant fashion. Subsequent to back-crossing, the transgenic plants can be evaluated for an increase in the weight percent of insecticidal activity, and / or normal to improved growth after exposure to insects. This can be done, for example, by immunofluorescence analysis of whole plant cell walls (e.g., by microscopy), or any of the assays described herein or available to those of skill in the art. Plants that can be improved include but are not limited to forage plants (e.g., alfalfa, clover, soybeans, turnips, bromegrass, bluestem, and fescue), starch plants (e.g., canola, potatoes, lupins, sunflower and cottonseed), grains (maize, wheat, barley, oats, rice, sorghum, millet and rye), grasses (switchgrass, prairie UC2023-989-2-PCT / / 3868.091WO1 grass, wheat grass, sudangrass, sorghum, straw-producing plants, miscanthus, switchgrass), sugar producing plants (sugarcane, beets), vegetable plants (e.g., cucumber, tomato), Brachypodium, Arabidopsis, bamboo, softwood, hardwood and other woody plants (e.g., those used for paper production such as poplar species, pine species, and eucalyptus). In some embodiments the plant is a forage crop species or a species useful for production of biofuels. Examples of plants useful for pulp and paper production include most pine species such as loblolly pine, Jack pine, Southern pine, Radiata pine, spruce, Douglas fir and others. Hardwoods that can be modified as described herein include aspen, poplar, eucalyptus, and others. Plants useful for making biofuels and ethanol include corn, Brachypodium, grasses (e.g., miscanthus, switchgrass, and the like), as well as trees such as poplar, aspen, willow, and the like. Plants useful for generating dairy forage include legumes such as alfalfa, as well as clover, soybeans, turnips, Brachypodium, Arabidopsis, and forage grasses such as bromegrass, and bluestem. Determination of Stably Transformed Plant Tissues: To confirm the presence of the introduced nucleic acids in the regenerating plants, or seeds or progeny derived from the regenerated plant, a variety of assays may be performed. Such assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analyzing the phenotype of the whole regenerated plant. Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues. PCR techniques may also be used for detection and quantification of RNA produced from introduced nucleic acids. PCR also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified by use of conventional PCR techniques. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be UC2023-989-2-PCT / / 3868.091WO1 determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species. While Southern blotting and PCR may be used to detect the nucleic acid in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced nucleic acids or evaluating the phenotypic changes brought about by their expression. Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the insecticidal protein such as evaluation by amino acid sequencing following purification. The Examples of this application also provide assay procedures for detecting and quantifying activity. Other procedures may be additionally used. The expression of a gene product can also be determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of preselected DNA segments encoding storage proteins which change amino acid composition and may be detected by amino acid analysis. Method of Isolating Fractions with Insecticidal Activity Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis kill insect hosts quickly. While infecting insects, EPNs spit out excretory / secretory products (ESPs) into the host. An arsenal of proteins is found UC2023-989-2-PCT / / 3868.091WO1 in EPN ESPs. In particular, EPNs release relatively large quantities (for their size) of a variety of proteins when stimulated by insect tissue (Figures 1 and 2). Of the two EPNs that were evaluated, S. carpocapsae and S. feltiae, it was found that they release different complex mixtures of proteins when exposed to the same host tissue, and that collectively, both ESP mixtures are highly toxic to insects. To identify candidate insecticidal proteins from these mixtures, activity- or bioassay-guided purification is employed followed by separation of extracted components based on differences in their physiochemical properties and biological activity, followed by another round of separation and assaying. In one embodiment, the biological activity is insecticidal activity, e.g., injecting small quantities of fractions into Drosophila melanogaster adults. To obtain a large amount of ESPs for multiple rounds of fractionation 100 million S. carpocapsae IJs were reared. All the IJs were activated in vitro as described in Lu et al., 2017. After activation, IJs are thoroughly washed and excreted / secreted proteins (ESPs) are then collected in PBS for 3 hours at 25 C. A total of 7.9 mg ESPs were collected, representing an average of .079 ng / IJ in the 3-hour period. Crude ESPs released by activated IJs of S. carpocapsae were highly toxic to fruit flies, killing them quickly (within hours) with as little as 20 nanograms (ng) (Figure 3) (Lu et al., 2017). The crude ESPs from S. carpocapsae contain ~500 different proteins and utilizing the published genome sequences and mass spectrometric data, all of the proteins present in the ESPs of S. carpocapsae were identified (Chang et al., 2019a; Dillman et al., 2015; Lu et al., 2017). EPN ESPs themselves were directly toxic to D. melanogaster. The crude ESPs of S. carpocapsae, S. feltiae, and S. hermaphroditum were tested in other insects including Bombyx mori, Manduca sexta, and Galleria mellonella and they were found to be highly toxic to all of these insects, indicating that EPN ESPs may have broad spectrum toxicity to insects. The invention will be further described by the following non-limiting example. EXAMPLE After having activated 100 million S. carpocapsae IJs and collected a large amount of crude ESPs (~8 mg), candidate insecticidal proteins were purified for further characterization and assessment for use in crop insect pest control. Purification of proteins was accomplished using different chromatographic UC2023-989-2-PCT / / 3868.091WO1 techniques. Anion exchange chromatography was used to separate the proteins. Crude ESPs were filtered through a 0.22 P filter to remove any particulate matter from the sample. A Mono-Q column (strong anion exchanger) was equilibrated with 20 mM Tris-Cl pH 8 buffer (Low salt buffer) and then proteins were applied to the column. Unbound proteins were washed with low salt buffer and bound proteins were eluted with 20 mM Tris-Cl pH 8 having 1 M NaCl (High salt buffer). SDS-PAGE followed by silver staining was performed to visualize the proteins in each fraction. The fractions eluted from anion exchange chromatography were dialyzed against 20 mM Tris-Cl pH 8 to remove the salt using 3 kDa snakeskin dialysis membrane. After dialysis all the fractions were injected into adult fruit flies and toxicity was assessed (Figure 4). The fraction with highest toxicity was further purified using size exclusion chromatography. In size exclusion chromatography the proteins are separated the basis of their molecular size. Fraction 37 was subjected to size exclusion followed by a silver-stained gel, and then fractions were dialyzed against 20 mM Tris-Cl pH 8. Then each fraction was tested for toxicity (Figure 5). The fraction with highest toxicity was collected and Mass Spectrometry analysis was performed to identify the proteins. After performing size exclusion, many of the resulting fractions were injected into adult flies. Some fractions had low but detectable amounts of protein, but others had too little to be detecting using a Qubit fluorometer. Nevertheless, 50 nanoliters of these fractions were injected into adult flies (Figure 5C). The size exclusion fraction found to be most toxic, fraction 56 (Figure 5C) was analyzed using mass spectrometry. For mass spectrometry, the fraction was concentrated. Peptides were quantified using BCA assay and a total of 1 ug of peptides were injected for LC-MS analysis. Trypsin-digested peptides were analyzed by ultra- high pressure liquid chromatography (UPLC) coupled with tandem mass spectroscopy (LC-MS / MS) using nano-spray ionization. Protein identification and label free quantification was carried out using Peaks Studio 8.5 (Bioinformatics solutions Inc.) Three proteins were detected from fraction 56: chymotrypsin-like protease (CLP), histone 3, and actin (Table 1). Considering histone 3 is a nucleosomal protein and actin is structural protein chances of being toxic of these proteins are low and chymotrypsin like protease is considered as the target protein for further characterization. UC2023-989-2-PCT / / 3868.091WO1 Table 1: Identification of proteins in fraction 56 resulting from size exclusion chromatography. This was the most toxic fraction to flies. (L596_023060) MRTLFVLFALIVTVSGIPLQGFAAPHVEQIFSEPALFPLDPEEFFRNLDKV NTTRFVYGGQEARPGQFPQHAFMLYKTAEGFFICGASLLSPTHALTAAH CVEGMMAPSQIMAGGLNRRDRRAPNAQWRSIHRATKHAEYKPPSKRN DIAIVEFHPPMTLNRDVQLTKIVEDDAELLQEKKSYVTGFGTYTFKNKQ AVTSDELLWAEIDLFDFSRCQQLWHRRLWQKQICAGAKNLGAGPGDSG GPLQVLHDGTLFQVGLTSYGTSDKFDDEFNQDRFPTVFTRVSSYCDFIAK VTDNAATCSSLSKKPTAKPDCRF (SEQ ID NO:1). (L596_012080) MGCPRARLIRFCITILAPLITVAKIPSSFLLLLLSSFILSPAQILSARAVLVVR VLISCLSYLPCINASLSFQNMARTKQTATKSTGGKSPIVSGSKPGSTGKSP KSLKQALAVKAARKTGGAPGVGTVKKAHRYRPGTVALREIRRYQKSTE LLLRKLPFQRLVREIAQEYKTDLRFQSSAVLALQEAAEAFLVGLFEDTNL CAIHAKRVTIMPRDIQLARRIRGDRF (SEQ ID NO:2). (L596_029743.2) MCDEEVAALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVG MGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNEL RVAPEEHPVLLTEAPLNPKANREKMTQIMFETFNTPAMYVAIQAVLSLY ASGRTTGVVLDSGDGVTHTVPIYEGYALPHAILRLDLAGRDLTDYLMKI LTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELP DGQVITVGNERFRCPEAMFQPSFLGMESAGIHETSYNSIMKCDIDIRKDL UC2023-989-2-PCT / / 3868.091WO1 SSPRSPPSNRCGSRSRSTTSPDPQSSTASASKKFPSFEVFLSTFFDYDDYYL RVDLRARGRHVFPLLSHFIVSVLPLKIFNFFCRTS (SEQ ID NO:3). The CLP encoding gene from S. carpocapsae (L596_023060) was synthesized and codon optimized for expression in E. coli: AGATTTGTATATGGTGGCCAAGAAGCTCGTCCAGGGCAGTTTCCACA ACATGCTTTCATGCTGTACAAAACTGCAGAGGGATTTTTCATATGTGG TGCTTCACTTCTGTCTCCAACTCATGCCCTTACAGCGGCTCATTGTGTT GAAGGAATGATGGCTCCTTCTCAAATAATGGCAGGAGGGTTGAACAG AAGGGACAGGAGAGCACCTAATGCACAATGGAGATCAATCCACCGT GCAACCAAACACGCAGAATATAAGCCTCCTAGTAAAAGAAATGATAT TGCAATAGTGGAGTTTCATCCCCCGATGACACTCAACAGAGATGTTC AGCTAACAAAGATTGTTGAAGATGATGCTGAACTGCTACAGGAGAAG AAAAGCTACGTGACTGGTTTTGGTACATATACCTTCAAGAACAAGCA AGCTGTTACGTCAGATGAATTATTATGGGCTGAAATTGATTTGTTTGA CTTTTCTAGGTGCCAGCAATTGTGGCATCGACGGCTTTGGCAGAAAC AGATCTGTGCCGGTGCAAAGAATCTTGGAGCTGGACCGGGTGATTCT GGCGGGCCACTCCAAGTGTTGCATGATGGAACTCTTTTTCAAGTTGGC TTGACGAGTTATGGAACATCTGATAAGTTTGATGACGAGTTCAATCA AGACCGATTCCCTACAGTATTTACCCGCGTCTCGAGCTATTGTGATTT TATTGCTAAAGTCACTGATAATGCTGCGACTTGCTCAAGTTTATCCAA AAAACCAACTGCCAAGCCTGATTGCAGGTTC (SEQ ID NO:4). The E. coli-optimized gene was cloned in pET30a expression vector with HIS-tag on N-terminal in BamHI and HindIII restriction sites and transformed into BL21 (DE3) cells. The recombinant CLP was applied to Ni-NTA resins for affinity purification followed by size exclusion chromatography. The protease activity of purified recombinant CLP using a protease assay kit was performed and found to be active (Figure 6). CLP was also assayed against various insect species for characterization of insecticidal potency. The purified recombinant CLP was fed orally to various insect species by soaking 250 ng / cm2 of the protein onto the diet. Spodoptera frugiperda and Plutella xylostella and found to be 100% toxic (Figure 7). Similarly, it was fed to Manduca sexta, and significant weight loss was observed in Manduca sexta UC2023-989-2-PCT / / 3868.091WO1 larvae, but no mortality was observed. The whiteflies (Bemisia tabaci) were fed CLP 250ng / ml of artificial diet orally following a previously published method with modifications (Upadhyay et al., 2011). The protein was sandwiched between two layers of stretched parafilm and 20 whiteflies were released into vial, and data was collected every 24 hours. The recombinant CLP was toxic to whiteflies and killed 85% within 72 hours. The CAFÉ assay was used to evaluate toxicity of recombinant CLP against Drosophila melanogaster. It was toxic to Drosophila melanogaster and killed 96.3% of them within 72 hours (Figure 7). Bibliography Chang, D.Z., Serra, L., Lu, D.H., Mortazavi, A., and Dillman, A.R. (2019). A core set of venom proteins is released by entomopathogenic nematodes in the genus Steinernema. Plos Pathogens 15. ARTN e1007626 Dillman, A.R., Macchietto, M., Porter, C.F., Rogers, A., Williams, B., Antoshechkin, I., Lee, M.M., Goodwin, Z., Lu, X., Lewis, E.E., et al. (2015). Comparative genomics of Steinernema reveals deeply conserved gene regulatory networks. Genome Biol 16, 200. 10.1186 / s13059-015-0746-6. Lu, D., Macchietto, M., Chang, D., Barros, M.M., Baldwin, J., Mortazavi, A., and Dillman, A.R. (2017). Activated entomopathogenic nematode infective juveniles release lethal venom proteins. Plos Pathog 13, e1006302. 10.1371 / journal.ppat.1006302. Upadhyay, S.K., Chandrashekar, K., Thakur, N., Verma, P.C., Borgio, J.F., Singh, P.K., and Tuli, R. (2011). RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J Biosci 36, 153-161. 10.1007 / s12038- 011-9009-1. All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

UC2023-989-2-PCT / / 3868.091WO1 WHAT IS CLAIMED IS:

1. A composition comprising an insecticidal amount of an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.

2. The composition of claim 1 further comprising other insecticidal compounds.

3. The composition of claim 2, wherein the other insecticidal compounds are proteins are from S. carpocapsae, S. feltiae, or S. hermaphroditum.

4. The composition of claim 1 further comprising a carrier.

5. The composition of claim 4, wherein the carrier is a water-soluble liquid.

6. The composition of claim 4, wherein the carrier provides for sustained release.

7. The composition of claim 1 which is aerosolized.

8. The composition of claims 1, wherein the polypeptide comprises SEQ ID NO:1, an isolated polypeptide comprising SEQ ID NO:1 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.

9. The composition of claim 1, wherein the polypeptide comprises SEQ ID NO:2, an isolated polypeptide comprising SEQ ID NO:2 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.UC2023-989-2-PCT / / 3868.091WO1 10. The composition of claim 1, wherein the polypeptide comprises SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.

11. A non-nematode host cell that expresses a polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.

12. The host cell of claim 11 which is a bacterial cell.

13. The host cell of claim 11 which is a plant cell.

14. The host cell of claim 13 which is a crop plant cell.

15. The host cell of claim 13 which is a vegetable cell.

16. A method of preparing an insect-resistant transgenic plant, comprising: introducing to plant cells isolated nucleic acid encoding a polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.

17. The method of claim 16, wherein the plant is a crop plant.

18. The method of claim 16, wherein the plant is a tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rape seed, pulse, or sunflower plant.UC2023-989-2-PCT / / 3868.091WO1 19. A transgenic plant, the genome of which comprises nucleic acid encoding a polypeptide comprising, SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, an isolated polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 that lacks the endogenous signal peptide, or a polypeptide with at least 90% amino acid sequence identity thereto.

20. The plant of claim 19 which is a crop plant.

21. The plant of claim 19 which is a tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rape seed, pulse, or sunflower plant.

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