Broad-spectrum insecticidal polypeptides against lepidopteran pests and methods of use thereof

Abstract

The present disclosure provides nucleic acids obtained from Bacillus thuringiensis strains, and variants and fragments thereof, which encode polypeptides having pesticidal activity against insect pests including Lepidoptera. Particular embodiments of the present disclosure provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, DNA constructs, and transformed microorganisms and plants comprising the nucleic acids of the embodiments. These compositions can be applied in methods for controlling pests, particularly plant pests.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 410045, filed October 19, 2016, which is incorporated herein by reference in its entirety.

[0003] References to sequence lists submitted electronically

[0004] The sequence list named "7157WOPCT_SequenceListing.txt" was created on September 19, 2017, and is 47 kilobytes in size. It is submitted with this specification in a computer-readable form. This sequence list is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to naturally occurring and recombinant nucleic acids obtained from novel Bacillus thuringiensis genes encoding pest-killing polypeptides, which possess pest-killing activity against insect pests. The compositions and methods of this disclosure utilize the disclosed nucleic acids and their encoded pest-killing polypeptides to control plant pests. Background Technology

[0006] Insect pests are a major contributor to crop losses worldwide. For example, armyworm feeding, black rootworm damage, or European corn borer damage can be economically devastating for agricultural producers. Insect pest-related crop losses from European corn borer attacks on fields and sweet corn alone amount to approximately one billion US dollars annually in losses and control costs.

[0007] Traditionally, the primary method for controlling insect pest populations has been the application of broad-spectrum chemical insecticides. However, consumers and government regulators are increasingly concerned about the environmental hazards associated with the production and use of synthetic chemical pesticides. As a result of these concerns, regulators have banned or restricted the use of some of the more dangerous pesticides. Consequently, there is considerable interest in developing alternative pesticides.

[0008] The use of microbial agents (such as fungi, bacteria, or other insect species) for the biological control of agricultural insect pests offers an environmentally friendly and commercially attractive alternative to chemically synthesized pesticides. Generally, the use of biopesticides carries a lower risk of pollution and environmental harm, and they offer greater target specificity than traditional broad-spectrum chemical insecticides. Furthermore, biopesticides are often less expensive to produce, thus increasing the economic yield of various crops.

[0009] Certain species of Bacillus microorganisms are known to possess biocidal activity against a wide range of insect pests, including Lepidoptera, Diptera, Coleoptera, and Hemiptera. Bacillus thuringiensis (Bt) and Bacillus papilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus (Harwook, ed., (1989) Bacillus (Plenum Press), 306), and Bacillus cereus (WO96 / 10083). Although biocidal proteins have been isolated from the vegetative growth stage of Bacillus species, their biocidal activity appears to be concentrated in parasporal crystal protein inclusion bodies. Several genes encoding these biocidal proteins have been isolated and characterized (see, for example, U.S. Patent Nos. 5,366,892 and 5,840,868).

[0010] Microbial insecticides, particularly those derived from Bacillus strains, play a vital role in agriculture as an alternative to the chemical control of pests. Recently, agricultural scientists have developed crop plants with enhanced insect resistance by genetically engineering them to produce pest-killing proteins from Bacillus strains. For example, corn and cotton plants have been genetically engineered to produce pest-killing proteins isolated from Bt strains (see, for example, Aronson (2002) Cell Mol. Life Sci. 59(3): 417-425; Schnepf et al. (1998) Microbiol Mol Biol Rev. 62(3): 775-806). These genetically engineered crops are now widely used in U.S. agriculture and provide farmers with an environmentally friendly alternative to traditional insect control methods. Additionally, genetically engineered potatoes containing the pest-killing Cry toxin have been sold to U.S. farmers. Although they have proven to be very commercially successful, these genetically engineered insect-resistant crop plants may only provide resistance to a narrow range of economically important insect pests.

[0011] Therefore, there remains a need for new Bt toxins with insecticidal activity against a wider range of insect pests, such as toxins active against a greater variety of insects from the Lepidoptera order. Additionally, there remains a need for biocides active against a variety of insect pests, as well as biocides with improved insecticidal activity. Summary of the Invention

[0012] Compositions and methods for influencing insect pests are provided. More specifically, this disclosure relates to methods for influencing insects by utilizing nucleotide sequences encoding insecticidal peptides to produce transformed microorganisms and plants expressing the insecticidal peptides of the embodiments. In some aspects, these nucleotide sequences encode peptides that are pest control agents for at least one insect belonging to the order Lepidoptera.

[0013] In some aspects, nucleic acids and their fragments and variants are provided that encode polypeptides with pest-killing activity against insect pests (e.g., SEQ ID NO: 1 encodes SEQ ID NO: 2). Wild-type (e.g., naturally occurring) nucleotide sequences obtained from Bt encode novel insecticidal peptides. These embodiments further provide fragments and variants of the disclosed nucleotide sequences encoding biologically active (e.g., insecticidal) polypeptides.

[0014] In some aspects, isolated pest-killing (e.g., insecticidal) polypeptides encoded by naturally occurring or modified (e.g., mutagenic or manipulated) nucleic acids of the embodiments are provided. In specific aspects, the pest-killing proteins of the embodiments comprise fragments of full-length proteins and polypeptides generated by mutagenic nucleic acids designed to introduce specific amino acid sequences into the polypeptides of these embodiments. In specific aspects, these polypeptides have enhanced pest-killing activity relative to the activity of naturally occurring polypeptides derived therefrom.

[0015] In some aspects, the nucleic acids of the embodiments can also be used to generate transgenic (e.g., transformed) monocotyledonous or dicotyledonous plants with a genome comprising at least one stably incorporated nucleotide construct containing the coding sequence of the embodiments, the coding sequence being operatively linked to a promoter driving the expression of a biocidal polypeptide. Thus, transformed plant cells, plant tissues, plants, and seeds thereof are also provided.

[0016] In one specific aspect, the transformed plants can be produced using nucleic acids that have been optimized for increased expression in host plants. For example, one of the pest-killing peptides of the embodiments can be reverse-translated to produce nucleic acids containing codons optimized for expression in a specific host, such as a crop plant like corn (Zea mays). Expression of the coding sequence of such a transformed plant (e.g., a dicotyledonous or monocotyledonous plant) will result in the production of the pest-killing peptide and confer increased insect resistance to the plant. Some embodiments provide transgenic plants expressing pest-killing peptides that can be used in methods affecting a variety of insect pests.

[0017] In another aspect, pest control compositions or insecticidal compositions comprising the insecticidal peptides of these embodiments are provided, and may optionally contain other insecticidal peptides. In yet another aspect, it is provided to apply such compositions to an environment in which insect pests exist to influence the insect pests. Attached Figure Description

[0018] Figure 1A-1B Display using Vector kit The amino acid sequences of modules Cry1Ea (SEQ ID NO: 6) and SEQ ID NO: 2 (amino acids 1-617, MP372) were aligned. Sequence diversity was highlighted. The connection point between domain I and domain II of the MP327-Mut5 chimera (SEQ ID NO: 4) in Example 3 is indicated by “…”. "express. Detailed Implementation

[0019] Embodiments of this disclosure relate to compositions and methods for influencing insect pests, particularly plant pests. More specifically, the isolated nucleic acids and their fragments and variants in the embodiments comprise nucleotide sequences encoding pest-killing polypeptides (e.g., proteins). The disclosed pest-killing proteins are biologically active (e.g., pest-killing) against insect pests (e.g., but not limited to lepidopteran insect pests).

[0020] The compositions of the embodiments comprise isolated nucleic acids and fragments and variants thereof encoding pest-killing polypeptides, expression cassettes containing the nucleotide sequences of the embodiments, isolated pest-killing proteins, and pest-killing compositions. Some embodiments provide modified pest-killing polypeptides characterized by improved insecticidal activity against lepidopteran insects, relative to the pest-killing activity of the corresponding wild-type proteins. The embodiments further provide plants and microorganisms transformed with these novel nucleic acids, and methods relating to using such nucleic acids, pest-killing compositions, transformed organisms, and their products to influence insect pests.

[0021] In some embodiments, the isolated nucleic acid molecule encoding the MP372 polypeptide has one or more variations in its nucleic acid sequence compared to the natural or genomic nucleic acid sequence. In some embodiments, variations in the natural or genomic nucleic acid sequence include, but are not limited to: variations in the nucleic acid sequence due to the degeneracy of the genetic code; variations in the nucleic acid sequence due to amino acid substitutions, insertions, deletions, and / or additions compared to the natural or genomic sequence; removal of one or more introns; deletion of one or more upstream or downstream regulatory regions; and deletion of 5′ and / or 3′ untranslated regions associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the MP372 polypeptide is a non-genomic sequence.

[0022] The nucleic acid and nucleotide sequences of the embodiments can be used to transform any organism to produce encoded pest-killing proteins. Methods relating to using such transformed organisms to influence or control plant pests are provided. The nucleic acid and nucleotide sequences of the embodiments can also be used to transform organelles such as chloroplasts (McBride et al. (1995) Biotechnology 13:362-365; and Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96:1840-1845).

[0023] The examples further relate to the identification of fragments and variants of naturally occurring coding sequences encoding bioactive pest-killing proteins. The nucleotide sequences of the examples can be directly applied to methods affecting pests, particularly insect pests such as lepidopteran pests. Therefore, the examples provide novel methods for affecting insect pests without relying on the use of conventional chemically synthesized insecticides. The examples also relate to the discovery of naturally occurring biodegradable pest-killing agents and the genes encoding them.

[0024] The embodiments further provide fragments and variants of naturally occurring coding sequences that also encode biologically active (e.g., pest-killing) polypeptides. The nucleic acids of the embodiments encompass nucleic acid or nucleotide sequences that have been optimized for expression by the cells of a particular organism, such as nucleic acid sequences that have been reverse-translated (i.e., back-translated) using plant-preferred codons based on the amino acid sequence of a polypeptide having enhanced pest-killing activity. The embodiments further provide mutations that confer improved or altered properties to the polypeptides of the embodiments. See, for example, U.S. Patent 7,462,760.

[0025] A large number of terms are used extensively in the following description. The following definitions are provided to aid in understanding these embodiments.

[0026] Units, prefixes, and symbols may be represented in their SI-accepted forms. Unless otherwise specified, nucleic acids are written from left to right in a 5′ to 3′ direction; amino acid sequences are written from left to right in an amino-to-carboxyl direction. Numerical ranges include the values ​​that define those ranges. Amino acids described herein may be represented by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Terminology. Similarly, nucleic acids may be represented by their commonly accepted single-letter codes. The terms defined above are generally defined more completely with reference to the specification.

[0027] As used herein, “nucleic acid” includes reference to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, covers known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides due to hybridization to single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides.

[0028] As used herein, when used in the context of a specified nucleic acid, the term "encoding" or "encoded" means that the nucleic acid contains the necessary information to directly translate the nucleotide sequence into the specified protein. The information used to encode the protein is described in detail by the use of codons. A nucleic acid that encodes a protein may contain untranslated sequences (e.g., introns) or untranslated sequences that may lack such insertions (e.g., in cDNA).

[0029] As used herein, references to the “full-length sequence” of a specific polynucleotide or its encoded protein refer to the entire nucleic acid sequence or amino acid sequence having a natural (non-synthetic) endogenous sequence. Full-length polynucleotides encode the full-length, catalytically active form of a specific protein.

[0030] As used herein, the term "antisense" in the context of nucleotide sequence orientation refers to a double-stranded polynucleotide sequence operatively linked to a promoter in the direction of transcription of the antisense strand. The antisense strand is highly complementary to the endogenous transcript, thus often repressing the translation of the endogenous transcript. Therefore, when the term "antisense" is used in the context of a specific nucleotide sequence, it refers to the complementary strand of the reference transcript.

[0031] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.

[0032] The terms “residue” or “amino acid residue” or “amino acid” are used interchangeably herein and refer to an amino acid incorporated into a protein, polypeptide, or peptide (collectively referred to as “protein”). An amino acid can be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of naturally occurring amino acids that function in a manner similar to that of naturally occurring amino acids.

[0033] The peptides of the embodiments can be generated from the nucleic acids disclosed herein or by using standard molecular biology techniques. For example, the proteins of the embodiments can be generated by expressing the recombinant nucleic acids of the embodiments in suitable host cells or alternatively by a combination of in vitro procedures.

[0034] As used herein, the terms “isolated” and “purified” are used interchangeably and refer to nucleic acids or polypeptides or their biologically active portions that are substantially or substantially free of components typically associated with or interacting with the nucleic acid or polypeptide as found in their natural environment. Therefore, isolated or purified nucleic acids or polypeptides are substantially free of other cellular material or the culture medium used when produced via recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0035] "Isolated" nucleic acids generally do not contain sequences naturally located flanking the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) (e.g., protein-coding sequences) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, isolated nucleic acids may contain nucleotide sequences of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb that are naturally located flanking the nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived.

[0036] As used herein, the terms “isolated” or “purified,” such as when used to refer to peptides in the examples, mean isolated proteins that are substantially free of cellular material and comprise protein formulations having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminated protein. When recombinantly producing the proteins of the examples or their bioactive portions, the culture medium contains less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-target protein chemicals.

[0037] Throughout this specification, the word “comprising” or variations thereof, such as “comprises” or “comprising”, shall be understood to imply the presence of one stated element, whole or step, or group of elements, whole or steps, but does not exclude any other element, whole or step, or group of elements, whole or steps.

[0038] In some embodiments, the MP372 insecticidal protein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 2. In some embodiments, the MP372 insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the MP372 insecticidal protein having SEQ ID NO: 2 is processed from a protoxin form to a mature toxin form. In some embodiments, the mature toxin comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with amino acids 1-617 of SEQ ID NO: 2. In some embodiments, the mature MP372 toxin is fused with a heterologous Cry protein protoxin region to form a chimeric protoxin form. In some embodiments, the mature MP372 toxin having amino acids 1-617 of SEQ ID NO: 2 is fused with a heterologous Cry protein protoxin region to form a chimeric protoxin form. In some embodiments, the MP372 insecticidal protein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 4. In some embodiments, the MP372 insecticidal protein comprises the amino acid sequence of SEQ ID NO: 4.

[0039] As used herein, the term "impacting insect pests" refers to changes that affect insect feeding, growth, and / or behavior at any stage of development, including but not limited to: killing insects; slowing growth; inhibiting reproductive capacity; and refusing to feed.

[0040] As used herein, the terms “pesticide activity” and “insecticide activity” are used synonymously to refer to the activity of an organism or substance (e.g., like a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest resistance, and other behavioral and physical changes in pests following adequate feeding and exposure. Thus, an organism or substance with pesticide activity adversely affects at least one measurable parameter of pest fitness. For example, a “pesticide protein” is a protein that, on its own or in combination with other proteins, exhibits pesticide activity.

[0041] As used herein, the term "effective pest control amount" refers to the amount of a substance or organism that is effective in killing pests when present in a pest environment. For each substance or organism, the effective pest control amount is determined empirically for each pest affected in a particular environment. Similarly, when the pest is an insect pest, the term "effective insect control amount" can be used to represent "effective pest control amount".

[0042] As used herein, the terms “recombinant engineered” or “engineering” refer to changes in protein structure introduced (e.g., engineered) using recombinant DNA technology based on an understanding and consideration of the protein’s mechanism of action, by introducing, deleting, or substituting amino acids.

[0043] As used herein, the terms “mutant nucleotide sequence” or “mutation” or “mutated nucleotide sequence” refer to a nucleotide sequence that has been mutated or altered to include one or more nucleotide residues (e.g., base pairs) not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions, substitutions, or replacements of nucleic acid residues. When mutation is performed by adding, removing, or replacing amino acids at proteolytic sites, such additions, removals, or substitutions can be within or near the proteolytic site motif, as long as the purpose of mutation is achieved (i.e., as long as protein hydrolysis at the site changes).

[0044] Mutant nucleotide sequences may encode mutant insecticidal toxins exhibiting improved or reduced insecticidal activity, or amino acid sequences that encode amino acid sequences conferring improved or reduced insecticidal activity to polypeptides containing them. As used herein, the term “mutant” or “mutation” in the context of a protein or polypeptide or amino acid sequence means a sequence that has been mutated or altered to include one or more amino acid residues not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of the addition, deletion, or substitution of one or more amino acid residues. Mutant polypeptides exhibit improved or reduced insecticidal activity, or amino acid sequences that confer improved insecticidal activity to polypeptides containing them. Thus, the term “mutant” or “mutation” refers to one or both of the mutant nucleotide sequence and the encoded amino acid. Mutants may be used alone or in combination with other mutants of the examples or with other mutants in any compatible combination. A “mutant polypeptide” may conversely exhibit reduced insecticidal activity. In cases where more than one mutation is added to a particular nucleic acid or protein, mutations may be added simultaneously or sequentially; if sequential, mutations may be added in any suitable order.

[0045] As used herein, the terms "improved insecticidal activity" or "improved pesticidal activity" refer to an insecticidal peptide of an embodiment that has enhanced insecticidal activity relative to the activity of its corresponding wild-type protein, and / or an insecticidal peptide effective against a wider range of insects, and / or an insecticidal peptide specific to insects insensitive to the toxicity of wild-type proteins. The discovery of improved or enhanced pesticidal activity requires demonstrating an increase of at least 10% in pesticidal activity against an insect target, or an increase of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, or 300% or higher in pesticidal activity relative to the wild-type insecticidal peptide identified against the same insect.

[0046] For example, improved pest-killing or insecticidal activity is provided, wherein a larger or narrower range of insects are affected by the polypeptide relative to the range of insects affected by wild-type Bt toxins. Where versatility is desired, a broader range of effects may be desired, while where, for example, beneficial insects might otherwise be affected by the use or presence of the toxin, a narrower range of effects may be desired. While the embodiments are not limited to any particular mechanism of action, improved pest-killing activity can also be provided by altering one or more characteristics of the polypeptide; for example, the stability or lifetime of the polypeptide in the insect gut may be increased relative to the stability or lifetime of the corresponding wild-type protein.

[0047] As used herein, the term "toxin" refers to a polypeptide that exhibits pest-killing or insecticidal activity or improved pest-killing or insecticidal activity. "Bt" or "Bacillus thuringiensis" toxins are intended to encompass a broader range of Cry toxins found in various Bt strains, including, for example, Cry1, Cry2, or Cry3 toxins.

[0048] The term "proteolytic site" or "cleavage site" refers to an amino acid sequence that confers sensitivity to a class or specific protease, thereby enabling the digestion of a polypeptide containing that amino acid sequence by that protease. A proteolytic site is considered to be "sensitive" to one or more proteases that recognize that site. It should be understood in the art that digestion efficiency will vary, and a decrease in digestion efficiency can lead to an increase in the stability or lifespan of the polypeptide in the insect gut. Therefore, a proteolytic site can confer sensitivity to more than one protease or class of proteases, but the digestive efficiency of various proteases at that site may differ. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites.

[0049] Studies have shown that intestinal proteases in lepidopteran insects include trypsin, chymotrypsin, and elastase. See, for example, Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212; and Hedegus et al. (2003) Arch. Insect Biochem. Physiol. 53: 30-47. For example, about 18 different trypsin species have been identified in the midgut of the cotton bollworm (Helicoverpa arnugera) larvae (see Gatehouse et al. (1997) Insect Biochem. Mol. Biol. 27: 929-944). Preferred proteolytic substrate sites for these proteases have been investigated. See Peterson et al. (1995) Insect Biochem. Mol. Biol. 25: 765-774.

[0050] Efforts have been made to understand the mechanisms of action of Bt toxins and to engineer toxins using improved performance. Insect intestinal proteases have been shown to influence the effects of Bt Cry proteins on insects. Some proteases activate Cry proteins by processing them from their “protoxin” form into a toxic form, or “toxin.” See Oppett (1999) Arch. Insect Biochem. Phys. 42: 1–12; and Carroll et al. (1997) J. Invertebrate Pathology 70: 41–49. This activation of the toxin can include the removal of N-terminal and C-terminal peptides from the protein and can also include internal cleavage of the protein. Other proteases can degrade Cry proteins. See Oppett, ibid.

[0051] Comparison of the amino acid sequences of different specific Cryotoxins revealed five highly conserved sequence blocks. Structurally, the toxins contain three distinct domains from the N-terminus to the C-terminus: a cluster of seven α-helices involved in pore formation (referred to as “domain 1”), three antiparallel β-sheets involved in cell binding (referred to as “domain 2”), and a β-sandwich (referred to as “domain 3”). The location and properties of these domains are known to those skilled in the art. See, for example, Li et al. (1991) Nature, 305: 815-821 and Morse et al. (2001) Structure, 9: 409-417. When referring to a particular domain, such as domain 1, it should be understood that the exact endpoints of the particular sequence domain are not critical, as long as its sequence or portion includes sequences that provide at least some of the function attributed to the particular domain. Thus, for example, when referring to “domain 1”, it is intended that the particular sequence includes a cluster of seven α-helices, but the exact endpoints of the sequences used or referenced by that cluster are not critical. Those skilled in the art are familiar with the identification of such endpoints and the evaluation of such functions.

[0052] To better characterize and improve Bt toxins, strains of the bacterium Bt were investigated. Crystallization formulations prepared from cultures of Bt strains were found to have biocidal activity against a wide range of lepidopteran pests (see, for example, Example 1). Efforts were made to identify the nucleotide sequences encoding crystal proteins from selected strains, and wild-type (i.e., naturally occurring) nucleic acids of examples were isolated from these bacterial strains, cloned into expression vectors, and transformed into *E. coli*. Depending on the characteristics of a given formulation, it was recognized that proof of biocidal activity sometimes requires trypsin pretreatment to activate the biocidal proteins. Therefore, it should be understood that some biocidal proteins require protease digestion (e.g., by trypsin, chymotrypsin, and the like) for activation, while others are bioactive (e.g., biocidal) without activation.

[0053] Such molecules can be modified using, for example, the methods described in U.S. Patent 7,462,760. Additionally, nucleic acid sequences can be engineered to encode polypeptides containing additional mutations that impart improved or altered cytotoxic activity relative to the naturally occurring polypeptides. The nucleotide sequences of such engineered nucleic acids contain mutations not found in the wild-type sequence.

[0054] The mutant peptides of the examples are generally prepared by a method involving the following steps: obtaining a nucleic acid sequence encoding a Cry family peptide; analyzing the structure of the peptide to identify specific “target” sites for mutagenesis of potential gene sequences based on considerations of the function of target domains proposed in the mode of action of toxins; introducing one or more mutations into the nucleic acid sequence to produce the desired change in one or more amino acid residues of the encoded peptide sequence; and determining the necrotic bioactivity of the resulting peptide.

[0055] Many Bt insecticidal toxins are related to varying degrees through similarities in their amino acid sequences and tertiary structures, and methods for obtaining the crystal structures of Bt toxins are well known. Exemplary high-resolution crystal structure solutions for both Cry3A and Cry3B peptides are available in the literature. The resolved structure of the Cry3A gene (Li et al. (1991) Nature 353: 815-821) provides insights into the relationship between the structure and function of the toxin. A comprehensive consideration of published structural analyses of Bt toxins and reported functions associated with specific structures, motifs, etc., suggests that specific regions of the toxin are associated with specific functions and discrete steps in the protein's mode of action. For example, many toxins isolated from Bt are generally described as comprising three domains: a seven-helical bundle involved in pore formation, a three-lamellar domain associated with receptor binding, and a β-sandwich motif (Li et al. (1991) Nature 305: 815-821).

[0056] As reported in U.S. Patent Nos. 7,105,332 and 7,462,760, the toxicity of Cry protein can be improved by targeting the region between α-helices 3 and 4 of domain 1 of the toxin. This theory is based on a knowledge system of insecticidal toxins, including: 1) the reported insertion of α-helices 4 and 5 of domain 1 of Cry3A toxin into the lipid bilayer of cells in the midgut of susceptible insects (Gazit et al. (1998) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 95: 12289-12294); 2) the inventors’ knowledge of the location of trypsin and chymotrypsin cleavage sites within the amino acid sequence of the wild-type protein; 3) the observation that the wild-type protein is more active against certain insects after in vitro activation by treatment with trypsin or chymotrypsin; and 4) reports that digestion of the toxin from the 3′ end leads to reduced toxicity to insects.

[0057] A series of mutations can be generated and placed in various background sequences to produce novel peptides with enhanced or altered cytotoxic activity. See, for example, U.S. Patent 7,462,760. These mutants include, but are not limited to: adding at least one more protease-sensitive site (e.g., a trypsin cleavage site) in the region between helices 3 and 4 of domain 1; replacing the original protease-sensitive site in the wild-type sequence with a different protease-sensitive site; adding multiple protease-sensitive sites at specific locations; adding amino acid residues near one or more protease-sensitive sites to alter the folding of the peptide and thus enhance digestion of the peptide at that one or more protease-sensitive sites; and adding mutations to protect the peptide from degradative digestion that reduces toxicity (e.g., creating a series of mutations in which wild-type amino acids are replaced with valine to protect the peptide from digestion). Mutations can be used alone or in any combination to provide the peptides of the examples.

[0058] In this way, the embodiments provide sequences containing a variety of mutations, such as mutations in additional or alternative protease-sensitive sites located between α-helices 3 and 4 of domain 1 of the encoded polypeptide.

[0059] Mutations that serve as alternative or substitute protease-sensitive sites may sensitize several classes of proteases, such as serine proteases (including trypsin and chymotrypsin), or enzymes such as elastase. Therefore, mutations can be engineered to serve as alternative or substitute protease-sensitive sites so that the site is readily recognized and / or cleaved by a class of proteases (e.g., mammalian or insect proteases). Protease-sensitive sites can also be engineered to be cleaved by specific classes of enzymes or specific enzymes known to be produced in organisms (e.g., chymotrypsin produced by the corn moth (Heliothis zea)) (Lenz et al. (1991) Arch. Insect Biochem. Physiol. [Archives of Insect Biochemistry and Physiology] 16: 201-212). Mutations can also confer resistance to proteolytic digestion, for example, resistance to digestion by chymotrypsin at the C-terminus of a peptide.

[0060] The presence of additional and / or alternative protease-sensitive sites in the amino acid sequence of the encoded polypeptide can improve the pesticidal activity and / or specificity of the polypeptide encoded by the nucleic acid of the examples. Therefore, the nucleotide sequences of the examples can be recombinantly engineered or manipulated to produce polypeptides with improved or altered insecticidal activity and / or specificity compared to unmodified wild-type toxins. Additionally, the mutations disclosed herein can be placed in or combined with other nucleotide sequences to provide improved properties. For example, protease-sensitive sites readily cleaved by insect chymotrypsin (e.g., chymotrypsin found in the Bertha armyworm or the corn ear moth) (Hegedus. (2003) Arch. Insect Biochem. Physiol. [Archives of Insect Biochemistry and Physiology] 53:30-47; and Lenz et al. (1991) Arch. Insect Biochem. Physiol. [Archives of Insect Biochemistry and Physiology] 16:201-212) can be placed in a Cry background sequence to provide improved toxicity to that sequence. In this way, the examples provide toxic peptides with improved properties.

[0061] For example, the mutagenized Cry nucleotide sequence may contain additional mutants that include additional codons that introduce a second trypsin-sensitive amino acid sequence (in addition to the naturally occurring trypsin site) into the encoded polypeptide. Alternative addition mutants of the examples include additional codons designed to introduce at least one additional, different protease-sensitive site into the polypeptide, such as a 5′ or 3′ chymotrypsin-sensitive site directly located at the naturally occurring trypsin site. Alternatively, substitution mutants may be generated in which at least one codon of the nucleic acid encoding the naturally occurring protease-sensitive site is disrupted, and an alternative codon is introduced into the nucleic acid sequence to provide a different (e.g., substituted) protease-sensitive site. Substitution mutants may also be added to the Cry sequence in which a naturally occurring trypsin cleavage site present in the encoded polypeptide is disrupted, and a chymotrypsin or elastase cleavage site is introduced at its position.

[0062] It has been recognized that any nucleotide sequence encoding an amino acid sequence that serves as a proteolytic site or a putative proteolytic site (e.g., sequences of RR or LKM, for example) can be used, and the exact identity of the codons used to introduce any of these cleavage sites into the variant polypeptide can vary depending on the intended use (i.e., expression in a particular plant species). It should also be recognized that any disclosed mutation can be introduced into any polynucleotide sequence of the embodiments containing codons of amino acid residues that provide a target natural trypsin cleavage site for modification. Therefore, variants of the full-length toxin or fragments thereof can be modified to include additional or alternative cleavage sites, and these embodiments are intended to be covered by the scope of the embodiments disclosed herein.

[0063] Those skilled in the art will understand that any useful mutation can be added to the sequence of the examples, provided that the encoded polypeptide retains its pest-killing activity. Therefore, the sequence can also be mutated to make the encoded polypeptide resistant to proteolytic digestion by chymotrypsin. More than one recognition site can be added to a particular location in any combination, and multiple recognition sites can be added to or removed from the toxin. Therefore, additional mutations can contain three, four, or more recognition sites. It should be recognized that multiple mutations can be engineered in any suitable polynucleotide sequence; thus, the full-length sequence or fragments thereof can be modified to include additional or alternative cleavage sites and make it resistant to proteolytic digestion. In this way, the examples provide Cry toxins containing mutations that improve pest-killing activity, as well as compositions and methods for improving pest control using other Bt toxins.

[0064] Mutations can protect peptides from protease degradation, for example, by removing putative proteolytic sites from different regions, such as putative serine protease sites and elastase recognition sites. Some or all of these putative sites can be removed or altered to reduce proteolysis at the original site location. Changes in proteolysis can be assessed by comparing mutant peptides to wild-type toxins or by comparing mutant toxins with different amino acid sequences. Putative proteolytic sites, and proteolytic sites, include, but are not limited to, the following sequences: RR, trypsin cleavage site; LKM, chymotrypsin site; and trypsin site. These sites can be altered by adding or deleting any number and type of amino acid residues, as long as the peptide's cytotoxic activity increases. Therefore, a polypeptide encoded by a mutated nucleotide sequence will contain at least one amino acid alteration or addition relative to the native or background sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 or more amino acid alterations or additions. As is known in the art, the biotoxic activity of a polypeptide can also be improved by truncating the native or full-length sequence.

[0065] In another embodiment, a fusion protein is provided whose amino acid sequence comprises the MP372 polypeptide or a chimeric MP372 polypeptide of this disclosure. Methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art. The polynucleotides encoding the MP372 polypeptide can be fused to signal sequences that will direct the MP372 polypeptide to a specific compartment of a prokaryotic or eukaryotic cell and / or direct the secretion of the MP372 polypeptide from embodiments of prokaryotic or eukaryotic cells. For example, in *E. coli*, it may be desirable to direct protein expression into the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) that can be fused to direct polypeptide expression into the bacterial periplasmic space include, but are not limited to: the pelB signal sequence, the maltose-binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the heat-labile enterotoxin B subunit of *E. coli* periplasmic protein, and the signal sequence of alkaline phosphatase. Several vectors for constructing fusion proteins that guide protein localization are commercially available, such as the pMAL series vectors (particularly the pMAL-p series) available from New England Biolabs. In specific embodiments, the MP372 peptide can be fused with the pelB pectic acid lyase signal sequence to increase the efficiency of expression and purification of these peptides in Gram-negative bacteria (see U.S. Patents 5,576,195 and 5,846,818). Plant plasmid transport peptide / peptide fusions are well known in the art. Apoplast transport peptides, such as rice or barley α-amylase secretion signals, are also well known in the art. The plasmid transport peptide is typically N-terminally fused to a target peptide (e.g., a fusion coupler). In one embodiment, the fusion protein consists essentially of the target plasmid transport peptide and the MP372 peptide. In another embodiment, the fusion protein comprises the target plasmid transport peptide and a peptide. In such embodiments, the plasmid transport peptide is preferably located at the N-terminus of the fusion protein. However, additional amino acid residues may be located at the N-terminus of the plasmid transport peptide, provided that the fusion protein at least partially targets the plasmid. In specific embodiments, the plasmid transport peptide is located at the half, one-third, or one-quarter of the N-terminus of the fusion protein. When inserted into the plasmid, most or all of the plasmid transport peptide is typically cleaved from the fusion protein. Due to specific intercellular conditions or the specific combination of transport peptide / fusion partner used, the cleavage site may vary slightly between plant species at different plant developmental stages. In one embodiment, the plasmid transport peptide cleavage is uniform, such that the cleavage site is identical throughout the fusion protein population. In another embodiment, the plasmid transport peptide is not uniform, such that the cleavage sites differ by 1-10 amino acids within the fusion protein population. The plasmid transport peptide can be recombinantly fused to a second protein in one of several ways.For example, restriction endonuclease recognition sites can be introduced into the nucleotide sequence of the transport peptide corresponding to its C-terminus, and identical or compatible sites can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminus. Care must be taken to design these sites to ensure that the coding sequences of the transport peptide and the second protein remain “in-the-box” to allow for the synthesis of the desired fusion protein. In some cases, when introducing new restriction sites, it is preferable to remove the initiation factor methionine of the second protein. Introducing restriction endonuclease recognition sites on two parent molecules, and their subsequent ligation via recombinant DNA technology, can result in the addition of one or more additional amino acids between the transport peptide and the second protein. This generally does not affect targeting activity, as long as the transport peptide cleavage site remains accessible, and the addition of these additional amino acids at its N-terminus does not alter the function of the second protein. Alternatively, those skilled in the art can use gene synthesis (Stemmer et al., (1995) Gene 164:49-53) or similar methods to generate precise cleavage sites (with or without their initiator methionine) between the transport peptide and the second protein. Additionally, transport peptide fusions can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of a mature protein can affect the ability of the transport peptide to target the protein to the plasmid and / or the cleavage efficiency after protein input. This may depend on the protein to be targeted. See, for example, Comai et al., (1988) J. Biol. Chem. 263(29): 15104-9. In some embodiments, the MP372 peptide is fused with a heterologous signal peptide or a heterologous transport peptide.

[0066] In some embodiments, the insecticidal protein is a chimeric Cry1E polypeptide comprising: a) domain I of a first Cry1E protein, and b) domains II and III of a second Cry1E protein or a fragment or variant thereof, as specified in SEQ ID NO: 2. In some embodiments, the insecticidal protein is a chimeric Cry1E polypeptide comprising: a) domain I of a Cry1Ea protein (accession number X53985) as specified in SEQ ID NO: 6, and b) domains II and III of a Cry1 protein as specified in SEQ ID NO: 2. In some embodiments, the insecticidal protein is a chimeric Cry1E polypeptide comprising: a) domain I having at least 95% sequence identity with amino acids 36-253 of SEQ ID NO: 6, and b) domains II and III having at least 95% sequence identity with amino acids 259-617 of SEQ ID NO: 2. In some embodiments, the insecticidal protein is a chimeric Cry1E polypeptide comprising amino acids 36-259 of SEQ ID NO: 6 and amino acids 259-617 of SEQ ID NO: 2. In some embodiments, the insecticidal protein is a chimeric Cry1E polypeptide comprising amino acids 1-259 of SEQ ID NO: 6 and amino acids 259-617 of SEQ ID NO: 2. In some embodiments, the chimeric Cry1E polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 4. In some embodiments, the chimeric Cry1E polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

[0067] The compositions of the embodiments comprise nucleic acids encoding biotoxic polypeptides and fragments and variants thereof. In particular, the embodiments provide isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4, or nucleotide sequences encoding said amino acid sequences (e.g., the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3) and fragments and variants thereof.

[0068] Also of interest are optimized nucleotide sequences encoding the biocidal proteins of the embodiments. As used herein, the phrase “optimized nucleotide sequence” refers to a nucleic acid optimized for expression in a particular organism, such as a plant. Optimized nucleotide sequences can be prepared for any target organism using methods known in the art. See, for example, U.S. Patent No. 7,462,760, which describes optimized nucleotide sequences encoding the disclosed biocidal proteins. Murray et al. (1989) Nucleic Acids Res. 17:477-498 describe the procedure in more detail. Optimal nucleotide sequences can be applied to increase the expression of biocidal proteins in plants, such as monocotyledonous plants of the Gramineae (or Poaceae) family, such as corn or maize plants.

[0069] The embodiments further provide isolated pest-killing (e.g., insecticidal) polypeptides encoded by naturally occurring or modified nucleic acids of these embodiments. More specifically, the embodiments provide polypeptides comprising the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4, as well as polypeptides encoded by nucleic acids described herein, such as those shown in SEQ ID NO: 1 or SEQ ID NO: 3, and fragments and variants thereof.

[0070] In specific embodiments, the pest-killing protein of the embodiments provides a full-length insecticidal polypeptide, fragments of the full-length insecticidal polypeptide, and variant polypeptides, which are generated from mutagenic nucleic acids designed to introduce specific amino acid sequences into the polypeptides of the embodiments. In specific embodiments, the amino acid sequence of the introduced polypeptide includes a sequence that provides a cleavage site for an enzyme, such as a protease.

[0071] It is known in the art that the pesticidal activity of Bt toxins is typically activated by cleavage of peptides in the insect gut by various proteases. Because peptides may not always be completely and efficiently cleaved in the insect gut, fragments of the full-length toxin may possess enhanced pesticidal activity compared to the full-length toxin itself. Therefore, some peptides in the examples comprise fragments of the full-length insecticidal peptide, and some peptide fragments, variants, and mutations will have enhanced pesticidal activity relative to the activity of their derived naturally occurring insecticidal peptides, particularly if the naturally occurring insecticidal peptide is not activated in vitro with proteases prior to activity screening. Therefore, this application covers truncated versions or fragments of the sequence.

[0072] Mutations can be placed in any background sequence (including such truncated peptides) as long as the peptide retains its necrotic activity. Those skilled in the art can readily compare the necrotic activity of two or more proteins using assays known in the art or described elsewhere herein. It should be understood that the peptides of the examples can be generated by expression of the nucleic acids disclosed herein or by using standard molecular biology techniques.

[0073] It has been recognized that pest-killing proteins can be oligomeric and will vary in molecular weight, residue number, component peptides, activity against specific pests, and other characteristics. However, the methods described herein allow for the isolation and characterization of proteins active against a wide variety of pests. The pest-killing proteins of the embodiments can be used in combination with other Bt toxins or other insecticidal proteins to increase the insect target range. Furthermore, the pest-killing proteins of the embodiments, when used in combination with other Bt toxins or other insecticidal mechanisms with different properties, have particular efficacy in preventing and / or managing insect resistance. Other insecticidal agents include protease inhibitors (both serine and cysteine ​​types), α-amylases, and peroxidases.

[0074] Fragments and variants of nucleotide and amino acid sequences, and the polypeptides encoded therefrom, are also covered in the examples. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of the examples. Fragments of nucleotide sequences may encode protein fragments that retain the biological activity of the native or corresponding full-length protein, and thus possess virulent biological activity. Therefore, it is generally accepted that some polynucleotide and amino acid sequences of the examples may be properly referred to as both fragments and mutants.

[0075] It should be understood that the term "fragment," as used to refer to the nucleic acid sequence of the embodiment, also encompasses sequences used as hybridization probes. These nucleotide sequences typically do not encode biologically active fragment proteins. Therefore, the range of nucleotide sequence fragments can be from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to the full-length nucleotide sequence encoding the protein of the embodiment.

[0076] Fragments of the nucleotide sequence of the embodiments encoding the bioactive portion of the biocidal protein of the embodiments will encode at least 15, 25, 30, 50, 100, 200, 250, or 300 consecutive amino acids, or up to the total number of consecutive amino acids present in the biocidal peptide of the embodiments (e.g., 1176 amino acids in SEQ ID NO: 2). Therefore, it should be understood that the embodiments also cover fragments of exemplary biocidal proteins as embodiments and having a length of at least 15, 25, 30, 50, 100, 200, 250, or 300 consecutive amino acids, or a length up to the total number of consecutive amino acids present in the biocidal peptide of the embodiments (e.g., 1176 amino acids in SEQ ID NO: 2). Fragments of the nucleotide sequence of the embodiments that can be used as hybridization probes or PCR primers generally do not need to encode the bioactive portion of the biocidal protein. Therefore, fragments of the nucleic acids of the embodiments may encode the bioactive portion of the biocidal protein, or may be fragments capable of being used as hybridization probes or PCR primers using the methods disclosed herein. The bioactive portion of a biocidal protein can be prepared by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the coding portion of the biocidal protein (e.g., by in vitro recombinant expression), and evaluating the activity of the coding portion of the biocidal protein.

[0077] The nucleic acid fragments of the nucleotide sequences used as examples comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 850, 900, or 950 nucleotides, or up to the number of nucleotides present in the nucleotide sequences disclosed herein (e.g., 3528 nucleotides of SEQ ID NO: 1). Specific embodiments contemplate fragments derived from (e.g., generated from) the first nucleic acid of the embodiments, wherein the fragment encodes a truncated toxin characterized by virulent biological activity. Truncated polypeptides encoded by the polynucleotide fragments of the embodiments are characterized by equivalent or improved virulent biological activity relative to the activity of a corresponding full-length polypeptide encoded by the first nucleic acid from which the fragment is derived. It is contemplated that such nucleic acid fragments of the embodiments may be truncated at the 3′ end of the native or corresponding full-length coding sequence. Nucleic acid fragments can also be truncated at both the 5′ and 3′ ends of the natural or corresponding full-length coding sequence.

[0078] As used herein, the term "variant" refers to substantially similar sequences. For nucleotide sequences, conserved variants include sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the biocidal polypeptides of the embodiments. Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, there are many nucleotide sequences that encode this disclosure.

[0079] Nucleic acids can be optimized to increase expression in the host organism when appropriate. Therefore, in the case of a plant host organism, synthetic nucleic acids can be synthesized using plant-preferred codons to improve expression. For a discussion of host-preferred codon usage, see, for example, Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92: 1-11. For example, while the nucleic acid sequences of the examples can be expressed in both monocot and dicot species, the sequences can be modified to address monocot or dicot-specific codon preferences and GC content preferences, as these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17: 477-498). Thus, corn-preferred codons for specific amino acids can be derived from known gene sequences of corn. Corn codon usage from 28 genes of the corn plant is listed in Table 4 of Murray et al. (ibid.). Methods for synthesizing plant-preferred genes are available in the art. See, for example, Murray et al., (1989) Nucleic Acids Res. 17: 477-498, and Liu H et al., Mol Bio Rep. 37: 677-684, 2010, which are incorporated herein by reference. A codon usage table for maize (Zea maize) can also be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=4577, accessible with the www prefix. Analysis of optimal codons for maize (adapted from Liu H et al., Mol Bio Rep. 37: 677-684, 2010).

[0080] The soybean codon usage table can be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=3847&aa=1&style=N, and can be accessed using the www prefix.

[0081] Those skilled in the art will further understand that changes can be introduced through mutations in the nucleotide sequence, resulting in alterations in the amino acid sequence encoding the polypeptide without changing the biological activity of the protein. Therefore, variant nucleic acid molecules can be generated by introducing one or more nucleotide substitutions, additions, and / or deletions into the corresponding nucleotide sequences disclosed herein, thereby introducing one or more amino acid substitutions, additions, or deletions into the encoded protein. Mutations can be introduced using standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also covered by this disclosure.

[0082] For example, these naturally occurring allelic variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined in this article.

[0083] Variant nucleotide sequences also include synthetically obtained nucleotide sequences, such as those generated by site-directed mutagenesis, but these sequences still encode the harmful biological protein of the embodiment, such as a mutant toxin. Typically, variants of a particular nucleotide sequence of the embodiment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with that particular nucleotide sequence, as determined by the sequence alignment procedure described elsewhere herein using default parameters. Variant nucleotide sequences of the embodiment may differ from that sequence by as few as 1-15 nucleotides, as few as 1-10, for example 6-10, as few as 5, as few as 4, 3, 2, or even 1 nucleotide.

[0084] Variants of a particular nucleotide sequence (i.e., exemplary nucleotide sequence) of an embodiment can also be evaluated by comparing the percentage sequence identity between a polypeptide encoded by a variant nucleotide sequence and a polypeptide encoded by a reference nucleotide sequence. Thus, for example, isolated nucleic acids encoding polypeptides having a given percentage sequence identity with the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 are disclosed. The percentage sequence identity between any two polypeptides can be calculated using the sequence alignment procedure described elsewhere herein with default parameters. In cases where any given polynucleotide pair of an embodiment is evaluated by comparing the percentage sequence identity common to the two polypeptides it encodes, the percentage sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, and typically at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0085] As used herein, the term "variant protein" includes polypeptides derived from natural proteins by: deleting (so-called truncating) one or more amino acids or adding one or more amino acids to the N-terminus and / or C-terminus of the natural protein; deleting or adding one or more amino acids at one or more sites in the natural protein; or substituting one or more amino acids at one or more sites in the natural protein. Therefore, the term "variant protein" encompasses a biologically active fragment of a natural protein containing a sufficient number of consecutive amino acid residues to retain the biological activity of the natural protein, i.e., possessing virulent bioactivity. Such virulent bioactivity may differ from or be improved relative to the natural protein, or may remain unchanged, as long as virulent bioactivity is retained.

[0086] The variant proteins covered by the examples are biologically active, meaning they retain the desired biological activity of the natural protein, i.e., the pest-killing activity as described herein. Such variants can be produced, for example, by genetic polymorphism or artificial manipulation. The biologically active variants of the natural pest-killing proteins of the examples will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the natural protein, as determined by the sequence alignment procedure described elsewhere herein using default parameters. The biologically active variants of the proteins of the examples may differ from the protein by as few as 1-15 amino acid residues, as few as 1-10, for example 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0087] In some embodiments, the polypeptide has modified physical properties. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "target physical property" and "target property" are used interchangeably to refer to the physical properties of the protein being studied and / or modified. Examples of physical properties include, but are not limited to: net surface charge and charge distribution on the protein surface, net hydrophobicity and distribution of hydrophobic residues on the protein surface, surface charge density, surface hydrophobic density, total count of ionized groups on the surface, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second inertial coefficient. Examples of physical properties also include, but are not limited to: solubility, folding, stability, and digestibility. In some embodiments, the polypeptide has increased digestibility of proteolytic fragments in the insect gut. Models that simulate gastric digestion are known to those skilled in the art (Fuchs, RL and JD. Astwood. Food Technology 50: 83-88, 1996; Astwood, JD, et al. Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al. J. Agric Food Chem. 50: 7154-7160, 2002).

[0088] The embodiments further cover microorganisms transformed with at least one nucleic acid of the embodiments, with an expression cassette containing the nucleic acid, or with a vector containing the expression cassette. In some embodiments, the microorganisms are microorganisms that reproduce on plants. Embodiments of this disclosure relate to encapsulated pest-killing proteins comprising transformed microorganisms capable of expressing at least one pest-killing protein of the embodiments.

[0089] Examples provide pest-killing compositions comprising the transformed microorganisms of the examples. In such examples, the transformed microorganisms are typically present in the pest-killing composition in a pest-killing effective amount together with a suitable carrier. Examples also cover pest-killing compositions comprising a pest-killing effective amount of isolated proteins of the examples (alone or in combination with the transformed organisms of the examples, and / or the encapsulated pest-killing proteins of the examples), together with a suitable carrier.

[0090] The embodiments further provide a method for increasing insect target range by using the pest-killing protein of the embodiments in combination with at least one other or "second" pest-killing protein. Any pest-killing protein known in the art can be used in the methods of the embodiments. Such pest-killing proteins include, but are not limited to, Bt toxins, protease inhibitors, α-amylases, and peroxidases.

[0091] The examples also cover transformed or transgenic plants comprising at least one nucleotide sequence of the examples. In some embodiments, plants are stably transformed using a nucleotide construct comprising at least one nucleotide sequence of the examples, the nucleotide sequence being operatively linked to a promoter driving expression in plant cells. As used herein, the terms “transformed plant” and “transgenic plant” refer to plants whose genomes contain heteropolynucleotides. Typically, heteropolynucleotides are stably integrated into the genome of transgenic or transformed plants, thus allowing the polynucleotide to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette.

[0092] It should be understood that, as used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of heterologous nucleic acids, including those transgenics that were originally altered in this way and those that were produced from the initial transgenics through sexual hybridization or asexual reproduction. As used herein, the term "transgenic" does not cover changes to the genome (chromosomal or extrachromosomal) resulting from conventional plant breeding methods or from naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0093] As used herein, the term "plant" includes the whole plant, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny. Parts of a transgenic plant are within the scope of the embodiments and include, for example, plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant masses, and intact plant cells in a plant or plant part (e.g., embryo, pollen, ovule, seed, leaf, flower, branch, fruit, kernel, ear, corn cob, outer shell, stem, root, root tip, anther, etc.) derived from a transgenic plant having the DNA molecules of the embodiments or its previously transformed progeny, and thus composed of at least a portion of transgenic cells. The plant species that can be used in the methods of the embodiments are generally as broad as the categories of higher plants suitable for transformation technologies, including monocots and dicots.

[0094] While the embodiments do not rely on specific biological mechanisms for increasing plant resistance to plant pests, expression of the nucleotide sequences of the embodiments in plants can lead to the production of pest-killing proteins of the embodiments and increased plant resistance to plant pests. The plants of the embodiments can be used in agriculture in methods affecting insect pests. Some embodiments provide transformed crop plants, such as maize plants, which can be used in methods affecting insect pests (e.g., lepidopteran pests) of plants.

[0095] "Subject plant or plant cell" refers to a plant or plant cell in which genetic alterations (e.g., transformations) have been affected regarding a target gene, or a plant or plant cell that has inherited from such an altered plant or cell and contains that alteration. "Control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the phenotypic characteristics of the subject plant or plant cell.

[0096] Control plants or plant cells may include, for example: (a) wild-type plants or cells, i.e., plants or plant cells with the same genotype as the starting material used to induce genetic changes in the subject plant or plant cell; (b) plants or plant cells with the same genotype as the starting material but transformed with a null construct (i.e., a construct that has no known effect on the target trait, such as a construct containing a marker gene); (c) plants or plant cells as untransformed isolates in the progeny of the subject plant or plant cell; (d) plants or plant cells that are genetically identical to the subject plant or plant cell but not exposed to conditions or stimuli that would induce expression of the target gene; or (e) the subject plant or plant cell itself under conditions that do not express the target gene.

[0097] Those skilled in the art will readily recognize that advances in the field of molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, offer a wide range of tools and protocols applicable to both altering or engineering the potential genetic sequences of proteins that are agriculturally advantageous.

[0098] Therefore, the proteins in the examples can be modified in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods of such manipulation are generally known in the art. For example, amino acid sequence variants of biotoxic proteins can be prepared by introducing mutations into synthetic nucleic acids (e.g., DNA molecules). Methods of mutagenesis and nucleic acid modification are well known in the art. For example, designed modifications can be introduced using oligonucleotide-mediated site-directed mutagenesis techniques. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. [Enzymological Methods] 154: 367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology [Techniques in Molecular Biology] (MacMillan Publishing Company, New York) and the references cited therein.

[0099] The mutagenic nucleotide sequence of the embodiments can be modified to alter about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more amino acids present in the primary sequence encoding the polypeptide. Alternatively, even further variations from the natural sequence can be introduced, such that the encoded protein can have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more of the codons changed or otherwise modified compared to the corresponding wild-type protein. In the same manner, the encoded protein may have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more of additional codons compared to the corresponding wild-type protein. It should be understood that the mutagenic nucleotide sequences of the examples are intended to encompass biofunctional, equivalent peptides with pest-killing activities (e.g., improved pest-killing activities, as determined by the antifeedant properties against European corn borer larvae). Such sequences may arise as a result of codon redundancy and functional equivalence known to be naturally present in nucleic acid sequences and the proteins thereby encoded.

[0100] Those skilled in the art will recognize that amino acid addition and / or substitution is generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, charge, size, etc. Exemplary amino acid substituents considering different foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0101] Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978), Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington), which is incorporated herein by reference. Conservative substitutions can be made, such as exchanging one amino acid with another amino acid that has similar properties.

[0102] Therefore, the gene and nucleotide sequences of the embodiments include both naturally occurring sequences and mutant forms. Similarly, the proteins of the embodiments encompass both naturally occurring proteins and variants (e.g., truncated polypeptides) and their modified forms (e.g., mutants). Such variants will retain the desired antimicrobial activity. Obviously, mutations made in the nucleotide sequence encoding the variant must not place that sequence outside the reading frame and will not create complementary regions capable of producing secondary mRNA structures. See European Patent Application Publication No. 75,444.

[0103] The deletions, insertions, and substitutions of protein sequences covered in this article are not expected to produce fundamental changes in protein characteristics. However, when it is difficult to predict the exact effects of substitution, deletion, or insertion in advance, those skilled in the art will understand that such effects will be evaluated through routine screening experiments (e.g., insect feeding assays). See, for example, Marrone et al. (1985) J. Econ. Entomol. [Journal of Economic Entomology] 78: 290-293, and Czapla and Lang (1990) J. Econ. Entomol. [Journal of Economic Entomology] 83: 2480-2485, which are incorporated herein by reference.

[0104] Variable nucleotide sequences and proteins also include sequences and proteins produced through mutagenesis and recombination methods, such as DNA shuffling. Using such procedures, one or more different coding sequences can be manipulated to create new biohazard-killing proteins possessing desired properties. In this way, recombinant polynucleotide libraries are generated from a population of related sequence polynucleotides, including sequence regions that have substantial sequence identity and can undergo homologous recombination in vitro or in vivo. For example, using this method, full-length coding sequences, sequence motifs encoding target domains, or any fragments of the nucleotide sequence of the example can be shuffled between corresponding portions of the nucleotide sequence of the example and other known Cry nucleotide sequences to obtain new genes encoding proteins with improved target properties.

[0105] The intended properties include, but are not limited to, the pest-killing activity per unit of pest-killing protein, protein stability, and toxicity to non-target species (particularly humans, livestock, and plants and microorganisms expressing the pest-killing peptides of the examples). The examples are not bound by any specific shuffling strategy, only at least one nucleotide sequence of the examples or a portion thereof is involved in such a shuffling strategy. Shuffling may involve only the nucleotide sequences disclosed herein, or it may additionally involve the shuffling of other nucleotide sequences known in the art. Strategies for DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 91: 10747-10751; Stemmer (1994) Nature [Nature] 370: 389-391; Crameri et al. (1997) Nature Biotech. [Nature Biotechnology] 15: 436-438; Moore et al. (1997) J. Mol. Biol. [Journal of Molecular Biology] 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 94: 4504-4509; Crameri et al. (1998) Nature [Nature] 391: 288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.

[0106] The nucleotide sequences of the examples can also be used to isolate corresponding sequences from other organisms, particularly other bacteria, and more specifically other Bacillus strains. In this way, such sequences can be identified using methods such as PCR, hybridization, etc. (based on their sequence homology with the sequences illustrated herein). The examples cover sequences selected based on sequence identity with all or fragments of the sequences illustrated herein. These sequences include sequences that are orthologs of the disclosed sequences. The term "ortholog" refers to a gene derived from a common ancestral gene and found in different species due to speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or the protein sequences they encode share substantial identity as defined elsewhere herein. The function of orthologs is generally highly conserved among species.

[0107] In PCR methods, oligonucleotide primers can be designed for PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any target organism. Methods for designing PCR primers and for PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter referred to as “Sambrook”. See also, Innis et al., ed. (1990) *PCR Protocols: A Guide to Methods and Applications* (Academic Press, New York); Innis and Gelfand, ed. (1995) *PCR Strategies* (Academic Press, New York); and Innis and Gelfand, ed. (1999) *PCR Methods Manual* (Academic Press, New York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, and partially mismatched primers.

[0108] In hybridization techniques, all or part of a known nucleotide sequence is used as a probe. This probe selectively hybridizes with other corresponding nucleotide sequences present in a cloned genomic DNA fragment or cDNA fragment group (i.e., a genome or cDNA library) from a selected organism. These hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be coupled with detectable groups such as... 32 P or any other detectable marker can be used for labeling. Therefore, for example, probes for hybridization can be prepared by synthesizing oligonucleotides based on sequence labeling from an embodiment. Methods for preparing probes for hybridization and constructing cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook.

[0109] For example, the complete sequences disclosed herein, or one or more portions thereof, can be used as probes capable of specifically hybridizing with the corresponding sequences and messenger RNA. To achieve specific hybridization under various conditions, such probes comprise sequences unique to the sequences in the examples and are typically at least about 10 or 20 nucleotides in length. Such probes can be used to amplify the corresponding Cry sequences from selected organisms via PCR. This technique can be used to isolate additional coding sequences from desired organisms or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of DNA libraries (plaques or colonies; see, for example, Sambrook).

[0110] Hybridization of such sequences can be performed under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" refers to conditions under which the probe hybridizes to its target sequence to a detectably higher degree than it hybridizes to other sequences (e.g., at least 2, 5, or 10 times the background). Stringent conditions are sequence-dependent and will vary under different conditions. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can also be tuned to allow for some mismatches in the sequences in order to detect a lower degree of similarity (heterologous detection). Typically, probes are less than about 1000 or 500 nucleotides in length.

[0111] Typically, stringent conditions are those where the salt concentration is less than about 1.5 M sodium ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M sodium ion concentration (or other salt), and the temperature is at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. Exemplary low stringent conditions include hybridization at 37 °C with a buffer solution of 30% to 35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50 °C to 55 °C in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate). Exemplary moderately stringent conditions include hybridization at 37°C in 40% to 45% formamide, 1.0M NaCl, and 1% SDS, followed by washing at 55°C to 60°C in 0.5X to 1X SSC. Exemplary highly stringent conditions include hybridization at 37°C in 50% formamide, 1M NaCl, and 1% SDS, followed by a final wash at 60°C to 65°C in 0.1X SSC for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The hybridization duration is typically less than about 24 hours, and is typically about 4 to about 12 hours.

[0112] Specificity typically depends on the function of post-hybridization washing, with key factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, T... m (The melting point) can be roughly estimated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. [Analytical Biochemistry] 138: 267-284: T m = 81.5℃ + 16.6(log M) + 0.41(%GC) - 0.61(%form) - 500 / L; where M is the molar concentration of the monovalent cation, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the base pair length of the heterozygote. T m The temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe (at the defined ionic strength and pH). Washing is typically performed at least until equilibration is reached and a low background hybridization level is achieved, for example, 2 hours, 1 hour, or 30 minutes.

[0113] For every 1% mismatch, T m The temperature drops by approximately 1°C; therefore, T can be adjusted. m Hybridization and / or washing conditions are used to hybridize with sequences of desired identity. For example, if a sequence with ≥90% identity is being sought, T can be used. m Lower by 10°C. Typically, stringent conditions are chosen to be Tf greater than that of a specific sequence and its complementary sequence at defined ionic strengths and pH. m Approximately 5°C lower. However, extremely demanding conditions can be achieved using temperatures higher than T. m Hybridization and / or washing at temperatures 1°C, 2°C, 3°C, or 4°C lower; moderately stringent conditions can utilize temperatures higher than T. m Hybridization and / or washing at temperatures 6°C, 7°C, 8°C, 9°C, or 10°C; low-stress conditions can utilize T... m Hybridization and / or washing at temperatures 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C lower.

[0114] Using equations, hybridization and washing compositions, and the required T m As will be understood by those skilled in the art, this essentially describes a change in the stringency of the hybridization and / or washing solutions. If the desired degree of mismatch results in T... mBelow 45°C (aqueous solution) or 32°C (formamide solution), the SSC concentration can be increased to allow for the use of higher temperatures. Comprehensive guidance on nucleic acid hybridization can be found in the following literature: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook. Therefore, the isolated sequence encoding the Cry protein of the examples and hybridizing under stringent conditions with the Cry sequence or fragments thereof disclosed herein is included in the examples.

[0115] The following terms are used to describe the sequence relationship between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “sequence identity percentage” and (e) “substantial identity”.

[0116] (a) As used herein, a “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset or the whole of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0117] (b) As used herein, a “comparison window” refers to a continuous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which does not include additions or deletions) used for optimal alignment of the two sequences. Typically, the length of the comparison window is at least 20 consecutive nucleotides, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art will understand that, due to the presence of vacancies in the polynucleotide sequence, a vacancy penalty is typically introduced and subtracted from the match number to avoid high similarity with the reference sequence.

[0118] The alignment methods used for comparing sequences are well known in the art. Therefore, the determination of percentage sequence identity between any two sequences can be performed using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is the work of Myers and Miller (1988) in CABIOS. Algorithms in 4:11-17; local alignment algorithms in Smith et al. (1981) Adv. Appl. Math. 2:482; global alignment algorithms in Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; search-based local alignment methods in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; algorithms in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872-264; and as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0119] Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (version 2.0); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in GCG Wisconsin Genetics Software Package version 10 (available from Accelrys, 9685 Scranton Road, San Diego, California, USA). Alignment using these programs can be performed using default parameters. The following fully describe the CLUSTAL procedure: Higgins et al. (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN procedure is based on the algorithm of Myers and Miller (1988) (ibid.). When comparing amino acid sequences, the ALIGN procedure can use the PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. The BLAST procedure described by Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403 is based on the algorithm described by Karlin and Altschul (1990) above. A BLAST nucleotide search can be performed using the BLASTN procedure with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleotide sequence encoding the protein of the example. A BLAST protein search can be performed using the BLASTX procedure with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein or polypeptide of the example. For obtaining vacancy-free alignments for comparative purposes, Gapped BLAST (in BLAST 2.0) as described by Altschul et al. (1997) Nucleic Acids Res. [Nucleic Acids Research] 25:3389 can be used.Alternatively, PSI-BLAST (in BLAST 2.0) can be used for iterative searches to detect distant relationships between molecules. See Altschul et al. (1997), ibid. When using BLAST, vacancy BLAST, or PSI-BLAST, the default parameters for each program can be used (e.g., BLASTN for nucleotide sequences, BLASTX for proteins). See the National Center for Biotechnology Information website, ncbi.hlm.nih.gov. Alignment can also be performed manually by inspection.

[0120] Unless otherwise stated, the sequence identity / similarity values ​​provided herein refer to values ​​obtained using GAP version 10 with the following parameters: % identity and % similarity of nucleotide sequences using GAP weight 50 and length weight 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity of amino acid sequences using GAP weight 8 and length weight 2 and the BLOSUM62 scoring matrix; or any equivalent procedure thereof. As used herein, the term “equivalent procedure” refers to any sequence comparison procedure that produces an alignment for any two sequences in discussion that, when compared to the corresponding alignment produced by GAP version 10, has the same nucleotide or amino acid residue pairing and the same percentage sequence identity.

[0121] GAP uses the Needleman and Wunsch (1970) algorithm described above to find an alignment between two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and produces an alignment with the maximum number of matching bases and the minimum number of gaps. It allows for gap generation penalties and gap extension penalties to be provided in units of matching bases. GAP must take a gain of the gap generation penalty for each gap it inserts based on the number of matches. If a gap extension penalty greater than zero is chosen, GAP must additionally take a gain of the gap length multiplied by the gap extension penalty for each inserted gap. In version 10 of the GCG Wisconsin Genetics software package, the default gap generation penalty and gap extension penalty values ​​for protein sequences are 8 and 2, respectively. For nucleotide sequences, the default gap generation penalty is 50, and the default gap extension penalty is 3. Gap generation and gap extension penalties can be represented as integers selected from the following group, consisting of numbers from 0 to 200. Therefore, for example, the penalty for creating and extending an open space can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0122] GAP represents a member of the best-aligned family. Many members of this family may exist, but others do not possess better quality. GAP exhibits four performance factors for alignment: quality, ratio, identity, and similarity. Quality is the measure maximized for aligning sequences. The ratio is quality divided by the number of bases in the shorter segment. The identity percentage is the percentage of actually matched symbols. The similarity percentage is the percentage of similar symbols. Symbols opposite gaps are ignored. A similarity score is given when the score matrix value of a pair of symbols is greater than or equal to the similarity threshold of 0.50. The score matrix used in version 10 of the GCG Wisconsin Genetics software package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 89: 10915).

[0123] (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences refers to identical residues in two sequences when aligned with maximum correspondence on a specified comparison window. When using a percentage of sequence identity for a protein, it is recognized that dissimilar residue positions are often associated with conserved amino acid substitutions, where an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ in terms of conserved substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservatism of that substitution. Sequences differing from these conserved substitutions are referred to as having “sequence similarity” or “identity.” The methods used to make this adjustment are well known to those skilled in the art. Typically, this involves scoring conserved substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, a score for a conserved substitution is between zero and 1 when identical amino acids score 1 and non-conservative substitutions score zero. Scores for conserved substitutions are calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0124] (d) As used herein, “sequence identity percentage” refers to the value determined by comparing two best-aligned sequences in a comparison window, where the polynucleotide sequence portion of the comparison window may contain additions or deletions (i.e., vacancies) to achieve optimal alignment of the two sequences compared to a reference sequence (which does not contain additions or deletions). This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to produce the sequence identity percentage.

[0125] (e)(i) The term “substantial identity” for a polynucleotide sequence means that the polynucleotide, when compared with a reference sequence using standard parameters with one of the alignment procedures described above, contains a sequence identity of at least 70%, 80%, 90%, or 95% or higher. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. For these purposes, substantial identity of the amino acid sequence generally means sequence identity of at least 60%, 70%, 80%, 90%, or 95% or higher.

[0126] Another indicator of substantially identical nucleotide sequences is whether the two molecules hybridize under stringent conditions. Typically, stringent conditions are chosen to be at specified ionic strengths and pH levels compared to the T0 of a particular sequence. m Approximately 5°C lower. However, the stringent conditions cover conditions beyond T. m Temperatures ranging from approximately 1°C lower to approximately 20°C, depending on the required stringency as limited elsewhere in this document. Nucleic acids that do not hybridize to each other under stringent conditions encode substantially identical polypeptides are considered substantially identical. This can occur, for example, when a single copy of a nucleic acid is produced using the maximum codon degeneracy allowed by genetic coding. One indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunely cross-reactive with the polypeptide encoded by the second nucleic acid.

[0127] (e)(ii) In the context of peptides, the term “substantially similar” means that the peptide contains a sequence within a specified comparison window that has at least 70%, 80%, 85%, 90%, 95%, or higher sequence identity with a reference sequence. Optimal alignment for these purposes can be performed using the global alignment algorithm of Needleman and Wunsch (1970) mentioned above. One indication that two peptide sequences are substantially identical is that one peptide is immunoreactive with an antibody against the second peptide. Thus, for example, when one peptide is not identical to a second peptide only because of conserved substitutions, the two peptides are substantially identical. Aside from the possibility that the positions of not entirely identical residues may differ due to conserved amino acid changes, “substantially similar” peptides share the sequence as described above.

[0128] The use of the term "nucleotide construct" herein is not intended to limit the embodiments to nucleotide constructs containing DNA. Those skilled in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides, as well as combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments further encompass all complementary forms of such constructs, molecules, and sequences. Furthermore, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences that can be used in the plant transformation methods of the embodiments, including but not limited to those composed of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc.

[0129] Further embodiments involve transformed organisms, such as those selected from the group consisting of plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism comprises: a DNA molecule of the embodiments, an expression cassette containing the DNA molecule, or a vector containing the expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0130] The DNA construct provides the sequence of the embodiment for expression in a target organism. The construct will include regulatory sequences operatively linked to the 5′ and 3′ of the sequence of the embodiment. As used herein, the term “operatively linked” refers to a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Generally, operatively linked means that the linked nucleic acid sequences are contiguous and, where necessary, contiguous and within the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into an organism. Alternatively, one or more additional genes may be provided on multiple DNA constructs.

[0131] These DNA constructs are equipped with multiple restriction sites for inserting Cryotoxin sequences to bring them under transcriptional regulation in these regulatory regions. The DNA constructs may additionally contain selective marker genes.

[0132] The DNA construct will include, in the transcriptional direction from 5′ to 3′: a transcription and translation initiation region (i.e., a promoter), the DNA sequence of the embodiment, and a transcription and translation termination region (i.e., a termination region) that functions in the host organism. The transcription initiation region (i.e., the promoter) may be natural, similar, exogenous, or heterologous, depending on the host organism and / or sequence of the embodiment. Furthermore, the promoter may be a natural sequence or, alternatively, a synthetic sequence. As used herein, the term "exogenous" means that the promoter is not found in the natural organism in which it is introduced. In the case where the promoter is "exogenous" or "heterogeneous" with respect to the sequence of the embodiment, it means that the promoter is not natural or naturally occurring with respect to the operably linked sequence of the embodiment. As used herein, chimeric genes contain a coding sequence operably linked to a transcription initiation region that is heterologous to that coding sequence. When the promoter is a natural or native sequence, the expression of the operably linked sequence changes from wild-type expression, resulting in a phenotypic alteration.

[0133] The termination region may be natural for the transcription start region, natural for the target DNA sequence to which it is operatively linked, natural for the plant host, or derived from another source (i.e., exogenous or heterologous to the promoter, target sequence, plant host, or any combination thereof).

[0134] Convenient termination regions can be obtained from Ti plasmids of Agrobacterium tumefaciens, such as the termination regions of octopaline synthase and carmine synthase. See also Guerineau et al. (1991) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. [Genes and Development] 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. [Nucleic Acid Research] 15: 9627-9639.

[0135] Nucleic acids can be optimized to increase expression in the host organism when appropriate. Therefore, in the case of a plant host organism, synthetic nucleic acids can be synthesized using plant-preferred codons to improve expression. For a discussion of host-preferred codon usage, see, for example, Campbell and Gowri (1990) Plant Physiol. [Plant Physiology] 92: 1-11. For instance, while the nucleic acid sequences of examples can be expressed in both monocot and dicot species, sequences can be modified to address monocot or dicot-specific codon preferences and GC content preferences, as these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17: 477-498). Thus, maize-preferred codons for specific amino acids can be derived from known maize gene sequences. Maize codon usage from 28 genes of maize plants is listed in Table 4 of Murray et al. (ibid.). Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0136] Other sequence modifications are known to enhance gene expression in the host cell. These include the removal of sequences encoding pseudopolyadenylation signals, exon-intron splicing site signals, transposon-like repeat sequences, and other well-characterized sequences that may be detrimental to gene expression. The GC content of a sequence can be adjusted to the average level for a given host cell, calculated with reference to known genes expressed in that host cell. As used herein, the term “host cell” refers to the cell that contains the vector and supports the replication and / or expression of the expression vector. Host cells can be prokaryotic cells such as *E. coli*, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a corn host cell. When possible, sequences are modified to avoid predictable hairpin secondary mRNA structures.

[0137] The expression cassette may additionally contain a 5′ leader sequence. These leader sequences can enhance translation. Translation leader sequences are known in the art and include: microRNA leader sequences, such as the EMCV leader sequence (5′ uncoding region of encephalomyocarditis) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 86: 6126-6130); potato Y virus leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al. (1995) Gene [Gene] 165(2): 233-238); MDMV leader sequences (corn dwarf mosaic virus), human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature [Nature] 353: 90-94); and untranslated leader sequences of the coat protein mRNA from alfalfa mosaic virus (AMV RNA). 4)(Jobling et al. (1987) Nature 325: 622-625); Tobacco mosaic virus leader sequence (TMV))(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and Maize chlorotic mottle virus leader sequence (MCMV))(Lommel et al. (1991) Virology 81: 382-385. See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

[0138] In preparing expression cassettes, various DNA fragments can be manipulated to provide DNA sequences that are in the appropriate orientation and, at the appropriate time, within the appropriate reading frame. For this purpose, adapters or linkers can be used to ligate the DNA fragments, or other manipulations can be involved to provide convenient restriction sites, remove redundant DNA, and remove restriction sites. For this purpose, in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, and substitution (e.g., conversion and transversion) can be employed.

[0139] Many promoters are available for implementing these embodiments. A promoter can be selected based on the desired results. Nucleic acids can be combined with constitutive promoters, tissue-preferred promoters, inducible promoters, or other promoters for expression in a host organism. Suitable constitutive promoters for plant host cells include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. Biol. 12: 619-632). Mol. Biol. [Plant Molecular Biology] 18: 675-689; pEMU (Last et al. (1991) Theor. Appl. Genet. [Theoretical and Applied Genetics] 81: 581-588); MAS (Velten et al. (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3: 2723-2730); ALS promoter (US Patent No. 5,659,026), etc. Other constitutive promoters include, for example, those discussed in US Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0140] Based on the desired results, gene expression from inducible promoters may be beneficial. Of particular interest in regulating nucleotide sequences expressed in plants is the wound-inducible promoter. This wound-inducible promoter may respond to damage caused by insect feeding and includes a potato proteinase inhibitor (PPI). II) Genes (Ryan (1990) Ann. Rev. Phytopath. [Annual Review of Plant Pathology] 28: 425-449; Duan et al. (1996) Nature Biotechnology 14: 494-498); wun1 and wun2 (US Patent Nos. 5,428,148); win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 215: 200-208); Systemin (McGurl et al. (1992) Science 225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. [Plant Molecular Biology] 22: 783-792; Eckelkamp et al. (1993) FEBS Letters [European Federation of Biochemical Societies Communications] 323: 73-76); MPI gene (Corderok et al. (1994) Plant J. [Plant Journal] 6(2): 141-150), etc., which are incorporated herein by reference.

[0141] Furthermore, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those derived from pathogenesis-associated proteins (PR proteins), which are induced upon pathogen infection; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. [Dutch Journal of Plant Pathology] 89: 245-254; Uknes et al. (1992) Plant Cell. 4: 645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99 / 43819, which is incorporated herein by reference.

[0142] Of particular interest are promoters expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. [Plant Molecular Biology] 9: 335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions [Molecular Plant-Microbe Interactions] 2: 325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 83: 2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 2: 93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 93: 14972-14977. See also Chen et al. (1996) Plant J. 10: 955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91: 2507-2511; Warner et al. (1993) Plant J. 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968; U.S. Patent No. 5,750,386 (nematode-induced promoter), and the references cited therein. Of particular interest is the inducible promoter of the maize PRms gene, whose expression is induced by the Fusarium moniliforme pathogen (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41: 189-200).

[0143] Chemically regulated promoters can be used to regulate gene expression in plants by applying exogenous chemical regulators. Depending on the objective, the promoter may be a chemically inducible promoter in the case of chemically induced gene expression, or a chemically repressive promoter in the case of chemically repressed gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter activated by a benzenesulfonamide herbicide safener, the corn GST promoter activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid. Other target chemical regulatory promoters include steroid response promoters (see, for example, glucocorticoid-inducible promoters in Schena et al. (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88: 10421-10425 and McNellis et al. (1998) Plant J [Journal of Plant Science] 14(2): 247-257) and tetracycline-inducible and tetracycline-inhibiting promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 227: 229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0144] Tissue-biased promoters can be used to target the enhanced expression of pest-killing proteins in specific plant tissues. Tissue-biased promoters include those discussed below: Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. [Plant Physiology] 112(2): 525-535; Canevascini et al. (1996) Plant Physiol. [Plant Physiology] 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. [Plant Cell Physiology] 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. [Results and Problems of Cell Differentiation] 20: 181-196; Orozco et al. (1993) Plant Mol Biol. [Plant Molecular Biology] 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90(20): 9586-9590; and Guevara-Garcia et al. (1993) Plant J. [Plant Journal] 4(3): 495-505. If necessary, such promoters can be modified for weak expression.

[0145] Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kwon et al. (1994) Plant Physiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Gotor et al. (1993) Plant J. 3: 509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590.

[0146] Root-biased or root-specific promoters are known and can be selected from many available promoters from the literature or de novo isolated from different compatible species. See, for example, Hire et al. (1992) PlantMol.Biol. 20(2): 207-218 (gene for soybean root-specific glutamine synthase); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of common bean); Sanger et al. (1990) Plant Mol.Biol. 14(3): 433-443 (root-specific promoter of mannitol synthase (MAS) in Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1): 11-22 (cloning of full-length cDNA encoding cytosolic glutamine synthase (GS) expressed in soybean roots and root nodules). See also Bogusz et al. (1990) Plant Cell 2(7): 633-641, which describes two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing non-leguminous plant Parasponia andersonii and the related non-nitrogen-fixing non-leguminous plant Trema tomentosa. The promoters of these genes are linked to β-glucuronidase reporter genes and have been introduced into both the non-leguminous crop Nicotiana tabacum and the leguminous crop Lotus corniculatus, and root-specific promoter activity has been preserved in both cases. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducible genes in Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1): 69-76). They concluded that enhancers and tissue-biased DNA determinants are dissociated in these promoters. Teeri et al. (1989) used gene fusions with lacZ to show that Agrobacterium tumefaciens T-DNA genes encoding octopus alkaloid synthase are active, particularly in the epidermis of root tips, and that the TR2′ gene is root-specific in intact plants and stimulated by wounds in leaf tissues, a particularly desirable combination of features for the combined use of insecticidal or larval-killing genes (see EMBO J. [Journal of the European Society for Molecular Biology] 8(2): 343-350). The TR1′ gene fused with nptII (neomycin phosphotransferase II) showed similar features.Other root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. [Plant Molecular Biology] 29(4): 759-772); and the rolB promoter (Capana et al. (1994) Plant Mol. Biol. [Plant Molecular Biology] 25(4): 681-691). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0147] Seed-preferred promoters include seed-specific promoters (those that are active during seed development, such as promoters of seed storage proteins) and seed germination promoters (those that are active during seed germination). See Thompson et al. (1989) BioEssays 10:108, which is incorporated herein by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced information); cZ19B1 (corn 19kDa zein); and milps (inositol-1-phosphate synthase) (see U.S. Patent No. 6,225,529, which is incorporated herein by reference). γ-zein and Glob-1 are endosperm-specific promoters. For dicotyledonous plants, seed-specific promoters include, but are not limited to, beta-coumarin, rapeseed protein, β-conglycinin, soybean lectin, cruciferous proteins, etc. For monocotyledons, seed-specific promoters include, but are not limited to, 15 kDa zeatin, 22 kDa zeatin, 27 kDa zeatin, g-zeatin, waxes, thymol 1, thymol 2, and globulin 1 in maize. See also WO 00 / 12733, which discloses seed-preferred promoters from the end1 and end2 genes; incorporated herein by reference. Promoters exhibiting “preferred” expression in a specific tissue are expressed at a higher level in that tissue than in at least one other plant tissue. Some tissue-preferred promoters are expressed almost exclusively in a specific tissue.

[0148] When low levels of expression are desired, weak promoters can be used. Generally, as used herein, the term "weak promoter" refers to a promoter that drives expression of the coding sequence at low levels. Low levels of expression are defined as approximately 1 / 1000 to approximately 1 / 100,000 to approximately 1 / 500,000 of the transcript. Alternatively, it should be recognized that the term "weak promoter" also encompasses promoters that drive expression only in a few cells but not in others, resulting in low overall expression. When a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to reduce expression levels.

[0149] Such weakly constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99 / 43838 and U.S. Patent No. 6,072,050), the core 35S CaMV promoter, etc. Other constitutive promoters include, for example, those disclosed in the following U.S. Patent Nos.: 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; which are incorporated herein by reference.

[0150] Typically, expression cassettes contain selective marker genes for selecting transformed cells. Selective marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicide compounds, such as glufosinate, bromosulfuron, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). Examples of suitable selective marker genes include, but are not limited to, genes encoding resistance to: chloramphenicol (Herrera Estrella et al. (1983) EMBOJ. [Journal of the European Society for Molecular Biology] 2: 987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303: 209-213; and Meije et al. (1991) Plant Mol. Biol. [Plant Molecular Biology] 16: 807-820); streptomycin (Jones et al. (1987) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 210: 86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. [Transgenic Research] 5: 131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. [Transgenic Research] 5: 131-137). Mol. Biol. [Plant Molecular Biology] 7: 171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. [Plant Molecular Biology] 15: 127-136); bromobenzonitrile (Stalker et al. (1988) Science 242: 419-423); glyphosate (Shaw et al. (1986) Science 233: 478-481; and US patents 7,709,702 and 7,462,481; glufosinate (DeBlock et al. (1987) EMBO) J. [Journal of the European Society for Molecular Biology] 6: 2513-2518). See also Yarranton (1992) Curr. Opin. Biotech. [Current Opinion on Biotechnology] 3: 506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89: 6314-6318; Yao et al. (1992) Cell [Cell] 71: 63-72; Reznikoff (1992) Mol. Microbiol.[Molecular Microbiology] 6: 2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg [Doctoral Dissertation, Heidelberg University]; Reines et al. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. [Molecular and Cell Biology] 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. [Nucleic Acid Research] 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. [Hot Topics in Molecular Structural Biology] 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. [Antimicrobial Agents and Chemotherapy] 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.[Antimicrobial Agents and Chemotherapy] 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill et al. (1988) Nature 334: 721-724. Such publications are incorporated herein by reference.

[0151] The list of selective marker genes above is not intended to be limiting. Any selective marker gene may be used in these embodiments.

[0152] The methods of these embodiments relate to introducing polypeptides or polynucleotides into plants. "Introduction" is intended to mean providing a polynucleotide or polypeptide to a plant in such a manner that the sequence enters the interior of the plant's cells. The methods of these embodiments are not dependent on the specific method used to introduce the polynucleotide or polypeptide into the plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing polynucleotides or polypeptides into plants are known in the art, including but not limited to stable transformation, transient transformation, and virus-mediated transformation.

[0153] "Stable transformation" refers to the integration of a nucleotide construct introduced into a plant into the plant's genome and its ability to be inherited by its offspring. "Transient transformation" refers to the introduction of a polynucleotide into a plant that does not integrate into the plant's genome, or the introduction of a polypeptide into the plant.

[0154] The transformation scheme and the scheme for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocot or dicot). Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include microinjection (Crossway et al. (1986) Biotechniques [Biotechnology] 4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 83: 5602-5606), Agrobacterium-mediated transformation (US Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3: 2717-2722) and ballistic particle acceleration (see, for example, US Patent Nos. 4,945,050, 5,879,918, 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental In Methods, edited by Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6: 923-926, and Lecl transformation (WO 00 / 28058). For potato transformation methods, see Tu et al. (1998) Plant Molecular Biology 37: 829-838 and Chong et al. (2000) Transgenic Research 9: 71-78. Other transformation procedures can be found in: Weissinger et al. (1988) Ann. Rev. Genet. [Annals of Genetics] 22: 421-477; Sanford et al. (1987) Particulate Science and Technology [Particle Science and Technology] 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. [Plant Physiology] 87: 671-674 (soybean); McCabe et al. (1988) Bio / Technology [Bio / Technology] 6: 923-926 (soybean); Finer and McMullen (1991) in Vitro Cell Dev. Biol. [In Vitro Cell and Developmental Biology] 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. [Theoretical and Applied Genetics] 96: 319-324 (Soybean); Datta et al. (1990) Biotechnology 8: 736-740 (Rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85: 4305-4309 (Corn); Klein et al. (1988) Biotechnology [Biotechnology] 6: 559-563 (Corn); US Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91: 440-444 (Corn); Fromm et al. (1990) Biotechnology 8: 833-839 (Corn); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; US Patent No. 5,736,369 (Cereal); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) In The Experimental Manipulation of Ovule Tissues, Chapman et al. (Longman, New York), pp. 197-209 (Pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255; Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (corn, Agrobacterium tumefaciens); all of these are incorporated herein by reference.

[0155] In certain embodiments, various transient conversion methods can be used to provide the sequences of these embodiments to plants. Such transient conversion methods include, but are not limited to, the direct introduction of the Cryotoxin protein or variants and fragments thereof into the plant or the introduction of Cryotoxin transcripts into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. [Molecular Genetics and General Genetics] 202: 179-185; Nomura et al. (1986) Plant Sci. [Plant Science] 44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 91: 2176-2180; and Hush et al. (1994) The Journal of Cell Science [Journal of Cell Science] 107: 775-784, all of which are incorporated herein by reference. Alternatively, Cryotoxin polynucleotides can be transiently converted into plants using techniques known in the art. Such techniques include viral vector systems and the precipitation of polynucleotides in a manner that prevents subsequent release of DNA. Therefore, transcription can proceed from the DNA bound to the microparticles, but the frequency with which it is released to integrate into the genome is greatly reduced. This approach involves the use of particles coated with polyethyleneimine (PEI; Sigma-Aldrich #P3143).

[0156] Methods for targeted insertion of polynucleotides at specific locations in a plant genome are known in the art. In one embodiment, a site-specific recombination system is used to achieve the insertion of the polynucleotide at the desired genomic location. See, for example, WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855, and WO 99 / 25853, all of which are incorporated herein by reference. Briefly, the polynucleotide of this embodiment can be contained within a transfer cassette flanked by two distinct recombination sites. The transfer cassette is introduced into a plant that has stably incorporated the target site into its genome, flanked by two distinct recombination sites corresponding to the transfer cassette site. A suitable recombinase is provided, and the transfer cassette is integrated into the target site. Thus, the target polynucleotide is integrated at a specific chromosomal location in the plant genome.

[0157] The transformed cells can be cultured into plants using conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be cultured and pollinated with the same or different transformed lines to identify hybrids with constitutive or inducible expression of the desired phenotypic trait. Two or more generations can be cultured to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and then the seeds are harvested to ensure that the expression of the desired phenotypic trait has been achieved.

[0158] Nucleotide constructs of the embodiments can be provided to plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve incorporating the target nucleotide construct into viral DNA or RNA molecules. It has been recognized that the recombinant proteins of the embodiments can initially be synthesized as part of a viral polyprotein, which can then be processed by in vivo or in vitro proteolytic hydrolysis to produce the desired pest-killing protein. It has also been recognized that such a viral polyprotein comprising at least a portion of the amino acid sequence of the pest-killing protein of the embodiments may possess the desired pest-killing activity. Such viral polyproteins and the nucleotide sequences encoding them are covered in these embodiments. Methods for providing nucleotide constructs to plants and producing encoded proteins in plants are known in the art and involve viral DNA or RNA molecules. See, for example, U.S. Patent Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931; which are incorporated herein by reference.

[0159] These embodiments further relate to plant propagation material of the transformed plants of the embodiments, including but not limited to seeds, tubers, bulbs, leaves, and cuttings of roots and buds.

[0160] These embodiments can be used to transform any plant species, including but not limited to monocots and dicots. Examples of target plants include, but are not limited to, corn (Zea mays), Brassica species (e.g., Brassica napus, Brassica rapa, Brassica juncea) (especially those Brassica species that can be used as a source of seed oil), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., Pennisetum glaucum), foxtail millet (Panicum miliaceum), millet (Setaria italica), sorghum (Eleusine coracana), sunflower (Helianthus annuus), and safflower (Carthamus). tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.).Avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), beet (Betavulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamental plants, and conifers.

[0161] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactucasativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (species of the genus Lathyrus spp.)), and members of the genus Cucumber such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and melon (C. melon). Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums. Conifers that can be used in implementing the embodiments include, for example, pine trees such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), American yellow pine (Pinus ponderosa), American black pine (Pinus contorta), and Montreal pine (Pinus radiata), Douglas fir (Pseudotsuga menziesii), western hemlock (Tsuga canadensis), North American spruce (Picea glauca), redwood (Sequoia sempervirens), fir trees such as silver fir (Abies amabilis) and balsam fir (Abies balsamea), and cedar trees such as western cypress (Thuja spp.). plicata)) and Alaskan yellow cedar (Chamaecyparis nootkatensis)).The plants in the examples include crop plants, including but not limited to: corn, alfalfa, sunflower, Brassica species, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, sugarcane, etc.

[0162] Turfgrasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); red fescue (Festuca rubra); meadow bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis). Glomerata; perennial ryegrass (Lolium perenne); red foxgrass (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvetbentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron) smithii); Cynodon spp. (Cynodon species); St. Augustine grass (Stenotaphrum secundatum); Zoysia spp. (Zoysia species)Bahia grass (Paspalum notatum); Carpet grass (Axonopus affinis); Centipede grass (Eremochloa ophiuroides); Kikuyu grass (Pennisetum clandesinum); Seashore paspalum (Paspalumvaginatum); Blue gramma (Bouteloua gracilis); Buffalo grass (Buchloe dactyloids); Sideoats gramma (Bouteloua curtipendula).

[0163] Target plants include cereal plants, oilseed plants, and legumes that provide the target seeds. Target seeds include cereal seeds such as corn, wheat, barley, rice, sorghum, rye, and millet. Oilseed plants include cotton, soybean, safflower, sunflower, brassica, maize, alfalfa, palm, coconut, flax, castor bean, and olive. Legumes include beans and peas. Beans include lentils, locust beans, fenugreek, soybean, green beans, cowpeas, mung beans, lima beans, broad beans, lentils, and chickpeas.

[0164] In some embodiments, the nucleic acid sequence of the embodiment can be stacked with any combination of the target polynucleotide sequence to produce a plant with the desired phenotype. For example, the polynucleotide of the embodiment can be stacked with any other polynucleotide that encodes a polypeptide with pest-killing and / or insecticidal activity, such as other Bt toxin proteins (described in U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109), pentin (described in U.S. Patent No. 5,981,722), etc. The resulting combination may also include multiple copies of any of the target polynucleotides. The polynucleotides of the embodiments can also be stacked with any other gene or combination of genes to produce plants with a variety of desired combinations of traits, including but not limited to traits desired for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g., hordothionin (U.S. Patent Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); high lysine in barley (Williamson et al. (1987) Eur. J. Biochem. [European Journal of Biochemistry] 165: 99-106; and WO 98 / 20122) and high-methionine proteins (Pedersen et al. (1986) J. Biol. Chem. [Journal of Biochemistry] 261: 6279; Kirihara et al. (1988) Gene [Gene] 71: 359; and Musumura et al. (1989) Plant Mol. Biol. [Plant Molecular Biology] 12: 123); increased digestibility (e.g., modified storage proteins (US Patent 6,858,778)) and; and thioredoxin (US Patent No. 7,009,087), the disclosure of which is incorporated herein by reference.

[0165] The polynucleotides in the embodiments can also be stacked with the following: traits desired for disease or herbicide resistance (these traits are, for example, fumonisin detoxification genes (US Patent No. 5,792,931); genes for nontoxicity and disease resistance (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); ethyl acetate leading to herbicide resistance Acyl-Lactate Synthase (ALS) mutants, such as S4 and / or Hra mutants; glutamine synthase inhibitors such as glufosinate or basta (e.g., the bar gene); and glyphosate resistance (EPSPS and GAT genes, as disclosed in U.S. Patent Nos. 7,709,702; and 7,462,481); and desired traits for processing or processed products, such as high oil content (e.g., U.S. Patent No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Patent No. 5,952,544; WO 94 / 11516); modified starches (e.g., ADPG pyrophosphorylase (AGPase), starch synthase (SS)), starch branching enzyme (SBE) and starch debranching enzyme (SDBE)); and polymers or bioplastics (e.g., U.S. Patent No. 5,602,321; β-ketothiolysis, polyhydroxybutyrate synthase and acetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. [Journal of Bacteriology] 170: 5837-5847) promoting polyhydroxyalkanoate (PHA) expression), the disclosure of which is incorporated herein by reference. The polynucleotides of the embodiments may also be combined with polynucleotides that provide agronomic traits (e.g., male sterility (see, for example, U.S. Patent No. 5,583,210), stem strength, flowering time, or transformational traits (e.g., cell cycle regulation or gene targeting (e.g., WO 99 / 61619; WO 00 / 17364; WO 99 / 25821)), the disclosure of which is incorporated herein by reference.

[0166] In some embodiments, the stacked traits may be traits or events that have already obtained regulatory approval (including, but not limited to, the following events), which can be found at the Environmental Risk Assessment Center (cera-gmc.org / ?action=gm_crop_database, which can be accessed using the www prefix) and at the International Service for the Application of Agri-biotech Technologies (isaaa.org / gmapprovaldatabase / default.asp, which can be accessed using the www prefix).

[0167] These stack combinations can be produced by any method, including but not limited to: any conventional or This method involves plant hybridization breeding or genetic transformation. If these traits are stacked by genetically transforming plants, the target polynucleotide sequences can be combined at any time and in any order. For example, a transgenic plant containing one or more desired traits can be used as a target to introduce additional traits through subsequent transformation. These traits can be introduced along with the target polynucleotides provided by any combination of transformation cassettes using a co-transformation protocol. For example, if two sequences are introduced, they can be contained in separate transformation cassettes (trans) or in the same transformation cassette (cis). Expression of these sequences can be driven by the same promoter or by different promoters. In some cases, it may be desirable to introduce a transformation cassette that will inhibit the expression of the target polynucleotide. This can be combined with any combination of other repressor or overexpression cassettes to produce the desired combination of traits in the plant. It should be further recognized that site-specific recombination systems can be used to stack polynucleotide sequences at desired genomic locations. See, for example, WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855, and WO 99 / 25853, all of which are incorporated herein by reference.

[0168] The compositions of the examples can be applied to protect plants, seeds, and plant products in a variety of ways. For example, the compositions can be used in methods involving placing an effective amount of the pest-killing composition into a pest environment through a procedure selected from the group consisting of spraying, dusting, sowing, or seed coating.

[0169] Plant propagation material (fruits, tubers, bulbs, corms, grains, seeds), especially seeds, is typically treated with herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these formulations before being sold as a commercial product. If necessary, this treatment may also be combined with additional carriers, surfactants, or application promoters commonly used in the formulation industry to provide protection against damage caused by bacteria, fungi, or animal pests. For seed treatment, protective coatings can be applied by impregnating tubers or grains with a liquid formulation or by coating them with a combination of wet or dry formulations. Additionally, in special cases, other methods of application to the plant are possible, such as treatment of buds or fruits.

[0170] Plant seeds of embodiments containing nucleotide sequences encoding the pest-killing proteins of the embodiments can be treated with a seed protectant coating comprising, for example, captan, carbendazim, thiram, metalaxyl, methyl pyrimiphos, and other agents commonly used in seed treatment. In one embodiment, the seed protectant coating comprising the pest-killing composition of the embodiments is used alone or in combination with one of the seed protectant coatings commonly used in seed treatment.

[0171] It has been recognized that genes encoding proteins that kill pests can be used to transform entomopathogenic protozoa. These organisms include baculoviruses, fungi, protozoa, bacteria, and nematodes.

[0172] Genes encoding the biotoxic protein of an embodiment can be introduced into a microbial host via a suitable vector, and the host can be applied to an environment, plant, or animal. In the context of inserting nucleic acids into cells, the term "introduction" means "transfection," "conversion," or "transduction" and includes the incorporation of nucleic acids into eukaryotic or prokaryotic cells, wherein the nucleic acids can be incorporated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), transformed into autonomous replicons, or transiently expressed (e.g., transfected mRNA).

[0173] Microbial hosts known to occupy the "phytosphere" (leaf surface, leaf margin, rhizosphere, and / or root surface) of one or more target crops can be selected. These microorganisms are selected so that they can successfully compete with wild-type microorganisms in a specific environment, provide stable maintenance and expression of genes expressing pest-killing proteins, and, hopefully, provide improved protection against environmental degradation and inactivation of pest-killing agents.

[0174] These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, including *Pseudomonas*, *Erwinia*, *Serratia*, *Klebsiella*, *Xanthomonas*, *Streptomyces*, *Rhizobium*, *Rhodopseudomonas*, *Methylius*, *Agrobacterium*, *Acetobacter*, and lactic acid bacteria. Genera of bacteria include Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, especially yeasts such as Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.Of particular interest are the plant-sphere bacterial species, such as *Pseudomonas syringae*, *Pseudomonas fluorescens*, *Serratia marcescens*, *Acetobacter xylinum*, *Agrobacterium*, *Rhodopseudomonas spheroides*, *Xanthomonas campestris*, *Rhizobium melioti*, *Alcaligenes entrophus*, *Clavibacter xyli*, and *Azotobacter*. *Vinelandii*, as well as plant-sphere yeast species, such as *Rhodotorularubra*, *R. glutinis*, *R. marina*, *R. aurantiaca*, *Cryptococcus albidus*, *C. diffluens*, *C. laurentii*, *Saccharomyces rosei*, *S. pretoriensis*, *S. cerevisiae*, *Sporobolomyces roseus*, *S. odorus*, *Kluyveromyces veronae*, and *Aureobasidium pollulans*. Of particular interest are the colored microorganisms.

[0175] Under conditions that allow for the stable maintenance and expression of genes, numerous methods are available to introduce genes expressing harmful organism proteins into microbial hosts. For example, expression cassettes can be constructed that include a target nucleotide construct operatively linked to transcriptional and translational regulatory signals for expressing the nucleotide construct, a nucleotide sequence homologous to a sequence in the host organism (which will be integrated here), and / or a replication system functional in the host (which will be integrated or stably maintained here).

[0176] Transcriptional and translation regulatory signals include, but are not limited to, promoters, transcription start sites, operons, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, and termination signals. See, for example, U.S. Patents 5,039,523 and 4,853,331; EPO 0480762 A2; Sambrook; Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Davis et al., ed. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and the references cited therein.

[0177] Suitable host cells (where the treated cells, when applied to an environment containing one or more target pests, are treated to prolong the activity of the pest-killing proteins within the cells) can include prokaryotes or eukaryotes, and are generally limited to those cells that do not produce toxic substances to higher organisms (e.g., mammals). However, organisms that produce toxic substances to higher organisms may be used, where the toxin is unstable or its application level is low enough to avoid any possibility of toxicity to a mammalian host. Prokaryotes and lower eukaryotes, such as fungi, are of particular interest as hosts. Illustrative prokaryotes (both Gram-negative and Gram-positive) include: Enterobacteriaceae, such as *Escherichia*, *Erwinia*, *Shigella*, *Salmonella*, and *Proteus*; Bacillaceae; Rhizobiaceae, such as *Rhizobium*; and Spirillaceae, such as *Photobacterium*. Genus *Zymomonas*, *Serratia*, *Aeromonas*, *Vibrio*, *Desulfovibrio*, and *Spirillum*; Family Lactobacillaceae; Family Pseudomonadaceae, such as *Pseudomonas* and *Acetobacter*; Family Azotobacteraceae and Family Nitrobacteraceae. In eukaryotes, these are fungi, such as those belonging to the classes Phycomycetes and Ascomycetes, including yeasts such as *Saccharomyces* and *Schizosaccharomyces*; and basidiomycetes yeasts such as *Rhodotorula*, *Aureobasidium*, and *Sporobolomyces*.

[0178] Particularly noteworthy features selected for use in host cells for the production of pest-killing proteins include ease of introduction of the pest-killing protein gene into the host, availability of the expression system, expression efficiency, protein stability in the host, and the presence of accessory gene functions. Features of interest for use as microcapsules of pest-killing agents include protective properties of the pest-killing agent, such as thick cell walls, pigmentation, and formation of intracellular packaging or inclusion bodies; leaf affinity; lack of mammalian toxicity; attraction for pest uptake; and ease of killing and remediation without damaging the toxin. Other considerations include ease of formulation and handling, economics, and storage stability.

[0179] Of particular interest are host organisms including yeasts, such as species of the genera *Rhodotorula*, *Bifidobacterium*, *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), and *Saccharomyces*; leaf organisms, such as species of the genera *Pseudomonas* (e.g., *Pseudomonas aeruginosa*, *Pseudomonas fluorescens*), *Erwinia*, *Flavobacterium* spp., and others including Bt, *Escherichia coli*, and *Bacillus subtilis*.

[0180] Genes encoding the pest-killing protein of an embodiment can be introduced into microorganisms (ectoparasites) that reproduce on plants to deliver the pest-killing protein to potential target pests. Ectoparasites can be, for example, Gram-positive or Gram-negative bacteria.

[0181] For example, root-colonizing bacteria can be isolated from the target plant using methods known in the art. Specifically, Bacillus cereus strains colonizing the roots of plants can be isolated (see, for example, Handelsman et al. (1991) Appl. Environ. Microbiol. [Applied Environmental Microbiology] 56: 713-718). Genes encoding the harmful organism-killing protein of the embodiment can be introduced into Bacillus cereus colonizing the roots using standard methods known in the art.

[0182] Genes encoding pest-killing proteins can be introduced into Bacillus species colonizing the roots, for example, via electroporation. Specifically, genes encoding pest-killing proteins can be cloned into shuttle vectors, such as pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. [FEMS Microbiology Communications] 60: 211-218). The shuttle vector pHT3101, containing the coding sequence of a specific pest-killing protein gene, can be transformed into Bacillus species colonizing the roots, for example, via electroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. [FEMS Microbiology Communications] 60: 211-218).

[0183] Expression systems can be designed to secrete cytotoxic proteins from the cytoplasm of Gram-negative bacteria such as Escherichia coli. The advantages of having secreted cytotoxic proteins are: (1) avoiding the potential cytotoxic effects of expressed cytotoxic proteins; and (2) improving the purification efficiency of cytotoxic proteins, including but not limited to improving the efficiency of protein recovery and purification per volume of cell culture medium, and reducing the time and / or cost of recovery and purification per unit of protein.

[0184] This can enable the secretion of biocidal proteins in *E. coli*, for example, by fusing a suitable *E. coli* signal peptide to the amino terminus of the biocidal protein. Signal peptides recognized by *E. coli* can be found in proteins known to be secreted in *E. coli* (e.g., OmpA protein) (Ghrayeb et al. (1984) EMBO J [Journal of the European Society for Molecular Biology] 3: 2437-2442). OmpA is a major protein of the *E. coli* outer membrane, and therefore its signal peptide is considered effective in the translocation process. Furthermore, the OmpA signal peptide does not require modification prior to treatment, as is the case with other signal peptides, such as lipoprotein signal peptides (Duffaud et al. (1987) Meth. Enzymol. [Enzymological Methods] 153: 492).

[0185] The biocidal proteins of the embodiments can be fermented in a bacterial host, and the resulting bacteria are treated in the same manner as Bt strains have been used as insecticide sprays and used as microbial sprays. In the case of one or more biocidal proteins secreted from Bacillus spp., the secretion signal is removed or mutated using procedures known in the art. Such mutations and / or deletions prevent the secretion of one or more biocidal proteins into the growth medium during the fermentation process. The biocidal proteins are retained intracellularly, and the cells are then treated to produce encapsulated biocidal proteins. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bt toxins as encapsulated proteins, and the resulting cells have been treated and sprayed as insecticides (Gaertner et al. (1993), in: Advanced Engineered Pesticides, edited by Kim).

[0186] Alternatively, pest-killing proteins can be produced by introducing a heterologous gene into a cell host. Expression of the heterologous gene directly or indirectly leads to the production and maintenance of the pest-killing agent within the cell. The cells are then treated under conditions that prolong the activity of the toxin produced within them when the cells are applied to an environment containing one or more target pests. The resulting product retains the toxicity of the toxin. These naturally encapsulated pest-killing proteins can then be formulated using conventional techniques for application to environments (e.g., soil, water, and plant leaves) hosting the target pests. See, for example, EP 0192319, and the references cited therein.

[0187] In embodiments, transformed microorganisms (including whole organisms, cells, one or more spores, one or more pest-killing proteins, one or more pest-killing components, one or more components affecting pests, one or more mutants, live or dead cells and cell components, including mixtures of live and dead cells and cell components, and including broken cells and cell components) or isolated pest-killing proteins can be formulated with an acceptable carrier into one or more pest-killing compositions (i.e., suspensions, solutions, emulsions, spray powders, dispersible granules or pellets, wettable powders and emulsifiable concentrates, aerosols or sprays, impregnated granules, adjuvants, coating pastes, colloids), and further encapsulated in, for example, a polymeric substance. Such formulations can be prepared by conventional methods such as drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, precipitating, or concentrating cell cultures containing peptides.

[0188] The compositions disclosed above can be obtained by adding surfactants, inert carriers, preservatives, wetting agents, feeding stimulants, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectants, buffers, flow agents, or fertilizers, micronutrient donors, or other agents that affect plant growth. One or more agrochemicals, including but not limited to herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, harvesting aids, and fertilizers, can be combined with carriers, surfactants, or adjuvants commonly used in formulation or other component fields to facilitate product treatment and application to specific target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to substances commonly used in formulation techniques, such as natural or recycled minerals, solvents, dispersants, wetting agents, thickeners, binders, or fertilizers. The active ingredients of the embodiments are typically applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions of the embodiments can be applied to grains during preparation or storage in barns or silos. The compositions of the examples may be applied simultaneously or sequentially with other compounds. Methods of applying the active ingredients of the examples or the agrochemical compositions of the examples (which contain at least one of the pest-killing proteins produced by the bacterial strains of the examples) include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of the corresponding pest infestation.

[0189] Suitable surfactants include, but are not limited to, anionic compounds, such as carboxylates of metals; carboxylates of long-chain fatty acids; N-acylsarcosine salts; monoesters or diesters of phosphates and fatty alcohol ethoxylates or salts of these esters; fatty alcohol sulfates, such as sodium dodecyl sulfate, sodium octadecyl sulfate, or sodium hexadecyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates, such as alkylbenzene sulfonates or lower alkylnaphthalene sulfonates, such as butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates, such as amide sulfonates, such as the sulfonated condensation product of oleic acid and N-methyl taurine; or dialkyl sulfosuccinates, such as sodium dioctyl succinate sulfonate. Nonionic surfactants include condensation products of fatty acid esters, fatty alcohols, fatty acid amides, or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide; fatty esters of polyol ethers, such as sorbitol fatty acid esters; condensation products of such esters with ethylene oxide, such as polyoxyethylene sorbitol fatty acid esters; block copolymers of ethylene oxide and propylene oxide; alkynyl diols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol or ethoxylated acetylenic diol. Examples of cationic surfactants include: aliphatic mono-, di-, or polyamines, such as acetates, naphthenates, or oleates; or oxyamines, such as amine oxides of polyoxyethylene alkylamines; amide-linked amines prepared by condensation of carboxylic acids with diamines or polyamines; or quaternary ammonium salts.

[0190] Examples of inert materials include, but are not limited to, inorganic minerals such as kaolin, layered silicates, carbonates, sulfates, and phosphates; or plant materials such as cork, powdered corn cobs, peanut shells, rice husks, and walnut shells.

[0191] The compositions of the examples may be in suitable forms for direct application or as a concentrate of the main composition, which requires dilution with an appropriate amount of water or other diluent before application. The pest-killing concentration will vary depending on the nature of the specific formulation (specifically, whether it is a concentrate or for direct application). The compositions contain 1% to 98% of a solid or liquid inert carrier and 0% to 50% or 0.1% to 50% of a surfactant. These compositions will be administered at the label rates of commercial products, for example, about 0.01 lb to 5.0 lb / acre when in dry form and about 0.01 pts. to 10 pts. / acre when in liquid form.

[0192] In another embodiment, the composition, as well as the transformed microorganisms and pest-killing proteins of the embodiments, may be treated prior to formulation to prolong pest-killing activity when applied to the environment of the target pest, provided that such pretreatment does not harm the pest-killing activity. Such treatment may be carried out by chemical and / or physical methods, provided that the treatment does not adversely affect the properties of one or more compositions. Examples of chemical agents include, but are not limited to, halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infective agents such as zephiran chloride; alcohols such as isopropanol and ethanol; and histological fixatives such as Bouin's fixative and Helly's fixative (see, for example, Humason (1967) Animal Tissue Techniques (WH Freeman and Co.)).

[0193] In other embodiments, treatment of the Cry toxin peptide with a protease (e.g., trypsin) may be beneficial to activate the protein before applying the pest-killing protein composition of the embodiments to the environment of the target pest. Methods for activating the protoxin via serine proteases are well known in the art. See, for example, Cooksey (1968) Biochem.J. [Journal of Biochemistry] 6:445-454, and Carroll and Ellar (1989) Biochem.J. [Journal of Biochemistry] 261:99-105, whose teachings are incorporated herein by reference. Suitable activation protocols include, for example, combining the peptide to be activated (e.g., a purified novel Cry peptide (e.g., having the amino acid sequence shown in SEQ ID NO: 2)) and trypsin at a protein / trypsin weight ratio of 1 / 100 in 20 nM NaHCO3 (pH 8), and digesting the sample at 36°C for 3 hours.

[0194] The composition (including the transformed microorganisms and pest-killing proteins of the examples) can be applied to an environment infested with insect pests, such as by spraying, atomizing, dusting, scattering, coating, or pouring, either as a protective measure when pests have already begun to appear or before they appear, by introducing it into irrigation water, by seed treatment, or by general application or dusting. For example, the pest-killing proteins and / or transformed microorganisms of the examples can be mixed with grains to protect them during storage. Generally, it is important to achieve good pest control in the early stages of plant growth, as this is when plants are most susceptible to damage. If deemed necessary, the compositions of the examples can conveniently contain another insecticide. In one embodiment, the composition is applied directly to the soil at planting time in particulate form as a carrier and a composition of dead cells of the Bacillus strain or transformed microorganisms of the examples. Another embodiment is a particulate form containing an agrochemical (e.g., like a herbicide, insecticide, fertilizer, inert carrier) and dead cells of the Bacillus strain or transformed microorganisms of the examples.

[0195] Those skilled in the art will recognize that not all compounds are equally effective against all pests. The compounds of the examples exhibit activity against insect pests that may include economically important pests in agronomy, forestry, greenhouses, nurseries, ornamental plants, food and fiber, public and animal health, residential and commercial buildings, and household and stored products. Insect pests include insects selected from the following orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., especially Coleoptera and Lepidoptera.

[0196] Lepidoptera insects include, but are not limited to, armyworms, noctuid moths, inchworms, and bollworms of the family Noctuidae: * *Agrotis ipsilon Hufnagel* (black cutworm); * *A. orthogonia Morrison* (western cutworm); * *A. segetum Denis & Schiffermüller* (turnip moth); * *A. subterranea Fabricius* (granulate cutworm); * *Alabama argillacea Hübner* (cotton leaf worm); * *Anticarsia gemmatalis Hübner* (velvetbean caterpillar); * *Athetis mindara Barnes and McDunnough* (rough skinned cutworm); * *Earias insulana* (cotton fruit moth). Boisduval (spiny bollworm); E. vitella Fabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoa messoria Harris (darksided cutworm); Helicoverpa armigera Hübner (American bollworm); corn earworm (corn earworm or cotton bollworm); Heliothis virescens Fabricius (tobacco budworm); Hypenascabra Fabricius (green cloverworm); Mamestra *Configurata Walker* (Bertha Armyworm); *M. cabbage noctuid moth*.* *Brassicae Linnaeus* (cabbage moth); *Melanchra picta Harris* (zebra caterpillar); *Pseudaletia unipuncta Haworth* (noctuar); *Pseudoplusia includens Walker* (soybean looper); *Richia albicosta Smith* or *Western bean cutworm*; *Spodoptera frugiperda JESmith* (fall armyworm); *S. exigua Hübner* (beetarmyworm); *S. litura Fabricius* (tobacco cutworm, cluster caterpillar); *Trichoplusiani* Hübner (cabbage looper); moths, sheath moths, web-forming insects, coneworms, and leaf-eating insects from the family Pyralidae; and grass moths such as Achroia grisella Fabricius (lesser wax moth); Amyelois transitella Walker (naval orange moth); Anagastakuehniella Zeller (Mediterranean flour moth); Cadracautella Walker (almond moth); Chilo partellus Swinhoe (stem moth); and C. suppressalis. Rice stem borer (C. terrenellus Pagenstecher); Rice moth (C. cephalonica Stainton); Corn root webworm (C. caliginosellus Clemens); Early maturing grass moth (C. )teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grapeleaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer); sugarcane borer (D.Sugarcane borer (saccharalis Fabricius); Lesser cornstalk borer (Elasmopalpus lignosellus Zeller); Mexican rice borer (Eoreuma loftini Dyar); Tobacco moth (Ephestia elutella Hübner); Great wax moth (Galleriamellonella Linnaeus); Sugarcane leafroller (Hedylepta accepta Butler); Sod webworm (Herpetogramma licarsisalis Walker); Sunflower electellum Hulst (sunflower moth); Loxostege sticticalis Linnaeus (beetwebworm); Maruca testulalis Geyer (bean pod borer); Orthaga thyrisalis Walker (tea tree web moth); Ostrinianubilalis Hübner (European corn borer); Plodiainterpunctella Hübner (Indian meal moth); Scirpophagaincertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leaf roller) Leaf tier); and leaf rollers, aphids, seed-bearing insects, and fruit-bearing insects in the Tortricidae family, including the Western blackheaded budworm (Acleris gloverana Walsingham) and the Eastern blackheaded budworm (A).variana Fernald (Eastern blackheaded budworm); Adoxophyes orana Fischer von. apple leafroller. (Summer fruit tortrix moth) ; Species of the genus *Archips* spp., including the fruit tree leaf roller (*A. argyrospila* Walker) and the European leaf roller (*A. rosana* Linnaeus). Species of the genus *Argyrotaenia* spp.; Brazilian apple leaf roller (*Bonagota salubricola* Meyrick) (Brazilian apple leaf roller) ; Species of the genus *Choristoneura* spp.; Banded sunflower moth (*Cochylis hospes* Walsingham) (banded sunflower moth) ; Filbert worm (*Cydialatiferreana* Walsingham) ; Codling moth (*C. pomonella* Linnaeus) (codling moth) ; Grape fruit moth (*Endopiza viteana*). Clemens (grape leafroller); Eupoecilia ambiguella Hübner (grape fruit borer); Grapholita molesta Busck (pear fruit moth); Lobesia botrana Denis & Schiffermüller (European grape vinemoth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Spilonota ocellaha Denis & Schiffermüller (eyespotted bud moth); and Suleima helianthaha (sunflower bud moth). Riley (sunflower bud moth).

[0197] Other agronomical pests selected from the Lepidoptera include, but are not limited to, the fall cankerworm (Alsophilapometaria Harris); the peach twig borer (Anarsia lineatella Zeller); and the orange-striped rhinoceros moth (Anisota senatoria JE).Smith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese oak moth); Bombyx mori Linnaeus (silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datanaintegerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth); Ennomos usbsignaria Hübner (elm looper). spanworm); linden looper (Erannistiliaria Harris); sugarcane bud moth (Erechthias flavistriata Walsingham or sugarcane bud moth); browntail moth (Euproctis chrysorrhoea Linnaeus); grasshopper moth (Harrisina americana Guérin-Méneville); subflexa Guenée; range caterpillar (Hemileucaoliviae Cockrell); fall webworm (Hyphantria cunea Drury); tomato pinworm (Keiferia lycopersicella Walsingham). pinworm); Eastern hemlock looper (Lambdina fiscellaria fiscellaria Hulst); Western hemlock looper (L.fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (snow moth); Lymantria dispar Linnaeus (gypsymoth); Malocosoma spp.; Manduca quinquemaculata Haworth (five-spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operaphtera brumata Linnaeus (winter moth); Orgyia spp.; Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orangedog); Phryganidia californica Packard (Californiaoakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. dark-veined cabbage white (…).* *Napi Linnaeus* (green veined whitebutterfly); *Platyptilia carduidactyla Riley* (artichokeplume moth); *Plutella xylostella Linnaeus* (diamondback moth); *Pectinophora gossypiella Saunders* (pink bollworm); *Pontia protodice Boisduval & Leconte* (Southern cabbageworm); *Sabulodes aegrotata Guenée* (omnivorous looper); *Schizura concinna JESmith* (red humped caterpillar); *Sitotroga cerealella* Olivier (grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothsmoth); Tuta absoluta Meyrick (tomato leafminer); and Yponomeuta padella Linnaeus (ermine moth).

[0198] Interesting are the larvae and adults of the order Coleoptera, including weevils from the families Anthribidae, Bruchidae, and Curculionidae, including but not limited to: the Mexican boll weevil (Anthonomus grandis Boheman); the sunflower stem weevil (Cylindrocopturus adspersus LeConte); the root weevil (Diaprepes abbreviatus Linnaeus); the clover leaf weevil (Hyperapunctata Fabricius); the rice water weevil (Lissorhoptrusoryzophilus Kuschel); and the West Indian cane weevil (Metamasius hemipterus hemipterus Linnaeus). Weevil; Sugarcane silk weevil (M. hempterus sericeus Olivier or silky cane weevil); Grain weevil (Sitophilus granarius Linnaeus); Rice weevil (S. oryzae Linnaeus); Red sunflower seed weevil (Smicronyx fulvus LeConte); Gray sunflower seed weevil (S. sordidus LeConte); Maize billbug (Sphenophorus maidis Chittenden); New Guinea sugarcane weevil (Rhabdoscelus obscurus Boisduval). sugarcaneweevil); flea beetles, cucumber leaf beetles, root worms, leaf beetles, potato leaf beetles, and leaf miners of the Chrysomelidae family, including but not limited to: Chaetocnema ectypa Horn (desert corn flea beetle); corn copper flea beetle (C.* *Pulicaria Melsheimer* (corn flea beetle); *Colaspis brunnea Fabricius* (grape colaspis); *Diabrotica barberi Smith & Lawrence* (northern corn rootworm); *D. undecimpunctata howardi Barber* (southern corn rootworm); *D. virgifera virgifera LeConte* (western corn rootworm); *Leptinotarsa ​​decemlineata Say* (Colorado potato beetle); *Oulema melanopus Linnaeus* (cereal leaf beetle); *Phyllotreta cruciferae* (cruciferous flea beetle). Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae, including but not limited to: Epilacachna varivestis Mulsant (Mexican bean beetle); scarab beetles and other beetles from the family Scarabaeidae, including but not limited to: Antitrogus parvulus Britton (Childers cane grub); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. spp. (C. spp.)*Immaculata Olivier* (Southern masked chafer, white grub); *Dermolepida albohirtum Waterhouse* (grey-backed cane beetle); *Euetheola humilis rugiceps LeConte* (cane beetle); *Lepidiota frenchi Blackburn* (French cane grub); *Tomarus gibbosus De Geer* (carrot beetle); *T. subtropicus Blatchley* (cane grub); *Phyllophaga crinita Burmeister* (white grub); *P. latifrons LeConte* (June beetle); *Popillia japomca Newman* (Japanese beetle); *Rhizotrogus* (European root-cutting grub). *Majalis Razoumowsky* (European chafer); *Carpet beetle* (from the family Dermestidae); *Hairy worms* (from the family Elateridae), including species of the genera *Eleodes* and *Melanotus* (including *M. communis Gyllenhal*); *Conoderus*; *Limonius*; *Agriotes*; *Ctenicera*; *Aeolus* (spp.); bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae, such as, but not limited to, *Migdolus fryanus* Westwood (longhorn beetle); and beetles from the family Buprestidae, including but not limited to: *Aphanisticuscochinchinae seminulum Obenberger* (leaf-mining buprestidbeetle).

[0199] Of particular interest are adult and immature Diptera, including the corn leafminer (Agromyza parvicornis Loew); chironomids (including but not limited to: sorghum midge (Contarinia sorghicola Coquillett); Hessian fly (Mayetiola destructor Say); wheat midge (Neolasioptera murtfeldtiana Felt); fruit flies (Tephritidae) and Swedish straw flies (Oscinella frit Linnaeus); and maggots (including but not limited to: species of the genus Delia). spp., including the gray ground seed fly (Delia platura Meigen) (seed fly); the wheat bulb fly (D. coarctata Fallen) (wheat bulb fly); the summer toilet fly (Fannia canicularis Linnaeus), the lesser housefly (F. femoralis Stein) (lesser housefly); the American wheat stem fly (Meromyza americana Fitch) (wheat stemmaggot); the house fly (Musca domestica Linnaeus) (house fly); the stable fly (Stomoxyscalcitrans Linnaeus) (stable fly); the autumn house fly, the hornfly, the green bottle fly, species of the genus Chrysomya (Chrysomya spp.); species of the genus Phormia (Phormia spp.); and other muscoid fly pests, and horseflies (Tabanus spp.). spp.); bot flies (Gastrophilus spp.); Oestrus spp.; cattle grubs (Hypoderma spp.); deer flies (Chrysops spp.).); sheep ticks (Melophagus ovinus Linnaeus) and other Brachycera species; mosquitoes (Aedes spp.); Anopheles spp.; Culex spp.; black flies (Prosimulium spp.); black midges (Simulium spp.); vampire midges, sand flies, sciarids, and other Nematocera species.

[0200] The target insects include insects of the order Hemiptera, such as, but not limited to, the following families: Aphididae, Whiteflyidae, Aphididae, Scale Scale, Spinyfly Scale, Leafhopper Scale, Cicadidae, Scale Scale, Scale Insect, Scale Insect, Plymanidae, Scale Insect ...

[0201] Important agricultural members from the order Hemiptera include, but are not limited to: *Acrosternum hilareSay* (green stink bug); *Acyrthisiphon pisum Harris* (pea aphid); *Adelges spp.* (adelgids); *Adelphocorisrapidus Say* (rapid plant bug); *Anasa tristis De Geer* (squash bug); *Aphis craccivora Koch* (cowpea aphid); *A. fabae Scopoli* (black bean aphid); *A. gossypii Glover* (cotton aphid); and *melon* aphid. aphid); Corn root aphid (A. maidiradicis Forbes); Apple yellow aphid (A. pomi De Geer); Spirea aphid (A. spiraecola Patch); Sugarcane scale insect (Aulacaspistegalensis Zehntner); Foxglove aphid (Aulacorthum solani Kaltenbach); Tobacco whitefly (Bemisia tabaci Gennadius); Sweet potato whitefly (B. argentifolii Bellows & Perring); American valley long bug (Blissus) *Leucopterus leucopterus Say* (chinch bug); a species of the family Blostomatidae (Blostomatidae spp.).); Cabbage aphid (Brevicoryne brassicae Linnaeus); Pear psylla (Cacopsyllapyricola Foerster); Potato capsid bug (Calocoris norvegicus Gmelin); Strawberry aphid (Chaetosiphon fragaefolii Cockerell); Species of the genus Cimicidae; Species of the family Coreidae; Corythuca gossypii Fabricius; Tomato bug (Cyrtopeltis modesta Distant); Suckfly (C. notatus Distant); Deois flavopicta. (Spittlebug); Citrus whitefly (Dialeurodes citri Ashmead); Honey locust plant bug (Diaphnocoris chlorionis Say); Russian wheat aphid (Diuraphis noxia Kurdjumov / Mordvilko); Armored scale (Duplachionaspis divergens Green); Rosy apple aphid (Dysaphis plantaginea Paaserini); Cotton bug (Dysdercus suturellus Herrich-) (cotton stainer) ; (dysmicoccus boninsis Kuwana) (gray sugarcane mealybug) ; (Empoasca fabae Harris) (potato leafhopper) ; (Eriosoma lanigerum Hausmann) (woolly apple aphid) ; (Erythroneoura spp.) (grapeleafhoppers) ; (Eumetopina flavipes Muir) (island sugarcane planthopper) ; (Eurygaster spp.) ; (Euschistus servus Say) (brown stink bug) ; (E. variolarius Palisot de) Beauvois (one-spotted stink bug); a species of the genus Graptostethus spp.(Fruit bug complex); and the peach plumaphid (Hyalopterus pruni Geoffroy); the cottony cushion scale (Icerya purchasi Maskell); the onion plant bug (Labopidicola allii Knight); the small brown planthopper (Laodelphaxstriatellus Fallen); the leaf-footed pine seed bug (Leptoglossus corculus Say); the sugarcane lace bug (Leptodictya tabida Herrich-Schaeffer); and the turnip aphid (Lipaphis erysimi Kaltenbach). Common green capsid (Lygocoris pabulinus Linnaeus); American tarnished plant bug (Lygus lineolaris Palisot de Beauvois); Western tarnished plant bug (L. Hesperus Knight); Common meadow bug (L. pratensis Linnaeus); European tarnished plant bug (L. rugulipennis Poppius); Potato aphid (Macrosiphum euphorbiae Thomas); Two-spotted leafhopper (Macrosteles quadrilineatus) Forbes (asterleafhopper); Magicicada septendecim Linnaeus (periodicalcicada); Mahanarva fimbriolata. (Sugarcane aphid); Sorghum aphid (Melanaphis sacchariZehntner or sugarcane aphid); Mealybug (Melanaspis glomerata Green) (black scale insect); Rose grain aphid (Metopolophium dirhodum Walker); Peach aphid (Myzus persicae Sulzer) (peach-potato aphid, green peachaphid); Lettuce aphid (Nasonovia ribisnigri Mosley) (lettuce aphid); Green leafhopper (Nephotettix cinticeps Uhler); Two-spotted black-tailed leafhopper (N. nigropictus) Rice leafhopper; rice green bug (Nezara viridula Linnaeus); southern green stink bug; brown planthopper (Nilaparvata lugens) Brown planthopper; false chinchbug (Nysius ericae Schilling); false chinchbug (Nysius raphanus Howard); rice stink bug (Oebalus pugnax Fabricius); large milkweed bug (Oncopeltus fasciatus Dallas); orthopscampestris Linnaeus; root aphids and gall aphids (Graptostethus spp.); corn planthopper (Peregrinus maidis Ashmead); sugarcane flat-horned planthopper (Perkinsiella saccharicida) Kirkaldy (sugarcane delphacid); Phylloxera devastatrix Pergande (pecan phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsus lineatus Fabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Pseudococcus spp. (other mealybug groups); Pulvinaria elongata Newstead (cottony grass scale); Pyrilla perpusilla Walker (sugarcane leafhopper); Pyrrhocoridae spp.); pear scale (Quadraspidiotus perniciosus Comstock) (San Jose scale); assassin bug species (Reduviidae spp.); corn aphid (Rhopalosiphum maidis Fitch); birdcherry-oat aphid (R. padi Linnaeus); sugar meal scale (Saccharicoccus sacchari Cockerell); green bug (Schizaphis graminum Rondani); yellow sugarcane aphid (Sipha flava Forbes); English grain aphid (Sitobion avenae Fabricius); white-backed planthopper (Sogatella furcifera) Horvath (white-backed planthopper); Sogatodes oryzicola Muir (rice planthopper); Spanagonicus albofasciatus Reuter (white-marked fleahopper); Therioaphis maculata Buckton (spotted alfalfaaphid); Tinidae spp.; Toxoptera aurantii Boyer deFonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Trialeurodes abutiloneus (bandedwinged whitefly) and greenhouse whitefly (T.*Vaporum vaporariorum* (greenhouse whitefly); *Trioza diospyri* Ashmead (persimmon psylla); and *Typhlocyba pomaria* McAtee (white apple leafhopper).

[0202] In addition, this includes adults and larvae of mites, such as *Aceriatosichella Keifer* (wheat curl mite); *Panonychus ulmi Koch* (European red mite); *Petrobia latens Müller* (brown wheat mite); *Steneotarsonemus bancrofti Michael* (sugarcane stem mite); spider mites and spider mites in the family Tetranychidae, including *Oligonychus grypus Baker & Pritchard*, *O. indicus Hirst* (sugarcane leaf mite), *O. pratensis Banks* (Banks grass mite), and *Oligonychus stickneyi McGregor* (sugarcane spider mite); and *Tetranychus urticae Koch* (two-spotted spider mite). mite); McDaniel mite (T. mcdanieli McGregor); carmine spider mite (T. cinnabarinus Boisduval); strawberry spider mite (T. turkestani Ugarov & Nikolski); flat mite and citrus flat mite (Brevipalpus lewisi McGregor) in the family Tenuipalpidae; rust mite and bud gall mite in the family Eriophyidae; other leaf-feeding mites and mites important to human and animal health, namely dust mites in the family Epidermoptidae, hair follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, and ticks in the family Ixodidae.The following ticks are included: *Ixodes scapularis Say* (deertick); *I. holocyclus Neumann* (Australian paralysis tick); *Dermacentor variabilis Say* (American dog tick); *Amblyomma americanum Linnaeus* (lone star tick); and itch mites and scabies mites in the families Psoroptidae, Pyemotidae, and Sarcoptidae.

[0203] Of particular interest are the insect pests of the order Thysanura, such as silverfish (Lepisma saccharina Linnaeus) and firebrat (Thermobia domestica Packard).

[0204] Other arthropod pests covered include spiders of the order Araneae, such as the brown recluse spider (Loxosceles reclusa Gertsch & Mulaik); the black widow spider (Latrodectus mactans Fabricius); and centipedes of the order Scutigeromorpha, such as the house centipede (Scutigera coleoptrata Linnaeus). Additionally, insect pests of the order Isoptera are of concern, including termites of the family termitidae, such as, but not limited to, Cylindrorermes nordenskioeldi Holmgren and Pseudacanthotermes militaris Hagen (cane termites). Insects of the order Thysanoptera are also of concern, including but not limited to thrips, such as Stenchaetothrips minutus van Deventer (cane thrips).

[0205] The biocidal activity of the compositions of the embodiments against insect pests can be tested at early developmental stages (e.g., as larvae or other immature forms). The insects can be reared in complete darkness at a temperature ranging from about 20°C to about 30°C and a relative humidity ranging from about 30% to about 70%. Bioassays can be performed as described in Czapla and Lang (1990) J. Econ. Entomol. [Journal of Economic Entomology] 83(6): 2480-2485. The methods for rearing insect larvae and performing bioassays are well known to those skilled in the art.

[0206] Various bioassay techniques are known to those skilled in the art. A typical procedure involves adding an experimental compound or organism to a feed source in a closed container. Pesticide activity can be measured, but is not limited to, changes in mortality, weight loss, attractiveness, repulsion, and other behavioral and physical changes following appropriate feeding and exposure. The bioassays described herein can be used for any feeding insect pest at the larval or adult stage.

[0207] The following examples are provided in an illustrative manner rather than in a restrictive manner.

[0208] experiment

[0209] Example 1 - Gene Identification and E. coli Expression

[0210] The polynucleotide encoding the insecticidal protein (SEQ ID NO: 2) named MP372, SEQ ID NO: 1, was obtained from an isolate of Bacillus thuringiensis, named DP250, which was derived from a proprietary collection of Bacillus thuringiensis isolates within DuPont. The polynucleotide (SEQ ID NO: 1) encoding the insecticidal polypeptide (SEQ ID NO: 2) was cloned into the pET28a vector (…). The culture was transformed into *E. coli* BL21 cells (Invitrogen). Large-scale 1.0 L cultures were grown to approximately 0.8 OD600 nm, and then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 16 °C for 16 h. The cell pellet was lysed with 50 mL of 500 mM NaCl / 20 mM Tris / 5 mM imidazole / pH 7.9 (containing 0.02% lysozyme (w / v), 0.1% Tween-20, and one tablet of Complete Protease Inhibitor (Roche). After lysis, the solution was sonicated, and the lysate was centrifuged at 25,000 rpm for 30 min. The supernatant containing the soluble protein fraction was filtered through a 0.45u vacuum filter, and then 1 ml of Talon (Clontech) slurry was added. The mixture was then incubated on a rotor at 100 rpm for 1 hour for binding. The lysate was then added to a column, and the bound protein was separated and washed with 20 ml of 50 mM NaCl / 20 mM Tris / 5 mM imidazole / pH 7.9, followed by elution with 1.5 ml of 50 mM NaCl / 20 mM Tris / 500 mM imidazole / pH 7.9. The purified protein was then dialyzed against 50 mM sodium carbonate buffer at pH 10. The purified protein was submitted for insecticidal activity in the Lepidoptera and Coleoptera in vitro feeding assay group. The insecticidal activity of MP372 (SEQ ID NO: 4) is shown in Table 1.

[0211] Example 2 - Cyr1Ea / MP327 Chimera

[0212] Chimeras were designed using domain I (amino acids 1-259 of SEQ ID NO: 6) from Cry1Ea (accession number X53985) and domains II and III (amino acids 259-617 of SEQ ID NO: 2) from MP372. The resulting chimera was named MP327-Mut 5, which has the polynucleotide sequence of SEQ ID NO: 3 encoding the polypeptide of SEQ ID NO: 4. For in vitro feeding assays of Lepidoptera and Coleoptera species, purified proteins were submitted for insecticidal activity. The insecticidal activity of MP372 (SEQ ID NO: 4) is shown in Table 1. Compared to MP327 (SEQ ID NO: 2), the MP372-Mut 5 (SEQ ID NO: 4) variant exhibits a broader insecticidal spectrum by introducing FAW activity and improved CEW, SBL, and VBC efficacy.

[0213] Example 3 - Insecticidal assay of purified protein

[0214] Insecticidal activity bioassays were conducted to screen for the effects of insecticidal proteins on a variety of species including: Lepidoptera, European corn borer (Ostrinia nubilalis), corn ear moth (Helicoverpazea), black rootworm (Agrotis ipsilon), fall armyworm (Spodopterafrugiperda), soybean looper (Pseudoplusia includens), and soybean looper (Anticarsia gemmatalis); and Coleoptera, western corn rootworm (Diabrotica virgifera).

[0215] Lepidoptera feeding assays were performed on artificial feed containing clarified lysates of bacterial strains in a 96-well plate apparatus. The clarified lysates were mixed with lepidoptera-specific artificial feed at a ratio of 20 μl clarified lysates to 40 μl feed mixture. Two to five newborn larvae were placed in each well and allowed to feed freely for 5 days. Results were expressed as positive larval responses (e.g., stunted growth) and / or mortality. A negative result was indicated if the larvae were similar to a negative control fed only the aforementioned buffer solution. Each clarified lysate was measured on the European corn borer, corn ear moth, black rootworm, fall armyworm (Spodoptera frugiperda), soybean looper (Pseudoplusia includens), and soybean looper (Anticarsia gemmatalis). Larval mortality or stunted growth severity was scored. Scores were recorded as death (3), severe stunted growth (2) (little or no growth but alive, equivalent to a first instar larva), stunted growth (1) (grown to the second instar but not equivalent to the control), or standard (0). The results are shown in Table 1.

[0216] Table 1

[0217]

[0218] For MP372-Mut 5 (SEQ ID NO: 4), a series of concentrations of purified protein samples were determined for SBL and VBC, and the concentration at which 50% mortality (LC50) or 50% inhibition (IC50) was calculated for each individual. The results are shown in Table 2.

[0219] Table 2

[0220] <![CDATA[ insect ]]> <![CDATA[ LC / IC ]]> <![CDATA[ ppm, 4d ]]> <![CDATA[ 95% CL lower limit ]]> <![CDATA[ 95% CL upper limit ]]> <![CDATA[ slope ]]> SBL LC50 1.28 0.28 2.73 4.24 VBC LC50 1.79 0.50 4.25 2.99

[0221] Example 4 - Transient expression and insect bioassay in transient corn leaf tissue

[0222] Two gene designs encoding MP372 (SEQ ID NO: 2) were each cloned into transient expression vectors under the control of a replicative version of the promoter from mirabilis mosaic virus (DMMV PRO; Dey and Maiti, (1999) Plant Mol. Biol., 40: 771-82). The Agrobacterium infiltration method, which introduces Agrobacterium cell suspensions into intact plant cells to allow for reproducible infection and subsequent measurement or study of plant-derived transgenic expression, is well-known in the art (Kapila et al., (1997) Plant Science, 122: 101-108). In short, young corn plant discs were infiltrated with Agrobacterium using standardized bacterial cell cultures of test and control strains. Leaf discs were infected with either the soybean looper (SBL) or the fall armyworm (FAW), along with appropriate controls. The degree of chlorophyll loss was assessed two days after infection.

[0223] Two days after feeding, the amount of leaf tissue consumed by SBL or FAW larvae was scored in 12 discs for each treatment. Compared with the negative control, the tissue damage with accumulated MP372 was significantly less in both FAW and SBL in the 12 discs (Table 3).

[0224] Table 3

[0225] Gene design FAW SBL MP372 V1 + + MP372 V2 + +

[0226] Example 5 - Transient expression and insect bioassay in transient dwarf bean leaf tissue

[0227] To confirm the activity of the insecticidal peptides disclosed herein, a transient expression system of *Phaseolus vulgaris* under the control of the AtUBQ10 promoter was used (Dav et al., (1999) Plant Mol. Biol. [Plant Molecular Biology] 40: 771-782; Norris SR et al. (1993) Plant Mol Biol. [Plant Molecular Biology] 21(5): 895-906). The Agrobacterium infiltration method, which introduces a suspension of *Agrobacterium* cells into intact plant cells to allow for repeated infection and subsequent measurement or study of plant-derived transgenic expression, is well known in the art (Kapila et al., (1997) Plant Science [Plant Science] 122: 101-108). In short, *Agrobacterium* infiltration was performed on *Phaseolus vulgaris* seedlings using one of two gene designs encoding MP372 (SEQ ID NO: 2) or MP372Mut 5 (SEQ ID NO: 4) and a control vector-transformed standardized bacterial cell culture. Four days later, leaf discs were harvested and infected individually with newly emerged soybean armyworm (SBL) (Chrysodeixis includens), velvetbean caterpillar (VBC) (Anticarsia gemmatalis), or fall armyworm (Spodoptera frugiperda). Control leaf discs were produced using plants soaked in buffer. The consumption of chlorophyll tissue was assessed two days post-infection (Table 4).

[0228] Table 4

[0229] Gene design FAW SBL VBC MP372 V1 - + + MP372 V2 - + + MP372 Mut 5 V1 + + + MP372 Mut 5 V2 + + +

[0230] Example 6 - Agrobacterium-mediated transformation of maize and regeneration of transgenic plants

[0231] For Agrobacterium-mediated transformation of maize using the nucleotide sequences of this disclosure (e.g., SEQ ID NO: 1 or SEQ ID NO: 3), Zhao's method (US Patent No. 5,981,840 and PCT Patent Publication WO 98 / 32326; the contents of which are incorporated herein by reference) can be used. In short, immature embryos are isolated from maize and contacted with a suspension of Agrobacterium spp. under conditions where the bacteria are able to transfer the nucleotide sequence (SEQ ID NO: 1 or SEQ ID NO: 3) into at least one cell of at least one immature embryo (Step 1: Infection Step). In this step, the immature embryos may be inoculated by immersing them in the Agrobacterium suspension. These embryos are co-cultured with Agrobacterium spp. for a period of time (Step 2: Co-culture Step). After the infection step, the immature embryos may be cultured on a solid medium. Following this co-culture period, an optional "resting" step is envisioned. In this resting step, the embryos are incubated in the presence of at least one antibiotic (without the addition of a plant transformant selector), known to inhibit the growth of Agrobacterium spp. (Step 3: Resting Step). Immature embryos are cultured on a solid medium containing antibiotics but without selective agents to eliminate Agrobacterium and allow for a quiescent phase of persistent cell infection. Next, the inoculated embryos are cultured on a medium containing a selective agent, and the resulting transformed callus is recovered (Step 4: Selection Step). These immature embryos are then cultured on a solid medium containing a selective agent, allowing for selective growth of the transformed cells. The callus is then regenerated into a plant (Step 5: Regeneration Step), and the callus grown on the selective medium is cultured on a solid medium to regenerate into a plant.

[0232] Example 7 - Transformation of soybean embryo

[0233] Soybean embryos are bombarded with plasmids containing the nucleotide sequences of this disclosure (e.g., SEQ ID NO: 1 or SEQ ID NO: 3) operably linked to a suitable promoter. To induce somatic embryos, cotyledons 3-5 mm in length are excised from surface-sterilized immature seeds of a suitable soybean cultivar and cultured on a suitable agar medium at 26°C in light or dark for six to ten weeks. The somatic embryos that produce secondary embryos are then excised and placed in a suitable liquid medium. After repeated screening of somatic embryo clusters amplified to the early globular stage, the suspension is maintained as described below.

[0234] Soybean embryogenic suspension cultures can be maintained on a rotating shaker at 26°C and 150 rpm in 35 mL of liquid medium, with a 16:8 hour day / night light schedule during flowering. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0235] Soybean embryonic suspension cultures can then be transformed by particle gun bombardment (Klein et al., (1987) Nature (London) 327:70-73; US Patent No. 4,945,050). These transformations can be performed using a DuPont Biolistic PDS1000 / HE instrument (helium-modified).

[0236] Selective marker genes that can be used to promote soybean transformation include, but are not limited to: the 35S promoter from cauliflower mosaic virus (Odell et al., (1985) Nature 313: 810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from Escherichia coli; Gritz et al., (1983) Gene 25: 179-188), and the 3′ region of the carmine synthase gene from the T DNA of the Ti plasmid from Agrobacterium tumefaciens. Expression cassettes containing toxin nucleotide sequences operatively linked to suitable promoters (e.g., SEQ ID NO: 1) can be isolated as restriction fragments. These fragments can then be inserted into the unique restriction enzyme sites of a vector carrying the marker gene.

[0237] Add the following to 50 μL of a 60 mg / mL 1 μm gold particle suspension (in sequence): 5 μL DNA (1 μg / μL), 20 μL spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). Stir the particle formulation for three minutes, centrifuge for 10 seconds, and remove the supernatant. Wash the DNA-coated particles once in 400 μL of 70% ethanol and resuspend in 40 μL of anhydrous ethanol. The DNA / particle suspension can be sonicated three times for one second each time. Then, add five μL of DNA-coated gold particles to each giant carrier disk.

[0238] Place approximately 300-400 mg of two-week-old suspension culture in an empty 60x15 mm culture dish and remove any residual liquid from the tissue using a pipette. For each transformation experiment, typically bombard approximately 5-10 tissue plates. Set the membrane disruption pressure to 1100 psi and evacuate the container to a vacuum of 28 inches of mercury. Place the tissue approximately 3.5 inches from the retardation sieve and bombard three times. After bombardment, the tissue can be halved and returned to the liquid for further culture as described above.

[0239] Five to seven days after bombardment, the liquid medium can be replaced with fresh medium, and eleven to twelve days after bombardment, it can be replaced with fresh medium containing 50 mg / mL hygromycin. This selective medium can be refreshed weekly. After seven to eight weeks of bombardment, transformed green tissue can be observed growing from untransformed necrotic embryogenic clusters. The isolated green tissue is taken and inoculated into individual shake flasks to produce new, asexually propagated, transformed embryogenic suspension cultures. Each new line can be treated as an independent transformation event. These suspensions can then be subcultured and maintained as immature embryogenic clusters, or regenerated into whole plants through the maturation and germination of individual somatic embryos.

[0240] All publications, patents, and patent applications mentioned in this specification indicate the level of skill of a person skilled in the art to which this disclosure pertains. All publications, patents, and patent applications are incorporated herein by reference to the extent that each individual publication, patent, or patent application is explicitly and individually cited in connection with the reference.

[0241] Although the foregoing disclosure has been described in considerable detail by means of illustration and examples for the purpose of clarity, it will be apparent that certain variations and modifications may be practiced within the scope of the embodiments.

Claims

1. A recombinant polynucleotide encoding a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

2. The recombinant polynucleotide of claim 1, operably linked to a heterologous regulatory element.

3. A DNA construct comprising the recombinant polynucleotide according to claim 1 or 2.

4. A method for producing a host cell, wherein the host cell comprises the DNA construct according to claim 3.

5. The method of claim 4, wherein the host cell is a bacterial cell.

6. The method of claim 4, wherein the host cell is a plant cell.

7. A method for producing a transgenic plant, wherein the transgenic plant comprises the DNA construct according to claim 3.

8. The method for producing a transgenic plant according to claim 7, wherein the plant is selected from the group consisting of: Corn, sorghum, wheat, cabbage, sunflower, tomato, cruciferous vegetables, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and rapeseed.

9. The method for producing transformed seeds of a plant according to claim 8, wherein the seeds comprise the DNA construct.

10. An agricultural composition comprising the polypeptide as defined in claim 1.

11. The composition of claim 10, wherein the composition is in the form of a powder, dust, pill, granule, spray, emulsion, colloid, or solution.

12. The composition of claim 10, comprising from 1% to 99% of the polypeptide by weight.

13. A method for controlling a population of lepidopteran pests, the method comprising contacting the population with a pest-killing amount of the polypeptide as defined in claim 1.

14. A method for killing lepidopteran pests, the method comprising contacting the pests with an effective amount of the peptide as defined in claim 1, or feeding the pests an effective amount of the peptide as defined in claim 1.

15. A method for protecting plants from lepidopteran pests, the method comprising expressing in the plant the polynucleotide according to claim 1 or 2.

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