BR112017007927B1 - Insecticidal polypeptides with improved spectrum of activity and their uses - Google Patents

Abstract

INSECTICIDAL POLYPEPTIDES WITH ENHANCED SPECTRUM OF ACTIVITY AND THEIR USES. The disclosure provides nucleic acids, and variants and fragments thereof, derived from strains of Bacillus thuringiensis encoding variant polypeptides with enhanced pesticidal activity against insect pests, including Lepidoptera and Coleopteran. Particular embodiments of the disclosure provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, DNA constructs, and microorganisms and transformed plants comprising a nucleic acid of the embodiments. These compositions find use in methods for controlling pests, especially plant pests.

Description

REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING

[001] The sequence listing with the file name "5409WOPCT_SequenceListing.txt" created on September 24, 2015 and with a size of 267 kilobytes is archived on computer media simultaneously with the descriptive report. The sequence listing is part of the descriptive report and is incorporated herein by reference in its entirety.

AREA OF INVENTION

[002] The present disclosure relates to recombinant nucleic acids encoding pesticidal polypeptides having insecticidal activity against corn earworm and/or fall armyworm and/or an enhanced spectrum of pesticidal activity against insect pests. The compositions and methods of the disclosure utilize the disclosed nucleic acids, and their encoded pesticidal polypeptides, to control plant pests.

BACKGROUND OF THE INVENTION

[003] Insect pests are a major factor in crop losses worldwide. For example, fall armyworm feeding, black cutworm damage, or European corn borer damage can be economically devastating to agricultural producers. Crop losses related to insect pests caused by European corn borer attacks on soil and corn alone have amounted to an estimated $1 billion per year in damages and control costs.

[004] Traditionally, the primary method for impacting 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. Because of these concerns, regulators have banned or limited the use of some of the most hazardous pesticides. Thus, there is substantial interest in the development of alternative pesticides.

[005] Biological control of agriculturally important insect pests using a microbial agent such as fungi, bacteria or another insect species provides an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. In general, the use of biopesticides presents a lower risk of pollution and environmental hazards, and biopesticides provide greater target specificity than that characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yields for a wide variety of crops.

[006] Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a wide range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera, and others. 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 B. larvae, B. lentimorbus, B. sphaericus (Harwook, ed., ((1989) Bacillus (Plenum Press), 306), and B. cereus (WO 96/10083). Pesticidal activity appears to be concentrated in parasporal crystal protein inclusions, although pesticidal proteins have also been isolated from the vegetative growth stage of Bacillus. Several genes encoding these pesticidal proteins have been isolated and characterized (See, e.g., U.S. Patent Nos. 5,366,892 and 5,840,868).

[007] Microbial insecticides, particularly those obtained from strains of Bacillus, have played an important role in agriculture as alternatives to chemical pest control. Recently, agricultural scientists have developed crop plants with increased insect resistance by genetically modifying crop plants to produce Bacillus pesticidal proteins. For example, corn and cotton plants have been genetically modified to produce pesticidal proteins isolated from Bt strains (see, e.g., Aronson (2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998) Microbiol Mol Biol Rev. 62(3):775-806). These genetically modified crops are now widely used in American agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect control methods. In addition, potatoes genetically modified to contain Cry pesticidal toxins have been sold to the American farmer. Although they have proven to be very successful commercially, these genetically modified insect-resistant crop plants provide resistance to only a narrow range of economically important insect pests.

[008] Accordingly, there remains a need for new Bt toxins with an improved spectrum of insecticidal activity against insect pests, e.g. toxins that are actively enhanced against insects of the order Lepidoptera and/or Coleoptera. Additionally, there remains a need for biopesticides with activity against a variety of insect pests and for biopesticides that have improved insecticidal activity.

SUMMARY OF THE INVENTION

[009] Compositions and methods for impacting insect pests are provided. More specifically, embodiments of the present disclosure relate to methods for impacting insects using nucleotide sequences encoding insecticidal peptides to produce transformed microorganisms and plants expressing an insecticidal polypeptide of the embodiments. In some embodiments, the nucleotide sequences encode polypeptides that are pesticidal to at least one insect belonging to the order Lepidoptera.

[0010] In some aspects, provided are nucleic acid molecules, and fragments and variants thereof, that encode polypeptides that have pesticidal activity against insect pests (e.g., SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46), and encoding the polypeptide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45, respectively). The wild-type (e.g., naturally occurring) nucleotide sequence of embodiments, which was obtained from Bt, encodes an insecticidal peptide. Embodiments further provide fragments and variants of the disclosed nucleotide sequence that encode biologically active polypeptides (e.g., insecticides).

[0011] In another aspect, variant Cry1B polypeptides encoded by a modified (e.g., mutagenized or engineered) nucleic acid molecule of the embodiments are provided. In particular examples, pesticidal proteins of the embodiments include fragments of full-length proteins and polypeptides that are produced from mutagenized nucleic acids designed to introduce particular amino acid sequences into the polypeptides of the embodiments. In particular embodiments, the polypeptides have increased pesticidal activity relative to the activity of the naturally occurring polypeptide from which they are derived.

[0012] In another aspect, the nucleic acids of the embodiments can also be used to produce transgenic (e.g., transformed) monocotyledonous or dicotyledonous plants that are characterized by genomes comprising at least one stably incorporated nucleotide construct comprising a coding sequence of the embodiments operably linked to a promoter that directs expression of the encoded pesticidal polypeptide. Accordingly, transformed plant cells, plant tissues, plants, and seeds are also provided.

[0013] In another aspect, the transformed plant can be produced using a nucleic acid that has been optimized for increased expression in a host plant. For example, one of the pesticidal polypeptides of embodiments can be back-translated to produce a nucleic acid comprising codons optimized for expression in a particular host, for example a crop plant such as a corn plant (Zea mays). Expression of a coding sequence by such a transformed plant (e.g., dicotyledonous or monocotyledonous) will result in the production of a pesticidal polypeptide and confer increased insect resistance to the plant. Some embodiments provide transgenic plants expressing pesticidal polypeptides that find use in methods for impacting various insect pests.

[0014] In another aspect, pesticidal or insecticidal compositions containing the variant Cry1B polypeptides of embodiments are provided, and the composition may optionally comprise additional insecticidal peptides. Embodiments encompass the application of such compositions to the insect pest environment in order to influence insect pests.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figures 1a-1g show an alignment of the amino acid sequences, using the ALIGNX® module of the Vector NTI® package, of Cry1Bd (SEQ ID NO: 1), IP1B-B1 (SEQ ID NO: 3), IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27(SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), IP1B-B34 (SEQ ID NO: 29), IP1B-B40 (SEQ ID NO: 31), IP1B-B41(SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), MP258 (SEQ ID NO: 47), and GS060 (SEQ ID NO: 49). The amino acid sequence diversity among Cry1B polypeptides is highlighted.

[0016] Figures 2a-2e show the amino acid sequence of MP258 with the leader region (*), Domain I (#), Domain II (&) and Domain III (!) indicated below the sequence.

[0017] Figure 3 shows an amino acid sequence alignment, using the ALIGNX® module of the Vector NTI® package, of the Cry1Be-like Domain I of Cry1Be (amino acids 35-276 of SEQ ID NO: 58) and the Cry1Be-like Domain I of MP258 (amino acids 36-276 of SEQ ID NO: 47). The amino acid sequence diversity among the Domain Is of the Cry1B polypeptides is highlighted.

[0018] Figure 4 shows an amino acid sequence alignment, using the ALIGNX® module of the Vector NTI® package, of Domain III of Cry1Ah (SEQ ID NO: 61), Cry1Bd, Cry1Bh (SEQ ID NO: 52), Cry1Bi (SEQ ID NO: 54), and MP258 (SEQ ID NO: 47). The amino acid sequence diversity among Domain III of the Cry1B polypeptides is highlighted.

[0019] Figures 5a-5c show an amino acid sequence alignment, using the ALIGNX® module of the Vector NTI® package, of Domain I and Domain II of MP258 (SEQ ID NO: 47), Cry1Be (SEQ ID NO: 58), Cry1Bi (SEQ ID NO: 54), Cry1Bg (SEQ ID NO: 60), Cry1Bf (SEQ ID NO: 59), Cry1Ba (SEQ ID NO: 55), Cry1Bh (SEQ ID NO: 52), Cry1Bd (SEQ ID NO: 1), Cry1Bb (SEQ ID NO: 56), and Cry1Bc (SEQ ID NO: 57). The amino acid sequence diversity between Domain I and Domain II of the Cry1B polypeptides is highlighted.

DETAILED DESCRIPTION

[0020] Embodiments of the disclosure are designed for compositions and methods for impacting insect pests, particularly plant pests. More specifically, the isolated nucleic acid of the embodiments, and fragments and variants thereof, comprise nucleotide sequences that encode pesticidal polypeptides (e.g., proteins). The disclosed pesticidal proteins are biologically active (e.g., pesticides) against insect pests such as, but not limited to, insect pests of the order Lepidoptera and/or Coleoptera.

[0021] Compositions of embodiments comprise isolated nucleic acids, and fragments and variants thereof, encoding pesticidal polypeptides, expression cassettes comprising nucleotide sequences of embodiments, isolated pesticidal proteins, and pesticidal compositions. Some embodiments provide modified pesticidal polypeptides with improved insecticidal activity against Lepidoptera relative to the pesticidal activity of the corresponding wild-type protein. Embodiments further provide plants and microorganisms transformed with these novel nucleic acids, and methods involving the use of such nucleic acids, pesticidal compositions, transformed organisms, and products thereof to impact insect pests.

[0022] The nucleic acid and nucleotide sequences of the embodiments can be used to transform any organism to produce the encoded pesticidal proteins. Methods involving the use of such transformed organisms to impact 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] Embodiments further relate to the identification of fragments and variants of the naturally occurring coding sequence that encode biologically active pesticidal proteins. The nucleotide sequences of the embodiments find direct use in methods for impacting pests, particularly insect pests such as pests of the order Lepidoptera. Accordingly, the embodiments provide novel approaches for impacting insect pests that do not rely on the use of traditional synthetic chemical insecticides. The embodiments involve the discovery of naturally occurring biodegradable pesticides and the genes that encode them.

[0024] Embodiments further provide variant fragments of the naturally occurring coding sequence that also encode biologically active polypeptides (e.g., pesticides). 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, for example nucleic acid sequences that have been back-translated (i.e., reverse translation) using plant-preferred codons based on the amino acid sequence of a polypeptide with increased pesticidal activity. Embodiments further provide mutations that confer improved or altered properties on the polypeptides of the embodiments. See, e.g., U.S. Pat. No. 7,462,760.

[0025] In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the embodiments.

[0026] Units, prefixes and symbols may be denoted in their accepted SI form. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in the amino to carboxy orientation, respectively. Numerical ranges include the numbers that define the range. Amino acids may be referred to herein either by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined above are more fully defined by reference to the specification as a whole.

[0027] As used herein, "nucleic acid" includes reference to a polymer of deoxyribonucleotide or ribonucleotide in single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides.

[0028] As used herein, the terms "coding" or "encoded" when used in the context of a specified nucleic acid mean that the nucleic acid comprises the information necessary for the direct translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) within translated regions of the nucleic acid or may have no such intervening untranslated sequences (e.g., as in cDNA).

[0029] As used herein, "full-length sequence" in reference to a specified polynucleotide or its encoded protein means having the complete nucleic acid sequence or the entire amino acid sequence of a native (non-synthetic) endogenous sequence. A full-length polynucleotide encodes the full-length, catalytically active form of the specified protein.

[0030] As used herein, the term "antisense" used in the context of the orientation of a nucleotide sequence refers to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation in which the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcript such that translation of the endogenous transcript is often inhibited. Thus, when the term "antisense" is used in the context of a particular nucleotide sequence, the term refers to the strand complementary to the reference transcript.

[0031] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids.

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

[0033] Polypeptides of the embodiments can be produced either from a nucleic acid disclosed herein or by using standard molecular biology techniques. For example, a protein of the embodiments can be produced by expression of a recombinant nucleic acid of the embodiments in an appropriate host cell or, alternatively, by a combination of ex vivo procedures.

[0034] As used herein, the terms "isolated" and "purified" are used interchangeably to refer to nucleic acids or polypeptides or biologically active portions thereof that are substantially or essentially free of components that normally accompany or interact with the nucleic acid or polypeptide as found in its natural environment. Thus, an isolated or purified nucleic acid or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0035] An "isolated" nucleic acid is generally free of sequences (such as, for example, protein-coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, isolated nucleic acids may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acids in the genomic DNA of the cell from which the nucleic acid is derived.

[0036] As used herein, the term "isolated" or "purified" as used to refer to a polypeptide of embodiments means that the isolated protein is substantially free of cellular material and includes protein preparations with less than about 30%, 20%, 10%, or 5% (by dry weight) contaminating protein. When the protein of embodiments or its biologically active portion is produced recombinantly, the culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) chemical precursors or non-protein chemicals of interest.

[0037] A "recombinant" nucleic acid (or DNA) molecule is used herein to refer to a nucleic acid (or DNA) sequence that is in a recombinant bacterial or plant host cell. In some embodiments, an "isolated" or "recombinant" nucleic acid is free of sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, "isolated" or "recombinant" when used to refer to nucleic acid molecules excludes isolated chromosomes.

[0038] As used herein, a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" refers to a nucleic acid molecule that has one or more changes in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion, and/or addition compared to the native or genomic sequence; removal of one or more introns associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5' and/or 3' untranslated region; and modification of a polyadenylation site. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0039] Throughout the specification, the word "comprising", or variations such as "comprises" or "comprise", will be understood to imply the inclusion of a declared element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0040] As used herein, the term "impact on insect pests" refers to effecting changes in the feeding, growth and/or behavior of the insect at any stage of development, including but not limited to: killing the insect; retarding growth; preventing reproductive capacity; anti-feeding activity; and the like.

[0041] As used herein, the terms "pesticidal activity" and "insecticidal activity" are used synonymously to refer to the activity of an organism or a substance (such as, for example, a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest repellency, and other behavioral and physical changes of a pest after feeding and exposure for an appropriate period of time. Thus, an organism or substance with pesticidal activity adversely influences at least one measurable parameter of pest condition. For example, "pesticidal proteins" are proteins that exhibit pesticidal activity by themselves or in combination with other proteins.

[0042] As used herein, the term "pesticidally effective amount" means an amount of a substance or organism that has pesticidal activity when present in the environment of a pest. For each substance or organism, the pesticidally effective amount is determined empirically for each affected pest in a specific environment. Similarly, an "insecticidally effective amount" may be used to refer to a "pesticidally effective amount" when the pest is an insect pest.

[0043] As used herein, the term "recombinant engineering" or "engineering" means the use of recombinant DNA technology to introduce (e.g., manipulate) a change in protein structure based on an understanding of the mechanism of action of the protein and a consideration of the amino acids introduced, deleted, or substituted.

[0044] As used herein, the term "mutant nucleotide sequence" or "mutation" or "mutagenized nucleotide sequence" means a nucleotide sequence that has been mutagenized or altered to contain one or more nucleotide residues (e.g., base pair) that are not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions, or alterations or substitutions of nucleic acid residues. When mutations are made by addition, removal, or substitution of an amino acid at a proteolytic site, such addition, removal, or substitution may be within or adjacent to the proteolytic site motif, provided that the object of the mutation is effected (i.e., provided that proteolysis at the site is altered).

[0045] A mutant nucleotide sequence may encode a mutant insecticidal toxin showing enhanced or diminished insecticidal activity, or an amino acid sequence that confers enhanced or diminished insecticidal activity over a polypeptide containing it. As used herein, the term "mutant" or "mutation" in the context of a protein, polypeptide or amino acid sequence refers to a sequence that has been mutagenized or altered to contain one or more amino acid residues that are not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions or alterations or substitutions of amino acid residues. A mutant polypeptide shows enhanced or diminished insecticidal activity, or represents an amino acid sequence that confers enhanced insecticidal activity over a polypeptide containing it. Thus, the term "mutant" or "mutation" refers to either or both of the mutant nucleotide sequences and the encoded amino acids. The mutants may be used alone or in any compatible combination with other mutants of the embodiments or with other mutants. A "mutant polypeptide" may instead show a decrease in insecticidal activity. When more than one mutation is added to a particular nucleic acid or protein, the mutations may be added simultaneously or sequentially; if sequentially, the mutations may be added in any suitable order.

[0046] As used herein, the term "enhanced insecticidal activity" or "enhanced pesticidal activity" refers to an insecticidal polypeptide of the embodiments that has increased insecticidal activity relative to the activity of its corresponding wild-type protein, and/or an insecticidal polypeptide that is effective against a broader range of insects, and/or an insecticidal polypeptide having specificity for an insect that is not susceptible to the toxicity of the wild-type protein. A finding of improved or increased pesticidal activity requires a demonstration of an increase in pesticidal activity of at least 10% against the target insect, or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, or 300% or more in pesticidal activity relative to the pesticidal activity of the wild-type insecticidal polypeptide determined against the same insect.

[0047] For example, improved pesticidal or insecticidal activity is provided when a broader or narrower range of insects are affected by the polypeptide relative to the range of insects that are affected by a wild-type Bt toxin. A broader range of impact may be desirable where versatility is desired, whereas a narrower range of impact may be desirable when, for example, beneficial insects may otherwise be affected by the use or presence of the toxin. Although the embodiments are not linked by any particular mechanism of action, improved pesticidal activity may also be provided by changes in one or more characteristics of a polypeptide; for example, the stability or longevity of a polypeptide in an insect gut may be increased relative to the stability or longevity of a corresponding wild-type protein.

[0048] The term "toxin" as used herein refers to a polypeptide that exhibits pesticidal activity or insecticidal activity or enhanced pesticidal activity or enhanced insecticidal activity. "Bt" or "Bacillus thuringiensis" toxin is intended to include the broader class of Cry toxins found in various strains of Bt, which includes toxins such as, for example, Cry1s, Cry2s, or Cry3s.

[0049] The terms "proteolytic site" or "cleavage site" refer to an amino acid sequence that confers sensitivity to a class of proteases or a particular protease such that a polypeptide containing the amino acid sequence will be digested by the class of proteases or particular protease. A proteolytic site is said to be "sensitive" to the protease(s) that recognize that site. It is appreciated in the art that the efficiency of digestion will vary, and that a decrease in the efficiency of digestion may lead to an increase in the stability or longevity of the polypeptide in an insect gut. Thus, a proteolytic site may confer sensitivity to more than one protease or class of proteases, but the efficiency of digestion at that site by the various proteases may vary. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites.

[0050] Research has shown that insect gut proteases from Lepidoptera include trypsins, chymotrypsins, and elastases. See, e.g., 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 trypsins have been found in the midgut of Helicoverpa armigera larvae (see Gatehouse et al. (1997) Insect Biochem. Mol. Biol. 27: 929-944). The preferred proteolytic substrate sites of these proteases have been investigated. See, e.g., Peterson et al. (1995) Insect Biochem. Mol. Biol. 25: 765-774.

[0051] Efforts have been made to understand the mechanism of action of Bt toxins and to engineer toxins with improved properties. It has been shown that insect gut proteases can affect the impact of Bt Cry proteins on the insect. Some proteases activate Cry proteins by processing them from a "protoxin" form to a toxic, or "toxin", form. See, Oppert (1999) Arch. Insect Biochem. Phys. 42: 1-12; and Carroll et al. (1997) J. Invertebrate Pathology 70: 41-49. This activation of the toxin may include removal of the N- and C-terminal peptides from the protein and may also include internal cleavage of the protein. Other proteases can degrade Cry proteins. See Oppert, ibid.

[0052] A comparison of the amino acid sequences of Cry toxins of different specificities reveals five highly conserved sequence blocks. Structurally, the toxins comprise three distinct domains that are, from the N- to C-terminus: a set of seven alpha helices involved in pore formation (referred to as "Domain I"), three antiparallel beta sheets involved in cell binding (referred to as "Domain 2"), and a beta 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 I, it is understood that the exact ends of the domain with respect to a particular sequence are not critical, so long as the sequence or portion thereof includes sequence that provides at least some function attributed to the particular domain. Thus, for example, when referring to "Domain I", it is intended that a particular sequence includes a cluster of seven alpha helices, but the exact ends of the sequence used or referred to with respect to that cluster are not critical. One skilled in the art is familiar with determining such ends and evaluating such functions.

[0053] In an effort to improve Cry2B toxins, an effort was undertaken to identify nucleotide sequences encoding crystal proteins from selected strains that had improved activity compared to the native toxin. Depending on the characteristics of a given preparation, it was recognized that demonstration of pesticidal activity sometimes required pretreatment with trypsin to activate the pesticidal proteins. Thus, it is understood that some pesticidal proteins require protease digestion (e.g., by trypsin, chymotrypsin, and the like) for activation, while other proteins are biologically active (e.g., pesticidal) in the absence of activation.

[0054] Such molecules can be altered by means described, for example, in U.S. Patent 7,462,760. In addition, the nucleic acid sequences can be engineered to encode polypeptides that contain additional mutations that confer improved or altered pesticidal activity relative to the pesticidal activity of the naturally occurring polypeptide. The nucleotide sequences of such engineered nucleic acids comprise mutations not found in the wild-type sequences.

[0055] Mutant polypeptides of embodiments are generally prepared by a process involving the steps of: obtaining a nucleic acid sequence encoding a Cry family polypeptide; analyzing the structure of the polypeptide to identify particular "target" sites for mutagenesis of the underlying genetic sequence based on a consideration of the proposed function of the target domain in the mode of action of the toxin; introducing one or more mutations into the nucleic acid sequence to produce a desired change in one or more amino acid residues of the encoded polypeptide sequence; and testing the produced polypeptide for pesticidal activity.

[0056] Many of the insecticidal toxins of Bt are related to varying degrees by similarities in their amino acid sequences and tertiary structure, and the means for obtaining crystal structures of Bt toxins are well known. An exemplary solution of the high-resolution crystal structure of both Cry3A and Cry3B polypeptides is available in the literature. The solved structure of Cry3A (Li et al. (1991) Nature 353:815-821) provides insight into the relationship between toxin structure and function. A combined consideration of the published structural analyses of Bt toxins and the reported function associated with particular structures, motifs, and the like indicates that specific regions of the toxin are correlated with particular 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-helix bundle that is involved in pore formation, a three-sheet domain that has been implicated in receptor binding, and a beta-sandwich motif (Li et al. (1991) Nature 305: 815–821).

[0057] As reported in U.S. Patent Nos. 7,105,332 and 7,462,760, the toxicity of Cry proteins can be enhanced by targeting the region located between alpha helices 3 and 4 of Domain I of the toxin. This theory was based on a body of knowledge about insecticidal toxins, including: 1) that alpha helices 4 and 5 of Domain I of Cry3A toxins have been reported to be inserted into the lipid bilayer of cells lining the midgut of susceptible insects (Gazit et al. (1998) Proc. Natl. Acad. Sci. USA 95:12289-12294); 2) the inventors' knowledge of the location of the trypsin and chymotrypsin cleavage sites within the amino acid sequence of the wild-type protein; 3) the observation that the wild-type protein was more active against certain insects after in vitro activation by trypsin or chymotrypsin treatment; and 4) reports that digestion of toxins from the 3' end resulted in reduced toxicity to insects.

[0058] A number of mutations can be engineered and placed into a variety of source sequences to create novel polypeptides with increased or altered pesticidal activity. See, e.g., U.S. Patent 7,462,760. Such mutants include, but are not limited to: the addition of at least one additional protease-sensitive site (e.g., trypsin cleavage site) in the region located between helices 3 and 4 of Domain I; the replacement of an original protease-sensitive site in the wild-type sequence with a different protease-sensitive site; the addition of multiple protease-sensitive sites at a specific location; the addition of amino acid residues near protease-sensitive site(s) to alter the folding of the polypeptide and thereby increase digestion of the polypeptide at the protease-sensitive site(s); and adding mutations to protect the polypeptide from degradative digestion that reduce toxicity (e.g., by making a series of mutations in which the wild-type amino acid is replaced with valine to protect the polypeptide from digestion). The mutations can be used alone or in any combination to provide polypeptides of the embodiments.

[0059] Homologous sequences were identified by similarity searching against the National Center for Bioinformatics Information (NCBI) non-redundant (nr) database using BLAST and PSI-BLAST. The homologous proteins consisted of Cry toxins primarily from Bacillus thuringiensis.

[0060] A mutation that is an additional or alternative protease-sensitive site may be sensitive to several classes of proteases such as serine proteases, which include trypsin and chymotrypsin, or enzymes such as elastase. Thus, a mutation that is an additional or alternative protease-sensitive site may be designed so that the site is readily recognized and/or cleaved by a class of proteases, such as mammalian proteases or insect proteases. A protease-sensitive site may also be designed to be cleaved by a particular class of enzymes or by a particular enzyme known to be produced in an organism, such as, for example, a chymotrypsin produced by the corn earworm Heliothis zea (Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212). Mutations may also confer resistance to proteolytic digestion, for example, to chymotrypsin digestion at the C-terminus of the peptide.

[0061] The presence of an additional and/or alternative protease-sensitive site in the amino acid sequence of the encoded polypeptide may improve the pesticidal activity and/or specificity of the polypeptide encoded by the nucleic acids of the embodiments. Accordingly, the nucleotide sequences of the embodiments may be recombinantly modified or engineered to produce polypeptides with improved or altered insecticidal activity and/or specificity compared to that of an unmodified wild-type toxin. Furthermore, the mutations described herein may be placed in or used in conjunction with other nucleotide sequences to provide improved properties. For example, a protease-sensitive site that is readily cleaved by insect chymotrypsin, e.g., a chymotrypsin found in the bertha caterpillar or corn earworm (Hegedus et al. (2003) Arch. Insect Biochem. Physiol. 53:30-47; and Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16:201-212), can be placed in a Cry origin sequence to provide improved toxicity for that sequence. In this way, embodiments provide toxic polypeptides with improved properties.

[0062] For example, a Cry mutagenized nucleotide sequence may comprise additional mutants comprising additional codons that present a second trypsin-sensitive amino acid sequence (in addition to the naturally occurring trypsin site) in the encoded polypeptide. An alternative addition mutant of embodiments comprises additional codons designed to introduce at least one additional different protease-sensitive site into the polypeptide, e.g., a chymotrypsin-sensitive site located immediately 5' or 3' of the naturally occurring trypsin site. Alternatively, substitution mutants may be created in which at least one codon of the nucleic acid encoding the naturally occurring protease-sensitive site is destroyed and alternative codons are introduced into the nucleic acid sequence so as to provide a different (e.g., replacement) protease-sensitive site. A substitution mutant can also be added to a Cry sequence in which the trypsin cleavage site naturally present in the encoded polypeptide is destroyed and a chymotrypsin or elastase cleavage site is introduced in its place.

[0063] It is recognized that any nucleotide sequence encoding amino acid sequences that are proteolytic sites or putative proteolytic sites (e.g., sequences such as RR, or LKM) may be used and that the exact identity of the codons used to introduce any such cleavage site into a variant polypeptide may vary depending on the use, i.e., expression in a particular plant species. It is also recognized that any of the disclosed mutations may be introduced into any polynucleotide sequence of the embodiments comprising the codons for amino acid residues that provide the native trypsin cleavage site that is targeted for modification. Accordingly, full-length toxin variants or fragments thereof may be modified to contain additional or alternative cleavage sites, and these embodiments are intended to be encompassed by the scope of the embodiments disclosed herein.

[0064] It will be appreciated by those skilled in the art that any useful mutation may be added to the sequences of the embodiments so long as the encoded polypeptides retain pesticidal activity. Thus, the sequences may also be mutated such that the encoded polypeptides are resistant to proteolytic digestion by chymotrypsin. More than one recognition site may be added at a particular location in any combination, and multiple recognition sites may be added or removed from the toxin. Thus, the additional mutations may comprise three, four, or more recognition sites. It is recognized that multiple mutations may be engineered into any suitable polynucleotide sequence; accordingly, full-length sequences or fragments thereof may be modified to contain additional or alternative cleavage sites as well as to be resistant to proteolytic digestion. Thus, the embodiments provide Cry toxins containing mutations that improve pesticidal activity as well as improved compositions and methods for impacting pests using other Bt toxins.

[0065] Mutations may protect the polypeptide from protease degradation, for example by removing putative proteolytic sites such as putative serine protease sites and elastase recognition sites from different areas. Some or all of the putative sites may be removed or altered so that proteolysis at the location of the original site is decreased. Changes in proteolysis may be assessed by comparing a mutant polypeptide to wild-type toxins or by comparing mutant toxins that differ in their amino acid sequence. Putative proteolytic sites and proteolytic sites include, but are not limited to, the following sequences: RR, a trypsin cleavage site; LKM, a chymotrypsin site; and a trypsin site. These sites may be altered by the addition or deletion of any number and type of amino acid residues, so long as the pesticidal activity of the polypeptide is increased. Thus, polypeptides encoded by nucleotide sequences comprising mutations will comprise at least one amino acid change or addition relative to the native or parent 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 changes or additions. The pesticidal activity of a polypeptide can also be improved by truncation of the native or full-length sequence, as is known in the art.

[0066] The compositions of embodiments include nucleic acids and their fragments and variants that encode pesticidal polypeptides. In particular, embodiments provide isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 45, or the nucleotide sequences encoding said amino acid sequence. amino acids, for example the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46, and their fragments and variants.

[0067] In particular, embodiments provide isolated nucleic acid molecules encoding the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8, or nucleotide sequences encoding said amino acid sequence, for example the nucleotide sequence set forth in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46, and their fragments and variants.

[0068] Equally interesting are optimized nucleotide sequences encoding the pesticidal proteins of the embodiments. As used herein, the phrase "optimized nucleotide sequences" refers to nucleic acids that are optimized for expression in a particular organism, e.g., a plant. Optimized nucleotide sequences can be prepared for any organism of interest using methods known in the art. See, e.g., U.S. Patent No. 7,462,760, which describes an optimized nucleotide sequence encoding a disclosed pesticidal protein. In this example, the nucleotide sequence was prepared by reverse-translating the amino acid sequence of the protein and altering the nucleotide sequence to comprise corn-preferred codons while still encoding the same amino acid sequence. This procedure is described in greater detail by Murray et al. (1989) Nucleic Acids Res. 17:477-498. Optimized nucleotide sequences find use in increasing the expression of a pesticidal protein in a plant, for example monocotyledonous plants of the family Gramineae (Poaceae) such as, for example, a grain plant or corn.

[0069] In some embodiments, provided are polypeptides comprising an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, and fragments and variants thereof.

[0070] In some embodiments, provided are polypeptides comprising an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, and fragments and variants thereof.

[0071] In some embodiments, provided are polypeptides comprising an amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29, and fragments and variants thereof.

[0072] In some embodiments, variant Cry1B polypeptides having an amino acid substitution compared to the corresponding reference Cry1B polypeptide are provided that have increased insecticidal activity against corn earworm and/or fall armyworm compared to the "corresponding reference Cry1B polypeptide". By "corresponding reference Cry1B polypeptide" is meant a native or wild-type Cry1B polypeptide or a variant Cry1B polypeptide of the present embodiments, which can serve as the amino acid sequence that is mutagenized to create the variant Cry1B polypeptide. In some embodiments, the corresponding reference Cry1B polypeptide comprises a Cry1Be-like Domain I and Cry1Ah-like Domain III. By "Cry1Be-like Domain I" is meant an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to amino acids 36-276 of SEQ ID NO: 58 (Cry1Be) or amino acids 35-276 of SEQ ID NO: 47. An amino acid sequence alignment of Domain I of Cry1Be (SEQ ID NO: 58) and MP258 (SEQ ID NO: 47) is shown in Figure 3. Similarly, other native Cry1B polypeptides can be aligned with Cry1Be (SEQ ID NO: 58) and MP258 (SEQ ID NO: 47) to identify other regions of Domain I-like Cry1Be. Cry1Be. By "Cry1Ah-type domain III" is meant an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to amino acids 483-643 of SEQ ID NO: 61 (Cry1Ah) or 494655 of SEQ ID NO: 47. An alignment of the amino acid sequence of Domain III of Cry1Ah (SEQ ID NO: 61), Cry1Bd (SEQ ID NO: 1), Cry1Bh (SEQ ID NO: 52), Cry1Bi (SEQ ID NO: 54), and MP258 (SEQ ID NO: 47) is shown in Figure 4. Similarly, other native Cry1B polypeptides can be aligned with Cry1Ah (SEQ ID NO: 61), Cry1Bd, Cry1Bh (SEQ ID NO: 52), Cry1Bi (SEQ ID NO: 54), and/or MP258 (SEQ ID NO: 47) to identify other Cry1Ah-like Domain III regions. In some embodiments, the corresponding reference Cry1B polypeptide comprises a Cry1Ba-like Domain I and Domain II. By "Cry1Ba-like Domain I and Domain II" is meant an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to any of the amino acids in the sequence. sequence with amino acids 30-489 of SEQ ID NO: 55 (Cry1Ba). An amino acid sequence alignment of Domain I and Domain II of MP258 (SEQ ID NO: 47), Cry1Be (SEQ ID NO: 58), Cry1Bi (SEQ ID NO: 54), Cry1Bg (SEQ ID NO: 60), Cry1Bf (SEQ ID NO: 59), Cry1Ba (SEQ ID NO: 55), Cry1Bh (SEQ ID NO: 52), Cry1Bd (SEQ ID NO: 1), Cry1Bb (SEQ ID NO: 56), and Cry1Bc (SEQ ID NO: 57) is shown in Figure 5. Similarly, other native Cry1B polypeptides can be aligned with Cry1Ba (SEQ ID NO: 55) and MP258 (SEQ ID NO: 47) to identify other Cry1Ba-like Domain I and Domain II regions.

[0073] In some embodiments, the corresponding reference Cry1B polypeptide comprises a Cry1Be-like Domain I and Domain II. By "Cry1Be-type Domain I and Domain II" is meant an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity sequence alignment with amino acids 35-494 of SEQ ID NO: 58 (Cry1Be) or amino acids 35-493 of SEQ ID NO: 47. An amino acid sequence alignment of Domain I and Domain II of MP258 (SEQ ID NO: 47), Cry1Be (SEQ ID NO: 58), Cry1Bi (SEQ ID NO: 54), Cry1Bg (SEQ ID NO: 60), Cry1Bf (SEQ ID NO: 59), Cry1Ba (SEQ ID NO: 55), Cry1Bh (SEQ ID NO: 52), Cry1Bd (SEQ ID NO: 1), Cry1Bb (SEQ ID NO: 56), and Cry1Bc (SEQ ID NO: 57) is shown in Figure 5. Similarly, other native Cry1B polypeptides can be aligned with Cry1Be (SEQ ID NO: 58) and MP258 (SEQ ID NO: 47) to identify other Cry1Be-like Domain I and Domain II regions.

[0074] By "improved activity" or "increased activity" is meant an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about of 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, by at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or more or at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times or at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about 2.9 times, at least about 3 times, at least about 3.1 times, at least about 3.2 times, at least about 3.3 times, at least about 3.4 times, at least about 3.5 times, at least about 3.6 times, at least about 3.7 times, at least about 3.8 times, at least about 3.9 times, at least about 4 times, at least about 4.1 times, at least about 4.2 times, at least about 4.3 times, at least about 4.4 times, at least about 4.5 times, at least about 4.6 times, at least about 4.7 times, at least about 4.8 times, at least about 4.9 times, at least about 5 times, at least about 5.1 times, at least about 5.2 times, at least about 5.3 times, at least about 5.4 times, at least about 5.5 times, at least about 5.6 times, at least about 5.7 times, at least about 5.8 times, at least about 5.9 times, at least about 6 times, at least about 6.1 times, at least about 6.2 times, at least about 6.3 times, at least about 6.4 times, at least about 6.5 times, at least about 6.6 times, at least about 6.7 times, at least 6.8 times, at least 6.9 times, at least about 7 times, at least about 7.1 times, at least about 7.2 times, at least about 7.3 times, at least about 7.4 times, at least about 7.5 times, at least about 7.6 times, at least about 7.7 times, at least about 7.8 times, at least about 7.9 times, at least about 8 times, at least about 8.1 times, at least about 8.2 times, at least about 8.3 times, at least about 8.4 times, at least about 8.5 times, at least about 8.6 times, at least about 8.7 times, at least about 8.8 times, at least about 8.9 times, at least about 9 times, at least about 9.1 times, at least about 9.2 times, at least about 9.3 times, at least about 9.4 times, at least about 9.5 times, at least about 9.6 times, at least about 9.7 times, at least about 9.8 times, at least about 9.9 times, at least about 10 times or more increase in the pesticidal activity of the variant protein compared to the activity of the corresponding reference Cry1B polypeptide.

[0075] In some embodiments, the improvement consists of a decrease in EC50 of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, or at least about 220%, or at least about 230%, or at least about 240%, or at least about 250%, or at least about 260%, or at least about 270%, or at least about 280%, or at least about 290%, or at least about 300%, or at least about 310%, or at least about 320%, or at least about 330%, or at least about 340%, or at least about 350%, or at least about 360%, or at least about 370%, or at least about 380%, or at least about 390%, or at least about 400%, or at least about 410%, or at least about 420%, or at least about 430%, or at least about 440%, or at least about 450%, or at least about 460%, or at least about 470%, or at least about 480%, or at least about 490%, or at least about about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or more or at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times or at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about 2.9 times, at least about 3 times, at least about 3.1 times, at least about 3.2 times, at least about 3.3 times, at least about 3.4 times, at least about 3.5 times, at least about 3.6 times, at least about 3.7 times, at least about 3.8 times, at least about 3.9 times, at least about 4 times, at least about 4.1 times, at least about 4.2 times, at least about 4.3 times, at least about 4.4 times, at least about 4.5 times, at least about 4.6 times, at least about 4.7 times, at least about 4.8 times, at least about 4.9 times, at least about 5 times, at least about 5.1 times, at least about 5.2 times, at least about 5.3 times, at least about 5.4 times, at least about 5.5 times, at least about 5.6 times, at least about 5.7 times, at least about 5.8 times, at least about 5.9 times, at least about 6 times, at least about 6.1 times, at least about 6.2 times, at least about 6.3 times, at least about 6.4 times, at least about 6.5 times, at least about 6.6 times, at least about 6.7 times, at least about 6.8 times, at least about 6.9 times, at least about 7 times, at least about 7.1 times, at least about 7.2 times, at least about 7.3 times, at least about 7.4 times, at least about 7.5 times, at least about 7.6 times, at least about 7.7 times, at least about 7.8 times, at least about 7.9 times, at least about 8 times, at least about 8.1 times, at least about 8.2 times, at least about 8.3 times, at least about 8.4 times, at least about 8.5 times, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or more reduction in the EC50 of the variant Cry1B polypeptide relative to the pesticidal activity of the corresponding reference Cry1B polypeptide.

[0076] In some embodiments, the EC50 of the Cry1Bvariant polypeptide is <100 ppm, <90 ppm, <80 ppm, <70 ppm, <60 ppm, <50 ppm, <45 ppm, <40 ppm, <35 ppm, <30 ppm, <25 ppm, <20 ppm, <19 ppm, <18 ppm, <17 ppm, <16 ppm, <15 ppm, <14 ppm, <13 ppm, <12 ppm, <11 ppm, <10 ppm, <9 ppm, <8 ppm, <7 ppm, <6 ppm, <5 ppm, <4 ppm, <3 ppm, <2 ppm, <1 ppm, <0.9 ppm, <0.8 ppm, <0.7 ppm, <0.6 ppm, <0.5 ppm, <0.4 ppm, <0.3 ppm, <0.2 ppm or <0.1 ppm.

[0077] In some embodiments, the improvement consists of an increase in the Average FAE Index of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, or at least about 220%, or at least about 230%, or at least about 240%, or at least about 250%, or at least about 260%, or at least about 270%, or at least about 280%, or at least about 290%, or at least about 300%, or at least about 310%, or at least about 320%, or at least about 330%, or at least about 340%, or at least about 350%, or at least about 360%, or at least about 370%, or at least about 380%, or at least about 390%, or at least about 400%, or at least about 410%, or at least about 420%, or at least about 430%, or at least about 440%, or at least about 450%, or at least about 460%, or at least about 470%, or at least about 480%, or at least about 490%, or at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or more or at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times or at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about 2.9 times, at least about 3 times, at least about 3.1 times, at least about 3.2 times, at least about 3.3 times, at least about 3.4 times, at least about 3.5 times, at least about 3.6 times, at least about 3.7 times, at least about 3.8 times, at least about 3.9 times, at least about 4 times, at least about 4.1 times, at least about 4.2 times, at least about 4.3 times, at least about 4.4 times, at least about 4.5 times, at least about 4.6 times, at least about 4.7 times, at least about 4.8 times, at least about 4.9 times, at least about 5 times, at least about 5.1 times, at least about 5.2 times, at least about 5.3 times, at least about 5.4 times, at least about 5.5 times, at least about 5.6 times, at least about 5.7 times, at least about 5.8 times, at least about 5.9 times, at least about 6 times, at least about 6.1 times, at least about 6.2 times, at least about 6.3 times, at least about 6.4 times, at least about 6.5 times, at least about 6.6 times, at least about 6.7 times, at least about 6.8 times, at least about 6.9 times, at least about 7 times, at least about 7.1 times, at least about 7.2 times, at least about 7.3 times, at least about 7.4 times, at least about 7.5 times, at least about 7.6 times, at least about 7.7 times, at least about 7.8 times, at least about 7.9 times, at least about 8 times, at least about 8.1 times, at least about 8.2 times, at least about 8.3 times, at least about 8.4 times, at least about 8.5 times, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or more increase in the Average FAE Index of the variant Cry1B polypeptide relative to the pesticidal activity of the corresponding reference Cry1B polypeptide.

[0078] “Mean FAE Index” (MFI) refers to the average of multiple FAEGNs, an arithmetic mean of FAEGNs. As used herein, “Mean Deviation Score” refers to the arithmetic mean of multiple Deviation Scores.

[0079] In some embodiments, the improvement consists of an increase in the Mean Deviation Score of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000% or more or at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times or at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about 2.9 times, at least about 3 times, at least about 3.1 times, at least about 3.2 times, at least about 3.3 times, at least about 3.4 times, at least about 3.5 times, at least about 3.6 times, at least about 3.7 times, at least about 3.8 times, at least about 3.9 times, at least about 4 times, at least about 4.1 times, at least about 4.2 times, at least about 4.3 times, at least about 4.4 times, at least about 4.5 times, at least about 4.6 times, at least about 4.7 times, at least about 4.8 times, at least about 4.9 times, at least about 5 times, at least about 5.1 times, at least about 5.2 times, at least about 5.3 times, at least about 5.4 times, at least about 5.5 times, at least about 5.6 times, at least about 5.7 times, at least about 5.8 times, at least about 5.9 times, at least about 6 times, at least about 6.1 times, at least about 6.2 times, at least about 6.3 times, at least about 6.4 times, at least about 6.5 times, at least about 6.6 times, at least about 6.7 times, at least about 6.8 times, at least about 6.9 times, at least about 7 times, at least about 7.1 times, at least about 7.2 times, at least about 7.3 times, at least about 7.4 times, at least about 7.5 times, at least about 7.6 times, at least about 7.7 times, at least about 7.8 times, at least about 7.9 times, at least about 8 times, at least about 8.1 times, at least about 8.2 times, at least about 8.3 times, at least about 8.4 times, at least about 8.5 times, at least about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at least about 10-fold or more increase in the Mean Deviation Score of the variant Cry1B polypeptide relative to the pesticidal activity of the corresponding reference Cry1B polypeptide.

[0080] In some embodiments, the improved activity of the variant Cry1B polypeptide is relative to the pesticidal activity of SEQ ID NO: 1 (Cry1Bd), SEQ ID NO: 47 (MP258), SEQ ID NO: 52 (Cry1Bh), SEQ ID NO: 54 (Cry1Bi), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.

[0081] In particular embodiments, the pesticidal proteins of the embodiments provide full-length insecticidal polypeptides, fragments of full-length insecticidal polypeptides, and variant polypeptides that are produced from mutagenized nucleic acids designed to introduce particular amino acid sequences into polypeptides of the embodiments. In particular embodiments, the amino acid sequences that are introduced into the polypeptides comprise a sequence that provides a cleavage site for an enzyme such as a protease.

[0082] It is known in the art that the pesticidal activity of Bt toxins is typically activated by cleavage of the peptide in the insect gut by various proteases. Since peptides cannot always be cleaved with full efficiency in the insect gut, fragments of a full-length toxin may have increased pesticidal activity compared to the full-length toxin itself. Thus, some of the polypeptides of the embodiments include fragments of a full-length insecticidal polypeptide, and some of the fragments, variants, and mutations of the polypeptide will have increased pesticidal activity relative to the activity of the naturally occurring insecticidal polypeptide from which they are derived, particularly if the naturally occurring insecticidal polypeptide is not activated in vitro with a protease prior to analysis for activity. Thus, the present application encompasses truncated versions or fragments of the sequences.

[0083] Mutations may be placed in any parent sequence, including such truncated polypeptides, as long as the polypeptide retains pesticidal activity. One skilled in the art can readily compare two or more proteins for pesticidal activity using assays known in the art, or described elsewhere herein. It should be understood that the polypeptides of the embodiments may be produced either by expression of a nucleic acid disclosed herein, or by using standard molecular biology techniques.

[0084] It is recognized that pesticidal proteins may be serooligomeric and will vary in molecular weight, number of residues, component peptides, activity against particular pests, and other characteristics. However, by the methods presented herein, proteins active against a variety of pests can be isolated and characterized. The pesticidal proteins of the embodiments can be used in combination with other Bt toxins or other insecticidal proteins to increase the range of target insects. Furthermore, the use of the pesticidal proteins of the embodiments in combination with other Bt toxins or other insecticidal principles of a distinct nature has particular utility for the prevention and/or management of resistance in insects. Other insecticidal agents include protease inhibitors (both serine and cysteine types), α-amylase, and peroxidase.

[0085] Fragments and variants of the nucleotide and amino acid sequences and the polypeptides encoded thereby are also encompassed by the embodiments. As used herein, the term “fragment” refers to a portion of a nucleotide sequence of a polynucleotide or a portion of an amino acid sequence of a polypeptide of the embodiments. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native or corresponding full-length protein and therefore possess pesticidal activity. Thus, it is recognized that some of the polynucleotide and amino acid sequences of the embodiments may correctly be referred to as fragments and mutants.

[0086] It should be understood that the term "fragment", as used to refer to nucleic acid sequences of the embodiments, also encompasses sequences that are useful as hybridization probes. This class of nucleotide sequences generally does not encode fragments of proteins that retain biological activity. Thus, fragments of a nucleotide sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the embodiments.

[0087] A fragment of a nucleotide sequence of the embodiments that encodes a biologically active portion of a pesticidal protein of the embodiments will encode at least 15, 25, 30, 50, 100, 200, 250, or 300 contiguous amino acids, or up to the total number of amino acids present in a pesticidal polypeptide of the embodiments (e.g., 651 amino acids for SEQ ID NO: 3). Thus, it is understood that the embodiments also encompass polypeptides that are fragments of the exemplary pesticidal proteins of the embodiments and that have lengths of at least 15, 25, 30, 50, 100, 200, 250, or 300 contiguous amino acids, or up to the total number of amino acids present in a pesticidal polypeptide of the embodiments (e.g., 651 amino acids for SEQ ID NO: 3). Fragments of a nucleotide sequence of the embodiments that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a pesticidal protein. Thus, a fragment of a nucleic acid of the embodiments may encode a biologically active portion of a pesticidal protein, or may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed herein. A biologically active portion of a pesticidal protein may be prepared by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the encoded portion of the pesticidal protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the pesticidal protein.

[0088] Nucleic acids that are fragments of a nucleotide sequence of embodiments 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 a nucleotide sequence disclosed herein (e.g., 1953 nucleotides for SEQ ID NO: 4). Particular embodiments contemplate fragments derived from (e.g., produced from) a first nucleic acid of embodiments, wherein the fragment encodes a truncated toxin with pesticidal activity. The truncated polypeptides encoded by the polynucleotide fragments of the embodiments have pesticidal activity that is equivalent to or improved over the activity of the corresponding full-length polypeptide encoded by the first nucleic acid from which the fragment is derived. It is anticipated that such nucleic acid fragments of the embodiments may be truncated at the 3' end of the native or corresponding full-length coding sequence. The nucleic acid fragments may also be truncated at either the 5' or 3' end of the native or corresponding full-length coding sequence.

[0089] The term "variants" is used herein to refer to substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the pesticidal polypeptides of the embodiments. Those skilled in the art will readily appreciate that due to the degeneracy of the genetic code there are a multiplicity of nucleotide sequences encoding the present disclosure.

[0090] In some embodiments, the nucleic acid molecule encoding the polypeptide is a non-genomic nucleic acid sequence. As used herein, a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" or "non-genomic polynucleotide" refers to a nucleic acid molecule that has one or more changes in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion, and/or addition compared to the native or genomic sequence; the removal of one or more introns associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5' and/or 3' untranslated region; and modification of a polyadenylation site. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0091] Where appropriate, a nucleic acid may be optimized to increase expression in the host organism. Thus, when the host organism is a plant, synthetic nucleic acids may be synthesized using plant-preferred codons for improved expression. See, e.g., Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. For example, although the nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, the sequences may be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons, as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the preferred maize codon for a particular amino acid can be derived from known maize gene sequences. The maize codon usage for 28 maize plant genes is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing preferred plant genes. 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, and Liu H et al. Mol Bio Rep 37:677684, 2010, incorporated herein by reference. A table of Zea maize codon usage can also be found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can be consulted using the prefix www.

[0092] A table of Glycine max codon usage is presented in Table 3 and can also be found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1&style=N, which can be accessed using the prefix www.

[0093] One skilled in the art will further appreciate that changes can be introduced by mutation of nucleic acid sequences, thereby leading to changes in the amino acid sequence of the encoded polypeptides, without altering the biological activity of the proteins. Thus, variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions and/or deletions into the corresponding nucleic acid sequence described herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by conventional techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0094] Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, such as, for example, polymerase chain reaction (PCR) and hybridization techniques as described herein.

[0095] In some embodiments, the polynucleotide encoding the polypeptide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45 is a non-genomic nucleic acid sequence.

[0096] Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, using site-directed mutagenesis, but which still encode a pesticidal protein of embodiments, such as a mutant toxin. Generally, variants of a particular nucleotide sequence of embodiments will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters. A variant of a nucleotide sequence of the embodiments may differ from that sequence by as few as 1-15 nucleotides, by as few as 1-10, such as 6-10, by as few as 5, by as few as 4, 3, 2, or even 1 nucleotide.

[0097] Variants of a given nucleotide sequence of embodiments (i.e., an exemplary nucleotide sequence) can also be assessed by comparing the percent sequence identity between the polypeptide encoded by a variant nucleotide sequence and the polypeptide encoded by the reference nucleotide sequence. Thus, for example, disclosed are isolated nucleic acids that encode a polypeptide having a given percent sequence identity to the polypeptides of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45. The percent sequence identity between two polypeptides can be calculated using sequence alignment programs described elsewhere herein using default parameters. When any given pair of polynucleotides of the embodiments is evaluated by comparing the percentage sequence identity shared by the two polypeptides they encode, the percentage sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least about 98%, 99% or more sequence identity.

[0098] As used herein, the term "variant protein" encompasses polypeptides that are derived from a native protein by: deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Accordingly, the term "variant protein" encompasses biologically active fragments of a native protein that comprise a sufficient number of contiguous amino acid residues to retain the biological activity of the native protein, i.e., to have pesticidal activity. Such pesticidal activity may be different or enhanced relative to the native protein or may remain unchanged, so long as the pesticidal activity is retained.

[0099] Variant proteins encompassed by embodiments are biologically active, i.e., they continue to possess the desired biological activity of the native protein, i.e., pesticidal activity as described herein. Such variants may result, for example, from genetic polymorphism or human manipulation. Biologically active variants of a native pesticidal protein of embodiments 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 more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a protein of the modalities may differ from that protein by as few as 1-15 amino acid residues, by as few as 1-10, such as 6-10, by as few as 5, by as few as 4, 3, 2, or even 1 amino acid residue.

[00100] In some embodiments the insecticidal polypeptide has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45.

[00101] In some embodiments the polypeptide has a modified physical property. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "physical property of interest" and "property of interest" are used interchangeably to refer to physical properties of proteins that are being investigated and/or modified. Examples of physical properties include, but are not limited to, net surface charge and charge distribution on the surface of the protein, effective hydrophobicity and distribution of hydrophobic residues on the surface of the protein, surface charge density, surface hydrophobicity density, total count of ionizable groups on the surface, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial 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 an insect gut. In some embodiments, the polypeptide has increased stability in an insect gut. Models for digestion by simulated gastric fluids are known to those skilled in the art (Fuchs, R.L. and J.D. Astwood. Food Technology 50:83-88, 1996; Astwood, J.D., et al. Nature Biotechnology 14:1269-1273, 1996; Fu T.J. et al. J. Agric Food Chem. 50:7154-7160, 2002).

[00102] Embodiments further encompass a microorganism that is transformed with at least one nucleic acid of the embodiments, with an expression cassette comprising the nucleic acid, or with a vector comprising the expression cassette. In some embodiments, the microorganism is one that multiplies in plants. One embodiment of the disclosure relates to an encapsulated pesticidal protein comprising a transformed microorganism capable of expressing at least one pesticidal protein of the embodiments.

[00103] Embodiments provide pesticidal compositions comprising a transformed microorganism of the embodiments. In such embodiments, the transformed microorganism is generally present in the pesticidal composition in a pesticidally effective amount, together with a suitable carrier. Embodiments also encompass pesticidal compositions comprising an isolated protein of the embodiments, alone or in combination with a transformed organism of the embodiments, and/or an encapsulated pesticidal protein of the embodiments, in an insecticidally effective amount, together with a suitable carrier.

[00104] Embodiments further provide a method for increasing the range of target insects using a pesticidal protein of the embodiments in combination with at least one other or "second" pesticidal protein. Any pesticidal protein known in the art can be employed in the methods of the embodiments. Such pesticidal proteins include, but are not limited to, Bt toxins, protease inhibitors, α-amylases, and peroxidases.

[00105] Embodiments also encompass transformed or transgenic plants comprising at least one nucleotide sequence of the embodiments. In some embodiments, the plant is stably transformed with a nucleotide construct comprising at least one nucleotide sequence of the embodiments operably linked to a promoter that drives expression in a plant cell. As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated into the genome of a transgenic or transformed plant such that the polynucleotide is passed to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant expression cassette.

[00106] 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 acid including those transgenics initially so altered, as well as those created by sexual crosses or asexual propagation of the initial transgenic. The term "transgenic", as used herein, does not cover the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by natural events, such as random cross-fertilization, non-recombinant viral infection, transformation of non-recombinant bacteria, non-recombinant transposition or spontaneous mutation.

[00107] As used herein, the term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof. Transgenic plant parts are within the scope of the embodiments and include, for example, plant cells, plant protoplasts, tissue cultures of plant cells from which plants can be regenerated, plant callus, plant clumps, and plant cells that are intact in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, husks, stems, roots, root tips, anthers, and the like, originating from transgenic plants or their progeny previously transformed with a DNA molecule of the embodiments and therefore consisting at least in part of transgenic cells. The class of plants that can be used in the methods of the embodiments is generally as broad as the class of higher plants susceptible to transformation techniques, including monocotyledonous and dicotyledonous plants.

[00108] Although the embodiments do not rely on a particular biological mechanism to increase a plant's resistance to a plant pest, expression of the nucleotide sequences of the embodiments in a plant can result in the production of the pesticidal proteins of the embodiments and an increase in the plant's resistance to a plant pest. The plants of the embodiments find use in agriculture in methods for impacting insect pests. Certain embodiments provide transformed crop plants, such as, for example, corn plants, that find use in methods for impacting insect pests of the plant, such as, for example, Lepidopteran pests.

[00109] A "plant or plant cell" is one in which genetic alteration, such as transformation, has been effected with respect to a gene of interest, or is a plant or plant cell that is descended from a plant or cell so altered and that comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the phenotype of the plant or plant cell in question.

[00110] A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration that resulted in the plant or cell in question; (b) a plant or plant cell of the same genotype as the starting material, but which has been transformed with a null construct (i.e., with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell that is an untransformed segregant among the progeny of a plant or plant cell in question; (d) a plant or plant cell genetically identical to the plant or plant cell in question, but which is not exposed to conditions or stimuli that induce the expression of the gene of interest; or (e) the plant or plant cell itself, under conditions in which the gene of interest is not expressed.

[00111] One 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 methodologies, and protein engineering techniques provide a large set of tools and protocols suitable for use in altering or manipulating the amino acid sequence and underlying genetic sequences of proteins of agricultural interest.

[00112] Thus, the proteins of the embodiments can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of pesticidal proteins can be prepared by introducing mutations into a synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and alterations of nucleic acids are well known in the art. For example, the engineered alterations can be introduced using an oligonucleotide-mediated site-directed mutagenesis technique. See, e.g., Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein.

[00113] The mutagenized nucleotide sequences of embodiments may be modified so as to alter about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more of the amino acids present in the primary sequence of the encoded polypeptide. Alternatively, further alterations can be introduced from the native sequence such that the encoded protein has 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 altered or otherwise modified as compared to the corresponding wild-type protein. Likewise, the encoded protein can have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or up to about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more additional codons compared to the corresponding wild-type protein. It is to be understood that the mutagenized nucleotide sequences of the embodiments are intended to encompass biologically functional equivalent peptides that have pesticidal activity, such as enhanced pesticidal activity as determined by anti-feeding properties against European corn borer larvae. Such sequences may arise as a consequence of the codon redundancy and functional equivalence that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.

[00114] One skilled in the art will recognize that amino acid additions and/or substitutions are generally based on the relative similarity of the amino acid side chain substituents, e.g., their hydrophobicity, charge, size, and the like. Examples of amino acid substitution groups that take into account several of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[00115] Recommendations for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), incorporated herein by reference. Conservative substitutions may be made, such as exchanging one amino acid for another with similar properties.

[00116] Thus, the genes and nucleotide sequences of the embodiments include both naturally occurring sequences and mutant forms. Likewise, the proteins of the embodiments encompass both naturally occurring proteins and variations (e.g., truncated polypeptides) and modified forms (e.g., mutants) thereof. These variants will continue to possess the desired pesticidal activity. Of course, the mutations that will be made in the nucleotide sequence encoding the variant must not place the sequence out of reading frame and will generally not create complementary regions that can produce mRNA secondary structure. See, EP Patent Application Publication No. 75,444.

[00117] Deletions, insertions and substitutions of the protein sequences covered herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion before making it, one skilled in the art will appreciate that the effect be assessed by routine analytical assays, such as insect feeding assays. See, for example, Marrone et al. (1985) J. Econ. Entomol. 78: 290-293 and Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485, incorporated herein by reference.

[00118] Variant nucleotide and protein sequences also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure, such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a novel pesticidal protein having desired properties. In this manner, recombinant polynucleotide libraries are generated from a population of sequence-related polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, full-length coding sequences, sequence motifs encoding a domain of interest, or any fragment of a nucleotide sequence of embodiments can be rearranged between nucleotide sequences of embodiments and corresponding portions of other known Cry nucleotide sequences to obtain a novel gene encoding a protein with an improved property of interest.

[00119] Properties of interest include, but are not limited to, pesticidal activity per unit of pesticidal protein, protein stability, and toxicity to non-target species, particularly humans, livestock, and plants and microbes expressing the pesticidal polypeptides of the embodiments. The embodiments are not linked by a particular rearrangement strategy, only that at least one nucleotide sequence of the embodiments, or portion thereof, is involved in such a rearrangement strategy. The rearrangement may involve only nucleotide sequences disclosed herein or may additionally involve the rearrangement of other nucleotide sequences known in the art. Strategies for DNA rearrangement are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Academic. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; y U.S. patents nos. 5,605,793 and 5,837,458.

[00120] The nucleotide sequences of the embodiments may also be used to isolate corresponding sequences from other organisms, particularly other bacteria, and more particularly other strains of Bacillus. In this manner, methods such as PCR, hybridization, and the like may be used to identify such sequences based on their sequence homology to the sequences presented herein. Sequences that are selected based on their sequence identity to the entire sequences presented herein or to fragments thereof are encompassed by the embodiments. Such sequences include sequences that are orthologs of the sequences disclosed. The term "orthologs" refers to genes derived from a common ancestral gene and that are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. The functions of orthologs are often highly conserved between species.

[00121] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook". See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfe, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfe, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods utilizing paired primers, nested primers, individual specific primers, degenerate primers, gene specific primers, vector specific primers, partially mismatched primers, and the like.

[00122] In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cloned cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides and can be labeled with a detectable group such as 32P or any other detectable marker. Thus, for example, hybridization probes can be made by labeling synthetic oligonucleotides based on the sequences of the embodiments. Methods for preparing hybridization probes and for constructing cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook.

[00123] For example, a full-length sequence disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to corresponding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique to the sequences of the embodiments and are generally at least about 10 or 20 nucleotides in length. Such probes can be used to amplify corresponding Cry sequences from a chosen organism by PCR. This technique can be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g., Sambrook).

[00124] Hybridization of such sequences can be performed under stringent conditions. The term "stringent conditions" or "stringent hybridization conditions" as used herein refers to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold, 5-fold, or 10-fold relative to baseline). Stringent conditions are sequence dependent and will be different under different circumstances. By controlling the stringency of the hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow for some mismatching in the sequences so that lower degrees of similarity are detected (heterologous probing). Typically, a probe is less than about 1000 or 500 nucleotides in length.

[00125] Typically, stringent conditions will be those where the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Examples of low-stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55 °C. Examples of moderate-stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 °C, and a wash in 0.5X to 1X SSC at 55 to 60 °C. Examples of high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 °C and a final wash in 0.1X SSC at 60 to 65 °C for at least about 20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

[00126] The following terms are used to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percent sequence identity", and (e) "substantial identity".(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used herein, "comparison window" refers to a specified, contiguous segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally may be 30, 40, 50, 100 or more. Those skilled in the art understand that to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and subtracted from the number of matches.

[00127] Sequence alignment methods for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the site search alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:58735877.

[00128] 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 the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Coupet 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 program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. Nucleotide BLAST searches can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding the protein of the embodiments. Protein BLAST searches can be performed with the BLASTX program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the embodiments. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs can be used (e.g., BLASTN for nucleotide sequences, BLASTX for proteins). See the National Center for Biotechnology Information website at ncbi.hlm.nih.gov. Alignment can also be performed manually by inspection.(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to those residues in the two sequences that are the same when aligned for maximum match over a specified comparison window. When using percent sequence identity for proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically this involves scoring a conservative substitution as a partial rather than a complete mismatch, thereby increasing the percent sequence identity. Thus, for example, when an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The score of conservative substitutions is calculated, for example, as implemented in the PC/GENE program (Intelligenetics, Mountain View, California). (d) As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in both sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the percent sequence identity. (e)(i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 80%, 90%, or 95% or more sequence identity when compared to a reference sequence using one of the alignment programs described using standard parameters. One skilled in the art will recognize that these values can be adjusted appropriately to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial amino acid sequence identity for these purposes generally means sequence identity of at least 60%, 70%, 80%, 90%, or 95% or more sequence identity.

[00129] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5 °C lower than the Tm for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1 °C to about 20 °C lower than the Tm, depending on the degree of stringency desired, as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.(e)(ii) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 80%, 85%, 90%, 95% or more sequence identity to a reference sequence over a specified comparison window. Optimal alignment for this purpose can be performed using the global alignment algorithm of Needleman and Wunsch (1970) supra. One indication that two peptide sequences are substantially identical is that one peptide is immunologically cross-reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, when the two peptides differ only by a conservative substitution. Peptides that are "substantially similar" share sequences as noted above, except that positions of residues that are not identical may differ through conservative amino acid changes.

[00130] The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those skilled in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed 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 employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised 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, forks, stem-loop structures, and the like.

[00131] An additional embodiment relates to a transformed organism such as an organism 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 comprising said DNA molecule, or a vector comprising said expression cassette, which can be stably incorporated into the genome of the transformed organism.

[00132] The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments. The term "operably linked" as used herein refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences that are linked are contiguous and, when necessary to join two protein coding regions, contiguous and in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) may be provided in multiple DNA constructs.

[00133] Such a DNA construct is provided with a plurality of restriction sites for insertion of the Cry toxin sequence to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[00134] The DNA construct will include in the 5' to 3' direction of transcription: a transcription and translation initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcription and translation termination region (i.e., termination region) functional in the host organism. The transcription initiation region (i.e., promoter) may be native, analogous, foreign, or heterologous to the host organism and/or the sequence of the embodiments. Additionally, the promoter may be the naturally occurring sequence or, alternatively, a synthetic sequence. The term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. When the promoter is "foreign" or "heterologous" to the sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked sequence of the embodiments. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. When the promoter is a native or naturally occurring sequence, expression of the operably linked sequence is altered from wild-type expression, resulting in a change in phenotype.

[00135] The termination region may be native to the transcriptional initiation region, may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host, or any combination thereof).

[00136] Convenient termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 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. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:96279639.

[00137] Where appropriate, a nucleic acid may be optimized to increase expression in the host organism. Thus, when the host organism is a plant, synthetic nucleic acids may be synthesized using plant-preferred codons for improved expression. See, e.g., Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. For example, although the nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, the sequences may be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons, as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the preferred maize codon for a particular amino acid can be derived from known maize gene sequences. The maize codon usage for 28 maize plant genes is listed in Table 4 of Murray et al., supra. Methods are available in the art for synthesizing preferred plant genes. 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, incorporated herein by reference.

[00138] Additional sequence modifications are known to enhance gene expression in a cellular host. These include the elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence may be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term "host cell" as used herein refers to a cell that contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may 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 monocot host cell is a maize host cell. When possible, the sequence is modified to avoid the predicted mRNA fork secondary structures.

[00139] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences may act to enhance translation. Translational leaders are known in the art and include: picornavirus leaders, e.g., EMCV (Encephalomyocarditis 5' noncoding region) leader (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); potyvirus leaders, e.g., TEV (Tobacco Cautery Virus) leader (Gallie et al. (1995) Gene 165(2): 233-238), MDMV (Maize Dwarf Mosaic Virus) leader, human immunoglobulin heavy chain-binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader of alfalfa mosaic virus coat protein mRNA (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622625); tobacco mosaic virus (TMV) leader (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237–256); and corn chlorotic mottle virus (MCMV) leader (Lommel et al. (1991) Virology 81: 382–385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965968.

[00140] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. To this end, adapters or linkers may be used to join the DNA fragments or other manipulations may be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. To this end, in vitro mutagenesis, primer repair, restriction, hybridization, new substitutions, e.g., transitions and transversions, may be involved.

[00141] Various promoters may be used in practicing the embodiments. Promoters may be selected based on the desired result. The nucleic acids may be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in the host organism. Constitutive promoters suitable for use in a plant host cell include, for example, the Rsyn7 promoter core promoter and other constitutive promoters described in WO 99/43838 and U.S. Patent No. 6,072,050; the CaMV 35S core 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. 18:675-689); pEMU (Last et al. (1991) Theou. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in 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.

[00142] Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, which can respond to damage caused by insect feeding, include the potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Patent No. 5,428,148; win1 and win2 (Stanfoud et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene (Couderok et al. (1994) Plant J. 6(2): 141-150); and the like, incorporated herein by reference.

[00143] Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those of pathogenesis-related proteins (PR proteins), which are induced upon infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, e.g., Redolfi et al. (1983) Neth. J. Plant Pathol. 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, incorporated herein by reference.

[00144] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular PlantMicrobe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 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 inducible); and references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, expression of which is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

[00145] Chemically regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending on the objective, the promoter can be a chemically inducible promoter, where application of the chemical induces gene expression, or a chemically repressed promoter, where application of the chemical represses gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the corn GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemically regulated promoters of interest include steroid-sensitive promoters (see, e.g., the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressed promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[00146] Tissue-preferred promoters can be used to direct increased pesticidal protein expression within a particular plant tissue. Tissue-preferred promoters include those discussed in 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. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513- 524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

[00147] Preferred leaf 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. Academic. Sci. USA 90(20):9586-9590.

[00148] Root-preferred promoters or root-specific promoters are known and can be selected from many available in the literature or isolated de novo from a variety of compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, in which two root-specific promoters isolated from hemoglobin genes of the non-legume nitrogen-fixing Parasponia andersonii and the non-legume nitrogen-fixing Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases the root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed root-inducing genes rolC and rolD from Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in these promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the root tip epidermis and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use as an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1' gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Other root preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 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.

[00149] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) and also "seed-germinating" promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108, incorporated herein by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milpas (myo-inositol-1-phosphate synthase). (See U.S. Patent No. 6,225,529, incorporated herein by reference). Gamma-zein and Glob-1 are endosperm-specific promoters. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, corn 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters of the end1 and end2 genes are disclosed; incorporated herein by reference. A promoter that has "preferred" expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue.

[00150] When low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives low level expression of a coding sequence at a low level. By low level expression levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts are intended. Alternatively, it is recognized that the term "weak promoters" also encompasses promoters that drive expression in only a few cells and not others to give a low overall level of expression. When a promoter drives expression at unacceptably high levels, portions of the promoter sequence may be deleted or modified to decrease expression levels.

[00151] Such constitutive weak promoters include, for example, the Rsyn7 promoter core promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the CaMV 35S core promoter, and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. 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.

[00152] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are used for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213; and Meijer et al. (1991) Plant Mol. Biol. 16:807-820); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15:127-136); bromoxanil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shaw et al. (1986) Science 233:478-481; and US Patent Nos. 7,709,702; and 7,462,481); phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513-2518). See generally, Yarranton (1992) Curr. Opinion. Biotech. 3: 506-511; Christopherson et al. (1992) Proc. Natl. Academic. Sci. USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 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. Academic. Sci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Academic. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248: 480483; Gossen (1993) PhD thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Academic. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Academic. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Academic. Sci. USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653; Hillene-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991)Antimicrob. AgentsChemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) PhD thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 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 disclosures are incorporated herein by reference.

[00153] The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene may be used in the embodiments.

[00154] The methods of embodiments involve introducing a polypeptide or polynucleotide into a plant. "Introduction" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a plant cell. The methods of embodiments do not rely on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gain access to the interior of at least one plant cell. Methods for introducing polynucleotides or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and viral-mediated methods.

[00155] "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the plant's genome and is capable of being inherited by the plant's progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the plant's genome or a polypeptide is introduced into a plant.

[00156] Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant or plant cell, i.e. monocotyledonous or dicotyledonous, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Patent Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Ougan Culture: Fundamental Methods, ed. Gamboug and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6: 923–926); and Lecl transformation (WO 00/28058). For potato transformation see Tu et al. (1998) Plant Molecular Biology 37: 829–838 and Chong et al. (2000) Transgenic Research 9: 71–78. Additional transformation procedures can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22: 421–477; Sanford et al. (1987) Particulate Science and Technology 5: 27–37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soy); McCabe et al. (1988) Bio/Technology 6: 923-926 (soy); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soy); Singh et al. (1998) Theou. Appl. Genet. 96: 319-324 (soy); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Academic. Sci. USA 85: 4305-4309 (corn); Klein et al. (1988) Biotechnology 6:559-563 (corn); U.S. patents 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-VanSlogteren et al. (1984) Nature (London) 311: 763-764; US Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Academic. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. 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 and Christou and Ford (1995) Annals of Botany 75: 407–413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745–750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference.

[00157] In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, introducing the Cry toxin protein or its variants and fragments directly into the plant or introducing the Cry toxin transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush et al. (1994) The Journal of Cell Science 107: 775-784, which are incorporated herein in their entirety by reference. Alternatively, the Cry toxin polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include the viral vector system and precipitation of the polynucleotide in a manner that prevents subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to integrate into the genome is greatly reduced. Such methods include the use of polyethylenimine (PEI; Sigma #P3143) coated particles.

[00158] Methods for the targeted insertion of a polynucleotide into a specific location in the plant genome are known in the art. In one embodiment, insertion of the polynucleotide into a desired genomic location is accomplished using a site-specific recombination system. See, e.g., WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are incorporated herein by reference. Briefly, the polynucleotide of the embodiments may be contained in a transfer cassette flanked by two non-identical recombination sites. The transfer cassette that is introduced into a plant has stably incorporated into its genome a target site that is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated into the target site. The polynucleotide of interest is thus integrated into a specific chromosomal position in the plant genome.

[00159] The transformed cells can be grown into plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be grown and pollinated with the same or different transformed strains, and the resulting hybrid with constitutive or inducible expression of the desired phenotypic trait identified. Two or more generations can be grown to ensure that expression of the desired phenotypic trait is stably maintained and inherited, and then seeds are harvested to ensure that expression of the desired phenotypic trait has been achieved.

[00160] The nucleotide sequences of the embodiments can be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that recombinant proteins of the embodiments can be initially synthesized as part of a viral polyprotein, which can later be processed by proteolysis in vivo or in vitro to produce the desired pesticidal protein. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a pesticidal protein of the embodiments, can have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences encoding them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in plants, which involve viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931; incorporated herein by reference.

[00161] The embodiments further relate to plant propagation material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves, and root and shoot cuttings.

[00162] The embodiments can be used for transformation of any plant species, including but not limited to monocotyledons and dicotyledons. Examples of plants of interest include but are not limited to corn (Zea mays), Brassica sp. (e.g. B. napus, B. rapa, B. juncea), in particular those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Ouyza sativa), rye (Secale cereale), sorghum (Soughum bicolou, Soughum vulgare), millet (e.g. pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), finger millet (Setaria italica), giant goosefoot (Eleusine couacana)), sunflower (Helianthus annuus), safflower (Carthamus tinctouius), wheat (Triticum aestivum), soya bean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (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), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[00163] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and melon (C. melo). Ornamental plants include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphoubia pulcherrima), and chrysanthemums. Conifers that may be used in the practice of the modalities include, for example, pines such as pinyon pine (Pinus taeda), American pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus conteta), and Monterey pine (Pinus radiata); Oregon pine (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis). Plants of the modalities include crop plants including, but not limited to: corn, alfalfa, sunflower, Brassica spp., soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, sugar cane, etc.

[00164] Lawns include, but are not limited to: poa-annual (Poaannua); annual ryegrass (Lolium multiflouum); Canadian poa (Poacompressa); red fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass(Agropyron desertuum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); fever weed (Poa pratensis); panasco (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); red grass (Agrostis alba); common poa (Poa trivialis); sheep fescue (Festuca ovina); bromine (Bromus inermis); tall fescue (Festuca arundinacea); cattail (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); Zoysia grass (Zoysia spp.); potato grass (Paspalum Notatum); carpet weed (Axonopus affinis); centipede grass (Eremochloa ophiuroides); Kikuio grass (Pennisetum cleesinum); seashore paspalum (Paspalum vaginatum); blue grass (Bouteloua gracilis); American grass (Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

[00165] Plants of interest include grain plants that provide seeds of interest, oilseed plants, and legumes. Seeds of interest include grain seeds such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oilseed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, castor, olive, etc. Legume plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden bean, cowpea, mung bean, lima bean, fava bean, lentils, chickpea, etc.

[00166] In certain embodiments, the nucleic acid sequences of the embodiments can be overlapped with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the polynucleotides of the embodiments can be overlapped with any other polynucleotides encoding polypeptides with pesticidal and/or insecticidal activity, such as other toxic Bt 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), and the like. The generated combinations may also include multiple copies of any of the polynucleotides of interest. The polynucleotides of the embodiments may also be overlapped with any other gene or combination of genes to produce plants with a variety of combinations of desired traits including, but not limited to, traits desirable for animal feed, such as high oil content genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. patent nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); high lysine barley (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and proteins with high methionine (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modified storage proteins (U.S. Patent No. 6,858,778); and thioredoxins (U.S. Patent No. 7,009,087), the disclosures of which are incorporated herein by reference.

[00167] The polynucleotides of the embodiments can also be overlapped with desirable traits for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Patent No. 5,792,931); virulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants leading to herbicide resistance such as the S4 and/or Hra mutations; glutamine synthase inhibitors such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene and GAT gene as disclosed in U.S. Patent Nos. 7,709,702; and 7,462,481; and desirable characteristics for processing or process products such as tall oil (e.g., U.S. Pat. No. 6,232,529); modified starches (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzymes (SBE), and starch familying enzymes (SDBE)), and polymers or bioplastics (e.g., U.S. Patent No. 5,602,321, beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:58375847) facilitate the expression of polyhydroxyalkanoates (PHA)), the disclosures of which are incorporated herein by reference. One could also combine the polynucleotides of the embodiments with polynucleotides that provide agronomic traits such as male sterility (see, e.g., U.S. Patent No. 5,583,210), stem strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are incorporated herein by reference.

[00168] In some embodiments the overlapping trait may be a trait or event that has received regulatory approval that is well known to one of skill in the art and that can be found at the Center for Environmental Risk Assessment (cera-gmc.org/?action=gm_crop_database, which can be found using the www prefix) and the International Service for the Acquisition of Agri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp, which can be found using the www prefix).

[00169] These overlapping combinations may be created by any method including, but not limited to, plant breeding by any conventional or TOPCROSS® methodology, or genetic transformation. If traits are to be overlapped by genetic transformation of plants, the polynucleotide sequences of interest may be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits may be used as the target for introducing additional traits by subsequent transformation. The traits may be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences are to be introduced, the two sequences may be contained in separate transformation cassettes (trans) or contained in the same transformation cassette (cis). Expression of the sequences may be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to produce the desired combination of traits in the plant. It is further recognized that polynucleotide sequences may be superimposed at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are incorporated herein by reference.

[00170] The compositions of the embodiments find use in protecting plants, seeds, and plant products in a variety of ways. For example, the compositions may be used in a method that involves placing an effective amount of the pesticidal composition into the pest environment by a procedure selected from the group consisting of spraying, dusting, broadcasting, or seed coating.

[00171] Before marketing the plant propagation product (fruit, tuber, bulb, corm, grain, seed), but especially seeds, is usually treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these preparations, if desired together with other carriers, surfactants or application-promoting adjuvants usually used in the formulation technique to provide protection against damage caused by bacterial, fungal or animal pests. To treat the seed, the protective coating can be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. Furthermore, in special cases, other methods of application to plants are possible, for example, treatment directed at the leaf buds or the fruit.

[00172] Plant seed of embodiments comprising a nucleotide sequence encoding a pesticidal protein of embodiments can be treated with a protective seed coating comprising a seed treatment compound, such as, for example, captan, carboxin, thiram, metalaxyl, pirimiphos-methyl, and others that are commonly used in seed treatment. In one embodiment, a protective seed coating comprising a pesticidal composition of embodiments is used alone or in combination with one of the protective seed coatings commonly used in seed treatment.

[00173] It is recognized that genes encoding pesticide proteins can be used to transform insect pathogenic organisms. Such organisms include baculoviruses, fungi, protozoa, bacteria and nematodes.

[00174] A gene encoding a pesticidal protein of the embodiments may be introduced via a suitable vector into a microbial host, and said host is applied to the environment or to plants or animals. The term "introduced" in the context of insertion of a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[00175] Microorganism hosts that are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere and/or rhizoplane) of one or more crops of interest may be selected. These microorganisms are selected so as to be able to compete successfully in the particular environment with wild-type microorganisms, provide stable maintenance and expression of the gene expressing the pesticidal protein, and desirably provide improved protection of the pesticide against environmental degradation and inactivation.

[00176] Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly yeasts, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as the yeast species Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vineleii and of phytosphere such as Rhodotouula rubra, 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 pigmented microorganisms.

[00177] There are several ways to introduce a gene expressing the pesticidal protein into the host microorganism under conditions that allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed that include the nucleotide constructs of interest operably linked with transcriptional and translational regulatory signals for expression of the nucleotide constructs and a nucleotide sequence homologous to a sequence in the host organism, whereby integration will occur, and/or a replication system that is functional in the host, whereby integration or stable maintenance will occur.

[00178] Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Patent Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook; Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and the references cited therein.

[00179] Suitable host cells, in which the pesticidal protein-containing cells will be treated to prolong the activity of the pesticidal proteins in the cell when the treated cell is applied to the environment of the target pest(s), may include prokaryotes or eukaryotes, typically being limited to those cells that do not produce substances toxic to higher organisms, such as mammals. However, organisms that produce substances toxic to higher organisms may be used, where the toxin is unstable or the level of application is sufficiently low to avoid any possibility of toxicity to a mammalian host. Of particular interest as hosts would be prokaryotes and lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and Gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae, such as Photobacteria, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Eukaryotes include fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and yeastBasidiomycetes, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

[00180] Characteristics of particular interest in selecting a host cell for the purpose of producing pesticidal proteins include ease of introduction of the pesticidal protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of ancillary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; foliar affinity; lack of toxicity to mammals; attractiveness of pests for ingestion; ease of killing and attachment without damaging the toxin; and the like. Other considerations include ease of formulation and handling, cost-effectiveness, storage stability, and the like.

[00181] Host organisms of particular interest include yeasts such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae), Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp. (such as P. aeruginosa, P. fluorescens), Erwinia spp., and Flavobacterium spp., and other such organisms including Bt, E. coli, Bacillus subtilis, and the like.

[00182] Genes encoding pesticidal proteins of the modalities can be introduced into microorganisms that multiply on plants (epiphytes) to provide pesticidal proteins to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria.

[00183] Root-colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a root-colonizing Bacillus cereus strain can be isolated from the roots of a plant (see, e.g., Heelsman et al. (1991) Appl. Environ. Microbiol. 56:713-718). The genes encoding the pesticidal proteins of the embodiments can be introduced into root-colonizing Bacillus cereus by conventional methods known in the art.

[00184] Genes encoding pesticidal proteins can be introduced, for example, into root-colonizing Bacillus by means of electrotransformation. Specifically, the genes encoding the pesticidal proteins can be cloned into a shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218. The shuttle vector pHT3101 containing the coding sequence for the particular pesticidal protein gene can, for example, be transformed into the root-colonizing Bacillus by means of electroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218).

[00185] Expression systems can be designed such that pesticidal proteins are secreted outside the cytoplasm of gram-negative bacteria, such as E. coli, for example. The advantages of having secreted pesticidal proteins are: (1) avoidance of the potential cytotoxic effects of the expressed pesticidal protein; and (2) improvement in the purification efficiency of the pesticidal protein, including, but not limited to, increased efficiency in recovery and purification of the protein per volume of cell broth and reduced recovery and purification time and/or costs per unit of protein.

[00186] Pesticidal proteins can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the pesticidal protein. Signal peptides recognized by E. coli can be found in proteins already known to be secreted in E. coli, for example the OmpA protein (Ghrayeb et al. (1984) EMBO J, 3:2437-2442). OmpA is a major outer membrane protein of E. coli, and thus its signal peptide is thought to be efficient in the translocation process. Furthermore, the OmpA signal peptide does not need to be modified prior to processing as might be the case for other signal peptides, for example lipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol. 153:492).

[00187] The pesticidal proteins of the embodiments can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bt strains have been used as insecticidal sprays. In the case of a pesticidal protein(s) that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the pesticidal protein(s) into the growth medium during the fermentation process. The pesticidal proteins are retained within the cell and the cells are then processed to produce the encapsulated pesticidal 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 processed and sprayed as an insecticide (Gaertner et al. (1993), in: Advanced Engineered Pesticides, ed. Kim).

[00188] Alternatively, pesticidal proteins are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest(s). The resulting product maintains the toxicity of the toxin. These naturally encapsulated pesticidal proteins can then be formulated according to conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and plant foliage. See, for example, EP0192319, and references cited therein.

[00189] In embodiments, a transformed microorganism (which includes whole organisms, cells, spore(s), pesticidal protein(s), pesticidal component(s), pest impact component(s), mutant(s), live or dead cells and cellular components, including mixtures of live and dead cells and cellular components, and including damaged cells and cellular components) or an isolated pesticidal protein can be formulated with an acceptable carrier into a pesticidal composition(s) that is, for example, a suspension, a solution, an emulsion, a spray powder, a dispersible granule or pellet, a wettable powder and emulsifiable concentrate, an aerosol or spray, an impregnated granule, an adjuvant, a coating paste, a colloid, and also encapsulated in, for example, polymeric substances. Such formulated compositions may be prepared by conventional means such as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of a cell culture comprising the polypeptide.

[00190] These compositions disclosed above can be obtained by adding a surfactant, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors or other preparations that influence plant growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, crop adjuvants and fertilizers, can be combined with carriers, surfactants or adjuvants commonly used in the formulation art or other components to facilitate the handling of the product and its application to specific pests. Suitable carriers and adjuvants may be solid or liquid and correspond to substances normally used in formulation technology, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. The active ingredients of the embodiments are normally applied in the form of compositions and may be applied to the crop area, plant or seed to be treated. For example, the compositions of the embodiments may be applied to grain in preparation for or during storage in a grain bin or silo, etc. The compositions of the embodiments may be applied simultaneously or in succession with other compounds. Methods of application of an active ingredient of the embodiments or an agrochemical composition of the embodiments containing at least one of the pesticidal proteins produced by the bacterial strains of the embodiments 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 infestation by the corresponding pest.

[00191] Suitable surfactants include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; a carboxylate of a long-chain fatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkylaryl sulfonates, such as alkylbenzene sulfonates or lower alkylnaphthalene sulfonates, e.g., butylnaphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g. the sulfonated condensation product of oleic acid and N-methyl taurine; or the alkyl sulfosuccinates, e.g. the sodium sulfonate of dioctyl succinate. Non-ionic surfactants include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or phenols with fatty alkyl or alkenyl substituted with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of these esters with ethylene oxide, e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol or ethoxylated acetylenic glycols. Examples of a cationic surfactant include, for example, an aliphatic mono-, di- or polyamine such as an acetate, naphthenate or oleate; or an oxygen-containing amine such as a polyoxyethylene alkylamine amine oxide; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

[00192] Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corn cobs, peanut shells, rice hulls, and walnut shells.

[00193] The compositions of the embodiments may be in a form suitable for direct application or as a primary composition concentrate that requires dilution with an appropriate amount of water or other diluent prior to application. The pesticide concentration will vary depending on the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 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 rate labeled for the commercial product, for example, about 0.01 lb-5.0 lb (0.0045 kg-2.27 kg) per acre when in dry form and at about 0.01 pts. - 10 pts. per acre when in liquid form.

[00194] In another embodiment, the compositions, as well as the transformed microorganisms and pesticidal proteins of the embodiments, may be treated prior to formulation to prolong pesticidal activity when applied to the environment of a target pest provided that the pretreatment is not detrimental to the pesticidal activity. Such treatment may be by chemical and/or physical means provided that the treatment does not detrimentally affect the properties of the composition(s). Examples of chemical reagents include but are not limited to halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infectives such as zephyran chloride; alcohols such as isopropanol and ethanol; and histological fixatives such as Bouin's fixative and Helly's fixative (see, e.g., Humason (1967) Animal Tissue Techniques (W.H. Freeman and Co.).

[00195] In other embodiments, it may be advantageous to treat Cry toxin polypeptides with a protease, e.g., trypsin, to activate the protein prior to application of a pesticidal protein composition of the embodiments to the target pest environment. Methods for activating the protoxin by a serine protease are well known in the art. See, e.g., Cooksey (1968) Biochem. J. 6:445-454 and Carroll and Ellar (1989) Biochem. J. 261:99-105, the teachings of which are incorporated herein by reference. For example, a suitable activation protocol includes, but is not limited to, combining a polypeptide to be activated, for example a novel purified Cry polypeptide (e.g., having the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8), and trypsin at a 1/100 by weight ratio of protein to trypsin in 20 nM NaHCO3, pH 8, and digesting the sample at 36 °C for 3 hours.

[00196] The compositions (including the transformed microorganisms and pesticidal proteins of the embodiments) can be applied to the environment of an insect pest by, for example, spraying, atomizing, dusting, dispersing, coating or pouring, introduction into or onto soil, introduction into irrigation water, by seed treatment or general application or dusting at the time the pest has started to appear or prior to the appearance of pests as a protective measure. For example, the pesticidal protein and/or the transformed microorganisms of the embodiments can be mixed with grain to protect the grain during storage. It is generally important to obtain good pest control in the early stages of plant growth, as this is the time when the plant can be most severely damaged. The compositions of the embodiments can conveniently contain another insecticide if this is considered necessary. In one embodiment, the composition is applied directly to the soil, at a planting height, in the granular form of a composition of a carrier and dead cells of a Bacillus strain or transformed microorganism of the embodiments. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, a herbicide, an insecticide, a fertilizer, an inert carrier, and dead cells of a Bacillus strain or transformed microorganism of the embodiments.

[00197] Those skilled in the art will recognize that not all compounds are equally effective against all pests. The compounds of the embodiments exhibit activity against insect pests, which may include pests of agronomics, forestry, greenhouses, nurseries, ornamentals, food and fiber, public and animal health, domestic and commercial structures, households, and stored products of economic importance. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Malofaga, Homoptera, Hemiptera, Orthoptera, Tisanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Tricoptera, etc., particularly Coleoptera and Lepidoptera.

[00198] Insects of the order Lepidoptera include, but are not limited to, fall armyworms, cutworms, false loopers, and bollworms in the family Noctuidae: Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Mourison (western cutworm); A. segetum Denis & Schiffermüller (turnip moth); A. subterranea Fabricius (tobacco cutworm); Alabama argillacea Hübner (cotton leafworm); Anticarsia gemmatalis Hübner (soybean armyworm); Athetis mindara Barnes and McDunnough (rough-skinned cutworm); Earias insulana Boisduval (spiny moth); E. vittella Fabricius (painted moth); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoa messoria Harris (black-flapped cutworm); Helicoverpa armigera Hübner (American moth); H. zea Boddie (corn earworm or cotton moth); Heliothis virescens Fabricius (tobacco bollworm); Hypena scabra Fabricius (green clover armyworm); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Melanchra picta Harris (zebra armyworm); Pseudaletia unipuncta Hawouth (armyworm); Pseudoplusia includens Walker (false looper); Richia albicosta Smith (western cutworm); Spodoptera frugiperda JE Smith (false armyworm); S. exigua Hübner (night butterfly); S. litura Fabricius (tobacco cutworm, almond moth); Trichoplusia ni Hübner (cabbageworm); borers, wallworms, lawn moths, pine coneworms, and American harrisina of the families Pyralidae and Crambidae such as Achroia grisella Fabricius (small wax moth); Amyelois transitella Walker (orange moth); Anagasta kuehniella Zeller (Mediterranean wax moth); Cadra cautella Walker (almond moth); Chilo partellus Swinhoe (stem borer); C. suppressalis Walker (free stem/rice borer); C. terrenellus Pagenstecher (sugarcane borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn rootworm); C. teterrellus Zincken (doghair caterpillar); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grapevine leafroller); Diaphania hyalinata Linnaeus (melon fruitworm); D. nitidalis Stoll (cucurbit borer); Diatraea grandiosella Dyar (sugarcane borer), D. saccharalis Fabricius (sugarcane borer); Elasmopalpus lignosellus Zeller (elasmo caterpillar); Eoueuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cocoa) moth); Galleria mellonella Linnaeus (large wax moth); Hedylepta accepta Butler (sugarcane leafroller); Herpetogramma licarsisalis Walker (turf moth); Homoeosoma electellum Hulst (sunflower moth); Loxostege sticticalis Linnaeus (beet armyworm); Maruca testulalis Geyer (bean borer); Outhaga thyrisalis Walker (tea tree moth); Ostrinia nubilalis Hübner (European corn borer); Plodia interpunctella Hübner (Indian corn moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (udea); and leafrollers, shootworms, seedworms, and fruitworms in the family Tortricidae Acleris gloverana Walsingham (Western black-headed shootworm); A. variana Fernald (Eastern black-headed shootworm); Adoxophyes orana Fischer von Rosslerstamm (tortrix fruit juice moth); Archips spp. including A. argyrospila Walker (fruit tree leafroller) and A. rosana Linnaeus (European leafroller); Argyrotaenia spp.; Bonagota salubricola Meyrick (Brazilian apple leafroller); Choristoneura spp.; Cochylis hospes Walsingham (sunflower band moth); Cydia latiferreana Walsingham (hazelnut moth); C. pomonella Linnaeus (apple moth); Endopiza viteana Clemens (grape moth); Eupoecilia ambiguella Hübner (grapevine moth); Grapholita molesta Busck (Oriental fruit moth); Lobesia botrana Denis & Schiffermüller (European grapevine moth); Platynota flavedana Clemens (diversified leafroller); P. stultana Walsingham (omnivorous leafroller); Spilonota ocellana Denis & Schiffermüller (budworm moth); and Suleima helianthana Riley (sunflower budworm moth).

[00199] Other selected agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (cankerworm); Anarsia lineatella Zeller (peach borer); Anisota senatouia J.E. Smith (orange woodworm); Antheraea pernyi Guérin-Méneville (Chinese woodworm); Bombyx mori Linnaeus (silkworm); Bucculatrix thurberiella Busck (cotton leafborer); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silkworm), Ennomos subsignaria Hübner (slippery elm caterpillar); Erannis tiliaria Harris (linden looper); Erechthias flavistriata Walsingham (sugarcane shoot moth); Euproctis chrysorrhoea Linnaeus (brown-tailed moth); Harrisina americana Guérin-Méneville (vine leaf strippers); Heliothis subflexa Guenée; Hemileuca oliviae Cockrell (bandworm); Hyphantria cunea Drury (summer cutworm); Keiferia lycopersicella Walsingham (tomato cutworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satina moth); Lymantria dispar Linnaeus (gypsy moth); Malacosoma spp.; Meuca quinquemaculata Hawouth (five-spotted moth, tomato hornworm); M. sexta Hawouth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Orgyia spp.; Paleacrita vernata Peck (spring caterpillar); Papilio cresphontes Cramer (giant Asian butterfly, orange dog); Phryganidia californica Packard (California woodworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (tent-shaped leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green-veined white butterfly); Platyptilia carduidactyla Riley (artichoque plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink earworm); Pontia protodice Boisduval & Leconte (southern cabbage looper); Sabulodes aegrotata Guenée (omnivorous false looper); Schizura concinna J.E. Smith (red arched looper); Sitotroga cerealella Olivier (Angoumois cereal moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (striped house moth); Tuta absoluta Meyrick (tomato leaf miner) and Yponomeuta padella Linnaeus (ermine moth).

[00200] Of interest are larvae and adults of the order Coleoptera including weevils of the families Anthribidae, Bruchidae, and Curculionidae including, but not limited to: Anthonomus greis Boheman (cotton boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (diaprepes root weevil); Hypera punctata Fabricius (clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); M. hemipterus sericeus Olivier (silk cane weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (corn weevil); Rhabdoscelus obscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leaf miners in the family Chrysomelidae including, but not limited to: Chaetocnema ectypa Houn (desert corn 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); Orlema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae including but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); Chafers and other beetles from the family Scarabaeidae including but not limited to: Antitrogus parvulus Britton (Childers' cane maggot); Cyclocephala borealis Arrow (northern masked beetle, white beetle); C. immaculata Olivier (southern masked beetle, white beetle); Dermolepida albohirtum Waterhouse (grey cane beetle); Euetheola humilis rugiceps LeConte (sugar cane beetle); Lepidiota frenchi Blackburn (French cane beetle); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugar cane beetle); Phyllophaga crinita Burmeister (white beetle); P. latifrons LeConte (St. John's beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European beetle); carpet beetles in the family Dermestidae; wireworms in the family Elateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal (wireworm); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles of the family Scolytidae; beetles of the family Tenebrionidae; beetles of the family Cerambycidae such as, but not limited to, Migdolus fryanus Westwood (long-horned beetle); and beetles of the family Buprestidae including, but not limited to, Aphanisticus cochinchinae seminulum Obenberger (buprestid miner beetle).

[00201] Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicounis Loew (maize leafminer); flies including but not limited to: Contarinia soughicola Coquillett (sorghum fruit fly); Mayetiola destructor Say (Hessian fruit fly); Neolasioptera murtfeldtiana Felt, (sunflower seed fly); Sitodiplosis mosellana Géhin (wheat fruit fly); fruit flies (Tephritidae), Oscinella frit Linnaeus (cereal fruit fly); worms including but not limited to: Delia spp. including Delia platura Meigen (maize seed maggot); D. coarctata Fallen (wheat bulb fly); Fannia canicularis Linnaeus, F. femoualis Stein (outdoor houseflies); Meromyza americana Fitch (wheat stemworm); Musca domestica Linnaeus (houseflies); Stomoxys calcitrans Linnaeus (barn flies); face flies, horn flies, blow flies, Chrysomya spp.; Phoumia spp.; and other fly pests, horse flies Tabanus spp.; flies of the family Oestridae Gastrophilus spp.; Oestrus spp.; cattle beetles Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.

[00202] Included as insects of interest are those of the order Hemipteratais such as, but not limited to, the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae, Delphacidae, Diaspididae, Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae, Membracidae, Miridae, Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.

[00203] Agronomically important members of the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Acyrthisiphon pisum Harris (pea aphid); Adelges spp. (adelgids); Adelphocoris rapidus Say (swift bug); Anasa tristis De Geer (squash bug); Aphis craccivora Koch (cattle aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Foubes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacaspis tegalensis Zehntner (sugarcane scale); Aulacorthum solani Kaltenbach (foxglove aphid); Bemisia tabaci Gennadius (tobacco whitefly, sweet potato whitefly); B. argentifolii Bellows & Perring (leaf whitefly); Blissus leucopterus leucopterus Say (grass bug); Blostomatidae spp.; Brevicoryne brassicae Linnaeus (cabbage aphid); Cacopsylla pyricola Foerster (pear psylla); Calocoris norvegicus Gmelin (potato capsid beetle); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Cimicidae spp.; Coreidae spp.; Corythuca gossypii Fabricius (cotton bug); Cyrtopeltis modesta Distant (tomato bug); C. notatus Distant (Asian fruit fly); Deois flavopicta Stâl (leafhoppers); Dialeurodes citri Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (Virginia hawthorn bug); Diuraphis noxia Kurdjumov/Moudvilko (Russian wheat aphid); Duplachionaspis divergens Green (armored scale); Dysaphis plantaginea Paaserini (pink apple aphid); Dysdercus suturellus Herrich- Schaffer (cotton dye bug); Dysmicoccus boninsis Kuwana (gray sugarcane mealy bug); Empoasca fabae Harris (potato grasshopper); Eriosoma lanigerum Hausmann (woolly apple aphid); Erythroneoura spp. (grape grasshoppers); Eumetopina flavipes Muir (Insular sugarcane bug); Eurygaster spp.; Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (seed bug complex); and Hyalopterus pruni Geoffroy (mealy plum aphid); Icerya purchasi Maskell (cottonwood scale); Labopidicola allii Knight (onion plant bug); Laodelphax striatellus Fallen (lesser brown leafhopper); Leptoglossus corculus Say (pine seed bug); Leptodictya tabida Herrich-Schaeffer (sugarcane aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Lygocoris pabulinus Linnaeus (common green capsid); Lygus lineolaris Palisot de Beauvois (lygus stink bug); L. Hesperus Knight (western lygus stink bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European lygus stink bug); Macrosiphum euphorbiae Thomas (potato aphid); Macrosteles quadrilineatus Foubes (grasshopper of the aster family); Magicicada septendecim Linnaeus (periodical cicada); Mahanarva fimbriolata Stâl (sugar cane leafhoppers); Melanaphis sacchari Zehntner (sugar cane aphid); Melanaspis glomerata Green (black scale); Metopolophium dirhodum Walker (pink cereal aphid); Myzus persicae Sulzer (peachy-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Nephotettix cinticeps Uhler (green grasshopper); N. nigropictus Stâl (rice grasshopper); Nezara viridula Linnaeus (southern green stink bug); Nilaparvata lugens Stâl (brown leafhopper); Nysius ericae Schilling (false stink bug); Nysius raphanus Howard (false stink bug); Oebalus pugnax Fabricius (rice bug); Oncopeltus fasciatus Dallas (large milky grass bug); Orthops campestris Linnaeus; Pemphigus spp. (root aphids and twig aphids); Peregrinus maidis Ashmead (corn leafhopper); Perkinsiella saccharicida Kirkaldy (sugarcane leafhopper); Phylloxera devastatrix Pergee (pecan phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsus lineatus Fabricius (four-lined grasshopper); Pseudatomoscelis seriatus Reuter (cotton aphid); Pseudococcus spp. (other mealybug complexes); Pulvinaria elongata Newstead (cottongrass scale); Pyrilla perpusilla Walker (sugarcane aphid); Pyrrhocoridae spp.; Quadraspidiotus perniciosus Comstock (St. Joseph scale); Reduviidae spp.; Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (oat aphid); Saccharicoccus sacchari Cockerell (sugarcane mealybug); Schizaphis graminum Rondani (green stinkbug); Sipha flava Foubes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grass aphid); Sogatella furcifera Houvath (white leafhopper); Sogatodes oryzicola Muir (rice delphid); Spanagonicus albofasciatus Reuter (white-labeled aphid); Therioaphis maculata Buckton (alfalfa aphid); Tinidae spp.; Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Trialeurodes abutiloneus (whitefly) and T. vaporariouum Westwood (greenhouse whitefly); Trioza diospyri Ashmead (persimmon psylla); and Typhlocyba pomaria McAtee (apple whitefly).

[00204] Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (garlic wilt mite); Panonychus ulmi Koch (European red mite); Petrobia latens Müller (wheat brown mite) Steneotarsonemus bancrofti Michael (sugarcane stem mite); spider mites and red mites in the family Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus Hirst (sugarcane leaf mite), O. pratensis Banks (Banks's grass mite), O. stickneyi McGregou (sugarcane spider mite); Tetranychus urticae Koch (two-spot spider mite); T. mcdanieli McGregou (McDaniel's mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite), flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregou (deer mite); rust and shoot mites in the family Eriophyidae and other foliar-feeding mites and mites important in human and animal health, i.e. dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick); and mange and itch mites in the families Psoroptidae, Pyemotidae, and Sarcoptidae.

[00205] Insect pests of the order Thysanura are of interest, such as Lepisma saccharina Linnaeus (moth); Thermobia domestica Packard (firebrat).

[00206] Other arthropod pests covered include: spiders in the order Araneae such as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); and Latrodectus mactans Fabricius (black widow spider); and centipedes in the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (house centipede). In addition, insect pests of the order Isoptera are of concern, including those of the family Termitidae, such as, but not limited to, Cylindrotermes nordenskioeldi Holmgren and Pseudacanthotermes militaris Hagen (sugarcane termite). Insects of the order Thysanoptera are also of concern, including but not limited to thrips, such as Stenchaetothrips minutus van Deventer (sugarcane thrips).

[00207] Insect pests can be tested for pesticidal activity of compositions of the embodiments at early stages of development, e.g., as larvae or other immature forms. Insects can be reared in complete darkness at from about 20°C to about 30°C and from about 30% to about 70% relative humidity. Bioassays can be performed as described in Czapla and Lang (1990) J. Econ. Entomol. 83(6): 2480-2485. Methods of rearing insect larvae and performing bioassays are well known to those skilled in the art.

[00208] A wide variety of bioassay techniques are known to those skilled in the art. General procedures include adding the experimental compound or organism to the dietary source in a closed container. Pesticidal activity can be measured by, but is not limited to, changes in mortality, weight loss, attraction, repellency, and other behavioral and physical changes following feeding and exposure for an appropriate period of time. The bioassays described herein can be used with any feeding insect pest in the larval or adult stage.

[00209] The following examples are presented by way of example and not as a limitation.

EXPERIMENTAL Example 1 - Production of Cry1B variants with improved spectrum of insecticidal activity

[00210] The insecticidal protein Cry1Bd with an amino acid of SEQ ID NO:1 (US 8,692,065) has high insecticidal activity (ILC50 = 1 ppm) against larvae of the European corn borer (Ostrinia nubilalis), but low insecticidal activity (ILC50 > 1000 ppm and ~ 400 ppm, respectively) against the corn earworm (Helicoverpa zea) and the fall armyworm (Spodoptera frugiperda). The insecticidal protein Cry1B, referred to as MP258 (Serial Number PCT/US14/49923) having an amino acid from SEQ ID NO: 47 has high insecticidal activity (ILC50 = 4 ppm) against larvae of the European corn borer (Ostrinia nubilalis), but lower insecticidal activity (ILC50 24 ppm and 62 ppm, respectively) against the corn earworm (Helicoverpa zea) and the fall armyworm (Spodoptera frugiperda). A series of variant Cry1B polypeptides derived from Cry1Bd (SEQ ID NO: 1) and MP258 were designed to improve insecticidal activity against corn earworm (CEW) and/or fall armyworm (FAW) compared to Cry1Bd (SEQ ID NO: 1) and/or MP258 (SEQ ID NO: 47) while maintaining ECB insecticidal activity. The variant Cry1B polypeptides with improved insecticidal activity that were produced include those listed in Table 1. The insecticidal activity of the Cry1B variants was determined as described in Example 4 and the insecticidal activity results are presented in Table 3. An amino acid sequence alignment of the variant Cry1b polypeptides is shown in Figure 1. Table 1

[00211] The percent amino acid sequence identity of the variant Cry1B polypeptides calculated using the Needleman-Wunsch algorithm as implemented in the Needle program (EMBOSS toolkit) is presented as a matrix table in Table 2a-2b. The empty portion of the matrix table is not shown. Table 2a Table 2b

Example 2 - Saturation mutagenesis at selected positions of variant Cry1B polypeptides from MP258 and IP-1B

[00212] The polynucleotides of SEQ ID NO: 48, SEQ ID NO: 6, SEQ ID NO: 14, and SEQ ID NO: 42 encoding MP258, IP1B-B21, IP1B-B25, and IP1B-B45 (SEQ ID NO: 47, SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 41, respectively) were used as templates for saturation mutagenesis at selected amino acid positions. A reverse mutagenesis primer and a complementary forward mutagenesis primer were designed to create the desired amino acid substitution(s) at the site(s) of interest. Typically, the mutagenesis primer is between 30 to 45 bases in length with two or more bases, usually 10 to 15, on either side of the site of interest. To perform saturation mutagenesis, degenerate primers covering all possible amino acid residues were used. Mutagenesis reactions were performed using the Agilent QuikChange™ Lightening Site-Directed Mutagenesis kit. The materials provided in the kit are QuikChange™ Lightening Enzyme, 10X QuikChange™ Lightning Buffer, dNTP mix, QuikSolution™ reagent, and Don restriction enzyme according to the manufacturer's instructions.

[00213] PCR amplifications were typically performed with the Expand™ High Fidelity PCR system (Roche, Switzerland) in 50 μL containing 50-100 ng of templates, 0.4-2 μM primer pair, 200 μM dNTPs and 2 Units of DNA polymerase. The mutagenesis reaction was initiated by preheating the reaction mixture at 94 °C for 3 min, followed by 16 cycles of the following cycling program: 94 °C for 1 min, 52 °C for 1 min and 68 °C for 8, 12, 16 or 24 min according to the template length. The mutagenesis reaction was completed by incubation at 68 °C for 1 h. PCR amplification products were evaluated by agarose gel electrophoresis. The PCR products were purified by QIAquick™ PCR purification kit (Qiagen, Germany) and subsequently treated with DpnI restriction enzyme. A 1 μL aliquot of the PCR product was typically transformed into BL21 (DE3) cells and inoculated onto Luria-Bertani (LB) plates containing 100 μg/mL ampicillin. Approximately 48 or more colonies were selected for saturation mutagenesis and plasmid DNA was isolated for sequencing. Two-step sequencing was used, first for specific mutation site(s) with a sequencing primer followed by full-length sequence confirmation with multiple sequencing primers. After all 19 amino acid mutations were confirmed by sequencing, these mutant genes were developed for protein expression and purification.

[00214] In the case of mutations made to cover the entire Domain III of IP1B-B25 spanning from T495 to E655, 48 mutant clones were harvested from each site and screened for CEW activity as described in Example 4. In order to sequence these mutant clones to determine mutated amino acids, among 151 amino acid residues subject to mutagenesis, 103 sites were sequenced based on the number of upstream and downstream mutations. Sites containing mutants that did not exhibit significant changes in activity were not sequenced.

Example 3 - Purification of cry1B variant insecticidal proteins

[00215] The cry1B variant insecticidal protein genes were expressed in a modified pMAL vector (Cat# E8000S from New England Biolabs) as a fusion with MBP (maltose-binding protein). The pMAL vector was modified to ligate a 6X His tag to the N-terminus of MBP after methionine at position 1. The plasmid containing the insecticidal protein gene was cloned into E. coli BL21 (DE3). BL21 cells were grown in MagicMedia™ (Life Technologies) in 96-deep-well plates or flasks on a shaker operating at 250 rpm at 37 °C for 8 h followed by 16 °C for 64 h. During incubation at 16 °C, the MBP-toxin fusion protein accumulated in the BL21 cell as a soluble protein.

[00216] To purify the fusion protein, E. coli cells were harvested by centrifugation and treated in a lysozyme solution consisting of 2mg/mL lysozyme in 50 mL of pH 8 sodium phosphate buffer containing 300 mM NaCl, 2U/mL endonuclease (Epicentre) and 5 mM MaCl2 for 3 hours at 37°C with gentle shaking. The lysozyme-treated E. coli cells were then disrupted with 1% Triton X100 and the clear lysate containing the IP-1B proteins was prepared by centrifugation at 4000 rpm, 30 min (96-well plates) or 9000 rpm (vial-produced samples). His-tagged MBP toxin proteins were purified from clear lysate by affinity chromatography using Qiagen™ NiNTA agarose following the manufacturer's standard procedure. For clear lysate samples made in 96-well plates, Pall Corporation™ (25 Harbor Park Drive Port Washington, NI 11050) 96-well deep filter plates were used as affinity chromatography columns. Purified toxin proteins eluted from NiNTA agarose were passed through Sephadex G25 to change the phosphate buffer to 25 mM HEPES-NaOH, pH 8, and used in an insect bioassay to determine insecticidality. MBP was digested with Factor Xa (New England Biolabs) 1/100 (w/w) at 25 °C overnight and removed from IP-1B proteins by Superdex 200 column chromatography using the size difference and weak affinity of MBP for Superdex.

[00217] Protein concentrations were determined by capillary electrophoresis with the LabChip™ GXII device (Caliper LifeSciences). Protein analysis was repeated at least 3 times until final concentrations were considered reliable within the predetermined deviation of less than 10%.

Example 4 - Determination of insecticidal activity of variant IP-1B proteins

[00218] The activity of Cry1B polypeptide variants against major maize pests European corn borer (ECB, Ostrinia nubilalis), corn earworm (ECW, Helicoverpa zea) and fall armyworm (FAW, Spodoptera frugiperda) was determined by a feeding assay described by Cong, R., et al. Proceedings of the 4th Pacific Rim Conferences on Biotechnology of Bacillus thuringiensis and its environmental impact, pp.118- 123, ed. by R. J. Akhurst, C. E. Beard and P. Hughes, published in 2002, Canberra, Australia. Briefly, the assays were conducted on an artificial diet containing the insecticidal proteins. Insecticidal proteins were prepared as described in Example 1, and 10 μL of protein samples were mixed with 40 μL of melted (40–50 °C) artificial insect diet prepared based on Southland Premix formulated for Lepidopteran insects (Southland Products, Lake Village, AR) with low-temperature melting agarose. The insecticidal diet-protein mixture was placed in each well of a 96-well microtiter plate. One or more neonate insect larvae were placed in each well for feeding for 4 days for CEW and FAW and 5 days for ECB at 28 °C.

[00219] Alternatively, insect eggs or larvae were sorted by Large Particle Flow Cytometry using COPAS™ (Complex Object Parametric Analyzer and Sorter) obtained from Union Biometrica (Holliston, MA) to place one egg or larva per well in a 96-well microtiter plate containing solidified artificial insect diet. When eggs were used for placement in the assay plates, only wells containing hatched larvae after 16 hours were used for assay data collection. Hatching rates of 90 to 95% were usually obtained due to the efficient COPAS sorting. After certain feeding periods, insect response to proteins was assessed using a 0-3 numerical scoring system based on larval size and mortality in each well. If no response was observed (or normal growth), a score of 0 was given. When growth was slightly retarded, a score of 1 was given. A score of 2 meant that the larvae were severely stunted in growth (near hatchling size). A score of 3 meant death for all larvae in the well. The percent response (Response) for each treatment was calculated by dividing the total score, a sum of the replicate well scores for each treatment, by the total of the highest possible scores. For example, if a treatment (one sample, one dose) had 6 replicate wells, the maximum possible score would be 3 X 6 = 18.

[00220] In order to identify variant Cry1B polypeptides that have increased levels of activity against those corn pests, significantly higher than the reference activity, such as the wild-type, non-mutated reference protein (e.g., MP258 SEQ ID NO: 47). The variant polypeptides at certain concentrations were assayed together with 4 doses of the reference protein within a 96-well assay plate. The concentrations of the insecticidal proteins were within 4 doses of the reference protein concentrations, preferably around the midpoint of the 4 dose concentrations. Each sample plate contained the reference protein in a significant number of wells such as 16 wells at 4 separate doses. Also on each plate, up to 80 mutant proteins were included for activity comparison to the reference protein. From a sample plate, 10 μL of samples from each well were collected by a multichannel pipette and distributed into an assay plate containing 40 μL of molten diet in each well and mixed on a shaker. This assay plate production process was repeated up to 6 times or more to produce a desired number of assay plates. After solidification of the diet and cooling to 4 °C, neonate insect larvae were placed in each well, sealed with perforated Mylar film and incubated in a constant temperature incubator at 28 °C. After a certain feeding period, the responses of the insects were scored under a magnifying glass. The sigmoid dose response values (Responses) were converted to linear probit dose response values using SAS-JMP®, (Generalized Linear Model, Binomial Response, Probit). The response for each protein in replicates was summed and compared to the probit dose-response line of the reference protein activity, creating a new number called the FAE (Fast Activity Evaluation) guide number. For example, if a mutant protein had a certain probit value at 40 ppm and the actual dose with the same probit value for the reference protein was 100 ppm, then the FAE value is 2.5 (100/40). This means that the mutant protein is 2.5 times more potent than the reference protein. This assay was performed with 2 different doses of mutant proteins each time and repeated 3 times generating 6 FAE guide number data points for each mutant. The average FAE guide number was called the FAE Index. For each protein, a two-sided t-test was performed comparing the 6 FAE guide numbers. The Bonferroni correction was used to evaluate p-values (number of new proteins/alpha) to determine if the FAE Index was statistically significant.

[00221] The other screening method used in this patent application is the High Dose Assay (HDA). In this method, test proteins at high concentrations (above EC50) were spotted onto insect assay plates as described above, along with a similar concentration of one or more reference proteins with a known level of activity. This HDA was often used in a tiered screen to quickly eliminate proteins with low or no activity.

[00222] Yet another screening method used was the High Throughput Functional Assay (HFA). This assay was similar to FAE but used only one dose instead of 2 doses. Otherwise, HFA, especially the way it calculates the index was identical to FAE. Thus, the HFA index has the same meaning as the FAE index.

[00223] The predicted point of 50% response in the scoring scheme is called the ILC50, since it is a combination of growth or feeding inhibition and lethal responses. To determine the ILC50 values, each treatment (one dose) was repeated 6 or more, typically 24, times. The insecticidal activity of the Cry1B variants is presented in Table 3.

[00224] Table 4 shows the insecticidal activity against corn earworm for the amino acid substitutions with increased activity (a FAE score > 1.2) compared to the reference polypeptide MP258 (SEQ ID NO: 47), IP1B-B21 (SEQ ID NO: 5), IP1B-B25 (SEQ ID NO: 13), or IP1B-B45 (SEQ ID NO: 41). Table 4 indicates the position and amino acid number corresponding to positions 50651 of MP258 (SEQ ID NO: 47); the predicted secondary structure and designation; solvent exposure classification; an amino acid sequence alignment of MP258 (SEQ ID NO: 47); IP1B-B21 (SEQ ID NO: 5), IP1B-B25 (SEQ ID NO: 13), IP1B-B45 (SEQ ID NO: 41), IP1B-B21 (SEQ ID NO: 5), Cry1Bd (SEQ ID NO: 1), Cry1Bh (SEQ ID NO: 52), and Cry1Bi (SEQ ID NO: 54); the polypeptide backbone in which the variant was made; the amino acid substitution variant (e.g., L50R); and the FAE insecticide classification against corn earworm compared to the corresponding polypeptide backbone (MP258 - SEQ ID NO: 47, IP1B-B21 - SEQ ID NO: 5, IP1B-B25 - SEQ ID NO: 13, or IP1B-B45 - SEQ ID NO: 41). Table 3

[00225] Table 5 shows the insecticidal activity against corn earworm for amino acid substitutions with a FAE score < 1.2 compared to the polypeptide backbone MP258 (SEQ ID NO: 47), IP1B-B21 (SEQ ID NO: 5), IP1B-B25 (SEQ ID NO: 13), or IP1B-B45 (SEQ ID NO: 41). Table 5 indicates the position and amino acid number corresponding to positions 50-651 of MP258 (SEQ ID NO: 47); the polypeptide backbone in which the variant was made; the amino acid substitution variant (e.g., L50R); and the insecticidal classification of FAE against corn earworm compared to the corresponding polypeptide backbone (MP258—SEQ ID NO: 47, IP1B-B21—SEQ ID NO: 5, IP1B-B25—SEQ ID NO: 13, or IP1B-B45—SEQ ID NO: 41.Table 4 Table 5

Example 5 - Transient expression in corn leaves and insect bioassay

[00226] Polynucleotides encoding variant Cry1B polypeptides have been cloned into transient expression vectors under the control of the maize ubiquitin promoter (Christensen and Quail, (1996) Transgenic Research 5:213-218) and a duplicated version of the mirabilis mosaic virus promoter (DMMV PRO; Dey and Maiti, (1999) Plant Mol. Biol., 40:771-82). The agroinfiltration method of introducing a cell suspension of Agrobacterium into intact plant tissue cells so that reproducible infection and subsequent expression of the plant-derived transgene can be measured or studied is well known in the art (Kapila, et. al., (1997) Plant Science 122:101-108). Briefly, young maize seedlings were agroinfiltrated with standardized bacterial cell cultures of the test and control strains. Leaf discs were generated from each seedling and infested with WCRW (Diabrotica virgifera) along with appropriate controls. The degree of consumption of green leaf tissues was scored after 2 days of infestation.

Example 6 - Transient expression in bush bean leaves and insect bioassay

[00227] For expression in soybean, optimized coding sequences can be designed. The agroinfiltration method of introducing a cell suspension of Agrobacterium into intact plant tissue cells so that reproducible infection and subsequent expression of the plant-derived transgene can be measured or studied is well known in the art (Kapila, et. al., (1997) Plant Science 122:101-108). Briefly, leaf discs removed from bush beans are agroinfiltrated with standardized bacterial cell cultures of test and control strains. After 4 days, leaf discs are infested with 2 neonates of either false looperworm (SBL) (Chrysodeixis includens), corn earworm (CEW) (Helicoverpa zea), soybean armyworm (VBC) (Anticarsia gemmatalis), or fall armyworm (Spodoptera frugiperda) only. Control leaf discs are generated with Agrobacterium containing only a DsRed2 fluorescence marker expression vector (Clontech™, 1290 Terra Bella Ave. Mountain View, CA 94043). Leaf discs from noninfiltrated plants are included as a second control. Green leaf tissue consumption is scored three days after infestation and given a score from 0 to 9.

Example 7 - Agrobacterium-mediated transformation of corn and regeneration of transgenic plants

[00228] For Agrobacterium-mediated transformation of corn with a polynucleotide sequence of the disclosure, the method of Zhao (U.S. Patent Number 5,981,840 and PCT Patent Publication WO98/32326; the contents of which are incorporated herein by reference) may be used. Briefly, immature embryos are isolated from corn and the embryos contacted with a suspension of Agrobacterium under conditions where the bacteria are capable of transferring the toxin nucleotide sequence to at least one cell of at least one of the immature embryos (step 1: the infection step). At this step the immature embryos may be immersed in a suspension of Agrobacterium to initiate inoculation. The embryos are co-cultured for a time with Agrobacterium (step 2: the co-cultivation step). Immature embryos can be cultured on solid media after the infection stage. After this period of co-cultivation, an optional "resting" stage is provided. In this resting stage, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (stage 3: resting). Immature embryos can be cultured on solid media with antibiotics but without a selection agent to eliminate Agrobacterium and to provide a resting phase for infected cells. The inoculated embryos are then cultured on medium containing a selective agent and the growing transformed callus is recovered (stage 4: the selection stage). Immature embryos are cultured on solid media with a selective agent that results in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step) and calli grown on selective media can be cultured on solid media to regenerate plants.

Example 8 - Transformation of Soybean Embryos

[00229] Soybean embryos are bombarded with a plasmid containing the toxin nucleotide sequence operably linked to a suitable promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length, dissected from surface-sterilized, immature seeds of a soybean cultivar are grown in light or dark at 26°C on an appropriate agar medium for six to ten weeks. Somatic embryos that produce secondary embryos are then removed and placed in a suitable liquid medium. After repeated selection of somatic embryo clusters that have multiplied as early globular stage embryos, the suspensions are maintained as described below.

[00230] Soybean embryogenic suspension cultures can be maintained in 35 mL of liquid medium on a rotary shaker, 150 rpm, at 26 °C with fluorescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[00231] Embryogenic soybean suspension cultures can then be transformed by the particle gun bombardment method (Klein, et al., (1987) Nature (London) 327:70-73, U.S. Patent No. 4,945,050). A Du Pont Biolistic PDS1000/HE (helium retrofit) instrument can be used for these transformations.

[00232] A selectable marker gene that can be used to facilitate the transformation of soybean includes, but is 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 E. coli, Gritz et al. Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising a toxin nucleotide sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 3, or an optimized sequence from corn) operably linked to a suitable promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector containing the marker gene.

[00233] To 50 μL of a 1 μm gold particle suspension at 60mg/mL is added (in order): 5 μL of DNA (1 μg/μL), 20 μL of spermidine (0.1 M), and 50 μL of CaCl2 (2.5 M). The particle preparation is then shaken for three minutes, centrifuged in a microcentrifuge for 10 seconds, and the supernatant removed. The DNA-coated particles are then washed once in 400 μL of 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded onto each macro support disk.

[00234] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60 x 15 mm Petri dish and residual liquid is removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 tissue plates are typically bombarded. The membrane burst pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches (71.12 cm) of mercury. The tissue is placed approximately 3.5 inches (8.89 cm) away from the retention screen and bombarded three times. After bombardment, the tissue can be split in half and placed back in liquid and cultured as described above.

[00235] Five to seven days after bombardment, the liquid medium can be exchanged with fresh medium and eleven to twelve days after bombardment with fresh medium containing 50 mg/mL hygromycin. This selective medium can be renewed weekly. Seven to eight weeks after bombardment, transformed, green tissue can be observed growing from untransformed necrotic embryogenic clumps. The isolated green tissue is removed and inoculated into individual flasks to generate new, clonally 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 embryo aggregates or regenerated into whole plants by maturation and germination of individual somatic embryos.

[00236] All publications, patents and patent applications mentioned in the specification are indicative of the level of skill in the art to which this disclosure pertains. All publications, patents and patent applications are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

[00237] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the embodiments.

Claims (25)

1. A variant Cry1B polypeptide comprising an amino acid sequence having the amino acid sequence as set forth by SEQ ID NO: 29. 2. The variant Cry1B polypeptide of claim 1, having increased insecticidal activity against fall armyworm, wherein the insecticidal activity is in comparison to the polypeptide of SEQ ID NO 47. 3. The variant Cry1B polypeptide of claim 2, wherein the increased insecticidal activity is quantified as an Average FAE Index. 4. Recombinant polynucleotide characterized by the fact that the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 30 and degenerate sequences thereof that encode a variant Cry1B polypeptide of SEQ ID NO:29. 5. Recombinant polynucleotide according to claim 4, characterized in that the nucleic acid sequence has been optimized for expression in a plant. 6. The recombinant polynucleotide of claim 5, wherein the nucleic acid sequence is a synthetic nucleotide sequence having plant-preferred codons that have been designed for expression in a plant. 7. Recombinant polynucleotide according to claim 6, characterized in that the nucleic acid sequence has been optimized for expression in corn or soybeans. 8. DNA construct characterized by comprising a polynucleotide, as defined by any one of claims 4 to 7, operably linked to a heterologous regulatory element. 9. A host cell comprising the DNA construct as defined in claim 8, wherein the host cell is a bacterial cell. 10. A method for producing a transgenic host cell comprising: (a) transforming a host cell with the DNA construct as defined in claim 8; (b) culturing said transformed host cell under host cell growth conditions. 11. The method of claim 10, wherein said transgenic host cell is a plant cell. 12. The method of claim 11, wherein said transgenic host cell is a corn or soybean cell. 13. A method for obtaining a transgenic plant, wherein said method comprises: (a) transforming a plant cell with the DNA construct as defined by claim 8; (b) cultivating said transformed plant cell under plant cell growth conditions; and (c) regenerating a transgenic plant from said transformed plant cell. 14. Method according to claim 13, characterized in that said transgenic plant is selected from the group consisting of corn, sorghum, wheat, cabbage, sunflower, tomato, a species of cruciferous plants, a species of pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley and oilseed rape. 15. Composition characterized by comprising the variant Cry1B polypeptide, as defined by claim 1. 16. Composition according to claim 15, characterized in that the composition comprises from 1% to 99% by weight of the variant Cry1B polypeptide. 17. A method for controlling a population of Lepidopteran pests on a plant comprising contacting said population with a plant having a pesticidally effective amount of the variant Cry1B polypeptide as defined by claim 1, or with a plant to which a pesticidally effective amount of the variant Cry1B polypeptide as defined by claim 1 has been directly applied. 18. A method for killing a Lepidopteran pest on a plant comprising contacting said pest with, or feeding said pest on, a plant having a pesticidally effective amount of the variant Cry1B polypeptide as defined by claim 1, or with a plant to which a pesticidally effective amount of the variant Cry1B polypeptide as defined by claim 1 has been directly applied. 19. A method for producing a polypeptide with pesticidal activity comprising culturing the host cell as defined by claim 9 under conditions in which the polynucleotide encoding the polypeptide is expressed in a plant. 20. Method of obtaining a transgenic plant characterized by the fact that said method comprises: (a) transforming a plant cell by stably incorporating into its genome the DNA construct, as defined by claim 8; (b) cultivating said transformed plant cell under plant cell growth conditions; and (c) regenerating a transgenic plant from said transformed plant cell. 21. Method of obtaining a transgenic plant cell characterized by the fact that said method comprises: (a) transforming a plant cell by stably incorporating into its genome the DNA construct, as defined by claim 8; (b) culturing said transformed plant cell under plant cell growth conditions. 22. A method of protecting a plant from an insect pest, comprising introducing into said plant the DNA construct as defined by claim 8. 23. The method of claim 22, wherein the variant Cry1B polypeptide has increased insecticidal activity against corn earworm and/or fall armyworm compared to the polypeptide of SEQ ID NO: 47. 24. The method of claim 22 or 23, wherein the insect pest is resistant to a non-Cry1B insecticidal polypeptide. 25. The method of claim 22, 23 or 24, wherein the insect pest is corn earworm, fall armyworm or European corn borer.