EA042693B1 - AXMI477, AXMI482, AXMI486 AND AXMI525 TOXIN GENES AND METHODS OF THEIR APPLICATION - Google Patents

Description

Link to related applications

This application claims priority of U.S. Provisional Application Ser. No. 61/913905, filed December 9, 2013, and U.S. Provisional Application Ser. No. 61/913911, filed December 9, 2013, the contents of which are incorporated herein by reference in their entirety .

Link to the electronically filed sequence listing

The official copy of the sequence listing is electronically submitted via EFS-Web as an ASCII sequence listing, with a 97 kilobyte file filed on November 14, 2014 under the name APA136054_ST25.txt at the same time as the specification. The sequence listing in this document in ASCII format is part of the specification and is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to the field of molecular biology. Provided herein are novel genes that encode pesticidal proteins. These proteins and their coding nucleotide sequences are used to prepare pesticide formulations and to obtain transgenic plants resistant to agricultural pests.

Background of the invention

Bacillus thuringiensis is a Gram-positive, spore-forming soil bacterium that has the ability to produce crystalline inclusions that exhibit specific toxicity to certain orders and insect species, but are harmless to plants and other non-target organisms. In this regard, compositions containing strains of Bacillus thuringiensis or their insecticidal proteins can be used as environmentally acceptable insecticides for the control of agricultural insect pests or insect vectors for the treatment of various human and animal diseases.

Crystalline (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against insect larvae, predominantly belonging to the orders Lepidoptera, Hemiptera, Diptera and Coleoptera. These proteins have also shown activity against the pest orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensis family tree. In Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, N. Y.) These proteins were originally classified as CryI-CryV, primarily on the basis of their insecticidal activity. The main classes were Lepidoptera-specific (I), Lepidoptera-specific and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and nematode-specific (V) and (VI). Proteins were further classified into subfamilies; the more closely related proteins within each family were assigned subgroup letters Cry1A, Cry1B, Cry1C, and so on. Even more closely related proteins within each subgroup were designated as Cry1C1, Cry1C2, and so on.

The nomenclature for the Cry genes has been described based on amino acid sequence homology rather than target insect specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). This classification assigns a unique name to each toxin, including a primary category (Arabic numeral), a secondary category (capital letter), a tertiary category (lower case letter), and a quaternary category (second Arabic numeral). Roman numerals have been replaced by Arabic numerals in the primary category. Proteins with less than 45% sequence identity have different primary categories, and the criteria for secondary and tertiary categories are 78% and 95% identity, respectively.

The crystalline protein does not exhibit insecticidal activity until it is ingested by the insect and dissolved in its midgut. The ingested protoxin undergoes hydrolysis by proteases in the digestive tract of insects to form active toxic molecules. (Hofte and Whiteley (1989) Microbiol. Rev. 53:242-255). This toxin binds to apical brush border receptors in the midgut of target larvae and inserts into the apical membrane, creating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain consists of seven alpha helices and is involved in membrane penetration and pore formation. Domain II consists of three pleated beta sheets that are ordered in a Greek key structure, while domain III consists of two anti-parallel pleated beta sheets in a jelly-roll structure (de Maagd et al., 2001, supra). Domains II and III are involved in the recognition and binding of receptors and are therefore considered de-expressionants of toxin specificity.

Due to the damage that insects can cause and the improvement in crop yields through pest control, there is a constant need to discover new forms of pesticide toxins.

Brief description of the invention

Compositions and methods are provided for imparting pesticidal activity to bacteria, plants,

- 1 042693 cells, tissues and seeds of plants. The compositions include nucleic acid molecules encoding pesticidal and insecticidal polypeptide sequences, vectors containing these nucleic acid molecules, and host cells containing such vectors. The compositions also contain pesticidal polypeptide sequences and antibodies to these polypeptides. Nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Nucleotide or amino acid sequences can be synthetic sequences that have been engineered for expression in an organism, including, but not limited to, a microorganism or plant. The compositions also include bacteria, plants, cells, tissues and plant seeds containing the nucleotide sequence of the present invention.

In particular, isolated or recombinant nucleic acid molecules are provided that encode a pesticidal protein. In addition, amino acid sequences corresponding to the pesticidal protein are included. In particular, the present invention provides an isolated or recombinant nucleic acid molecule that contains a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5-26, or the nucleotide sequence set forth in SEQ ID NO: 1-4, as well as their biologically -active options and fragments. Nucleotide sequences that are complementary to the nucleotide sequence of the present invention, or that hybridize to the sequence of the present invention or its complementary strand, are also covered. Additionally provided are vectors, host cells, plants, and seeds containing the nucleotide sequences of the present invention, or nucleotide sequences encoding the amino acid sequences of the present invention, as well as biologically active variants and fragments thereof.

Methods are provided for preparing the polypeptides of the present invention and using these polypeptides to control or kill insects belonging to the orders Lepidoptera, Hemiptera, Coleoptera, Nematodes, or Diptera. Also included are methods and kits for detecting nucleic acids and polypeptides of the present invention in a sample.

The compositions and methods of the present invention are suitable for producing organisms with increased or resistance to pests. These organisms and compositions containing the organisms are suitable for agricultural purposes. The compositions of the present invention are also useful for creating altered or improved proteins that have pesticidal activity, or for detecting the presence of pesticidal proteins or nucleic acids in products or organisms.

Detailed description

The present invention relates to compositions and methods for controlling resistance or resistance to pests in organisms, in particular plants or plant cells. By resistance is meant that the pest (eg, insect) dies after ingestion or other contact with the polypeptides of the present invention. By tolerance is meant a violation or inhibition of the functions of movement, nutrition, reproduction or other functions of the organism of the pest. The methods include transformation of organisms using a nucleotide sequence encoding a pesticidal protein of the present invention. In particular, the nucleotide sequences of the present invention are used to obtain plants and microorganisms that have pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions are pesticidal nucleic acids and proteins from Bacillus or other species. The sequences find use in constructing expression vectors for subsequent transformation into organisms of interest, as probes for isolating other homologous (or partially homologous) genes, and for generating altered pesticidal proteins using methods known in the art such as domain block shuffling. or DNA shuffling, for example using members of the Cry1, Cry2 and Cry9 endotoxin families. The proteins find use in controlling or inducing the death of populations of pests from the orders Lepidoptera, Hemiptera, Coleoptera, Diptera, and Nematodes and in the preparation of compositions with pesticidal activity.

By the term pesticidal toxin or pesticidal protein is meant a toxin that has toxic activity against one or more pests, including, but not limited to, members of the orders Lepidoptera, Diptera, Hemiptera, and Coleoptera, or the Nematoda phylum, or a protein that is homologous to such a protein. Pesticide proteins have been isolated from organisms which include, for example, Bacillus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticide proteins contain amino acid sequences derived from the full length nucleotide sequences disclosed herein and amino acid sequences that are shorter than the full length sequences, either through the use of an alternative downstream translation start site or through processing that results in a shorter protein. , characterized by pesticidal activity. Processing can take place in the organism in which the protein is expressed, or in the organism of the pest after ingestion of the protein.

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Thus, novel isolated or recombinant nucleotide sequences are provided herein that provide pesticidal activity. These nucleotide sequences encode polypeptides with homology to known delta-endotoxins or binary toxins. Also provided herein are the amino acid sequences of the pesticidal proteins. The protein synthesized as a result of the translation of this gene allows cells to control or cause the death of pests when they enter their digestive tract.

Isolated nucleic acid molecules and their variants and fragments

One aspect of the present invention relates to isolated or recombinant nucleic acid molecules that contain nucleotide sequences encoding pesticidal proteins and polypeptides or their biologically active parts, as well as nucleic acid molecules suitable for use as hybridization probes to identify nucleic acid molecules that encode proteins with regions of sequence homology. Also encompassed herein are nucleotide sequences capable of hybridizing to the nucleotide sequences of the present invention under stringent conditions, as defined elsewhere in this document. As used herein, the expression nucleic acid molecule includes DNA molecules (eg, recombinant DNA, cDNA, or genomic DNA) and RNA molecules (eg, mRNA) and DNA or RNA analogs created using nucleotide analogs. The nucleic acid molecule may be single or double stranded, but is preferably double stranded DNA.

The expression isolated or recombinant nucleotide sequence (or DNA) is used herein to refer to a nucleotide sequence (or DNA) that is no longer in its natural environment, such as in an in vitro system or in a recombinant bacterial or plant host cell. In some embodiments, the isolated or recombinant nucleic acid does not contain sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (i.e., sequences that are located at the 5' and 3' ends of the nucleic acid) in the organism's genomic DNA from which the nucleic acid is derived. In the context of the present invention, the expression isolated, when used in relation to nucleic acid molecules, excludes isolated chromosomes. For example, in various embodiments, an isolated delta-endotoxin encoding a nucleic acid molecule may comprise nucleotide sequences less than about 5 kb, 4 kb, 3 kb .o., 2 kb, 1 kb, 0.5 kb or 0.1 kb, which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid was derived. In various embodiments, a delta-endotoxin protein that is substantially free of cellular material includes protein formulations containing less than about 30%, 20%, 10%, or 5% (by dry weight) of delta-endotoxin protein (also referred to herein as contaminant protein) .

Nucleotide sequences encoding the proteins of the present invention include the sequence set forth in SEQ ID NO: 1-4, and its variants, fragments and complementary chains. By complementary is meant a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to a given nucleotide sequence to form a stable duplex. The corresponding amino acid sequences of the pesticide proteins that are encoded by these nucleotide sequences are set forth in SEQ ID NOs: 5-26.

Nucleic acid molecules that are fragments of these nucleotide sequences encoding pesticidal proteins are also included in the present invention. By fragment is meant a portion of the nucleotide sequence encoding a pesticidal protein. A fragment of the nucleotide sequence may encode a biologically active part of the pesticidal protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods described below. Nucleic acid molecules, which are fragments of a nucleotide sequence encoding a pesticidal protein, contain at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 contiguous nucleotides, or a number of nucleotides up to the number of nucleotides present in the full length nucleotide sequence encoding the pesticidal protein disclosed herein, depending on the intended use. By adjacent nucleotides is meant nucleotide residues that are directly adjacent to each other. Fragments of the nucleotide sequences of the present invention will encode protein fragments that retain the biological activity of the pesticidal protein and therefore retain the pesticidal activity. Thus, biologically active fragments of the polypeptides disclosed herein are also included in the present invention. By retaining activity is meant that the fragment will have at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or more of the pesticidal activity of the pesticidal protein. In one embodiment, the pesticidal activity is

- 3 042693 activity relative to Coleoptera. In another embodiment, the pesticidal activity is activity against Lepidoptera. In another embodiment, the pesticidal activity is activity against nematodes. In another embodiment, the pesticidal activity is activity against Diptera. In another embodiment, the pesticidal activity is activity against hemipterans. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and US Pat. No. 5,743,477, all of which are incorporated herein in their entirety by reference.

A fragment of the nucleotide sequence encoding a pesticidal protein that encodes the biologically active portion of the protein of this invention will encode at least about , 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 contiguous amino acids or up to the total number of amino acids present in the full length protein of the pesticidal protein of the present invention. In some embodiments, the implementation of the fragment is a fragment resulting from proteolytic cleavage. For example, a fragment resulting from proteolytic cleavage may have an N-terminal or C-terminal truncation of at least about 30 amino acids, at least about 40 amino acids, at least about 50, at least about 100 amino acids, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 350, about 400, about 450, about 500, or about 550 amino acids relative to SEQ ID NO: 2, 3, 4, 5, 6, or 7. In some embodiments, the fragments that are included herein are formed by removing the C-terminal crystallization domain, for example , by proteolysis, or by inserting a stop codon into the coding sequence. In some embodiments, the implementation of the fragments that are included in this document, formed by removing the N-terminal signal peptide. N-terminal truncations may further include a methionine residue at the N-terminus.

Preferred pesticidal proteins of the present invention are encoded by a nucleotide sequence that is sufficiently identical to the nucleotide sequence of SEQ ID NO: 1-4, or the pesticidal proteins are sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 5-26. By sufficiently identical is meant an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99% or higher sequence identity compared to the reference sequence as shown using one of the alignment programs described herein using standard parameters. One skilled in the art will appreciate that these values can be adjusted accordingly to determine the respective identity of the proteins that are encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). In one embodiment, the two sequences are the same length. In another embodiment, percent identity is calculated over the entire length of the reference sequence (ie, the sequence disclosed herein as any of SEQ ID NOs: 1-26). Percent identity between two sequences can be determined using techniques similar to those described below, allowing gaps in the sequence or not. When calculating percent identity, the number of exact matches is usually counted. Pass, i.e. an alignment position in which a residue is present in one sequence but not in the other is considered a position with non-identical residues.

Determining the percent identity between two sequences can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm given in Karlin and Altschul (1990) Proc. Natl. Acad. sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. sci. USA 90:5873-5877. Such an algorithm is built into the BLASTN and BLASTX programs by Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the pesticide-like nucleic acid molecules of the present invention. Searches for BLAST proteins can be carried out using the program

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BLASTX, score=50, wordlength=3, to obtain amino acid sequences homologous to the pesticidal protein molecules of the present invention. To obtain gapped alignments for comparison purposes, the Gapped BLAST program (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic acids Res. 25:3389. Alternatively, the PSI-Blast program can be used to perform a re-searching that finds distant bonds between molecules. See Altschul et al. (1997) supra. When working with the BLAST, Gapped BLAST and PSIBlast programs, the default parameters set for the respective programs (eg BLASTX and BLASTN) can be applied. Alignment can also be done manually by reconciliation.

Another non-limiting example of a mathematical algorithm used for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entire length of an amino acid or DNA sequence and thus can provide conservatism data for the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module included in the Vector NTI software package (Invitrogen Corporation, Carlsbad, CA). After aligning amino acid sequences using the ClustalW program, the percent amino acid identity can be estimated. A non-limiting example of computer programs that are used to analyze ClustalW alignments include GENEDOC™. GENEDOC™ (Karl Nicholas) evaluates the amino acid (or DNA) similarity and identity of numerous proteins. Another non-limiting example of a mathematical algorithm used for sequence comparison is Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is integrated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics, Version 10 software package (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When working with the ALIGN program, a PAM120 residue substitution table, a gap continuation penalty of 12, and a gap opening penalty of 4 can be used to compare amino acid sequences.

Unless otherwise noted, GAP Version 10, which uses the algorithm described in Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453 will be used to determine sequence identity or similarity using the following parameters: % identity and % similarity for the nucleotide sequence using a gap opening penalty of 50 and a continuation penalty of 3 and a matrix alignment weights nwsgapdna.cmp; % identity or % similarity for an amino acid sequence using a gap opening penalty of 8 and a continuation penalty of 2 and the BLOSUM62 scoring program. Equivalent programs may also be used. By equivalent program is meant any sequence comparison program that, for any two compared sequences, produces an alignment that has identical nucleotide residue matches and an identical percent sequence identity compared to the corresponding alignment obtained using GAP Version 10.

The present invention also encompasses variants of nucleic acid molecules. Nucleotide sequence variants encoding pesticidal proteins include those sequences that encode the pesticidal proteins disclosed herein but that differ conservatively due to the degeneracy of the genetic code, as well as those sequences that are sufficiently identical, as discussed above. Naturally occurring allelic variants can be identified using well known molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques, which are described below. Nucleotide sequence variants also include synthetically generated nucleotide sequences that have been generated, for example, using site-directed mutagenesis a, but which still encode the pesticidal proteins disclosed herein, as discussed below. The protein variants that are included in the present invention are biologically active, which means that they continue to exhibit the desired biological activity of the native protein, ie pesticidal activity. By retaining activity is meant that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 24802485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and US Patent No. 5,743,477, all of which are incorporated herein in their entirety by reference.

The skilled artisan will also appreciate that changes can be made by mutating the nucleotide sequences of the present invention, thereby leading to changes in the amino acid sequence encoding the pesticidal proteins without altering the biological activity of the proteins. Therefore, variants of isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, insertions, or deletions at the appropriate

- 5 042693 nucleotide sequence disclosed in this document, so that one or more substitutions, insertions or deletions of amino acids were introduced into the encoded protein. Mutations can be caused using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variants of the nucleotide sequences are also included in the present invention.

For example, conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. A non-essential amino acid residue is a residue that can be changed from a wild-type pesticidal protein sequence without changing biological activity, while an essential amino acid residue is required for biological activity to occur. A conservative amino acid substitution is one where an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine).

Delta-endotoxins typically have five conserved sequence domains and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain contains seven alpha helices and is associated with membrane incorporation and pore formation.

Domain II consists of three pleated beta sheets that are ordered in a Greek key structure, while domain III consists of two anti-parallel pleated beta sheets in a jelly-roll structure (de Maagd et al, 2001, supra). Domains II and III are involved in receptor recognition and binding and are therefore considered to be determinants of toxin specificity.

Amino acid substitutions can be made at non-conserved sites that retain function. In general, such substitutions are not made for conserved amino acid residues, or for amino acid residues within a conservative motif where such residues are essential to the activity of the protein. Examples of residues that are conserved and that may be indispensable for the activity of a protein include, for example, residues that are identical for all proteins included in the sequence alignment of similar or related toxins with respect to the sequence of the present invention (for example, residues that are identical when aligning homologous proteins). Examples of residues that are conservative, but that can allow conservative amino acid substitutions and still retain activity, include, for example, residues that have only conservative substitutions for all proteins included in the sequence alignment of similar or related toxins relative to the sequence of the present invention ( for example, residues that have only conservative substitutions for all homologous proteins included in the alignment). However, one of skill in the art will appreciate that functional variants may have minor conservative or non-conservative differences in conservative residues.

Alternatively, variants of the nucleotide sequences can be generated by randomly inducing mutations over all or part of the coding sequence, such as using saturation mutagenesis, and the resulting mutants can be screened for their ability to confer pesticidal activity to detect mutants. that remain active. After induction of mutagenesis, the encoded protein can be expressed recombinantly, and the activity of this protein can be determined using standard assay techniques.

Using methods such as PCR, hybridization, and the like. suitable pesticidal sequences can be identified, such sequences that have significant identity to the sequence of the present invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).

When using the hybridization method, all or part of the pesticidal nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as 32P or any other detectable marker such as other radioactive isotopes, a fluorescent compound, an enzyme or enzyme cofactor. Hybridization probes can be generated by labeling synthetic oligonucleotides based on the known nucleotide sequence encoding the pesticidal protein disclosed herein. Additionally, degenerate primers designed based on conservative nucleotides or amino acid residues in the nucleotide sequence or encoded amino acid sequence can be used. The probe typically contains a region of a nucleotide sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of the nucleotide sequence encoding the pesticidal protein. of the present invention, or a fragment or variant thereof. Methods for making probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra, incorporated herein by reference.

For example, the entire pesticidal sequence disclosed herein, or one or more parts thereof, can be used as a probe capable of specific hybridization with corresponding pesticidal protein-like sequences and messenger RNA molecules. To achieve specific hybridization under various conditions, such probes contain sequences that are unique and preferably are at least about 10 nucleotides long, or at least about 20 nucleotides long. Such probes can be used to amplify appropriate pesticide sequences from a selected 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 a coding sequence in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (as plaques or colonies; see e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

Thus, the present invention includes hybridization probes as well as nucleotide sequences capable of hybridizing all or part of the nucleotide sequence of the present invention (e.g., at least about 300 nucleotides in length, at least about 400, at least about 500, 1000, 1200 , 1500, 2000, 2500, 3000, 3500 or up to the full length of the nucleotide sequence disclosed herein). Hybridization of such sequences can be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is meant conditions under which a probe will hybridize to its target sequence to a higher detectable extent than to other sequences (eg, at least 2 times background). Strict conditions are sequence dependent and will vary under different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, the stringency conditions can be adjusted to allow mismatches to occur in the sequence so that lower degrees of similarity can be detected (heterologous probing). In general, the probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ions, typically from about 0.01 to 1.0 M Na ions (or other salts) at a pH of 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (eg, 10 to 50 nucleotides) and at least about 60° C. for long probes (eg, more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution containing 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and washing in SSC from 1X to 2X (20X SSC = 3.0M NaCl/ 0.3 M trisodium citrate) at 50-55°C. Exemplary medium stringency conditions include hybridization with a solution containing 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37°C, and washing in SSC from 0.5X to 1X SSC at 55-60°C. Typical high stringency conditions include hybridization with a solution containing 50% formamide, 1M NaCl, 1% SDS at 37°C, and washing in 0.1X SSC at 60-65°C. Optionally, wash buffers may include from about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, typically from about 4 to about 12 hours.

Specificity generally depends on washes after hybridization, in which the most important factors are the ionic strength and temperature of the final wash solution. For DNA/DNA hybrids, the Tm value can be approximated from the Meinkoth and Wahl (1984) Anal equation. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine-containing and cytosine-containing nucleotides in DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in a base pair. Tm is the temperature (at a given ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm decreases by about 1°C for every 1% mismatch; so

- 7 042693 Thus, Tm, hybridization and/or washing conditions can be adjusted to hybridize with sequences with the desired degree of identity. For example, when searching for sequences with >90% identity, Tm can be reduced by 10°C. In general, stringent conditions are chosen such that the temperature is about 5° C. below the melting point (Tm) for a particular sequence and its complementary strand at given ionic strength and pH. However, very stringent conditions may use hybridization and/or washing at a temperature of 1, 2, 3 or 4°C below the melting point (Tm); conditions of medium stringency can use hybridization and/or washing at a temperature of 6, 7, 8, 9 or 10°C below the melting point (Tm); low stringency conditions may use hybridization and/or washing at 11, 12, 13, 14, 15, or 20° C. below the melting point (Tm). Using the equation, the hybridization and wash compositions, and the required Tm, one of ordinary skill in the art will appreciate that variations in the degree of stringency of hybridization conditions and/or washes are essentially described. If the desired degree of mismatch results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), then increasing the concentration is preferable so that a higher temperature can be used. Detailed guidance on nucleic acid hybridization is provided in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

Isolated proteins and their variants and fragments

Pesticide proteins are also covered in the present invention. By pesticidal protein is meant a protein having the amino acid sequence set forth in SEQ ID NOs: 5-26. Fragments, biologically active parts and variants thereof are also provided and can be used to practice the methods of the present invention. The term isolated protein or recombinant protein is used to refer to a protein that is no longer in its natural environment, but is, for example, in an in vitro system or in a recombinant bacterial or plant host cell.

Fragments or biologically active parts include fragments of a polypeptide that contains amino acid sequences that are sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 5-26, and exhibit pesticidal activity. The biologically active portion of the pesticidal protein may be a polypeptide of length, e.g. 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids. Such biologically active parts can be obtained using recombinant techniques and evaluated for pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and US Pat. No. 5,743,477, all of which are incorporated herein in their entirety by reference. As used herein, the fragment contains at least 8 contiguous amino acids from SEQ ID NO: 5-26. However, the present invention encompasses other fragments, such as any fragment in a protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids.

Variants are understood to mean proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90, 91, 92, 93, 94 , 95, 96, 97, 98 or 99% identical to the amino acid sequence of any of the sequences of SEQ ID NO: 5-26. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO: 1-4 or its complementary strand under stringent conditions. Variants include polypeptides that differ in amino acid sequences as a result of mutagenesis. The protein variants that are encompassed by the present invention are biologically active, that is, they continue to retain the desired biological activity of the native protein, that is, they retain pesticidal activity. In some embodiments, the variants have improved activity relative to the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and US Pat. No. 5,743,477, all of which are incorporated herein in their entirety by reference.

Bacterial genes, such as the axmi genes of the present invention, quite often have several methionine start codons located close to the start of the open reading frame. Often, translation initiation at one or more of these start codons will result in the formation of a functional protein. These start codons may include ATG codons. However, bacteria such as Bacillus sp. also recognize the GTG codon as a start codon,

- 8 042693 and proteins that initiate translation at GTG codons contain methionine as the first amino acid. In rare cases, translation in bacterial systems can start at the TTG codon, although in this case TTG codes for methionine. In addition, it is often not initially determined which of these codons are used naturally in bacteria. Thus, it is understood that the use of one of the alternative methionine codons may also lead to the creation of pesticidal proteins. These pesticidal proteins are encompassed by the present invention and can be used in the methods of the present invention. It will be understood that when expressed in plants, it will be necessary to change the alternative start codon to ATG in order to properly translate.

In various embodiments of the present invention, the pesticidal proteins comprise sequences derived from the full length nucleotide sequences disclosed herein and amino acid sequences that are shorter than the full length sequences by using a downstream alternative translation start site. Thus, the nucleotide sequence of the present invention and/or vectors, host cells and plants containing the nucleotide sequence of the present invention (and methods of obtaining and using the nucleotide sequence of the present invention) may contain a nucleotide sequence that encodes an amino acid sequence corresponding to SEQ ID NO: 6 , 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 25 and 26.

Also covered are antibodies to the polypeptides of the present invention, or variants or fragments thereof. Methods for making antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; US Pat. No. 4,196,265).

Thus, one aspect of the present invention relates to antibodies, single-chain antigen-binding molecules or other proteins that specifically bind to one or more proteins or peptide molecules of the present invention and their homologues, fusions or fragments. In a particularly preferred embodiment, the antibody specifically binds to a protein having the amino acid sequence set forth in SEQ ID NO: 5-26, or a fragment thereof. In another embodiment, the antibody specifically binds to a fusion protein containing an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5-26, or a fragment thereof.

The antibodies of the present invention may be used to quantify or quantify the content of a protein or peptide molecule of the present invention, or to detect post-translational modifications of proteins. As used herein, an antibody or peptide is said to specifically bind to a protein or peptide molecule of the present invention if such binding is not completely inhibited by the presence of unrelated molecules.

The antibodies of the present invention may be contained in a kit used to detect protein or peptide molecules of the present invention. The invention further encompasses a method for detecting a molecule of a protein or peptide of the present invention (in particular, a protein encoded by the amino acid sequence set forth in SEQ ID NOs: 5-26, including variants or fragments thereof capable of specifically binding to an antibody of the present invention), comprising: contacting the sample with an antibody of the present invention; and determining whether the sample contains a protein or peptide molecule of the present invention. The method of using antibodies to detect a protein or peptide of interest is known in the art.

Changed or improved options

It is known that the DNA sequences of a pesticidal protein can be altered using various methods and that these alterations may result in a DNA sequence encoding proteins with different amino acid sequences from those encoding the pesticidal proteins of the present invention. This protein can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions of one or more amino acid sequences from SEQ ID NO: 5-26, including up to about 2, about 3, about 4, about 5, about 6, about 7 about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155 or more amino acid substitutions, deletions, or inserts. Methods for carrying out such manipulations are generally known in the art. For example, variants of the amino acid sequence of a pesticidal protein can be generated using mutations in the DNA. This can also be achieved using one of several varieties of mutagenesis and/or by directed development. In some aspects, the changes encoded by the amino acid sequences will not significantly affect the function of the protein. Such variants will have the desired pesticidal activity. However, it is understood that the ability of a pesticidal protein to provide pesticidal activity can be improved by applying such techniques to the compositions of the present invention. For example, one can express

- 9 042693 pesticidal protein in host cells that exhibit high levels of base insertion errors in DNA replication, such as XL-1 Red (Stratagene, La Jolla, CA). After propagation in such strains, DNA can be isolated (for example, by obtaining plasmid DNA or by amplifying by PCR and cloning the resulting PCR fragment into a vector), culturing the pesticidal protein with mutations in a non-mutagenic strain, and identifying the mutated pesticidal genes. activity, for example by conducting an assay to investigate pesticidal activity. In general, protein is mixed and applied in feeding analyses. See, for example, Marrone et al. (1985) J. of Economic Entomology 78:290-293. Such assays may include bringing the plant into contact with one or more pests and determining the plant's ability to survive and/or kill the pest. Examples of mutations that lead to increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.

Alternatively, changes can be made to the sequence of many proteins in the region of the amino or carboxy terminus without significantly affecting the activity. Such changes may include insertions, deletions, or changes induced using modern molecular techniques such as PCR, including PCR amplifications that alter or extend the protein coding sequence by inserting amino acid coding sequences into oligonucleotides used in PCR amplifications. Alternatively, the added protein sequences may comprise entire protein-coding sequences, such as those commonly used in the art to produce fusion proteins. Such fusion proteins are often used to (1) increase the expression of a protein of interest, (2) introduce a binding domain, enzymatic activity, or epitope in order to facilitate protein purification, protein detection, or for other experimental applications known in the art, (3) directing the secretion or translation of the protein to a subcellular organelle such as the periplasmic space of Gram-negative bacteria, the endoplasmic reticulum of eukaryotic cells, the latter often resulting in glycosylation of the protein.

Variants of the nucleotide and amino acid sequences of the present invention also cover sequences obtained using procedures that lead to recombination and cause the formation of mutations, such as shuffling in DNA. In carrying out such a technique, one or more different regions encoding pesticidal proteins can be used to create a new pesticidal protein with the desired properties. Thus, libraries of recombinant polynucleotides are generated from a population of related polynucleotide sequences that contain regions of sequence with significant identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest can be swapped between the pesticidal activity gene of the present invention and other known pesticide genes to produce a new gene encoding a protein sequence of interest with an improved property, such as with high insecticidal activity. Strategies for such 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. Acad. sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and US Pat. Nos. 5,605,793 and 5,837,458.

Domain shuffling or shuffling is another mechanism for creating altered pesticidal proteins. Domains can move between pesticidal proteins, leading to the formation of hybrid or chimeric toxins with improved pesticidal activity or different target characteristics. Methods for making recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl Environ Microbiol 62:1537-1543 Geetal (1991) J Biol Chem 266:17954-17958 Schnepf et al (1990) J Biol Chem 265:20923-20930 Rang et al.91999) Appl. Environ. microbiol. 65:2918-2925).

Vectors

The pesticidal sequence of the present invention may be presented in an expression cassette for expression in a plant of interest. By plant expression cassette is meant a DNA construct that is capable of leading to the expression of an open reading frame protein in a plant cell. They usually contain a promoter and a coding sequence. Often such constructs will also contain a 3' untranslated region. Such constructs may contain a signal sequence or leader sequence to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi complex.

By a signal sequence is meant a sequence known or suspected to result in co-translational or post-translational transport

- 10 042693 tids through the cell membrane. In eukaryotes, it is usually associated with secretion in the Golgi apparatus, with some degree of glycosylation occurring as a result. Bacterial insecticidal toxins are often synthesized as protoxins that are proteolytically activated in the gut of the target insect (Chang (1987) Methods Enzymol. 153:507-516). In some embodiments, the implementation of the present invention, the signal sequence is localized in the native sequence, or can be obtained from the sequence of the present invention. By leader sequence is meant any sequence which, when translated, results in an amino acid sequence sufficient to drive the co-translational transport of the peptide chain to a subcellular organelle. Thus, leader sequences include leader sequences that have a targeted effect on transport and/or glycosylation by passing into the endoplasmic reticulum, passing into vacuoles, plastids, including chloroplasts, mitochondria, and the like.

By plant transformation vector is meant a DNA molecule that is necessary for efficient transformation of a plant cell. Such a molecule may contain one or more plant expression cassettes and may be organized into more than one vector DNA molecule. For example, binary vectors are plant transformation vectors that use two non-contiguous DNA vectors to encode all required functions with both cis and trans activity for plant cell transformation (Hellens and Mullineaux (2000) Trends in Plant Science 5:446- 451). The term vector refers to a nucleic acid construct intended to be transferred between different host cells. An expression vector is a vector that has the ability to introduce, integrate and express heterologous DNA sequences or fragments thereof in a foreign cell. The cassette will contain 5' and/or 3' regulatory sequences operably linked to the sequence of the present invention. By operably linked is meant a functional linkage between a promoter sequence and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. In general, the expression operably linked means that the linked nucleotide sequences are contiguous and, in cases where two protein-coding regions need to be joined, they are contiguous and in the same reading frame. The cassette may further comprise at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) may be delivered by multiple expression cassettes.

In various embodiments, the nucleotide sequence of the present invention is operably linked to a promoter, such as a plant promoter. A promoter is a nucleotide sequence whose function is to direct the transcription of the underlying coding sequence. A promoter, along with other transcriptional and translational regulatory nucleotide sequences (also referred to as control sequences), are required for the expression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restriction sites for the insertion of a pesticidal sequence subject to transcriptional regulation by regulatory regions.

The expression cassette will comprise, in the 5'-3' direction of transcription, a transcription and translation initiation site (i.e., a promoter), a DNA sequence of the present invention, and a translation and transcription termination site (i.e., a termination site) functioning in plant. The promoter may be native or similar, or may be foreign or heterologous to the host plant and/or to the DNA sequence of the present invention. Additionally, the promoter may be a natural sequence or, alternatively, a synthetic sequence. Where a promoter is native or homologous to a host plant, the promoter is meant to be found in the native plant into which the promoter is introduced. Where a promoter is foreign or heterologous to a DNA sequence of the present invention, it is understood that the promoter is not native or naturally occurring to the operably linked DNA sequence of the present invention.

The expression site may be native with a transcription initiation site, may be native with an operably linked DNA sequence of interest, may be native with the host plant, or may be derived from another source (i.e. foreign or heterologous to the promoter, DNA sequence of interest to the host plant, or any combination thereof). Suitable termination sites can be obtained from the A. tumefaciens Tiplasmid, such as the octopine synthase and nopaline synthase termination sites. 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 91:151158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-11 042693

9639.

Where appropriate, the gene(s) can be optimized to increase expression in the transformed host cell. Thus, genes can be synthesized using host cell-preferred codons to improve expression, or can be synthesized using codons at a host cell-preferred codon frequency. In general, the GC content of the gene will increase. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of the use of host cell preferred codons. There are methods in the art for synthesizing plant-preferred genes. See, for example, US Pat. Nos. 5,380,831 and 5,436,391, US Patent Publication No. 20090137409, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

In one embodiment, the pesticidal protein is directed to the chloroplast for expression. Thus, in cases where the pesticidal protein is not introduced directly into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide that directs the pesticidal protein to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

The gene for pesticidal activity to be directed to the chloroplast can be optimized for expression in the chloroplast to account for the difference in codon usage between the plant nucleus and that organelle. Thus, nucleic acids of interest can be synthesized using chloroplast-preferred codons. See, for example, US patent No. 5380831, which is incorporated into this document by reference.

plant transformation

The methods of the present invention include introducing a nucleotide construct into a plant. By introducing is meant providing the plant with a nucleotide construct in such a way that the construct gains access to the interior of the plant cell. The methods of the present invention do not require the use of a specific method for introducing the nucleotide construct into the plant, but only for the nucleotide construct to gain access to the interior of at least one plant cell. Methods for introducing nucleotide constructs into plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

By plant is meant whole plants, plant organs (eg leaves, stems, roots, etc.), seeds, plant cells, shoots, embryos and progeny of these plants. Plant cells may be differentiated or undifferentiated (eg callus, cell culture suspension, protoplasts, leaf cells, root cells, phloem cells, pollen).

Transgenic plants or transformed plants or stably transformed plants or cells or tissues refer to plants that have introduced or integrated into the plant cell exogenous nucleotide sequences or DNA fragments. These nucleotide sequences include those that are exogenous, or not present in the non-transformed plant cell, as well as those that may be endogenous, or present in the non-transformed plant cell. The term heterologous generally refers to nucleotide sequences that are not endogenous to the cell or are not part of the native genome in which they are present and that have been introduced into the cell by infection, transfection, microinjection, electroporation, microparticle bombardment, and the like. .

The transgenic plants of the present invention express one or more of the novel toxin sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional insect resistance genes (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, and Cry1F families; Cry2, such as a member of the Oy2A family; Cry9, such as members of the Oy2A family; families Oy9A, Cry9B, Cry9C, Cry9D, Oy9E and Cry9F, etc.). One of skill in the art will appreciate that a transgenic plant may contain any gene conferring an agricultural trait of interest.

Transformation of plant cells can be carried out using one or more methods known in the art. The pesticidal activity gene of the present invention can be modified to obtain or increase expression in plant cells. Typically, a construct that expresses such a protein contains a promoter that regulates the transcription of the gene, as well as a 3' untranslated region that provides for transcriptional termination and polyadenylation. The structure of such constructs is well known in the art. In some cases, it may be desirable to construct a gene in such a way that it will result in the secretion of the resulting peptide or other targeted action in the plant cell. For example, a gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. It may also be preferred to design an intron-containing expression cassette such that mRNA processing of the intron is required for expression.

Typically, this plant expression cassette will be introduced into a plant transformation vector. This plant transformation vector may contain one or more DNA vectors necessary to achieve transformation of the plant. For example, it is common practice in the art to use plant transform vectors that contain more than one contiguous DNA segment. In the art, these vectors are often referred to as binary vectors. Binary vectors, as well as vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation, where the size and total length of the DNA segments required to achieve efficient transformation are large enough that it is advisable to separate the functions into individual DNA molecules. Binary vectors typically contain a plasmid vector that contains cis-acting sequences necessary for T-DNA transfer (such as those located at the left border and at the right border of the fragment), a selectable marker that is designed to be able to to be expressed in a plant cell, and a gene of interest (a gene designed to be capable of being expressed in a plant cell, which requires the creation of transgenic plants). Also in this plasmid vector there are sequences necessary for bacterial replication. The cis-acting sequences are organized in a way that allows for efficient transfer to and expression in plant cells. For example, a selectable marker gene and a gene that determines pesticidal activity are located between the left and right borders. Often, the second plasmid vector contains transposition factors that mediate the transfer of T-DNA from Agrobacterium into plant cells. The plasmid often contains virulence genes (Vir genes) that allow for infection of Agrobacterium plant cells and DNA transfer by cleavage at border sequences and vir-mediated DNA transfer as known in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (eg LBA4404, GV3101, EHA101, EHA105, etc.) can be used to transform plants. The second plasmid vector is not necessary for plant transformation using other methods such as microparticle bombardment, microinjection, electroporation, polyethylene glycol transfection, etc.

In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by the application of appropriate selection with a maximum threshold level of appropriate selection (in selectable marker gene) in order to isolate the transformed plant cells from the group of non-transformed cell mass. The explants are usually transferred to a fresh portion of the same culture medium and cultured in a standard manner. Further, the transformed cells are differentiated into shoots after they are placed on a regeneration medium, to which a selection agent is added at a concentration of the maximum threshold level. The shoots are then transferred to a selective plant rooting substrate to establish the shoot or seedling. The transgenic seedling then grows into a mature plant and produces fertile seeds (eg, Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). The explants are usually transferred to a fresh batch of the same culture medium and cultured in a standard manner. For a general description of techniques and methods for producing transgenic plants, see Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Since the transformed material contains numerous cells; both transformed and non-transformed cells are present in any part of the treated target callus or tissue or group of cells. The ability to cause the death of non-transformed cells and allow the proliferation of transformed cells allows to obtain cultures of transformed plants. Often, the ability to remove non-transformed cells is a limitation for the rapid isolation of transformed plant cells and the successful creation of transgenic plants.

Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant or plant cell, i. monocotyledonous or dicotyledonous, intended for transformation. Generation of transgenic plants can be accomplished using one of several methods including, but not limited to, microinjection, electroporation, targeted gene transfer, Agrobacterium-assisted introduction of heterologous DNA into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with particle-conjugated heterologous foreign DNA, accelerated particle method, transformation using an aerosol beam injector (US Published Application No. 20010026941; US Patent No. 4945050; International Publication No. WO 91/00915; US Patent Application Published No. 2002015066), Lec1 transformation and various other methods DNA transfer,

- 13 042693 not mediated by the direct introduction of particles.

Methods for transforming chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. This method is based on the use of a gene gun for the delivery of DNA containing a selectable marker and the targeted introduction of DNA into the plastid genome through homologous recombination. Additionally, plastid transformation can be achieved by trans-activating a silent, plastid transgene through tissue-preferential expression of a plastid, nuclear RNA polymerase. Such a system is described in McBride et al. (1994) Proc. Natl. Acad. sci. USA 91:7301-7305.

Once the heterologous foreign DNA has been integrated into plant cells, appropriate selection is used, with a maximum threshold level of appropriate selection in the medium, to induce the death of the N-transformed cells and to separate and proliferate putatively transformed cells that survive this selection treatment, producing regular cell transfer into fresh environment. Through continuous passage and the introduction of an appropriate selection agent, the cells that have been transformed with the plasmid vector are identified and stimulated to proliferate. Molecular and biochemical methods can then be used to confirm the presence of an integrated heterologous gene of interest in the genome of the transgenic plant.

Cells that have been transformed 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 either pollinated with the same transformed line or with a different line, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic can be 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. Thus, the present invention provides transformed seeds (also referred to as transgenic seeds) containing the nucleotide construct of the present invention, for example, the expression cassette of the present invention, stably integrated into their genome.

Assessment of plant transformation

After introducing the heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid technique for screening transformed cells, tissue, or shoots for the presence of an introduced gene at an earlier stage prior to transplantation into soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY). PNR is carried out using oligonucleotide primers specific for the gene of interest or Agrobacterium vector background, etc.

Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated on an agarose gel, and transferred to a nitrocellulose or nylon membrane. The membrane or blot is then examined with, for example, a radioactive P labeled target DNA fragment to confirm the integration of the introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from special transformant tissues, fractionated on an agarose gel containing formaldehyde, and transferred to a nylon filter according to standard methods routinely used in the art (Sambrook and Russell, 2001, supra). The expression of the RNA encoded by the pesticidal activity gene is then examined by filter hybridization with a radioactive probe derived from the pesticide gene by methods known in the art (Sambrook and Russell, 2001, supra).

Confirmation of the presence of a protein encoded by a gene that determines pesticidal activity can be performed on transgenic plants using standard Western blot procedures, biochemical assays and the like on transgenic plants (Sambrook and Russell, 2001, supra) using antibodies that bind to one or multiple epitopes present on the pesticidal protein.

Pesticide activity in plants

In another aspect of the present invention, transgenic plants can be created that express a pesticidal protein that has pesticidal activity. The exemplary methods described above can be used to generate transgenic plants, but the method by which transgenic plant cells are obtained is not critical to the present invention. At the discretion of the experimenter, methods known or described in the art may be used, such as Agrobacterium-mediated transformation, biolistic transformation

- 14 042693 non-particle mediated methods and methods. Plants expressing the pesticidal protein can be isolated by well-known methods described in the art, for example, by transforming the callus, selecting the transformed callus, and regenerating a fruitful plant from such transgenic callus. Any gene can be used as a selectable marker in such a method, provided that its expression in plant cells allows the identification or selection of transformed cells.

A number of markers have been developed for use in plant cells, such as resistance to chloramphenicol, aminoglycoside G418, hygromycin, or the like. Other genes that encode a product involved in chloroplast metabolism can also be used as selectable markers. For example, genes that confer resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use. Such genes have been described in the literature (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (nitrilase bromoxynil resistance gene); and Sathasivan et al. Acids Res. 18:2188 (AHAS imidazolinone resistance gene) Additionally, the genes disclosed herein are useful as markers for assessing the transformation of bacterial or plant cells.Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell , sprout, embryo, or progeny thereof are well known in the art In one embodiment, the presence of the transgene is detected by testing for pesticidal activity.

Fruit-bearing plants that express the pesticidal protein can be tested for pesticidal activity, and plants can be selected for further crosses that exhibit optimal activity. Methods for assaying for pesticidal activity are available in the art. Typically the protein is mixed and used in feeding assays. See for example Marrone et al. (1985) J. of Economic Entomology 78:290-293.

The present invention can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley and oilseed rape, Brassica sp. ., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee tree, coconut, pineapple, citrus trees, cocoa tree, tea bush, banana, avocado, fig tree, guava, mango tree, olive tree, papaya, cashews, macadamia, almonds, oats, vegetables, ornamental plants and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, pea species, and members of the Curcumis genus such as cucumber, cantaloupe, and muskmelon. Ornamental plants include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, yellow daffodils, petunia, carnation, poinsettia, and chrysanthemum. Preferably, the plants of the present invention are crop plants (e.g., maize, sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley, oilseed rape, etc. ).

Application in pest control

General methods of using strains containing the nucleotide sequence of the present invention, or a variant thereof, are known in the art as pesticides in pest control or in the creation of other organisms. See, for example, US patent No. 5039523 and EP 0480762 A2.

Bacillus strains containing the nucleotide sequence of the present invention or a variant thereof, or microorganisms that are genetically modified to contain the gene for pesticidal activity of the present invention and a protein, can be used to protect crops and products from pests. In one aspect of the present invention, integral, i. non-lysed cells of the toxin-producing organism (pesticide) are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is placed in the environment of the target pest(s).

Alternatively, the pesticide is produced by introducing a gene for pesticidal activity into the host cell. Expression of a gene that determines pesticidal activity directly or indirectly leads to intracellular production and maintenance of the pesticide level. In one aspect of the present invention, these cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is placed in the environment of the target pest(s). The resulting product retains the toxicity characteristic of the toxin. These naturally encapsulated pesticides can then be formulated according to conventional techniques for application to the target pest's habitat, such as soil, water, and plant foliage. See, for example, EPA 0192319 and the references cited therein. Alternatively, it is possible to formulate a preparation of cells expressing the gene of this invention, in such a way as to ensure the use of the resulting material as a pesticide.

The active ingredients of the present invention are usually applied in the form of compositions and can be

- 15 042693 apply simultaneously or sequentially with other compounds to the cultivated area or the plant to be treated. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, pesticidal soaps, dormancy oils, polymers and/or slow release or biodegradable carrier formulations that allow long-term dosed release in the target area after a single dose. adding the composition. They may also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematocides, molluscicides, or mixtures of several of these preparations, if required, together with additional agriculturally acceptable carriers, surfactants or auxiliary agents. application aids that are commonly used in the formulation art. Suitable carriers and adjuvants may be solid or liquid and correspond to those commonly used in formulation technology, such as natural or reconstituted minerals, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Similarly, the formulations may be in the form of edible baits, or may be shaped into pest traps to allow the pesticide formulation to be fed to or ingested by the target pest.

Methods for applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention include foliar application, seed coating, and soil application. The number of applications and application rate depends on the intensity of the infestation of the respective pest.

The composition may be formulated as a powder, dust, pellet, granule, aerosol emulsion, colloid, solution, or the like, and may be prepared by conventional methods such as drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or cell culture concentration, including polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present at a concentration (w/w) of from about 1% to about 99%.

With the methods of the present invention, Lepidoptera, Hemiptera, Diptera or Coleoptera pests can be killed or reduced in a given area, or can be used prophylactically in the environment to prevent infestation by a susceptible pest. Preferably, the pest ingests or comes into contact with a pesticidally effective amount of the polypeptide. By pesticidally effective amount is meant an amount of a pesticide that is capable of causing the death of at least one pest or markedly restricting the pest's growth, nutrition, or normal physiological development. This amount will vary depending on factors such as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop or agricultural area to be treated, environmental conditions and method, rate, concentration, stability and amount of application. a polypeptide composition providing a pesticidal effect. The formulations may also be varied to suit climatic conditions, environmental impact and/or frequency of application and/or severity of pest infestation.

The described pesticidal compositions can be prepared by formulating either a bacterial cell, a crystal and/or a suspension of spores, or an isolated protein component with the desired agriculturally acceptable carrier. The compositions may be formulated prior to administration by appropriate means such as lyophilization, freeze-drying, drying, or in an aqueous vehicle, vehicle, or suitable diluent such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or suspension in oil (vegetable or mineral), or aqueous or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for use in the field of agriculture. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, inerts, dispersants, surfactants, tackifiers, binders, etc., which are commonly used in pesticide formulation technology; they are well known to those skilled in the art of pesticide formulation. These formulations can be mixed with one or more solid or liquid adjuvants and obtained in various ways, for example, by uniformly mixing, mixing and/or grinding the pesticide composition with suitable adjuvants using conventional formulation techniques. Suitable compositions and application methods are described in US patent No. 6468523, which is incorporated into this document by reference.

The pest includes, but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Der- 16 042693 maptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., in particular Coleoptera , Lepidoptera and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga. The suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea, while the suborder Polyphaga includes the superfamilies Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerymbycoidea, and Chculionoidea. The superfamily Caraboidea includes the families Cicindelidae, Carabidae, and Dytiscidae. The superfamily Gyrinoidea includes the family Gyrinidae. The superfamily Hydrophiloidea includes the family Hydrophilidae. The superfamily Staphylinoidea includes the families Silphidae and Staphylinidae. The superfamily Cantharoidea includes the families Cantharidae and Lampyridae. The superfamily Cleroidea includes the families Cleridae and Dermestidae. The superfamily Elateroidea includes the families Elateridae and Buprestidae. The superfamily Cucujoidea includes the family Coccinellidae. The superfamily Meloidea includes the family Meloidae. The superfamily Tenebrionoidea includes the family Tenebrionidae. The superfamily Scarabaeoidea includes the families Passalidae and Scarabaeidae. The superfamily Cerambycoidea includes the family Cerambycidae. The superfamily Chrysomeloidea includes the family Chrysomelidae. The superfamily Curculionoidea includes the families Curculionidae and Scolytidae.

The order Diptera includes the suborders Nematocera, Brachycera, and Cyclorrhapha. The suborder Nematocera includes the families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. The suborder Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. The suborder Cyclorrhapha includes sections Aschiza and Aschiza. Section Aschiza includes the families Phoridae, Syrphidae, and Conopidae. Section Aschiza includes subsections Acalyptratae and Calyptratae. The section Acalyptratae includes the families Otitidae, Tephritidae, Agromyzidae, and Drosophilidae. The section Calyptratae includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae.

Nematodes include parasitic nematodes such as nodule, cyst and attack nematodes including Heterodera spp., Meloidogyne spp. and Globodera spp.; in particular, representatives of cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (grain cyst nematode); and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Infecting nematodes include Pratylenchus spp.

Hemiptera pests (which include species referred to as Hemiptera, Homoptera, or Heteroptera) include, but are not limited to, Lygus spp. such as western meadow bug (Lygus hesperus), meadow bug (Lygus lineolaris), and alfalfa bug (Lygus elisus); plant aphids such as green peach aphid (Myzus persicae), cotton aphid (Aphis gossypii), cherry or cherry aphid (Myzus cerasi), soybean aphid (Aphis glycines Matsumura); black leafhopper (Nilaparvata lugens) and rice green leafhopper (Nephotettix spp.); and stink stink bugs such as green stink stink (Acrosternum hilare), marble stink stink (Halyomorpha halys), southern green stink stink (Nezara viridula), rice stink stink (Oebalus pugnax), red-legged stink stink (Pentatoma rufipes), European stink stink (Rhaphigaster nebulosa) and troilus stink stink Troilus luridus.

Insect pests of the present invention for major crops include: maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, epsilon cutworm; Helicoverpa zea, armyworm; Spodoptera frugiperda, grass cutworm; Diatraea grandiosella, southwestern corn moth; Elasmopalpus lignosellus, grain grinder; Diatraea saccharalis, sugar cane moth; Diabrotica virgifera, the western corn root beetle; Diabrotica longicornis barberi, northern corn root beetle; Diabrotica undecimpunctata howardi, southern corn root beetle; Melanotus spp., click beetles; Cyclocephala borealis, northern beetle (beetle larva); Cyclocephala immaculata, southern beetle (beetle larva); Popilliajaponica, Japanese beetle; Chaetocnema pulicaria, ground corn flea; Sphenophorus maidis, corn weevil; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, North American wheat bug; Melanoplus femurrubrum, the red-footed filly; Melanoplus sanguinipes, a migratory filly; Hylemya platura, the larva of the sprout fly; Agromyza parvicornis, corn moth; Anaphothrips obscrurus, cereal thrips; Solenopsis milesta, thief ant; Tetranychus urticae, the common spider mite; Sorghum: Chilo partellus, sorghum grinder; Spodoptera frugiperda, grass cutworm; Helicoverpa zea, armyworm; Elasmopalpus lignosellus, a small corn stalk grinder; Feltia subterranea, the caterpillar of the winter cutworm; Phyllophaga crinita, Maybug larva; Eleodes, Conoderus and Aeolus spp., wireworms; Oulema melanopus, red-breasted leech; Chaetocnema pulicaria, corn flea; Sphenophorus maidis, corn weevil; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, the yellow sugar cane aphid; Blissus leucopterus leucopterus, North American wheat bug; Contarinia sorghicola, sorghum gall midge; Tetranychus cinnabarinus, the red spider mite;

- 17 042693

Tetranychus urticae, the common spider mite; wheat: Pseudaletia unipunctata, cutworm; Spodoptera frugiperda, grass cutworm; Elasmopalpus lignosellus, a small corn stalk grinder; Agrotis orthogonia, rectangular cutworm; Elasmopalpus lignosellus, a small corn stalk grinder; Oulema melanopus, red-breasted leech; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn root beetle; Russian wheat aphid; Schizaphis graminum, common grass aphid; Macrosiphum avenae, large grass aphid; Melanoplus femurrubrum, the red-footed filly; Melanoplus differentialis, the distinctive filly; Melanoplus sanguinipes, a migratory filly; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, orange grass midge; Meromyza americana, the larva of the American meromyza; Hylemya coarctata, winter fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, the grain sawfly; Aceria tulipae, the tulip bulb mite; Sunflower: Suleima helianthana, sunflower bud; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower exclamation bollworm; Bothyrus gibbosus, the carrot beetle; Neolasioptera murtfeldtiana, sunflower seed gall; Cotton plant: Heliothis virescens, cotton leaf roller; Helicoverpa zea, cotton bollworm; Spodoptera exigua, little cutworm; Pectinophora gossypiella, the pink cotton bollworm; Anthonomus grandis, cotton weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton bug; Trialeurodes autilonea, cotton whitefly; Lygus lineolaris, grass bug; Melanoplus femurrubrum, the red-footed filly; Melanoplus differentialis, the distinctive filly; Thrips tabaci, tobacco thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, the red spider mite; Tetranychus urticae, the common spider mite; rice: Diatraea saccharalis, sugar cane grinder; Spodoptera frugiperda, grass cutworm; Helicoverpa zea, armyworm; leafworm of the species Colaspis brunnea; Lissorhoptrus oryzophilus, the rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, North American wheat bug; Acrosternum hilare, the green stink bug; soy: Pseudoplusia includens, soy moth; Anticarsia gemmatalis, velvet bean cutworm; Plathypena scabra, a green clover pest; Ostrinia nubilalis, the European corn borer; Agrotis ipsilon, epsilon cutworm; Spodoptera exigua, little cutworm; Heliothis virescens, cotton leaflet; Helicoverpa zea, armyworm; Epilachna varivestis, the Mexican bean weevil; Myzus persicae, the green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, the green stink bug; Melanoplus femurrubrum, the red-footed filly; Melanoplus differentialis, the distinctive filly; Hylemya platura, the larva of the sprout fly; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, the Turkestan spider mite; Tetranychus urticae, the common spider mite; barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, epsilon cutworm; Schizaphis graminum, common grass aphid; Blissus leucopterus leucopterus, North American wheat bug; Acrosternum hilare, the green stink bug; Euschistus servus, the brown stink bug; Delia platura, the larva of the sprout fly; Mayetiola destructor, Hessian fly; Petrobia latens, petrobia polyphagous; oilseed rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, cruciferous flea; Mamestra configurata, lettuce cutworm; Plutella xylostella, cabbage cutworm; Delia ssp., spring cabbage fly larvae.

Nematodes include parasitic nematodes such as nodule, cyst and attack nematodes including Heterodera spp., Meloidogyne spp. and Globodera spp.; in particular, representatives of cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (grain cyst nematode); and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Infecting nematodes include Pratylenchus spp.

Ways to increase plant yield

Methods for increasing plant yields are provided. The methods include providing a plant or plant cell that expresses a polynucleotide encoding the sequence of a pesticidal polypeptide disclosed herein and growing the plant or seed derived from it in a field infested (or susceptible to infestation) by a pest against which said polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a lepidoptera, hemipteran, dipteran, hemipteran, or nematode pest and said field is infested with a lepidoptera, hemipteran, hemipteran, dipteran, or nematode pest. As defined herein, plant yield refers to the quality and/or amount of biomass produced by a plant. By biomass is meant any valued product of plant origin. An increase in biomass production is any improvement in the measurable yield of a plant product. Increasing crop yields has several commercial applications. For example, increasing the biomass of plant leaves can increase the yield of leafy vegetables for human or animal consumption. Additionally, the increase in leaf biomass can be used to increase the production of pharmaceutical or industrial plant products. The increase in yield may include any statistically significant increase

at least 1% increase, at least 3% increase, at least 5% increase, at least 10% increase, at least 20% increase , at least 30%, at least 50%, at least 70%, at least 100% or more significant yield increase compared to a plant that does not express the pesticidal protein sequence. When using specific methods, plant yield is increased as a result of increased pest resistance of a plant that expresses the pesticidal protein disclosed herein. Expression of the pesticidal protein results in a reduction in the pest's ability to infect or eat.

Plants can also be treated with one or more chemical compositions containing one or more herbicides, insecticides or fungicides. Illustrative chemical compositions include: fruit/vegetable herbicides: atrazine, bromacyl, diuron, glyphosate, linuron, metribuzin, simazine, trifluralin, fluazifop, glufosinate, Gowan halosulfuron, paraquat, propizamide, sethoxydim, butafenacil, halosulfuron, indaziflam; fruit/vegetable insecticides: aldicarb, Bacillus thuriengiensis, carbaryl, carbofuran, chlorpyrifos, cypermethrin, deltamethrin, abamectin, cyfluthrin/beta-cyfluthrin, esfenvalerate, lambda-cyhalothrin, acequinocil, biphenazate, methoxyfuroside, novaluron, chromapane, fludinothiacri- noside , спиродиклофен, гаммацигалотрин, спиромезифен, спиносад, ринаксипир, циазипир, трифлумурон, спиротетрамат, имидаклоприд, флубендиамид, тиодикарб, метафлумизон, сульфоксафлор, цифлуметофен, цианопирафен, клотианидин, тиаметоксам, спинеторам, тиодикарб, флоникамид, метиокарб, эмамектин бензоат, индоксакарб, фенамифос, pyriproxyfen, fenbutatin oxide; fruit/vegetable fungicides: amethoctradin, azoxystrobin, benthiavalicarb, boscalid, captan, carbendazim, chlorothalonil, copper, cyazofamide, cyflufenamide, cymoxanil, cyproconazole, cyprodinil, difenoconazole, dimethomorph, dithianone, fenamidone, fenhexamid, fluazinam, fluopyramid, fluoxastrobin, fluxapiroxad, folpet, fosetil, iprodione, iprovalicarb, isopyrazam, kresoxim-methyl, mancozeb, mandipropamide, metalaxyl/mefenoxam, metiram, metrafenone, myclobutanil, penconazole, penthiopyrad, picoxystrobin, propamocarb, propiconazole, propineb, proquinazid, pyraclostrobin, pyrimethanil, quinoxifene, spiroxamine, sulfur, tebuconazole, thiophanate-methyl, trifloxystrobin; cereal herbicides: 2,4-D, amidosulfuron, bromoxynil, carfentrazone-ethyl, chlorotoluron, chlorsulfuron, clodinafop-, clopyralid, dicamba, diclofop-methyl, diflufenican, fenoxaprop, florasulam, flucarbazon sodium, flufenacet, flupirosulfuron-M, fluroxypyr, flurtamone , glyphosate, iodosulfuron, ioxynil, isoproturon, MCPA, mesosulfuron, metsulfuron, pendimethalin, pinoxaden, propoxycarbazone, prosulfocarb, pyroxulam, sulfosulfuron, tifensulfuron, tralkoxydim, triasulfuron, tribenuron, trifluralin, tritosulfuron; cereal fungicides: azoxystrobin, bixaphene, boscalid, carbendazim, chlorothalonil, cyflufenamid, cyproconazole, cyprodinil, dimoxystrobin, epoxyconazole, fenpropidine, fenpropimorph, fluopyram, fluoxastrobin, fluquinconazole, fluxapiroxad, isopyrazam, kresoxim-methyl, picoxystropyradinone, metraconazole , propiconazole, proquinazid, prothioconazole, pyraclostrobin, quinoxifene, spiroxamine, tebuconazole, thiophanate-methyl, trifloxystrobin; cereal insecticides: dimethoate, lambda-cyhalothrin, deltamethrin, alpha-cypermethrin, β-cyfluthrin, bifenthrin, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, chlorpyrifos, pyrimicarb, methiocarb, sulfoxaflor; maize herbicides: atrazine, alachlor, bromoxynil, acetochlor, dicamba, clopyralid, (S-) dimethenamid, glufosinate, glyphosate, isoxaflutol, (S-) metolachlor, mesotrione, nicosulfuron, primisulfuron, rimsulfuron, sulcotrione, foramsulfuron, topramesone, tembotrione, saflufenacil, thiencarbazone, flufenacet, pyroxasulfone; insecticides for maize: carbofuran, chlorpyrifos, bifenthrin, fipronil, imidacloprid, lambda-cyhalothrin, tefluthrin, terbufos, thiamethoxam, clothianidin, spiromesifen, flubendiamide, triflumuron, rinaxipyr, deltamethrin, thiodicarb, β-cyfluthrin, cyperomethrin, lufenumethrin, bibufenumethrin ethiprole, ciazipyr, thiacloprid, acetamiprid, dinetofuran, avermectin; maize fungicides: azoxystrobin, bixaphene, boscalid, cyproconazole, dimoxystrobin, epoxiconazole, fenitropan, fluopyram, fluoxastrobin, fluxapiroxad, isopyrazam, metconazole, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, tebuconazole, trifloxystrobin; herbicides for rice: butachlor, propanil, azimsulfuron, bensulfuron, cyhalofop, daimuron, fentrazamide, imazosulfuron, mefenacet, oxaziclomefon, pyrazosulfuron, pyributicarb, quinclorac, thiobencarb, indanophane, fluphenacet, fentrazamide, halosulfuron, oxazicycloxuloximide, benzobicyclomide oxadiargyl, ethoxysulfuron, pretilachlor, mesotrione, tefuryltrione, oxadiazon, fenoxaprop, pyrimisulfan; rice insecticides: diazinon, fenobucarb, benfuracarb, buprofezin, dinotefuran, fipronil, imidacloprid, isoprocarb, thiacloprid, chromafenoside, clothianidin, ethiprole, flubendiamide, rinaxipyr, deltamethrin, acetamiprid, thiamethoxam, cyazipyr, spinoperosad, cymcytin-atem chlorpyrifos, etofenprox, carbofuran, benfuracarb, sulfoxaflor; rice fungicides: azoxystrobin, carbendazim, carpropamide, diclocimet, difenoconazole, edifenphos, ferimzone, gentamicin, hexaconazole, gimexazole, iprobenphos (IBP), isoprothiolane, isothianil, kasugamycin, mancozeb, methominostrobin, orysastrobin, pencycuron, probena

- 19 042693 zol, propiconazole, propineb, pyroquilon, tebuconazole, thiophanate-methyl, thiadinil, tricyclazole, trifloxystrobin, validamycin; cotton herbicides: diuron, fluometuron, MSMA, oxyfluorfen, promethrin, trifluralin, carfentrazone, cletodym, fluazifop-butyl, glyphosate, norflurazon, pendimethalin, pyrithiobac-sodium, trifloxysulfuron, tepraloxydim, glufosinate, flumioxazine, thidiazuron; cotton insecticides: acephate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, abamectin, acetamiprid, emamectin benzoate, imidacloprid, indoxacarb, lambda-cyhalothrin, spinosad, thiodicarb, gamma-cyhalothrin, spiromesifen, pyridalyl, flonicamid, betaspiraspira, flubendiamide, flubendiamide, -cyfluthrin, spirotetramat, clothianidin, thiamethoxam, thiacloprid, dinetofuran, flubendiamide, cyazipyr, spinosad, spinotoram, gamma-cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2difluoroethyl)amino]furan-2 (5H)-one, thiocarb, avermectin, flonicamid, pyridalil, spiromesifen, sulfoxaflor; cotton fungicides: azoxystrobin, bixaphene, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, dimoxystrobin, epoxiconazole, fenamidone, fluazinam, fluopyram, fluoxastrobin, fluxapiroxad, iprodione, isopyrazam, isothianil, mancozeb, maneb, methopyradinostrobin, penoxystrobin, propineb, prothioconazole, pyraclostrobin, quintozen, tebuconazole, tetraconazole, thiophanate-methyl, trifloxystrobin; soybean herbicides: alachlor, bentazone, trifluralin, chlorimuron-ethyl, chloransulam-methyl, fenoxaprop, fomesafen, fluazifop, glyphosate, imazamox, imazakvin, imazethapyr, (S-)metolachlor, metribuzin, pendimethalin, tepraloxydim, glufosinate; soybean insecticides: lambda-cyhalothrin, methomyl, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, flubendiamide, rinakipir, cyazipyr, spinosad, spinetoram, emamectin benzoate, fipronil, ethiprol, deltamethrin, β-cyfluthrin, gamma and lambda -cyhalothrin, 4-[[(6chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, spirotetramat, spinodiclofen, triflumuron, flonicamid, thiodicarb, beta-cyfluthrin; soybean fungicides: azoxystrobin, bixaphene, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, dimoxystrobin, epoxiconazole, fluazinam, fluopyram, fluoxastrobin, flutriafol, fluxapiroxad, isopyrazam, iprodione, isothianil, mancozeb, maneb, metconazole, methominoclotan, penthiopyrad, picoxystrobin, propiconazole, propineb, prothioconazole, pyraclostrobin, tebuconazole, tetraconazole, thiophanate-methyl, trifloxystrobin; sugar beet herbicides: chloridazone, desmedifam, etofumezat, phenmedifam, triallat, clopyralid, fluazifop, lenacil, metamitron, quinmerac, cycloxidim, triflusulfuron, tepraloxydim, chizalofop; sugar beet insecticides: imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, deltamethrin, β-cyfluthrin, gamma/lambda-cyhalothrin, 4[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl) amino]furan-2(5H)-one, tefluthrin, rinaxipyr, cyaxipyr, fipronil, carbofuran; herbicides for canola: clopyralid, diclofop, fluazifop, glufosinate, glyphosate, metazachlor, trifluralin etametsulfuron, quinmerac, quizalofop, Clethodim, tepraloxydim; canola fungicides: azoxystrobin, bixaphene, boscalid, carbendazim, cyproconazole, difenoconazole, dimoxystrobin, epoxiconazole, fluazinam, fluopyram, fluoxastrobin, flusilazole, fluxapiroxad, iprodione, isopyrazam, mepiquat chloride, metconazole, methominostrobin, piclobyratazole, prochlorazstrobutrazol, pentiopyrabutrazol. , prothioconazole, pyraclostrobin, tebuconazole, thiophanate-methyl, trifloxystrobin, vinclozolin; canola insecticides: carbofuran, thiacloprid, deltamethrin, imidacloprid, clothianidin, thiamethoxam, acetamiprid, dinetofuran, B-cyfluthrin, gamma- and lambda-cyhalothrin, tau-fluvaleriate, ethiprole, spinosad, spinetoram, flubendiamide, rinaxipir, cyazipyr, 4[[(6 -chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

The following examples are offered by way of illustration and not by way of limitation.

Experimental examples

Example 1. Discovery of new genes that determine pesticidal activity in Bacillus thuringiensis.

New pesticide genes were identified from bacterial strains ATX47307 and ATX56002 using the following steps.

Obtaining all DNA from a strain. Total DNA contains both genomic DNA and extrachromosomal DNA. Extrachromosomal DNA contains a mixture of some or all of the following: plasmids of various sizes; phage chromosomes; other uncharacterized extrachromosomal molecules.

DNA sequencing. The total DNA is sequenced using next generation sequencing methods.

Identification of putative toxin genes by homology and/or other computational analyses.

Obtain the final refined sequence of the gene of interest by one of several PCR or cloning strategies (eg, TAIL-PCR), if necessary.

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Table 1

New gene identified in strain ATX47307

Gene name Molecular weight (kDa) Closest homologue Nucleotide SEQID NO Amino acid SEQ ID NO Axmi477 132 75% Cry9Ba1 1 5 Axmi477.2 6 Axmi477.3 7 Aht1477(truncated) 60% Cry9Bal (truncated) 8 Axgsh477.2(truncated) 9 Aht1477.3(truncated) 10

Axmi477.2 and Axmi477.3 are proteins encoded from downstream of Axmi477 the translation start site.

table 2

New gene identified in strain ATX47307

Gene name Molecular weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino acid SEQ ID NO Axmi482 37 94% Axmi486; 51% Mtx3 2 eleven Axmi482.2 12 Axmi482.3 13 Axmi482.4 14

Axmi482.2, Axmi482.3 and Axmi482.4 are proteins encoded from downstream of Axmi482 the translation start region.

Table 3

New gene identified in strain ATX65002

Gene name Molecular weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino acid SEQ ID NO Axmi486 38 94% Axmi482; 49% Mtx3 3 15 Axmi486.2 16 Axmi486.3 17 Axmi486.4 18 Axmi486.5 19

Axmi486.2, Axmi486.3, Axmi486.4 and Axmi486.5 are proteins encoded from downstream of Axmi486 the translation start site.

Table 4

New gene identified in strain ATX65002

Gene name Molecular weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino acid SEQ ID NO >Axmi525 >Axmi525.2 38 97% Axmi486; 51% Mtx3 4 20 21 >Axmi525.3 22 >Axmi525.4 23 >Axmi525.5 24 >Axmi525.6 25 >Axmi525.7 26

Axmi525.2, Axmi525.3, Axmi525.4, Axmi525.5, Axmi525.6 and Axmi525.7 are proteins encoded from the downstream Axmi525 translation start region.

Example 2 Pesticide Activity Assays.

The nucleotide sequences of the present invention can be tested for their ability to produce pesticidal proteins. The ability of a pesticidal protein to act as a pesticide on a pest is often analyzed using a number of methods. One method well known in the art is to perform a fed assay. In this feeding assay, the pest is exposed to a sample containing either the compounds to be tested or control samples. This is often done by placing the material to be tested, or a suitable solution of such material, on a material that the pest will ingest, such as an artificial breeding ground. The material to be examined may be liquid, solid, or slurry. The material to be examined can be placed on the surface and then allowed to dry. Alternatively, the material to be tested can be mixed with molten artificial culture medium and then distributed in the analysis chamber. The assay chamber may be, for example, a cup, plate, or well of a microtiter plate.

Assays with sucking pests (eg, aphids) may involve separating the test material from the insect with a septum, ideally a section that the sucking insect can pierce with parts of the sucking mouthparts to allow absorption of the test material. Often, the test material is mixed with a feeding stimulant such as sucrose to promote absorption of the test compound.

Other types of assays may include microinjection of test material into the mouth or gut of the pest, as well as development of transgenic plants and then testing the pest's ability to feed on the transgenic plant. Plant research may involve isolating parts of plants that are normally consumed, for example, in small chambers attached to a leaf, or isolating whole plants in chambers that contain insects.

Other methods and approaches for assessing pests are known in the art and can be found in, for example, Robertson and Preisler, eds. (1992) Pesticide bioassays with arthropods CRC, Boca Raton, FL. Alternatively, the assays are usually described in the Arthropod Management Tests and Journal of Economic Entomology, or discussed with members of the Entomological Society of America (ESA).

In some embodiments, DNA regions encoding a toxin region in the pesticide proteins disclosed herein are cloned into the E. coli pMAL-C4x expression vector behind the malE gene encoding a maltose binding protein (MBP). These in-frame fusions result in the expression of MBP-Axmi fusion proteins in E. coli.

For expression in E. coli, BL21*DE3 is transformed with separate plasmids. Individual colonies are inoculated into Luria-Bertani medium supplemented with carbenicillin and glucose and grown overnight at 37°C. The following day, fresh medium is inoculated with 1% daily culture and grown at 37° C. to a logarithmic growth phase. Cultures are then induced with 0.3 mM IPTG overnight at 20°C. Each cell pellet is suspended in 20 mM Tris-Cl buffer, pH 7.4 + 200 mM NaCl + 1 mM DTT + protease inhibitors and sonicated. SDS-PAGE analysis can be used to confirm the expression of fusion proteins.

All cell-free extracts are then run through an amylose column attached to a rapid protein liquid chromatography (FPLC) chromatograph for affinity purification of the MBP-axmi fusion proteins. The bound fusion proteins are eluted from the resin with a 10 mM maltose solution. The purified fusion proteins are then digested with either factor Xa or trypsin to remove the amino-terminal MBP tag from the Axmi protein. Protein digestion and solubility can be determined using SDS-PAGE.

Example 3 Expression and purification.

Truncated versions of Axmi477 (which is described herein as SEQ ID NO: 8), Axmi482 (which is described herein as SEQ ID NO: 13), Axmi486 (which is described herein as SEQ ID NO: 16), and Axmi525 (which is set forth herein as SEQ ID NO: 26) were expressed and analyzed for biological activity. The genes were amplified by PCR from their respective strains using HERCULASE® II fused DNA polymerase with primers inserted with AscI linkers at the 3' end. The amplified PCR product was digested with AscI and ligated into the pMa1C4X vector. Clonings were confirmed by sequencing and transformed into competent B121 cells. Each individual colony was inoculated into Luria-Bertani medium and grown at 37°C to log-phase and induced with 0.5 mM IPTG at 20°C for 18 hours. The purified protein was digested with factor Xa at a ratio of 1:50 at room temperature overnight. The purified protein was subjected to a biological activity assay against selected insect pests according to a standard protocol. The results are shown in table. 5-8.

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Table 5

Mortality and stunting rates for Axmi477

pest group Stunting indicator Mortality, in percent Plutella xylostella (DBM) 4 100 Anticarsia gemmatalis (VBC) 4 100 Diatraea grandiosella (SWCB) 3.5 25 Diatraea saccharalis (SCB) 3.5 0 Heliothis virescens (Hv) 4 75 Heliocoverpa zea (Hz) 2 25 Ostrinia nublialis (ECB) 3 25 Spodoptera frugiperda (FAW) 1 0 Spodoptera exigua (BAW) 3 0 Agrotis ipsilon (BCW) 4 0 Pseudoplusia includens (SBL) 4 100 See indicators pest group Plutella xylostella (DBM) Anticarsia gemmatalis (VBC) Diatraea grandiosella (SWCB) Diatraea saccharalis (SCB) Heliothis virescens (Hv) Heliocoverpa zea (Hz) Ostrinia nublialis (ECB) Spodoptera frugiperda (FAW) See indicators pest group Plutella xylostella (DBM) Anticarsia gemmatalis (VBC) Diatraea grandiosella (SWCB) stunting and stunting for Stunting rate 4 4 4 3 1 4 3 3 stunting and stunting for Stunting rate 4 4 3.5 Table 6 i Axmi482 Mortality, in percent 100 75 50 0 0 0 1 0 Table 7 i Axmi486 Mortality, in percent 100 37 50 Diatraea saccharalis (SCB) 2.5 25 Heliothis virescens (Hv) 3.5 0 Heliocoverpa zea (Hz) 3 0 Pseudoplusia includens (SBL) 1 0

Table 8

Mortality and stunting rates for Axmi525

pest group Stunting indicator Mortality, in percent Spodoptera frugiperda (FAW) 1 0% Heliothis virescens (Hv) 1.5 0% Helicoverpa zea (Hz) 3 0% Anticarsia gemmatalis (VBC) 4 42% Spodoptera eridania (SAW) 3.2 70% Plutella xylostella (DBM) 4 100% Diatraea grandiosella (SWCB) 4 83% Diatraea crambidoides (SCB) 4 66%

Stunting Index:

- there is no activity;

- heterogeneous growth retardation;

- slight growth retardation;

- severe growth retardation;

- a sharp growth retardation.

Example 4. Vectoring of Genes for Plant Expression.

The coding regions of the present invention are linked to appropriate promoter and terminator sequences for expression in plants. Such sequences are well known in the art and may include the rice actin promoter or maize ubiquitin promoter for expression in monocots, the Arabidopsis UBQ3 promoter, or the CaMV promoter.

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35S for expression in dicots and nos or PinII terminators. Methods for obtaining and validating promoter-gene-terminator constructs are also well known in the art.

In one aspect of the present invention, synthetic DNA sequences are designed and created. These synthetic sequences have an altered nucleotide sequence from the original sequence but encode proteins that are substantially identical to the original sequence.

In another aspect of the present invention, modified synthetic gene variants are designed such that the resulting peptide is targeted to a plant organelle, such as the endoplasmic reticulum or apoplast. Peptide sequences are known in the art which are known to result in targeting of fusion proteins to the plant organelle. For example, it is known in the art that the N-terminal region of the protein encoded by the acid phosphatase gene of the white lupine Lupinus albus (GENBANK® ID GI: 14276838, Miller et al. (2001) Plant Physiology 127: 594-606) leads to the targeting of heterologous proteins on the endoplasmic reticulum. If the resulting fusion protein also contains at the C-terminus an endoplasmic reticulum retention sequence containing lysine-aspartic acid-glutamic acid-leucine at the N-terminus of the peptide (i.e., KDEL motif, SEQ ID NO: 27), then the fusion protein will target the endoplasmic reticulum. If the fusion protein lacks an endoplasmic reticulum-targeting sequence at the C-terminus, the protein will target the endoplasmic reticulum but will eventually be bound in the apoplast.

Thus, this gene encodes a fusion protein that contains at the N-terminus thirty-one amino acids from the acid phosphatase gene of the white lupine Lupinus albus (GENBANK® ID GI: 14276838, Miller et al, 2001, see above), fused to the N-terminus amino acid sequence of the present invention, as well as the KDEL sequence (SEQ ID NO: 27) at the C-terminus. Thus, the resulting protein is expected to target the plant endoplasmic reticulum when expressed in the plant cell.

The plant expression cassettes described above are combined with a suitable plant selectable marker to facilitate selection of transformed cells and tissues and ligated into plant transformation vectors. These may include binary vectors for Agrobacterium mediated transformation or simple plasmid vectors for aerosol or biolistic transformation.

Example 5 Transformation of maize cells with the pesticidal protein genes described herein.

Maize cobs are best harvested 8-12 days after pollination. Embryos are isolated from cobs and embryos 0.8-1.5 mm in size are preferably used for transformation. Embryos are plated upside down in a suitable incubation medium such as DN62A5S medium (3.98 g/l N6 salts; 1 ml/l (1000x stock solution), vitamins N6; 800 mg/l L-asparagine; 100 mg/l myoinositol ; 1.4 g/l L-proline; 100 mg/l casamino acids; 50 g/l sucrose; 1 ml/l (1 mg/ml stock solution) 2,4-D). However, media and salts other than DN62A5S and known in the art are suitable. Embryos are incubated overnight at 25° C. in the dark. However, overnight incubation of embryos is not necessary as such.

The resulting explants are transferred to grid cells (30-40 per dish), transferred to an osmotic medium for about 30-45 minutes, then transferred to a beam exposure dish (see, for example, PCT Publication No. WO/0138514 and US Patent No. 5240842).

DNA constructs designed to express the genes of the present invention in plant cells are accelerated into plant tissue using an aerosol beam accelerator using conditions essentially as described in PCT Publication No. WO/0138514. After exposure to the beam, the embryos are incubated for about 30 min on osmotic medium and placed in the incubation medium overnight at 25° C. in the dark. To avoid unnecessary damage to beam-exposed explants, they are incubated for at least 24 hours before being transferred to recovery medium. The embryos are then distributed in the medium for a recovery period of approximately 5 days at 25° C. in the dark, then transferred to a selective medium. The explants are incubated in the selective medium for up to eight weeks, depending on the nature and characteristics of the particular selection method used. After a selection period, the resulting callus is transferred to embryo maturation medium and cultured until mature somatic embryo formation is observed. The resulting mature somatic embryos are then placed in low light conditions and the regeneration process is initiated using methods known in the art. The resulting shoots are allowed to root on the rooting medium and the resulting plants are transferred to seedling pots and propagated as transgenic plants.

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Claims (29)

Materials Medium DN62A5S Components per liter Source A mixture of basic salts N6 according to Chu (product number C 416) 3.98 g/l Phytotechnology Labs Chu Vitamin N6 Solution (Product No. C 149) 1 ml/l (1000x stock solution) Phytotechnology Labs L-asparagine 800 mg/l Phytotechnology Labs myoinositol 100 mg/l Sigma L-proline 1.4 g/l Phytotechnology Labs casamino acids 100 mg/l Fisher Scientific sucrose 50 g/l Phytotechnology Labs 2,4-D (product number D7299) 1 ml/l (stock solution with a concentration of 1 mg/ml) The Sigma solution was adjusted to pH 5.8 with IN KOH/1N KCl, Gelrite (Sigma) was added at a concentration of up to 3 g/l and the medium was autoclaved. After cooling to 50° C., a 2 ml/l stock solution of silver nitrate at a concentration of 5 mg/ml (Phytotechnology Labs) is added. Example 6 Transformation of Plant Cells with the Genes of the Invention by Agrobacterium Mediated Transformation. Cobs are best harvested 8-12 days after pollination. Embryos are isolated from cobs and embryos 0.8-1.5 mm in size are preferably used for transformation. The embryos are plated upside down in a suitable incubation medium and incubated overnight at 25° C. in the dark. However, overnight incubation of embryos is not necessary as such. Embryos are contacted with an Agrobacterium strain containing vectors suitable for Ti-plasmid mediated transfer for about 5-10 minutes and then placed in co-culture medium for about 3 days (25° C. in the dark). After cocultivation, the explants are transferred to the medium for a recovery period of approximately five days (at 25°C in the dark). The explants are incubated in the selective medium for a period of eight weeks, depending on the nature and characteristics of the particular selection method used. After a selection period, the resulting callus is transferred to embryo maturation medium and cultured until mature somatic embryos are observed. The resulting mature somatic embryos are then placed in low light conditions and the regeneration process is initiated, as is known in the art. All publications and patent applications referred to in this specification are indicative of the skill of those skilled in the art to which the present invention belongs. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application is specifically and individually incorporated by reference. Although the foregoing invention has been described in some detail for purposes of illustration and also by way of example for clarity of understanding, it will be appreciated that certain changes and modifications may be made in practice within the scope of the appended claims. CLAIM 1. A recombinant nucleic acid molecule that contains a nucleotide sequence encoding an amino acid sequence that has pesticidal activity, characterized in that said nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 3; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 15-19; c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 15. 2. A recombinant nucleic acid molecule that contains a nucleotide sequence encoding an amino acid sequence that has pesticidal activity, characterized in that said nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 4; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 20-26; c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence - 25 042693 with the sequence of any of SEQ ID NOs: 20-26. 3. A recombinant nucleic acid molecule according to claim 1 or 2, characterized in that said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant. 4. A recombinant nucleic acid molecule according to claim 1 or 2, characterized in that said nucleotide sequence is operably linked to a promoter capable of directing the expression of said nucleotide sequence in a plant cell. 5. A vector containing a recombinant nucleic acid molecule according to claim 1 or 2. 6. The vector of claim 5, which further comprises a nucleic acid molecule encoding a heterologous polypeptide. 7. A host cell that contains a recombinant nucleic acid according to claim 1 or 2. 8. The host cell of claim 7, which is a bacterial host cell. 9. The host cell according to claim 7, which is a plant cell. 10. A transgenic plant that contains a host cell according to claim 9. 11. Transgenic plant according to claim 10, characterized in that said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane , tobacco, barley and oilseed rape. 12. Transgenic seed containing the nucleic acid molecule according to claim 1 or 2. 13. A recombinant pesticidal polypeptide selected from the group consisting of a) a polypeptide containing the amino acid sequence of any one of SEQ ID NOs: 15-19, and b) a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 15. 14. A recombinant pesticidal polypeptide selected from the group consisting of a) a polypeptide containing the amino acid sequence of any one of SEQ ID NOs: 20-26, and b) a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence of any of SEQ ID NOs: 20-26. 15. A polypeptide according to claim 13 or 14, which further comprises heterologous amino acid sequences. 16. A pesticidal composition containing a polypeptide according to claim 13 or 14. 17. Composition according to claim 16, characterized in that said composition is selected from the group consisting of powder, dust, pellet, granule, aerosol, emulsion, colloid and solution. 18. Composition according to claim 16, characterized in that said composition is obtained by drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of the bacterial cell culture. 19. The composition according to claim 16, containing a mass fraction of from about 1% to about 99% of the specified polypeptide. 20. A method for controlling a population of a lepidopteran, hemipteran, hemipteran, nematode, or dipteran pest, comprising contacting said population with a pesticidally effective amount of a polypeptide according to claim 13 or 14. 21. A method for controlling a lepidopteran, hemipteran, hemipteran, nematode, or dipteran pest, comprising bringing said pest into contact with or feeding said pest a pesticidally effective amount of a polypeptide according to claim 13 or 14. 22. A method for producing a polypeptide with pesticidal activity, comprising culturing a host cell according to claim 7 under conditions under which a nucleic acid molecule encoding the polypeptide is expressed. 23. A plant cell with a DNA construct stably integrated into its genome containing a nucleotide sequence that encodes a protein with pesticidal activity, characterized in that the specified nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 3; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 15-19; And c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 15. 24. A plant cell with a DNA construct stably integrated into its genome, containing a nucleotide sequence that encodes a protein with pesticidal activity, characterized in that the specified nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 4; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 20-26; And c) a nucleotide sequence that encodes a polypeptide containing an amino acid - 26 042693 sequence with at least 95% sequence identity with the amino acid sequence of any of SEQ ID NO: 20-26. 25. A method for protecting a plant from a pest, comprising the expression in a plant or its cell of a nucleotide sequence that encodes a pesticidal polypeptide, characterized in that said nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 3; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 15-19; And c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 15. 26. A method for protecting a plant from a pest, comprising the expression in a plant or its cell of a nucleotide sequence that encodes a pesticidal polypeptide, characterized in that said nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 4; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 20-26; And c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence of any of SEQ ID NOs: 20-26. 27. The method according to claim 25 or 26, wherein said plant produces a pesticidal polypeptide having pesticidal activity against a lepidopteran, hemipteran, beetle, nematode or dipterous pest. 28. A method for increasing the yield of a plant, which includes growing in the field a plant or its seed with a DNA construct stably integrated into its genome containing a nucleotide sequence that encodes a protein with pesticidal activity, characterized in that the specified nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 3; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 15-19; And c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, and said field is infested with a pest against which said polypeptide exhibits pesticidal activity. 29. A method for increasing the yield of a plant, which includes growing in the field a plant or its seed with a DNA construct stably integrated into its genome, containing a nucleotide sequence that encodes a protein with pesticidal activity, characterized in that the specified nucleotide sequence is selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 4; b) a nucleotide sequence that encodes a polypeptide containing the amino acid sequence of any of SEQ ID NOs: 20-26; And c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence with at least 95% sequence identity with the amino acid sequence of any of SEQ ID NO: 20-26, and said field is infested with a pest against which said polypeptide exhibits pesticidal activity. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2