CN111616159A

CN111616159A - Control of hemipteran insects using AXMI-011

Info

Publication number: CN111616159A
Application number: CN202010206316.1A
Authority: CN
Inventors: K.S.桑普森; D.莱赫蒂宁
Original assignee: Bayer CropScience LP
Current assignee: BASF SE; BASF Agricultural Solutions Seed US LLC
Priority date: 2013-11-25
Filing date: 2014-11-21
Publication date: 2020-09-04
Anticipated expiration: 2034-11-21
Also published as: PH12016500965A1; WO2015077525A1; CN105764343B; CN111616159B; CN105764343A

Abstract

The present invention relates to the control of hemipteran insects using AXMI-011. Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. In particular, methods of killing or controlling hemipteran pest populations, particularly planthopper pest populations, are provided. The methods comprise contacting the hemipteran pest with a pesticidally-effective amount of a polypeptide comprising a hemipteran toxin, particularly a planthopper toxin. Further included are methods of increasing yield in plants by expressing the toxins of the invention.

Description

Control of hemipteran insects using AXMI-011

The present application is a divisional application of the invention patent application entitled "control of hemipteran insects using AXMI-011" on application date 2014, 21/11, application No. 201480064309.5.

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application serial No. 61/908,392 filed on 25/11/2013, the contents of which are hereby incorporated by reference in their entirety.

Reference to electronically submitted sequence Listing

A formal copy of the ASCII formatted sequence list is submitted electronically via EFS-Web with a file name "APA 136053_ st25. txt", created on 11 months and 14 days 2014, with a file size of 9 kilobytes, and submitted concurrently with this specification. The sequence listing contained in this ASCII formatted document is part of this specification and is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to the field of molecular biology. Provided are novel genes encoding pesticidal proteins. These proteins and the nucleic acid sequences encoding them are useful in the preparation of pesticidal formulations and in the production of transgenic pest-resistant plants.

Background

Transgenic crops engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) were grown in 2010 around the world for 5800 more than ten thousand hectares (James C) (2010) Global Status of commercial biotechnology/transgenic crops (Global Status of commercial Biotech/GMCrops): 2010. isama abstract number 42. isaka, new york state: isama). Advantages of Bt crops include reduced regional suppression of the use of harmful insecticides and some important agricultural pests (Carrie re et al (2003) Proc Natl Acad Sci USA 100: 1519-.

Sap feeding insects, including aphids, whiteflies, lygus bugs and stink bugs, have become major agricultural pests. Hemiptera causes direct damage by eating crops and, in some cases, indirectly damage through the transmission of plant viruses. Current management relies almost exclusively on the application of traditional chemical pesticides. Although the development of transgenic crops expressing Bt-derived toxins provides effective plant protection against some pests, commercially available Bt toxins exhibit little toxicity to sap-feeding insects.

These insects, which were originally thought to be only unimportant or minor pests, have become major pests, in part, due to changes in agricultural practice such as increased use of transgenic and classically screened plant species resistant to major pests, and decreased application of chemical pesticides.

Several species of Lygus bugs (Lygus spp.) are the major agricultural pests, including western Lygus pratensis, Lygus legrinus (Lygus hesperus) knight, Lygus lineolaris, Lygus americanus (Lygus lineolaris), and Lygus lucorum (Lygus de Beauvois). Lygus hesperus and lygus pratensis are the major Pests of a wide range of agronomic and horticultural crops throughout the united states and canada (esyvalel and Mowery) (2007) environmental insects (environ. entomol.)36: 725-730; Wheeler (Wheeler a. g., Jr.) lygus (Hemiptera: lygus family) Biology of Pests, natural enemies, principals (Biology of the plantat bugs (Hemiptera: Pests, Predators, opanks. congkok Publishing company (Comstock Publishing Associates), sakaka, usa, cout et al (patent D.R.) (american society 1977) publication of american puzzling (ento. 19819; american lous.: 3579) insects (american society of america, 1987) 3519. Lygus has been reported to feed on 117 non-crop plants and over 25 cultivated plants.

Stink bugs include a complex of pests of critical significance affecting 12 major crops worldwide (McPherson J.E.), McPherson R.M., the economically important stink bugs in Mexico, North USA (StickBugs of Economic Improportion in America North of Mexico) CRC Press, Bocardon, Florida, 2000. More than 50 closely related species of stink bugs affect crops including fruits, vegetables, nuts, fiber, and grains. The most abundant and important species include green bugs ((e.g., (Acrosternum hirare); rice green bugs (nezaravicularia (L.))); and brown stinkbugs ((e.g., Euschistus servus). Stink bugs cause losses estimated at $6400 million in U.S. cotton in 2005 and $3100 million in 2008, while losses in soybeans (Glycine maxl. merrill) reach $1300 million (reyi-Jones f.p.f.) (2010) environmental insects (environ. entomol.)39: 944-.

Aphids are one of the most economically important pests of exclusive phloem consumers and temperate agriculture (Blackman R.L.) (2000) Aphids, Identification and Information Guide on world Crops (Aphids on the Crops, An Identification and Information Guide), John Wiley & Sons, Inc., New York, N.Y.). Aphids cause significant economic losses in almost all crops and account for a large proportion of 13% of the estimated losses in agricultural yield caused by insect Pests (emmden H.), Harrington (Harrington R.) aphid as a Crop pest (aphis as Crop pest), CABI press, london, uk 2007, p.717; Faria (Faria c.a.), Wackers (Wackers F.L.), priricharde (pricard J.), Barrett D.A ], turls (Turlings t.c.) Bt transgenic maize has a High sensitivity to Aphids that enhances the performance of lepidopteran pest parasites (High sensitivity of maize to Aphids of the pest parasites of the lepidopteran pest pair of ploss 2007: PLoS 2. 2007).

Two major pests of Rice, Brown Planthopper (BPH) (Brown Rice lice) and Rice black tail hopper (black tail hopper genus), cause serious physiological damage to Rice plants and are economically important vectors for the Rice dongfru, grass dwarf and ragged dwarf virus (pedigree et al (Mochida) (1979) Some of the Considerations of Screening Rice disease resistant varieties/Lines against Brown planthoppers (delvatales) (Delphacidae) (Some Considerations of Rice Considerations on Screening reagents cuvars/Lines of Rice Plant to the Brown Planthopper, nilava Lugens (holm., delphadae)). philippine ross: IRRI, multi.1-9; saxone, r.c. and Khan (Khan, Z.R) (1983.132), review of agriculture). A key limiting factor in the invention for increasing rice yield in south east asia is Brown Planthopper (BPH), which causes "hopper" and is a carrier of viral diseases. Several factors, including the ability of rice planthoppers to develop resistance to common pesticides and the ecological imbalance of predation versus predation caused by pesticide abuse in rice fields, lead to BPH ash death and reignition.

Disclosure of Invention

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. In particular, methods of killing or controlling hemipteran pest populations, particularly rice planthopper pest populations, are provided. The method comprises contacting a hemipteran pest with a pesticidally-effective amount of a polypeptide comprising a hemipteran toxin, particularly a planthopper toxin. In various embodiments, the hemipteran toxin comprises the amino acid sequence of SEQ ID No. 3 or 4, or a pesticidally effective variant or fragment thereof. In some embodiments, a method for protecting a plant or cell thereof from a hemipteran pest population, particularly a rice planthopper pest, comprises expressing in the plant or cell thereof a nucleic acid sequence encoding SEQ ID No. 3 or 4, or a variant or fragment thereof, wherein the nucleic acid is operably linked to a promoter capable of directing expression of the nucleic acid in a plant cell.

Further included is a method of increasing yield in a plant, the method comprising growing in a field a plant or seed thereof having stably integrated in its genome a DNA construct comprising a promoter operably linked to a promoter capable of directing expression of the nucleic acid in a plant, wherein the nucleic acid encodes SEQ ID No. 3 or 4, or a pesticidally effective variant or fragment thereof.

The compositions and methods of the present invention may be used in the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions include organisms for desirable agricultural uses.

Detailed Description

The present invention describes methods for modulating pest resistance or tolerance of an organism, particularly a plant or plant cell. By "resistant" is meant that the pest (e.g., insect) is killed upon absorption or contact with the polypeptide of the invention. "tolerance" refers to an impairment or reduction in the motility, feeding, reproduction, or other function of a pest. These methods involve transforming organisms with nucleotide sequences encoding pesticidal proteins of the invention. In particular, the nucleotide sequences of the present invention are useful for the preparation of plants and microorganisms having pesticidal activity. The methods described herein may be used to control or kill a hemipteran pest population and to produce compositions having pesticidal activity against hemipteran pests.

"pesticidal toxin" or "pesticidal protein" refers to a toxin that has toxic activity against one or more hemipteran pests, including, but not limited to, Lygus pratensis, such as western Lygus pratensis (Lygus legrinus), Lygus pratensis (Lygus americanus), and Lygus lucorum (Lygus elsus); aphids, such as the peach aphid (Myzus persicae), cotton aphid (Aphisgossypii), cherry aphid or black cherry aphid (Myzus cerasi), soybean aphid (Aphis vitamins Matsumura); brown rice planthopper (brown rice planthopper), rice leafhopper (black tail cicada), Sogatella furcifera and brown small brown planthopper (laodelphax striatellus); and stink bugs such as green stinkbug (lygus lucorum), brown marbled stinkbug (tea wing stinkbug), southern green stinkbug (Nezara viridula), rice stinkbug (american rice stinkbug), forest red stinkbug (Pentatoma rufipepes), european stinkbug (rhaphigater nebulosa), and stinkbugs bugs (Troilus lucidus). Pesticidal proteins include deduced amino acid sequences of the full-length nucleotide sequences of the disclosure, as well as amino acid sequences that are shorter than the full-length sequences, either due to the use of alternative downstream initiation sites, or due to processing to produce a shorter protein with pesticidal activity. Processing may occur in the organism in which the protein is expressed, or in the pest after absorption of the protein.

In particular embodiments, the pesticidal protein comprises the Axmi011 protein set forth in SEQ ID NO 3 or 4, as well as variants and fragments thereof.

Accordingly, provided herein are methods for killing or controlling a hemipteran pest population comprising contacting the pest, or exposing the pest to a composition comprising a pesticidal toxin of the present invention.

Isolated nucleic acid molecules and variants and fragments thereof

One aspect of the invention relates to isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding pesticidal proteins and polypeptides or biologically active portions thereof, and to nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins having regions of sequence homology. Also included herein are nucleotide sequences that hybridize to the nucleotide sequences of the present invention under the strict definitions defined elsewhere herein. The term "nucleic acid molecule" as used herein is intended to include both DNA molecules (e.g., recombinant DNA, cDNA, or genomic DNA) and RNA molecules (e.g., mRNA) as well as analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

An "isolated" or "recombinant" nucleic acid sequence (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, e.g., in vitro or in a recombinant bacterial or plant host cell. In some embodiments, an isolated or recombinant nucleic acid is a sequence (preferably a protein coding sequence) that does not naturally flank a nucleic acid (i.e., a sequence located 5 'and 3' to the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For the purposes of the present invention, isolated chromosomes are not included when "isolated" is used to describe nucleic acid molecules. For example, in various embodiments, an isolated-endotoxin encoding a nucleic acid molecule may comprise less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. In various embodiments, an endotoxin protein that is substantially free of cellular material includes a protein preparation having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-endotoxin protein (also referred to herein as a "contaminating protein").

The nucleotide sequence of the encoded protein of the invention comprises the sequences shown in SEQ ID NO 1 and 2, and variants, fragments and complements thereof. "complement" refers to a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence that it can hybridize to the given nucleotide sequence, thereby forming a stable duplex. The corresponding amino acid sequence for the pesticidal protein is encoded by the nucleotide sequence shown in SEQ ID NO 3 or 4.

The invention also includes nucleic acid molecules that are fragments of nucleotide sequences encoding pesticidal proteins. "fragment" refers to a portion of a nucleotide sequence that encodes a pesticidal protein. A fragment of the nucleotide sequence may encode a biologically active portion of a pesticidal protein, or it may be a fragment that is used as a hybridization probe or PCR primer using the methods described below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a pesticidal protein may include at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 consecutive nucleic acids, or up to the number of nucleotides present in the full-length nucleotide sequence encoding a pesticidal protein, depending on the desired use. "contiguous" nucleotides refer to nucleotide residues that are immediately adjacent to each other. The nucleotide sequence fragments of the present invention encode protein fragments that retain the biological activity of the pesticidal protein and, therefore, the pesticidal activity. Thus, biologically active fragments of the disclosed polypeptides are also included herein. By "retains 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 coleopteran killing. In another embodiment, the pesticidal activity is a lepidopteran activity. In another embodiment, the pesticidal activity is nematicidal activity. In another embodiment, the pesticidal activity is dipteran activity. In another embodiment, the pesticidal activity is hemipteran activity. Methods for measuring pesticidal activity are well known in the art. See, e.g., Czapla and Lang (1990) journal of insects of the economic nature (J.Econ. Entomol.)83: 2480-2485; andrews (Andrews) et al (1988) journal of biochemistry (biochem. J.)252: 199-; maloen (Marrone) et al (1985) journal of Economic insects 78: 290-293; and U.S. Pat. nos. 5,743,477; and Heong et al (2013) Research methods for the monitoring of rice planthoppers with respect to toxicological and insecticidal resistance, Research methods in toxicological and insecticidal resistance monitoring, second edition, Philippine Rosesnei Olympus, International Rice Research institute, the contents of which are hereby incorporated by reference in their entirety.

A fragment of a nucleotide sequence encoding a pesticidal protein encodes a biologically active portion of a protein of the invention, which fragment encodes at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300 consecutive amino acids, or up to the number of all amino acids present in a full-length pesticidal protein of the invention. In some embodiments, the fragment is a proteolytically cleavable fragment. For example, a proteolytically cleavable fragment 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 amino acids, at least about 100 amino acids, at least about 120, about 130, about 140, about 150, about 160 amino acids involving SEQ ID NO 3 or 4. In some embodiments, fragments encompassed herein are the result of removal of the C-terminal crystallization region, e.g., by proteolysis, or by insertion of a stop codon in the coding sequence. In some embodiments, the fragments encompassed herein are the result of removal of the N-terminal signal peptide (e.g., SEQ ID NO: 4). The N-terminal truncation may further comprise a methionine at the N-terminus.

Preferred pesticidal proteins of the invention are encoded by nucleotide sequences that are sufficiently identical to the nucleotide sequences of SEQ ID NO. 1-2, or pesticidal proteins that are sufficiently identical to the amino acid sequences shown in SEQ ID NO. 3 or 4. "sufficiently identical" refers to 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 more sequence identity to a reference sequence using alignment procedures described herein using standard parameters. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by the two nucleotide sequences by 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 of two nucleic acids is a function of the number of identical positions shared by the sequences (i.e., percent identity-the number of identical positions/total number of positions (e.g., overlapping positions) × 100). In one embodiment, the two sequences have the same length. In another embodiment, percent identity is calculated across the entire reference sequence (e.g., the sequence of any one of SEQ ID NOs: 1-3 disclosed herein). The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, exact matches are typically calculated. A gap (i.e., a position in an alignment in which a residue is present in one sequence but not in the other) is considered to be a position having non-identical residues.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm in Carlin (Karlin) and Alchuru (Altschul) (1990) Proc. Natl. Acad. Sci. USA 87:2264, as amended in Carlin (Karlin) and Alchuru (Altschul) (1993) Proc. Natl. Acad. Sci. Am.)90: 5873-. Such an algorithm is incorporated into the BLASTN and BLASTX programs, which are described in Archie (Altschul) et al (1990) journal of molecular biology (J.mol.biol.)215: 403. BLAST nucleotide searches can be performed using the BLASTN program (score 100, word length 12) to obtain nucleotide sequences homologous to the pesticidally-analogous nucleic acid molecules of the invention. BLAST protein searches can be performed using the BLASTX program (score 50, word length 3) to obtain amino acid sequences homologous to the pesticidal protein of the present invention. For purposes of obtaining gap alignment, Gapped BLAST (in BLAST 2.0) can be used, as described by Altschul et al (1997) Nucleic Acids research (Nucleic Acids Res.)25: 3389. (Altschul et al, (1997) Nucleic Acids Res.25:3389) Gapped BLAST can be used as described. Alternatively, PSI-Blast can be used for iterative search to detect distant relationships between molecules. See altuchul et al (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection.

Another non-limiting example of a mathematical algorithm for comparing sequences is the ClustalW algorithm (Seggins et al (1994): Nucleic Acids research 22: 4673-4680). ClustalW compares sequences and aligns the entirety of amino acid or DNA sequences, and thus can provide data on sequence conservation for the complete amino acid sequence. The ClustalW algorithm has been used in several commercially available DNA/amino acid analysis software packages, such as the align x module of the vector NTI program group (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences using ClustalW, percent amino acid identity can be assessed. A non-limiting example of a software program that can be used to analyze the ClustalW alignment is GENEDOC^TM。GENEDOC^TM(Karl Nicholas) allow the evaluation of amino acid (or DNA) similarity and identity between multiple proteins. For comparison of sequencesAnother non-limiting example of a mathematical algorithm listed is the algorithm of computer application 4:11-17 in Miller and Meiers (1988) biosciences (Myers and Miller (1988) CABIOS 4: 11-17). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, version 10 (available from Accelrys, Inc., of 9685 Schrandon, san Diego, Calif., USA). When comparing amino acid sequences using the ALIGN program, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

GAP version 10 uses the Needleman and Wunsch algorithms (Needleman and Wunsch (1970) journal of molecular biology (J.mol.biol.)48(3): 443. sup. 453. 443. will use the parameters of% identity and% similarity of nucleotide sequences using the GAP weight of 50, the length weight of 3, and the nwsgapdna. cmp scoring matrix, the% identity or% similarity of amino acid sequences using the GAP weight of 8, the length weight of 2, and the BLOSUM62 scoring program.

The invention also encompasses various nucleic acid molecules. "variants" of a pesticidal protein encoding a nucleotide sequence include those sequences disclosed herein that encode pesticidal proteins but which differ conservatively because of the degeneracy of the genetic code, and those of sufficient identity as discussed above. Naturally occurring allelic variants can be identified by using well known molecular biology techniques, such as Polymerase Chain Reaction (PCR) and hybridization techniques as outlined below. Different nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis, but that still encode the pesticidal proteins of the present disclosure as discussed below. The different proteins encompassed by the present invention have biological activity, i.e. they continue to possess the desired biological activity of the native protein, i.e. pesticidal activity. By "retains 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, e.g., Czapla and Lang (1990) journal of insects of the economic nature (J.Econ. Entomol.)83: 2480-2485; andrews (Andrews) et al (1988) journal of biochemistry (biochem. J.)252: 199-; maloen (Marrone) et al (1985) journal of Economic insects 78: 290-293; and U.S. Pat. nos. 5,743,477; and Heong et al (2013) Research methods for the monitoring of rice planthoppers with respect to toxicological and insecticidal resistance (Research methods in toxicological and insecticidal resistance monitoring of rice planthoppers), second edition, Philippines Rossinian Oerson: the international rice institute, the contents of which are hereby incorporated by reference in their entirety.

It will be further understood by those skilled in the art that changes may be introduced by mutation of the nucleotide sequences of the present invention, thereby resulting in a change in the amino acid sequence of the encoded pesticidal protein, without altering the biological activity of the protein. Thus, variants of an isolated nucleic acid molecule can be created by introducing one or more nucleotide substitutions, additions, deletions into the corresponding nucleotide sequences disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. These nucleotide sequence variants are also encompassed by the present invention.

For example, conservative amino acid substitutions may be made at one or more predicted insignificant amino acid residues. An "unimportant" amino acid residue is one that can be altered from the wild-type sequence of the pesticidal protein without altering the biological activity, where the "important" amino acid residue is essential for biological activity. A "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following side chains: 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 (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

Amino acid substitutions may be made in non-conserved regions that retain function. In general, such substitutions will not be made for conserved amino acid residues, or amino acid residues that reside in conserved motifs, which are important for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of toxins similar or related to the sequences of the invention (e.g., residues that have only conservative substitutions between all proteins contained in homologous proteins of the alignment). However, one skilled in the art will appreciate that functional variations may have slight conservative or non-conservative changes in conserved amino acid residues.

Alternatively, variant nucleotide sequences may be generated by randomly introducing mutations along all or part of the coding sequence, for example by saturation mutagenesis, and among the resulting mutants, mutants capable of conferring pesticidal activity may be screened to identify mutants that retain activity. Following mutagenesis, the encoded protein may be expressed recombinantly and the activity of the protein may be determined by standard assay techniques.

Corresponding pesticidal sequences may be identified using methods such as PCR, hybridization, and the like, such sequences being substantially identical to the sequences of the invention. See, e.g., Sambrook and Russell (2001) molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual) (Cold spring harbor Laboratory Press, Cold spring harbor, N.Y.), and Innis et al (1990) PCR protocols: guidance for methods and Applications (PCR Protocols: A Guide to methods and Applications) (Academic Press, New York).

In the hybridization method, all or part of the pesticidal nucleotide sequence may be used to screen a cDNA or genomic library. Methods for constructing these cDNA and genomic libraries are generally known in the art and are disclosed above in Sambrook and Russell,2001 (Sambrook and Russell, 2001). The hybridization probe may be a genomic DNA fragment, cDNA fragment, RNA fragment or other oligonucleotide, and may be labeled with a detectable group (e.g., a probe labeled with a probe-specific probe, a³²P) or any other detectable label, such as other radioisotopes, fluorescent compounds, enzymes or enzyme cofactors. Probes for hybridization can be made by labeling synthetic oligonucleotides based on known pesticidal protein-encoding nucleotide sequences disclosed herein. In addition, degenerate primers designed based on nucleotides or amino acid residues that are conserved in the nucleotide sequence or encoded amino acid sequence may be used. Probes typically comprise a nucleotide sequence region 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, or fragments or variants thereof, of a nucleotide sequence of the present invention encoding a pesticidal protein. Methods of preparation of hybridization probes are generally known in the art and are disclosed above in Sambrook and russell,2001 (Sambrook and russell,2001), which is incorporated herein by reference.

For example, the entire pesticidal sequence disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to a corresponding pesticidal protein-like sequence and messenger RNA. To achieve specific hybridization under a variety of conditions, these probes comprise unique, and preferably at least about 10 nucleotides long or at least about 20 nucleotides long sequences. These probes can be used to amplify the corresponding pesticidal sequences from a selected organism by PCR. This technique can be used to isolate additional coding sequences from an organism of interest, or as a diagnostic test to determine whether coding sequences are present in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (plaques or clones; see, e.g., Sambruk (Sambrook) et al (1989) Molecular Cloning: A laboratory Manual (2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.).

Thus, the invention includes probes for hybridization, as well as nucleotide sequences capable of hybridizing to all or a portion of a nucleotide sequence of the invention (e.g., at least about 300 nucleotides, at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or up to the full-length nucleotide sequences disclosed herein). Hybridization of these sequences can be performed under stringent conditions. "stringent conditions" or "stringent hybridization conditions" refer to conditions under which a probe will hybridize to its target sequence to a greater extent than to other sequences (e.g., at least two-fold greater than background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow for some mismatches in the sequence, allowing for lower similarity to be detected (heterologous probing). Generally, probes are less than about 900 nucleotides in length, preferably less than 450 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5M Na ion, usually about 0.01 to 1.0M Na ion concentration (or other salt) at a pH of 7.0 to 8.3 and the temperature of the short probes (e.g., 10 to 50 nucleotides) is at least about 30 ℃ and the temperature of the long probes (e.g., greater than 50 nucleotides) is at least about 60 ℃. Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with 30% -35% formamide buffer, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ℃, and washing in 1X to 2X SSC (20X SSC ═ 3.0M NaCl/0.3M trisodium citrate) at 50 ℃ to 55 ℃. Exemplary moderately stringent conditions include hybridization in 40% -45% formamide, 1.0M NaCl, 1% SDS at 37 ℃ and washing in 0.5X to 1X SSC at 55 ℃ to 60 ℃. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37 ℃ and washing in 0.1 XSSC at 60 ℃ to 65 ℃. The wash buffer may comprise about 0.1% to about 1% SDS. Typically, the duration of hybridization is less than about 24 hours, typically from about 4 to about 12 hours.

Specificity typically refers to the function of post-hybridization wash, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybridization, T_mIt can be roughly estimated from the equation for the analysis of biochemistry 138:267-284(Meinkoth and Wahl (1984) anal. biochem.138:267-284) by Menixx and Wall (1984): t is_m81.5 ℃ +16.6(log M) +0.41 (GC%) -0.61 (% form) -500/L; where M is the molar concentration of monovalent cations, GC% is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of hybridization in a base pair. T is_mIs the temperature (at defined ionic strength and pH) at which 50% of the complementary target sequences hybridize to the perfectly matched probe. 1% per mismatch, T_mA reduction of about 1 ℃; thus, T can be adjusted_mHybridization and/or washing conditions to hybridize to sequences of the desired identity. For example, if a sequence with an identity of > 90% is obtained, T_mThe reduction by 10 c is possible. Generally, stringent conditions are selected to be more than the thermodynamic melting point (T) of the specific sequence and its complement at a defined ionic strength and pH_m) About 5 deg.c lower. However, highly stringent conditions may be at the specific thermodynamic melting point (T)_m) Hybridization and/or washing at 1 deg.C, 2 deg.C, 3 deg.C or 4 deg.C; moderately stringent conditions may be at the specific thermodynamic melting point (T)_m) Hybridization and/or washing at 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C or 10 deg.C lower;the low stringency conditions can be at the specific thermodynamic melting point (T)_m) Hybridization and/or washing is carried out at 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃ or 20 ℃. Using equation, hybridization, washing compositions and predicted T_mOne of ordinary skill will understand that variations in stringency of hybridization and/or wash conditions are essentially described. If the expected degree of mismatch results in T_mBelow 45 ℃ (aqueous solution) or 32 ℃ (formamide solution), it is preferable to increase the SSC concentration so that higher temperatures can be used. Extensive guidelines for Nucleic Acid Hybridization can be found in dijkson (1993) biochemical and molecular Biology experimental Techniques — Nucleic Acid probe Hybridization Part I, Chapter 2 (New York, einzel publishers) (Tijssen (1993) Laboratory Techniques in Biochemistry and molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York)); and, Osubel et al (1995) modern molecular Biology techniques, Chapter 2(Green groin Press and Wei interchange science Press, N.Y.) (Ausubel et al, eds. (1995) Current Protocols in molecular Biology, Chapter 2(Greene Publishing and Wiley-Interscience, New York.) see Aluchu et al (Altschul) (1989) molecular cloning: A Laboratory Manual (2 nd edition, Cold spring harbor Laboratory Press, Cold spring harbor, N.Y.).

Isolated proteins and variants and fragments thereof

Pesticidal proteins are also included in the present invention. "pesticidal protein" refers to a protein having the amino acid sequence shown in SEQ ID NO. 3 or 4. Fragments of biologically active portions and variants thereof are also provided and can be used to practice the methods of the invention. An "isolated protein" or "recombinant protein" is used to refer to a protein that is no longer in its natural environment, e.g., in vitro or in a recombinant bacterial or plant host cell.

"fragments" or "biologically active portions" include polypeptide fragments comprising an amino acid sequence having sufficient identity to the amino acid sequence set forth in SEQ ID NO 3 or 4 and exhibiting pesticidal activity. The biologically active portion of a pesticidal protein can be a polypeptide, i.e., for example, 10, 25, 50, 100, 150, 200, 250, 300, 350 or more amino acids in length. Such biologically active moieties may be prepared by recombinant techniques and used for the assessment of pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, e.g., Czapla and Lang (1990) journal of insects of the economic nature (J.Econ. Entomol.)83: 2480-2485; andrews (Andrews) et al (1988) journal of biochemistry (biochem. J.)252: 199-; maloen (Marrone) et al (1985) journal of Economic insects 78: 290-293; and U.S. Pat. nos. 5,743,477; and Heong et al (2013) Research methods for monitoring rice planthoppers for toxicological and pesticide resistance (Research methods and pesticide resistance monitoring of rice planthoppers), second edition, Philippines Rosebiana Oerson: the international rice institute, the contents of which are hereby incorporated by reference in their entirety. As used herein, a fragment includes at least 8 contiguous amino acids of SEQ ID NO 3 or 4. The invention includes other fragments, however, such as any fragment of a protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300 or more amino acids in length.

A "variant" refers to a protein or polypeptide 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 SEQ ID NO 3 or 4. Variants also include polypeptides encoded by nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecules of SEQ ID Nos. 1-2 or their complementary strands. Variants include polypeptides that differ in amino acid sequence due to mutation. The variant proteins encompassed by the present invention are biologically active, i.e., they continue to possess the biological activity expected of the native protein, i.e., retain pesticidal activity. In some embodiments, the variant has improved activity relative to the native protein. Methods for measuring pesticidal activity are well known in the art. See, e.g., Czapla and Lang (1990) journal of insects of the economic nature (J.Econ. Entomol.)83: 2480-2485; andrews (Andrews) et al (1988) journal of biochemistry (biochem. J.)252: 199-; maloen (Marrone) et al (1985) journal of economic insects (J. of Economatic Entomology)78: 290-293; and U.S. Pat. nos. 5,743,477; and Heong et al (2013) Research methods for the monitoring of rice planthoppers with respect to toxicological and pesticide resistance (Research methods in toxicological and insecticidal resistance monitoring of rice planthoppers), second edition, Philippine Rossinian Oerson: the international rice institute, the contents of which are hereby incorporated by reference in their entirety.

Bacterial genes, such as the axmi gene of the invention, typically possess multiple methionine start codons near the start of the open reading frame. Typically, initiation of translation at one or more of these initiation codons will result in the production of a functional protein. These initiation codons may include ATG codons. However, bacteria such as Bacillus (Bacillus sp.) also recognize GTG as the start codon, and the protein that initiates translation at the GTG codon includes a methionine at the first amino acid. In rare cases, translation in bacterial systems may be initiated at the TTG codon, although in this case TTG encodes methionine. Furthermore, it is often uncertain which of these codons was inherently utilized in bacteria. It is therefore understood that the use of an alternative methionine codon may also result in the production of pesticidal proteins. These pesticidal proteins are included in the present invention and may be utilized in the methods of the present invention. It will be appreciated that when expressed in plants, it may be necessary to alter the alternative initiation codon to ATG for correct translation.

In various embodiments of the invention, pesticidal proteins include amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, as well as amino acid sequences that are shorter than the full-length sequences due to the use of alternate downstream initiation sites.

Antibodies to the polypeptides of the invention, or variants or fragments thereof, are also included. Methods for producing Antibodies are well known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,196,265).

Accordingly, one aspect of the invention relates to antibodies, single chain antigen binding molecules, or other proteins that specifically bind to one or more of the protein or peptide molecules of the invention and homologs, fusions, or fragments thereof. In a particularly preferred embodiment, the antibody specifically binds to an amino acid sequence having the sequence shown in SEQ ID NO 3 or 4 or a fragment thereof. In another embodiment, the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the amino acid sequences set forth in SEQ id nos 3 or 4, or fragments thereof.

The antibodies of the invention may be used to detect the protein or peptide molecules of the invention quantitatively or qualitatively, or to detect post-translational modifications of the protein. As used herein, an antibody or peptide is said to "specifically bind" to a protein or peptide molecule of the invention if binding is not competitively inhibited by the presence of the unrelated molecule.

Altered or improved variants

It will be appreciated that the DNA sequence of a pesticidal protein may be altered by various means, and that such alterations may result in DNA sequences encoding proteins having amino acid sequences that differ from the amino acid sequence encoded by the pesticidal protein of the present invention. Such proteins may be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions of one or more of the amino acids in SEQ ID NOs 3 or 4, 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 insertions. Methods of doing so are well known in the art. For example, amino acid sequence variants of pesticidal proteins may be prepared by mutations in DNA. This may be done by one of several forms of mutation and/or in direct evolution. In some aspects, the changes encoded in the amino acid sequence will not materially affect the function of the protein. Such variants will possess the desired pesticidal activity. However, it will be appreciated that the ability of pesticidal proteins to confer pesticidal activity may be improved by the use of such techniques on the compositions of the present invention. For example, pesticidal proteins, such as XL-1Red (Stratagene, La Jolla, Calif.) can be expressed in host cells that exhibit a high proportion of base misinsertions during DNA replication. After propagation in such strains, the DNA may be isolated (e.g., by preparing plasmid DNA, or PCR fragments produced by PCR and clonal amplification in a vector), the pesticidal protein mutants cultured in non-mutant strains, and the mutant genes identified as having pesticidal activity, e.g., by conducting an experiment to test pesticidal activity. Typically, the proteins are mixed and used in feeding trials. See, for example, Marron et al (1985) J.Economic Entomology 78: 290-293. Such testing may include contacting one or more pests with a plant and determining the survival of the plant and/or the ability to cause death of the pest. Examples of mutations that result in increased toxicity are described in Snell (Schnepf) et al (1998) review of microbial molecular biology (Microbiol. mol. biol. Rev.)62: 775-806.

Alternatively, it is also possible to alter the protein sequence at the amino or carboxy terminus of many proteins without substantially affecting activity. This may include the introduction of insertions, deletions or alterations by modern molecular biological methods such as PCR, including PCR amplification, which alters or extends the protein coding sequence by virtue of the inclusion of amino acid coding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the added protein sequence may include the coding sequence of the entire protein, such as those commonly used in the art to produce protein fusions. Such fusion proteins are commonly used (1) to increase the expression of a protein of interest (2) to introduce binding domains, enzymatic activities, or epitopes to facilitate protein purification, protein detection, or other experimental uses known in the art (3) target secretion or protein translation to subcellular organelles, such as the periplasmic space of gram-negative bacteria, or the endoplasmic reticulum of eukaryotes, which typically results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the invention also include sequences derived from mutant and recombinant genetic programs such as DNA shuffling. By such a procedure, one or more different pesticidal protein encoding regions may be used to create a new pesticidal protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from populations of related sequence polynucleotides comprising regions of sequences that have substantial sequence identity and that can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding domains of interest can be shuffled between the pesticidal genes of the invention and other known pesticidal genes to obtain new genes encoding proteins with improved properties of interest, such as increased pesticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Schlemmer (Stemmer) (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; schlemur (Stemmer) (1994) Nature 370: 389-391; crameri et al (1997) Nature Biotech 15: 436-; moore et al (1997) journal of molecular biology 272:336- & 347); zhang et al (1997) Proc. Natl. Acad. Sci. USA 94: 4504-; crameri et al (1998) Nature 391: 288-291; and U.S. Pat. nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for producing altered pesticidal proteins. The domains may be exchanged between pesticidal proteins resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectra. Methods for producing recombinant proteins and testing their pesticidal activity are well known in the art (see, e.g., Naimov et al (2001) applied and environmental microbiology (Appl. Environ. Microbiol.)67: 5328-5330; de Maagd et al (1996) applied and environmental microbiology (Appl. Environ. Microbiol.)62: 1537-1543; Ge et al (1991) journal of molecular biology (J.Mol.Chem.)266: 17954-17958; Sneptff (Schnepf) et al, (1990) journal of molecular biology (J.Mol.Chem.)265: 20923-20930; Rang et al, 91999) applied and environmental microbiology (Appl. Environ.Biol.Chem.) 298: 2925).

Carrier

The pesticidal sequences of the present invention may be provided in an expression cassette for expression in a plant of interest. By "plant expression cassette" is meant a DNA construct capable of causing expression of a protein from an open reading frame in a plant cell. Typically these include promoters and coding sequences. These constructs also typically contain a 3' untranslated region. These constructs may contain a "signal sequence" or "leader sequence" to facilitate co-or post-translational transport of the peptide to certain intracellular structures, such as the chloroplast (or other plastid), endoplasmic reticulum, or golgi apparatus.

"Signal sequence" refers to a sequence that is known or that may result in co-translational or post-translational peptide transport across a cell membrane. In eukaryotic cells, it typically involves secretion into the golgi apparatus, some of which can lead to glycosylation. Bacterial insecticidal toxins are generally synthetic toxins which hydrolyze protein activation in the intestine of target pests (Chang (1987) Methods in enzymology 153: 507-. In some embodiments of the invention, the signal sequence is in a native sequence, or may be derived from a sequence of the invention. "leader sequence" refers to a sequence that, when translated, produces an amino acid sequence sufficient to trigger cotranslational transport of the peptide chain to subcellular organelles. Thus, it includes leader sequences that target transport and/or glycosylation by entry into the endoplasmic reticulum, the vacuolar channel, including the plastids of chloroplasts, mitochondria, and the like.

"plant transformation vector" refers to a DNA molecule necessary for the efficient transformation of plant cells. Such molecules may include one or more plant expression cassettes, and may be organized into more than one "vector" DNA molecule. For example, a binary vector is a Plant transformation vector that encodes all the necessary cis-and trans-acting functions for transforming Plant cells using 2 non-contiguous DNA vectors (Hellens and Mullinkes (2000) Trends in Plant Science,5: 446-. "vector" refers to a nucleic acid construct designed to be transferred between different host cells. "expression vector" refers to a vector capable of binding, integrating and expressing a heterologous DNA sequence or fragment in a foreign cell. The cassette will include 5 'and/or 3' regulatory sequences operably linked to the sequences of the invention. "operably linked" refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame, although this is not always the case. In addition, the cassette may contain at least one additional gene to be co-transformed into an organism. Alternatively, additional genes may be provided on multiple expression cassettes.

In various embodiments, the nucleotide sequence of the present invention is operably linked to a promoter, e.g., a plant promoter. "promoter" refers to a nucleic acid sequence that functions to direct the transcription of a downstream coding sequence. These elements, along with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "control sequences"), are necessary for expression of the DNA sequence of interest.

Such expression cassettes are provided with multiple restriction sites for insertion of pesticidal sequences under the transcriptional control of the regulatory regions.

The expression cassette will include the functions of a transcription, transcription and translation initiation region (i.e., promoter), a DNA sequence of the present invention, and a translation and transcription termination region (i.e., termination region) in the 5'-3' direction in plants. The promoter may be native or analogous, or foreign or heterologous to the plant host and/or DNA sequence of the invention. In addition, the promoter may be a natural sequence or alternatively a synthetic sequence. When a promoter is "native" or "homologous" to a plant host, it means that the promoter is found in the native plant into which it is introduced. When a promoter is "foreign" or "heterologous" to a DNA sequence of the invention, it is intended that the promoter is not native or naturally occurring to the operably linked DNA sequence of the invention.

The termination region may be native to the transcriptional initiation region, may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host, or any combination thereof). Suitable termination regions may be obtained from the Ti-plasmid of Agrobacterium tumefaciens, for example the octopine synthase and nopaline synthase termination regions. See also Guerineau et al (1991) journal of molecular Gene and genetics (J.mol.Gen.Genet.262: 141. sup.144; Proudfoot (1991) Cell 64: 671. sup.674; Sanfacon et al (1991) Gene development (Genes Dev.)5: 141. sup.149; MMogen et al (1990) Plant Cell 2: 1261. sup.1272; Munroe et al (1990) Gene 91: 151. sup.158; Balas et al (1989) Nucleic Acid research (Nucleic Acids Res.)17: 7891. sup.7903; and Joshi et al (1987) Nucleic Acid research (Nucleic Acids Res. 15: 9639. sup.9639).

Where appropriate, one or more genes may be optimized for increased expression in the transformed host cell. That is, the gene may be synthesized using codons preferred by the host cell to improve expression, or may be synthesized using codons according to the codon usage frequency preferred by the host. Generally, the GC content of the gene will increase. See, e.g., Campbell and Gowri (1990) Plant physiology 92:1-11 for discussion of host preferred codon usage. Methods for synthesizing plant-preferred genes are known in the art. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, U.S. patent application No. 20090137409, and Murray et al (1989) Nucleic acids Res (Nucleic acids) 17:477-498, incorporated herein by reference.

In one embodiment, the pesticidal protein is expressed targeted to a chloroplast. In this way, when the pesticidal protein is not inserted directly into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the pesticidal protein to the chloroplast. These transit peptides are known in the art. See, for example, Von Heijne et al (1991) Plant molecular biology guide (Plant mol. biol. Rep.)9: 104-126; clark et al (1989) journal of molecular biology (J.mol.Chem.264: 17544-17550; Della-Cioppa et al (1987) Plant physiology (Plant physiology.) 84: 965-968; Romer et al (1993) communication of biochemistry and biophysical studies (biochem.Biophys.Res.Commun.)196: 1414-1421; and Shah et al (1986) Science (Science)233: 478-481.

Pesticidal genes targeted to the chloroplast can be optimized for expression in chloroplasts to account for differences in codon usage between the plant nucleus and the organelle. In this manner, chloroplast-preferred codons can be used to synthesize a nucleic acid of interest. See, for example, U.S. Pat. No. 5,380,831, which is incorporated herein by reference.

Plant transformation

The methods of the invention involve introducing a nucleotide construct into a plant. By "introducing" is meant providing the nucleotide construct to the plant in a manner such that the construct is able to reach the interior of the plant cell. The methods of the invention do not require the use of a particular method for introducing the nucleotide construct into the plant, but only require that the nucleotide construct be able to reach the interior of at least one cell of the plant. Methods known in the art for introducing nucleotide constructs into plants include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

"plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, vegetative propagules, embryos, and progeny thereof. Plant cells may be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

A "transgenic plant" or "transformed plant" or "stably transformed" plant or cell or tissue refers to a plant into which an exogenous nucleic acid sequence or DNA fragment has been incorporated or integrated into the plant cell. These nucleic acid sequences include those sequences that are not present in the exogenous or untransformed plant cell, as well as those sequences that may be endogenous or already present in the untransformed plant cell. "heterologous" generally refers to a nucleic acid sequence that is not endogenous to the cell or a portion of the native genome in which it is located and has been added to the cell by infection, transfection, microinjection, electroporation, micrographs, and the like.

The transgenic plants of the invention express one or more of the novel toxin sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes that are insect-resistant (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, and Cry1F families; Cry2, such as members of the Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.). One skilled in the art will appreciate that a transgenic plant may include any gene capable of expressing an agronomic trait of interest.

Transformation of plant cells can be accomplished by one of a variety of techniques known in the art. The pesticidal gene of the present invention may be modified to obtain or enhance expression in a plant cell. Typically, constructs expressing such proteins will contain a promoter to drive gene transcription and a 3' untranslated region to terminate transcription and polyadenylation. The organization of these constructs is well known in the art. In some cases, it may be useful to design genes such that the peptides produced thereby are secreted or otherwise targeted for localization within a plant cell. For example, the 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 process the plant expression cassette to contain an intron such that its expression requires mRNA processing of the intron.

The "plant expression cassette" will typically be inserted into a "plant transformation vector". The plant transformation vector may include one or more DNA vectors required to effect plant transformation. For example, it is common practice in the art to use plant transformation vectors that include more than one contiguous DNA segment. These vectors are commonly referred to in the art as "binary vectors". Binary vectors as well as vectors with helper plasmids are most commonly used for agrobacterium-mediated transformation, where the size and complexity of the DNA fragments required to achieve efficient transformation are very large and it is advantageous to isolate functions on isolated DNA molecules. Binary vectors typically contain a plasmid vector, a selectable marker, and a "gene of interest"; plasmid vectors contain cis-acting sequences required for T-DNA transfer (e.g., left and right borders); the selectable marker is designed to be expressed in a plant cell; by "gene of interest" is meant a gene that is designed to be expressed in plant cells in order to form a generation of transgenic plants. Sequences required for bacterial replication are also present on this plasmid vector. The cis-acting sequence is arranged in a suitable manner to allow efficient transfer into and expression in a plant cell. For example, a selectable marker gene and a pesticidal gene are located between the left and right borders. Typically, the second plasmid vector contains trans-acting factors that mediate T-DNA transfer from the Agrobacterium to the plant cell. This plasmid usually contains a virulence (viral gene), causes infection of Plant cells by Agrobacterium, and allows DNA transfer by cleavage at border sequences and virus-mediated DNA transfer, as is known in the art (Hellen and Mulleneaux (2000) Plant Science,5: 446-. Several types of agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) may be used for plant transformation. For transformation of plants by other means, such as micrographs, microinjection, electroporation, polyethylene glycol, and the like, a second plasmid vector is not necessary.

Generally, plant transformation methods involve the transfer of heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by the application of a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover transformed plant cells from a set of untransformed shaped cell clumps. Typically, explants are transferred to a fresh supply of the same medium and cultured routinely. Subsequently, transformed cells are differentiated into shoots after being placed on regeneration medium supplemented with a maximum threshold level of a selection agent. The shoots are then transferred to a selective rooting medium for recovery of rooted shoots or plantlets. The transgenic plants are then grown to mature plants and fertile seeds are produced (e.g.Hiei et al (1994) Plant Journal 6: 271-282; Ishida et al (1996) Natural Biotechnology 14: 745-750). Typically, explants are transferred to a fresh supply of the same medium and cultured routinely. General descriptions of techniques and methods for generating transgenic plants are found in the Plant Science reviews by Els and Park (1994) 13:219-239(Ayres and Park (1994) clinical reviews in Plant Science 13:219-239) and Bominini and fur Ha (1997) maize 42:107-120(Bommineni and Jauhar (1997) Maydica 42: 107-120). Since the transformed material contains many cells; both transformed and untransformed cells are present in any piece of callus or tissue or cell population of interest. The untransformed cells are killed and the transformed cells are allowed to proliferate to produce a transformed plant culture. Generally, the ability to remove untransformed cells limits the rapid recovery of transformed plant cells and the successful production of transgenic plants.

The transformation methods and methods of introducing nucleotide sequences into plants can vary with the type of plant or plant cell targeted for transformation, i.e., monocots or dicots. Transgenic plants can be generated by one of a number of methods including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA into plant cells by agrobacterium (agrobacterium-mediated transformation), bombardment of plant cells with heterologous exogenous DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation (U.S. published application No. 20010026941, U.S. patent No. 4,945,050, international publication No. WO91/00915, U.S. published application No. 2002015066), Lec1 transformation, and other different non-particulate direct-mediated methods of transforming DNA.

Methods for transforming chloroplasts are known in the art. See, e.g., Svab et al (1990) Proc. Natl. Acad. Sci. USA 87: 8526-; svab and Maliga (1993) journal of national academy of sciences of the United states (Proc. Natl. Acad. Sci. USA)90:913 + 917; svab and Maliga (1993) journal of European molecular biology organization (EMBO J.)12: 601-606. The method relies on gene gun delivery of DNA containing a selectable marker and targeting of the DNA into the plastid genome by homologous recombination. In addition, trans-activation of silent plastid carrier transgenes is achieved through tissue-preferred expression of nuclear-encoded, plastid-localized RNA polymerase, and plastid transformation can be accomplished. Such a system has been reported in McBride et al (1994) Proc. Natl. Acad. Sci. USA 91: 7301-.

After integration of the heterologous foreign DNA into the plant cells, a maximum threshold level is then applied in the selection medium to kill the untransformed cells, and by periodically transferring into fresh medium, the cells that can survive the selection process and be presumed to have been transformed are isolated and propagated. Cells transformed with the plasmid vector can be identified and propagated by serial passage and challenged with the appropriate selection. Molecular and biochemical methods can then be used to confirm whether the heterologous gene of interest is integrated into the genome of the transgenic plant.

The cells which have been transformed can be cultured into plants according to a conventional method. See, for example, McCormick et al (1986) Plant Cell Reports 5: 81-84. These plants are then grown and pollinated with the same transformed line or a different line, and the resulting hybrid has constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved. In this way, the invention provides transformed seeds (also referred to as "transgenic seeds") having stably incorporated into their genome a nucleotide construct of the invention (e.g., an expression cassette of the invention).

Evaluation of plant transformation

After introduction of the heterologous foreign DNA into the plant cell, transformation or integration of the heterologous gene into the plant genome can be confirmed by various methods, for example, analysis of nucleic acids or proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method of screening transformed cells, tissues or shoots for the presence of bound genes at an early stage prior to transplantation into soil (Sambruke and Lassel, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.) (Sambrook and Russell,2001.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is performed using oligonucleotide primers specific to the gene of interest or the background of the Agrobacterium vector, etc.

Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell,2001, supra). Generally, total DNA is extracted from transformants, digested with appropriate restriction enzymes, fractionated on agarose gels, and transferred to nitrocellulose or nylon membranes. Then labelled, for example, with radioactivity according to standard techniques (Sambruke and Lassel, 2001(Sambrook and Russell,2001), supra)³²The P target DNA fragment probes the membrane or "blot" to confirm the integration of the introduced gene in the plant genome.

For Northern blot analysis, RNA was isolated from specific tissues of transformants, fractionated in formaldehyde agarose gel, and blotted onto nylon filters according to standard procedures routinely used in the art (Sambrook and Russell,2001, supra). Expression of RNA encoded by the pesticidal gene is then detected by hybridizing the filter to a radioactive probe derived from the pesticidal gene using methods known in the art (Sambrook and Russell, supra, 2001).

Western blotting, biochemical assays, etc. can be performed on the transgenic plants using antibodies that bind to one or more epitopes present on the pesticidal protein to confirm the presence of the protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell,2001, Sambrook and Russell, supra).

Pesticidal activity in plants

In another aspect of the invention, transgenic plants expressing pesticidal proteins having pesticidal activity can be produced. The methods described above by way of example may be used to produce transgenic plants, but the manner in which the transgenic plant cells are produced is not critical to the present invention. Methods known or described in the art, such as Agrobacterium-mediated transformation, biolistic transformation, and non-particle-mediated methods can be used at the discretion of the experimenter. Plants expressing pesticidal proteins can be isolated by general methods described in the art, for example by callus transformation, selection of transformed calli, and generation of fertile plants from such transgenic calli. In such a process, any gene can be used as a selectable marker as long as its expression in a plant confers the ability to identify and select transformed cells.

Many markers have been developed for use in plant cells, such as resistance to chloramphenicol, aminoglycoside G418, hygromycin, and the like. Other genes encoding products involved in chloroplast metabolites may 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 reported (Stokes et al (Stalker) 1985 J.Biol.chem.)263:6310-6314 (bromoxynil-resistant nitrilase gene) and Sathasivan et al 1990 Nucleic acid research 18:2188(AHAS imidazolinone-resistant gene) in addition, the genes disclosed herein can be used as markers for assessing bacterial or plant cell transformation methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaf, stem, root, etc.), seed, plant cell, propagule, embryo or progeny thereof are well known in the art.

Fertile plants expressing pesticidal proteins can be used for pesticidal activity tests, and plants showing the best activity are selected for further breeding. Methods are available in the art to determine pest activity. Typically, the proteins are mixed and used in feeding trials. See, for example, Marron et al (1985) J.Economic Entomology 78: 290-293.

The present invention can be used for transformation of any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to, corn, sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, brassica, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, nectarine, fig, guava, mango, olive, papaya, cashew, macadamia nut, apricot, oat, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, green beans, kidney beans, peas, and members of the cucumis genus (e.g., cucumbers, cantaloupes, and melons). Ornamental plants include, but are not limited to, azalea, rust ball, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. Preferably, the plants of the present invention are crop plants (e.g., corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, oilseed rape, etc.).

Use in pest control

General methods for using strains comprising the nucleotide sequences of the present invention or variants thereof in pest control or engineering other organisms as pesticides are known in the art. See, for example, U.S. patent No. 5,039,523 and EP 0480762 a 2.

Bacillus strains containing the nucleic acid sequences of the invention or variants thereof, or microorganisms genetically altered to contain the pesticidal genes and proteins of the invention, are useful for protecting crops and products against pests. In one aspect of the invention, intact, i.e., unlysed, cells of a toxin (pesticide) -producing organism are treated with an agent that prolongs the activity of the toxin produced in the cells when the cells are applied to the environment of one or more target pests.

Alternatively, the pesticide is produced by introducing a pesticidal gene into a cellular host. Expression of the pesticidal gene directly or indirectly results in the production and maintenance of the pesticide within the cell. In one aspect of the invention, when the cells are applied to the environment of one or more target pests, the cells are then treated under conditions that prolong the activity of the toxins produced in the cells. The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for use in environments having target pests, such as soil, water, and leaves of plants. See, for example, EPA 0192319, and incorporated herein by reference. Alternatively, cells expressing the genes of the invention may be formulated so as to allow the use of the resulting substances as pesticides.

The active ingredients of the invention are generally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or sequentially with other compounds. The compounds may be fertilizers, herbicides, cryoprotectants, surfactants, detergents, insecticidal soaps, dormant oils, polymers, and/or timed release or biodegradable carrier formulations that allow for long-term administration of a target area following a single application of the formulation. They may also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these formulations, if desired, further together with agriculturally acceptable carriers, surfactants or application-promoting adjuvants conventionally employed in the art of formulation. Suitable carriers and adjuvants may be solid or liquid and correspond to substances customarily employed in the art of formulation, for example natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise, these formulations may be prepared as edible "baits" or fashioned into pest "traps" to allow for feeding or digestion by the target pest of the pesticidal formulation.

Methods of applying the active ingredient of the invention or the agronomic chemical composition of the invention comprising at least one pesticidal protein produced by the bacterial strain of the invention include foliar, seed coating and soil applications. The number of applications and the application ratio depend on the intensity of the infestation by the corresponding pests.

The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or the like, and may be prepared by conventional methods such as drying, freeze-drying, homogenizing, extracting, filtering, centrifuging, settling, or concentrating a cell culture comprising the polypeptide. In all of these compositions comprising at least one such pesticidal polypeptide, the polypeptide may be present at a concentration (by weight) of from about 1% to about 99%.

Hemipteran pests may be killed or reduced in number in a given area by the method of the invention, or may be applied prophylactically to an environmental area to prevent infestation by susceptible pests. Preferably, the polypeptide is ingested or contacted with a pesticidally effective amount. By "pesticidally effective amount" is meant an amount of a pesticide that is capable of killing at least one pest, or significantly reducing the growth, feeding, or normal physiological development of the pest. This amount will vary depending on such factors as the particular target pest to be controlled, the particular environment, location, plant, crop, or agricultural site to be treated, environmental conditions, and the method, ratio, concentration, stability, and number of applications of the pesticidally effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.

The pesticide composition may be prepared by formulating a suspension of bacterial cells, crystals and/or spores, or an isolated protein fraction with the desired agriculturally acceptable carrier. These compositions may be formulated prior to administration by any suitable method, such as lyophilization, freeze-drying, dehydration, or in an aqueous carrier, medium, or suitable diluent, such as physiological saline or other buffer. The formulated composition may be in the form of a dust or granular material, or a suspension in an oil (vegetable or mineral), or in the form of an aqueous or oil/water emulsion, or as a wettable powder, or in combination with any other carrier material suitable for agricultural applications. Suitable agricultural carriers may be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, and the like that are commonly used in pesticide formulation techniques; these are well known to those skilled in the art of pesticide formulation. These formulations may be mixed with one or more solid or liquid adjuvants and prepared by various methods, for example by homogeneously mixing, stirring and/or grinding the pesticidal composition using conventional formulation techniques and suitable adjuvants. Suitable formulations and methods of application are described in U.S. patent No. 6,468,523, incorporated herein by reference.

Pests of the order hemiptera, including, but not limited to, Lygus bugs, such as western Lygus pratensis (Lygus legrinus), Lygus lucorum (Lygus americanus), and Lygus lucorum (Lygus elsus); aphids, such as the peach aphid (Myzus persicae), cotton aphid (Aphisgossypii), cherry aphid or black cherry aphid (Myzus cerasi), soybean aphid (Aphis vitamins Matsumura); brown rice planthopper (brown rice planthopper), rice leafhopper (black tail cicada), Sogatella furcifera and brown small brown planthopper (laodelphax striatellus); and stink bugs such as green stinkbug (lygus lucorum), brown marbled stinkbug (tea wing stinkbug), southern green stinkbug (Nezara viridula), rice stinkbug (american rice stinkbug), forest red stinkbug (Pentatoma rufipepes), european stinkbug (rhaphigater nebulosa), and stinkbugs bugs (Troilus lucidus). In some embodiments, the hemipteran pest is a rice planthopper pest, such as brown planthopper.

Method for increasing plant yield

Methods for increasing plant yield are provided. The method comprises the following steps: plants or plant cells are provided that express a polynucleotide encoding a pesticidal polypeptide sequence disclosed herein, and plants or seeds thereof are planted in a field that is infected with (or susceptible to being infected by) a pest for which the polypeptide has pesticidal activity. In some embodiments, the Axmi011 polypeptide described herein has pesticidal activity against a hemipteran pest, and the field is infected with the hemipteran pest. In various embodiments, the hemipteran pest is a rice planthopper pest, such as brown planthopper (brown rice planthopper). As defined herein, "yield" of a plant refers to the quality and/or quantity of biomass produced by the plant. "Biomass" refers to any measured plant product. An increase in biomass yield is any increase in yield of the measured plant product. There are several commercial applications for increasing plant yield. For example, increasing the biomass of plant leaves can increase the yield of green leaf vegetables for human or animal consumption. In addition, increased leaf biomass can be used to increase the yield of plant-derived pharmaceutical or industrial products. An increase in yield may include any statistically significant increase, including, but not limited to, an increase in yield of at least 1%, at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, or more, as compared to a plant not expressing a pesticidal sequence. In particular methods, the increase in plant yield is due to an increase in pest resistance of a plant expressing a pesticidal protein disclosed herein. Expression of pesticidal proteins results in pest infection or reduced feeding ability.

Plants may also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides. Typical chemical compositions include:fruit/vegetable herbicides:atrazine, bromacil, diuron, glyphosate, linuron, metribuzin, simazine, trifluralin, fluazifop-p-butyl, glufosinate, clomiphene, paraquat, pentyne, sethoxydim, butafenacil, clopyralid, indoxachlor;fruit/vegetable insecticides:aldicarb, bacillus thuringiensis, carbaryl, carbofuran, chlorpyrifos, cypermethrin, deltamethrin, abamectin, cyfluthrin/lambda-cyhalothrin, esfenvalerate, lambda-cyhalothrin, fenaminoquinone, bifenazate, methoxyfenozide, novaluron, chromafenozide, thiacloprid, dinotefuran, fluacrypyrim, flufenozide, thiacloprid, flufenozide,spirodiclofen, gamma-cyhalothrin, spiromesifen, spinosad, bromocyantraniliprole, triflumuron, spirotetramat, imidacloprid, flubendiamide, thiodicarb, metaflumizone, sulfoxaflor, cyflumetofen, cyenopyrafen, clothianidin, thiamethoxam, snotom (spinooram), thiodicarb, flonicamid, methiocarb, emamectin benzoate, indoxacarb, fenamiphos, pyriproxyfen, phenylbutoxide;fruit/vegetable fungicides:ametoctradin, azoxystrobin, benthiavalicarb-isopropyl, boscalid, captan, carbendazim, chlorothalonil, copper, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, difenoconazole, gadolinate morpholine, dithianon, fenamidone, fenhexamid, fluazinam, fludioxonil, fluopyram, fluoxastrobin, fluxapyroxastrobin, folpet, fosetyl acid, iprodione, iprovalicarb, kresoxim-methyl, mancozeb, two-piece unitary bactericidal amine, metalaxyl/mefenoxam, metiram, metrafenone, myclobutanil, penconazole, penthiopyrad, picoxystrobin, propamocarb, propiconazole, imazaquin, iodoquinconazole, prothioconazole, pyraclostrobin, pyrimethanil, quindox, spiroxamide, thifenthiobac, tebuconazole, thiophanate, trifloxystrobin;cereal herbicides:2,4-D, acylcrylonine, bromoxynil, carfentrazone-E, chlortoluron, chlorsulfuron, clomazone-P, clopyralid, dicamba, diclofop-M, diflufenican, fenoxaprop-P, diflufenzopyr, flucarbazone-NA, flufenacet, flupyrsulfuron-M, fluroxypyr, flerazine, glyphosate, iodosulfuron, ioxynil, isoproturon, MCPA, mesosulfuron, metsulfuron-methyl, pendimethalin, pinoxaden, propoxycarbazone, prosulfocarb, pyroxsulam, sulfonylurea, thifensulfuron, oxadiazon, triasulfuron, tribenuron, trifluralin, triflumuron;cereal fungicides:azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, cyflufenamid, cyproconazole, cyprodinil, dimoxystrobin, epoxiconazole, fenbutamol, fenpropimorph, fluopyram, fluoxastrobin, fluquinconazole, fluxapyroxad, isopimam, kresoxim-methyl, metconazole, metrafenone, penthiopyrad, picoxystrobinProchloraz, propiconazole, iodoquinazolinone, prothioconazole, pyraclostrobin, quindox, spiroxamine, tebuconazole, thiophanate-methyl, trifloxystrobin;grain insecticides:dimethoate, lambda-cyhalothrin, deltamethrin, cis-cypermethrin, β -cyfluthrin, bifenthrin, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinotefuran, chlorpyrifos, pirimicarb, methiocarb and sulfoxaflor;corn herbicide:atrazine, alachlor, bromoxynil, acetochlor, dicamba, clopyralid, (S-) thiophenylamine, glufosinate, glyphosate, isoflurane, (S-) metolachlor, mesotrione, nicosulfuron, primisulfuron, rimsulfuron, sulcotrione, foramsulfuron, topramezone, terbutazone, pyribenzoxim-methyl, thiencarbazone-methyl, flufenacet, bishomoxafon (pyroxasulfofon);corn insecticides:carbofuran, chlorpyrifos, bifenthrin, fipronil, imidacloprid, lambda-cyhalothrin, tefluthrin, terbufos, thiamethoxam, clothianidin, methoprene, flubendiamide, triflumuron, chlorantraniliprole, deltamethrin, thiodicarb, β -cyfluthrin, cypermethrin, bifenthrin, lufenuron, butylpyrimidine, ethiprole, cyantraniliprole, thiacloprid, acetamiprid, dinotefuran, abamectin;corn fungicides:azoxystrobin, bixafen, boscalid, cyproconazole, dimoxystrobin, epoxiconazole, pyraoxystrobin, fluoxastrobin, fluxapyroxad, isopimam, metconazole, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, tebuconazole, trifloxystrobin;herbicide for rice:butachlor, propanil, pyrosulfuron, bensulfuron, cyhalofop-butyl, prosulfuron, fentrazamide, imazasulfuron, mefenacet, oxaziclomefone, pyrazosulfuron, pyributicarb, quinclorac, prosulfocarb, benzofenazamide, indanone, flufenacet, fentrazamide, halosulfuron-methyl, oxaziclomefone, benzobicyclon, pyriftalid, penoxsulam, bispyribac, oxadiargyl, ethoxysulfuron, pretilachlor, mesotrione, tefuracanone, oxadiazon, fenoxaprop-p-ethyl, pyritinol (Pyrimisulfan);rice insecticides:diazinon, fenobucarb and prothiockCarbofuran, buprofezin, dinotefuran, fipronil, imidacloprid, isoprocarb, thiacloprid, chromafenozide, clothianidin, ethiprole, flubendiamide, chlorantraniliprole, deltamethrin, acetamiprid, thiamethoxam, cyantraniliprole, spinosad, spinosyn (spinooram), emamectin benzoate, beta-cypermethrin, chlorpyrifos, ethofenprox, carbofuran, benfuracarb, sulfoxaflor;rice fungicides:azoxystrobin, carbendazim, cyprodinil, diclorocyanid, difenoconazole, edifenphos, azozone, gentamicin, hexaconazole, hymexazol, Iprobenfos (IBP), isoprothiolane, isotianil, kasugamycin, mancozeb, metominostrobin, orysastrobin, pencycuron, thiabendazole, propiconazole, propineb, pyroquilon, tebuconazole, thiophanate methyl, dichotomamide, tricyclazole, trifloxystrobin, validamycin;cotton herbicide:diuron, fluometuron, MSMA, oxyfluorfen, prometryn, trifluralin, carfentrazone-ethyl, clethodim, fluazifop-butyl, glyphosate, norflurazon, pendimethalin, pyrithiobac-sodium, trifloxysulfuron, pyrone, glufosinate, flumioxazin and thifensulfuron-methyl;cotton insecticides:acephate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, abamectin, acetamiprid, emamectin benzoate, imidacloprid, indoxacarb, lambda-cyhalothrin, spinosad, thiodicarb, gamma-cyhalothrin, spiromesifen, pyridalyl, flonicamid, flubendiamide, triflumuron, chlorantraniliprole, β -cyfluthrin, spirotetramat, clothianidin, thiamethoxam, thiacloprid, dinotefuran, flubenamide, cyantraniliprole, spinosad, snotom (spinoram), gamma-cyhalothrin, 4- [ [ (6-chloropyridin-3-yl) methyl](2, 2-Difluoroethyl) amino]Furan-2 (5H) -one, thiodicarb, abamectin, flonicamid, pyridalyl, spiromesifen and sulfoxaflor;cotton Flower insecticides:azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, dimoxystrobin, epoxiconazole, fenamidone, fluazinam, fluopyram, fluoxastrobin, fluxapyroxad, iprodione, isopyrazam, isotianil, mancozeb, maneb, fenpyroxene, fenbuconazole, flutriafol,metominostrobin, penthiopyrad, picoxystrobin, propineb, prothioconazole, pyraclostrobin, quintozene, tebuconazole, tetraconazole, thiophanate-methyl, trifloxystrobin;soybean herbicide:alachlor, bentazone, trifluralin, chlorimuron-ethyl, cloransulam-methyl, fenoxaprop-p-ethyl, fomesafen, fluazifop-butyl, glyphosate, imazamox, imazaquin, imazethapyr, (S-) metolachlor, metribuzin, pendimethalin, pyroxene and glufosinate;soybean insecticide:lambda-cyhalothrin, methomyl, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinotefuran, flubendiamide, chlorantraniliprole, cyantraniliprole, spinosad, spinosyn (spinotam), emamectin benzoate, fipronil, ethiprole, deltamethrin, β -cyfluthrin, gamma and lambda-cyhalothrin, 4- [ [ (6-chloropyridin-3-yl) methyl ] methyl](2, 2-Difluoroethyl) amino]Furan-2 (5H) -one, spirotetramat, spirodiclofen, triflumuron, flonicamid, thiodicarb, β -cyfluthrin;soybean fungicide:azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, dimoxystrobin, epoxiconazole, fluazinam, fluopyram, fluoxastrobin, flutriafol, fluxapyroxad, isopimam, peimine, isotianil, mancozeb, maneb, metconazole, metominostrobin, myclobutanil, penthiopyrad, picoxystrobin, propiconazole, propineb, prothioconazole, pyraclostrobin, tebuconazole, tetraconazole, thiophanate-methyl, trifloxystrobin;beet herbicide:chlorphenamine, desmedipham, furbenflurane, phenmedipham, triallate, clopyralid, fluazifop-p-butyl, lenacil, quinmerac, cycloxydim, triflusulfuron-methyl, pyroxydim, quizalofop-p-ethyl;sugar beet insecticides:imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinotefuran, deltamethrin, β -cyfluthrin, gamma-/lambda-cyhalothrin, 4- [ [ (6-chloropyridine-3-yl) methyl](2, 2-Difluoroethyl) amino]Furan-2 (5H) -one, tefluthrin, chlorantraniliprole, sesamel (Cyaxypyr), fipronil, carbofuran;kanuolashi And (3) preparing a herbal agent:clopyralid, diclofop-methyl, fluazifop-butyl and glufosinate-ammoniumGlyphosate, metazachlor, triflumuron-methyl, quinclorac, quizalofop-p-ethyl, clethodim and pyrone;canola fungicides:azoxystrobin, bixafen, boscalid, carbendazim, cyproconazole, difenoconazole, dimoxystrobin, epoxiconazole, fluazinam, fluopyram, fluoxastrobin, flusilazole, fluxapyroxad, prochloraz, isopimam, mepiquat chloride, metconazole, metominostrobin, paclobutrazol, penthiopyrad, picoxystrobin, prochloraz, prothioconazole, pyraclostrobin, tebuconazole, thiophanate-methyl, trifloxystrobin, vinclozolin;canola insecticides:carbofuran, thiacloprid, deltamethrin, imidacloprid, clothianidin, thiamethoxam, acetamiprid, dinotefuran, β -cyfluthrin, gamma and lambda cyhalothrin, tau-fivelet (tau-fluvalinate), ethiprole, spinosad, spinosyn (spinotam), flubendiamide, chlorantraniliprole, cyantraniliprole, 4- [ [ (6-chloropyridin-3-yl) methyl ] methyl [ ]](2, 2-Difluoroethyl) amino]Furan-2 (5H) -one.

The following examples are illustrative and not limiting in any way.

Examples

Example 1 Axmi011 expression and Activity

The Axmi011 gene (SEQ ID NO:1) was isolated from a strain of Bacillus thuringiensis (enriched from ARS cultures from the United states department of agriculture) and described in U.S. patent publication 20080070829. The AXMI011 amino acid sequence (SEQ ID NO:3) has 23% sequence identity to Mtx 2.

Full-length Axm011 and a truncated version of Axm011 (the nucleotide sequence encoding the putative signal sequence was removed and replaced with the N-terminal codon encoding methionine) were expressed and analyzed for biological activity. The amino acid sequence of the truncated Axmi011 protein is shown in SEQ ID NO 4. PCR amplification of the gene was performed using a primer having a NotI linker at the 5 'end and an AscI linker at the 3' end and Herculase polymerase. The amplified PCR product was digested with AscI and NotI and ligated into the pMalC4X vector to generate plasmid pAX 7606. This clone was confirmed by sequencing and was transformed into pAX7606 in BL21 competent cells. Individual colonies of each were inoculated in LB medium and grown at 37 ℃ until log phase and induced with 1.0mM IPTG for 16 hours at 16 ℃. Purified Axmi011 and truncated Axmi011 were submitted for bioassay against selected pests according to standard protocols. Both Axmi011 and truncated Axmi011 were used to demonstrate severe dwarfing and a mortality rate of greater than 75% for Brown Planthopper (BPH).

Example 2 delivery of genes for plant expression

The coding regions of the present invention are linked to suitable promoter and terminator sequences for expression in plants. Such sequences are well known in the art. Techniques for generating and validating promoter-gene-terminator constructs are also well known in the art.

In one aspect of the invention, synthetic DNA sequences are designed and generated. These synthetic sequences have altered nucleotide sequences relative to the parent sequence, but encode proteins that are essentially identical to the parent sequence. In some embodiments, the synthetic DNA sequence comprises SEQ ID NO 2.

In another aspect of the invention, the modified version of the synthetic gene is designed such that the resulting peptide is targeted to a plant organelle, such as the endoplasmic reticulum or apoplast. Peptide sequences known to result in targeting of fusion proteins to plant organelles are known in the art. For example, the N-terminal region of the acid phosphatase gene derived from Lupinus albus (Lupinus albus) ((

ID GI 14276838, Miller et al (2001) Plant Physiology 127:594-606) is well known in the art to result in endoplasmic reticulum targeting of heterologous proteins. If the resulting fusion protein also contains an endoplasmic reticulum retention sequence at the C-terminus, which comprises the peptide N-terminal-lysine-aspartic acid-glutamic acid-leucine (i.e., the "KDEL" motif, SEQ ID NO:5), 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 be targeted to the endoplasmic reticulum, but will eventually be sequestered in the apoplast.

Thus, this gene encodes a fusion protein comprising the N-terminal 31 amino groups of the acid phosphatase geneAcid (from Lupinus albus (Lupinus albus) (L))

ID GI:14276838, supra, Miller et al, 2001)) is fused to the N-terminus of the amino acid sequence of the invention and comprises a KDEL (SEQ ID NO:5) sequence at the C-terminus. Thus, the protein produced is predicted to target the endoplasmic reticulum after expression in a plant cell.

The plant expression cassettes described above are combined with a suitable plant selectable marker to aid in the selection of transformed cells and tissues, and ligated into a plant transformation vector. These may include binary vectors from Agrobacterium-mediated transformation or simple plasmid vectors for aerosol or gene gun transformation.

In the present invention, the expression cassette comprises a promoter region encoding the synthetic gene Axmi011(SEQ ID NO:2) operably linked to the sucrose synthase 1 gene of rice (Oryza sativa) (Wang et al (1992) Plant Molecular Biology, 19, 881-42-. The expression cassette further comprises the 3 'untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al (1982) Journal of molecular and Applied Genetics 1,561-573), operably linked to the 3' end of the Axmi011 sequence.

Example 4 transformation of Rice

Immature rice seeds (containing embryos at the correct developmental stage) were collected from donor plants grown under greenhouse conditions. Following sterilization of the seeds, immature embryos were excised and pre-induced on solid medium for 3 days. After pre-induction, embryos are soaked for a few minutes in agrobacterium suspension with the desired vector. Embryos were then co-cultured in solid medium containing acetosyringone and cultured in the dark for 4 days. The explants are then transferred to a first selection medium containing glufosinate as a selection agent. After about 3 weeks, the cuticle scales with callus development were cut into multiple pieces and transferred to the same selective medium. Subsequent subcultures were performed approximately every 2 weeks. After each subculture, actively growing callus was cut into smaller pieces and cultured on a second selective medium. After several weeks, calli with significant resistance to glufosinate were transferred to selective regeneration medium. The resulting plants were cultured under half-intensity MS for full elongation. Plants were finally transferred to soil and grown in the greenhouse.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

The present invention provides the following:

1.a method for controlling a hemipteran pest population, the method comprising contacting said population with a pesticidally-effective amount of a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3 or 4, wherein said polypeptide has pesticidal activity against the hemipteran pest population.

2. A method for controlling a plant-hopper pest population, the method comprising contacting said population with a pesticidally-effective amount of a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3 or 4, wherein said polypeptide has pesticidal activity against the plant-hopper pest population.

3. The method of item 2, wherein the planthopper pest is a brown planthopper and the plant is a rice plant.

4. A method of protecting a plant against a hemipteran pest, the method comprising expressing in a plant or cell thereof a nucleotide sequence operably linked to a promoter capable of directing expression of the nucleotide sequence in a plant cell, wherein said nucleotide sequence is selected from the group consisting of:

a) a nucleotide sequence shown in SEQ ID NO. 1 or 2; and

b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3 or 4, wherein said polypeptide has pesticidal activity against a hemipteran pest.

5. A method of increasing yield in a plant, the method comprising growing in a field a plant or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence operably linked to a promoter capable of directing expression of the nucleotide sequence in a plant cell, wherein said nucleotide sequence is selected from the group consisting of:

a) a nucleotide sequence shown in SEQ ID NO. 1 or 2; and

b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3 or 4, wherein said polypeptide has pesticidal activity against a hemipteran pest;

wherein said field is infected with the hemipteran pest.

6. The method of item 5, wherein the pest is a brown planthopper pest, and wherein the plant is a rice plant.

7. The method of clauses 4-6, wherein the plant further comprises one or more nucleotide sequences encoding one or more additional insect toxins.

8. A plant or plant cell comprising the nucleotide sequence of SEQ ID NO 2.

9. An expression cassette comprising the nucleotide sequence of SEQ ID NO 2.

10. A nucleic acid comprising the nucleotide sequence of SEQ ID NO 2.

Sequence listing

<110> Samson, Kimberly (Sampson)

Leltisen, Dow (Lehtinen, Duane)

Wanweilite, atti Zhui (van Vliet, Adri)

Wan Rui, Jie Ruo En (van Rie, Jeroon)

Bottz, Daniela (Portz, Daniela)

<120> use of AXMI-011 for controlling hemipteran insects

<130>APA13-6053

<160>5

<170> PatentIn version 3.5

<210>1

<211>957

<212>DNA

<213> Bacillus thuringiensis

<400>1

atgaataaaa aacctatggt agcgttgata ttagccactt cgattggtat accttgtaca 60

tttacacctg gaagtgcatt agcagcagaa aatattcaga ctagtgttaa tgaaaatgta 120

aaagttggta ttacagatgt tcaatctgaa ttgaataaga taggagacta ttattatagt 180

aataacttag caaatacgac tataaaacct cctcatcatt gggattatac acttaaaaaa 240

aatcctgata aagttggaac aaatttggat tttagtatta ctggtactgc tagtaaacta 300

aattatgata gtgtaactcc tatatacatt gggcataatg aatttaataa tgattcagat 360

cagcctcaaa aatttacaac ttctaaattt actaaagctg taacagaggg aacaacaagt 420

accgtaacaa atggatttag attaggaaat ccaggtttaa acttatttac tattccatta 480

attttaagtg atggtatgaa aattaatgcg gaatttaact cttctacttc agaatctcaa 540

caaaaatcgg aaacaaaaac aatagaagca tcacctcaaa acatagaagt tccagcacat 600

aaaaaatata aagtagatgt tgtattggaa caaacaagct attgggcaga tgttacattt 660

acaggtgaag gaattaatct taatactact ataaatgcaa ctggaataca tactgggcat 720

atgggaatgc aggagtcaag aaaattttct tggaacaaaa ataccattga attatttaat 780

ggactaaaac aagagcaaaa aaataatata catgggatta aatttagtaa tgggaaaatg 840

aatgcaaacg gaacaggtaa agttgaaggt atttttggta gtaatctagt tgtaaaggta 900

aatgatgtta cagatccatt aaatcctatc ctagtaatga ctaaaagttt aaaataa 957

<210>2

<211>876

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic gene encoding Axmi011

<400>2

atggcggaga acatccagac ctccgtcaat gaaaatgtta aggtgggcat cacagatgtg 60

cagagcgagc tgaacaagat cggagattat tattattcaa acaacctggc caacaccacc 120

atcaagccgc cgcaccactg ggactacacc ctcaagaaga acccggacaa ggtgggcacc 180

aacctggact tctccatcac cggcaccgcc agcaagctga actatgacag cgtcaccccc 240

atctacatcg gccacaacga gttcaacaat gattcagacc agccgcagaa gttcaccacc 300

agcaagttca ccaaggccgt cacagaaggc accaccagca ccgtcaccaa tggcttccgg 360

ctgggcaacc ccggcctcaa cctcttcacc atccccctca tcctctctga tggcatgaag 420

atcaatgcag agttcaacag cagcacctca gagagtcagc agaagagcga gacaaagacc 480

atcgaggcct cgccgcagaa catcgaggtg ccggcgcaca agaagtacaa ggtggatgtg 540

gtgctggagc agaccagcta ctgggctgat gtcaccttca ccggcgaggg catcaacctg 600

aacaccacca tcaacgccac cggcatccac accggccaca tgggcatgca agaatcaagg 660

aagttcagct ggaacaagaa caccattgag ctcttcaacg gcctcaagca ggagcagaag 720

aacaacattc atggcatcaa gttcagcaac ggcaagatga acgccaatgg caccggcaag 780

gtggagggca tcttcggcag caacctggtg gtgaaggtga atgatgtcac agatcccctc 840

aaccccatcc tggtgatgac caagagcctc aagtga 876

<210>3

<211>318

<212>PRT

<213> Bacillus thuringiensis

<400>3

Met Asn Lys Lys Pro Met Val Ala Leu Ile Leu Ala Thr Ser Ile Gly

1 5 10 15

Ile Pro Cys Thr Phe Thr Pro Gly Ser Ala Leu Ala Ala Glu Asn Ile

20 25 30

Gln Thr Ser Val Asn Glu Asn Val Lys Val Gly Ile Thr Asp Val Gln

35 40 45

Ser Glu Leu Asn Lys Ile Gly Asp Tyr Tyr Tyr Ser Asn Asn Leu Ala

50 55 60

Asn Thr Thr Ile Lys Pro Pro His His Trp Asp Tyr Thr Leu Lys Lys

65 70 75 80

Asn Pro Asp Lys Val Gly Thr Asn Leu Asp Phe Ser Ile Thr Gly Thr

85 90 95

Ala Ser Lys Leu Asn Tyr Asp Ser Val Thr Pro Ile Tyr Ile Gly His

100 105 110

Asn Glu Phe Asn Asn Asp Ser Asp Gln Pro Gln Lys Phe Thr Thr Ser

115 120 125

Lys Phe Thr Lys Ala Val Thr Glu Gly Thr Thr Ser Thr Val Thr Asn

130 135 140

Gly Phe Arg Leu Gly Asn Pro Gly Leu Asn Leu Phe Thr Ile Pro Leu

145 150 155 160

Ile Leu Ser Asp Gly Met Lys Ile Asn Ala Glu Phe Asn Ser Ser Thr

165 170 175

Ser Glu Ser Gln Gln Lys Ser Glu Thr Lys Thr Ile Glu Ala Ser Pro

180 185 190

Gln Asn Ile Glu Val Pro Ala His Lys Lys Tyr Lys Val Asp Val Val

195 200 205

Leu Glu Gln Thr Ser Tyr Trp Ala Asp Val Thr Phe Thr Gly Glu Gly

210 215 220

Ile Asn Leu Asn Thr Thr Ile Asn Ala Thr Gly Ile His Thr Gly His

225 230 235 240

Met Gly Met Gln Glu Ser Arg Lys Phe Ser Trp Asn Lys Asn Thr Ile

245 250 255

Glu Leu Phe Asn Gly Leu Lys Gln Glu Gln Lys Asn Asn Ile His Gly

260 265 270

Ile Lys Phe Ser Asn Gly Lys Met Asn Ala Asn Gly Thr Gly Lys Val

275 280 285

Glu Gly Ile Phe Gly Ser Asn Leu Val Val Lys Val Asn Asp Val Thr

290 295 300

Asp Pro Leu Asn Pro Ile Leu Val Met Thr Lys Ser Leu Lys

305 310 315

<210>4

<211>291

<212>PRT

<213> Artificial sequence

<220>

<223> truncated version of Axmi011

<400>4

Met Ala Glu Asn Ile Gln Thr Ser Val Asn Glu Asn Val Lys Val Gly

1 5 10 15

Ile Thr Asp Val Gln Ser Glu Leu Asn Lys Ile Gly Asp Tyr Tyr Tyr

20 25 30

Ser Asn Asn Leu Ala Asn Thr Thr Ile Lys Pro Pro His His Trp Asp

35 40 45

Tyr Thr Leu Lys Lys Asn Pro Asp Lys Val Gly Thr Asn Leu Asp Phe

50 55 60

Ser Ile Thr Gly Thr Ala Ser Lys Leu Asn Tyr Asp Ser Val Thr Pro

65 70 75 80

Ile Tyr Ile Gly His Asn Glu Phe Asn Asn Asp Ser Asp Gln Pro Gln

85 90 95

Lys Phe Thr Thr Ser Lys Phe Thr Lys Ala Val Thr Glu Gly Thr Thr

100 105 110

Ser Thr Val Thr Asn Gly Phe Arg Leu Gly Asn Pro Gly Leu Asn Leu

115 120 125

Phe Thr Ile Pro Leu Ile Leu Ser Asp Gly Met Lys Ile Asn Ala Glu

130 135 140

Phe Asn Ser Ser Thr Ser Glu Ser Gln Gln Lys Ser Glu Thr Lys Thr

145 150 155 160

Ile Glu Ala Ser Pro Gln Asn Ile Glu Val Pro Ala His Lys Lys Tyr

165 170 175

Lys Val Asp Val Val Leu Glu Gln Thr Ser Tyr Trp Ala Asp Val Thr

180 185 190

Phe Thr Gly Glu Gly Ile Asn Leu Asn Thr Thr Ile Asn Ala Thr Gly

195 200 205

Ile His Thr Gly His Met Gly Met Gln Glu Ser Arg Lys Phe Ser Trp

210 215 220

Asn Lys Asn Thr Ile Glu Leu Phe Asn Gly Leu Lys Gln Glu Gln Lys

225 230 235 240

Asn Asn Ile His Gly Ile Lys Phe Ser Asn Gly Lys Met Asn Ala Asn

245 250 255

Gly Thr Gly Lys Val Glu Gly Ile Phe Gly Ser Asn Leu Val Val Lys

260 265 270

Val Asn Asp Val Thr Asp Pro Leu Asn Pro Ile Leu Val Met Thr Lys

275 280 285

Ser Leu Lys

290

<210>5

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> endoplasmic reticulum-targeting peptide

<400>5

Lys Asp Glu Leu

1

Claims

1.A method for controlling a hemipteran pest population, the method comprising contacting said population with a pesticidally-effective amount of a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3, wherein said polypeptide has pesticidal activity against the hemipteran pest population.

2. A method for controlling a plant hopper pest population, the method comprising contacting the population with a pesticidally-effective amount of a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3, wherein the polypeptide has pesticidal activity against the plant hopper pest population.

3. The method of claim 2, wherein the planthopper pest is brown planthopper and the plant is a rice plant.

a)1, the nucleotide sequence shown in SEQ ID NO; and

b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3, wherein said polypeptide has pesticidal activity against a hemipteran pest.

a)1, the nucleotide sequence shown in SEQ ID NO; and

b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 3, wherein said polypeptide has pesticidal activity against a hemipteran pest;

wherein said field is infected with the hemipteran pest.

6. The method of claim 5, wherein said pest is a brown planthopper pest, and wherein said plant is a rice plant.

7. The method of claims 4-6, wherein the plant further comprises one or more nucleotide sequences encoding one or more additional insect toxins.

8. A plant or plant cell comprising the nucleotide sequence of SEQ ID NO. 1.

9. An expression cassette comprising the nucleotide sequence of SEQ ID NO. 1.

10. A nucleic acid comprising the nucleotide sequence of SEQ ID NO. 1.