Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance

Definitions

• sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "APA136053_ST25.txt", created on November 14, 2014, and having a size of 9 kilobytes and is filed concurrently with the specification.
• sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
• This invention relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest- resistant plants.
• Bt crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) were planted on more than 58 million hectares worldwide in 2010 (James C (2010) Global Status of Commercialized Biotech/GM Crops: 2010. ISAAA Brief No. 42. Ithaca, New York: ISAAA).
• Benefits of Bt crops include reduced use of harmful insecticides and regional suppression of some key agricultural pests (Carriere et al. (2003) Proc Natl Acad Sci U S A 100: 1519-1523; Cattaneo et al. (2006) Proc Natl Acad Sci U S A 103 : 7571-7576; Huang et al. (2005) Science 308: 688-690; Wu (2008) Science 321 : 1676-1678; and Hutchison et al. (2010) Science 330: 222-225).
• the sap sucking insects which include aphids, whiteflies, plant bugs and stink bugs, have emerged as major agricultural pests.
• the hemiptera cause direct damage by feeding on crops, and in some cases indirect damage by transmission of plant viruses.
• Current management relies almost exclusively on application of classical chemical insecticides. While the development of transgenic crops expressing toxins derived from Bt has provided effective plant protection against some insect pests, commercially available Bt toxins exhibit little toxicity against sap sucking insects.
• insects Initially recognized as only minor or secondary pests, these insects have become primary pests due in part to changes in agricultural practices, such as the increased use of transgenic and classically selected plant varieties resistant to primary pests, and the decline in application of chemical insecticides.
• Lygus spp. are reported to feed on 117 non-crop plants and over 25 cultivated plants.
• Stink bugs comprise a pest complex of critical importance, impacting 12 major crops worldwide (McPherson J.E., McPherson R.M. Stink Bugs of Economic Importance in
• stink bugs affect crops including fruit, vegetable, nut, fiber, and cereals.
• the most abundant and important species include the green stink bug, Acrosternum hilare (Say); the Southern green stink bug, Nezara viridula (L.); and the brown stink bug,
• Aphids are exclusive phloem feeders and are among the most economically important pest insects of temperate agriculture (Blackman R.L. (2000) Aphids on the Worlds Crops, An Identification and Information Guide, John Wiley & Sons; New York, NY). Aphids cause major economic losses on almost all crops, and account for a large part of the 13% of agricultural output estimated to be lost to insect pests (van Emden FL, Harrington R. Aphids as Crop Pests. CABI Publishing; London, UK: 2007. p. 717; Faria C.A., Wackers F.L., Pritchard J., Barrett D.A., Turlings T.C. High susceptibility of Bt maize to aphids enhances the performance of parasitoids of lepidopteran pests. PLoS One. 2007;2:e600).
• BPH rice brown planthopper
• compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided.
• methods for killing or controlling a hemipteran pest population, particularly a planthopper pest population 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.
• the hemipteran toxin comprises the amino acid sequence of SEQ ID NO:3 or 4, or pesticidally-effective variants or fragments thereof.
• planthopper pest comprises expressing in a plant or cell thereof a nucleic acid 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.
• compositions and methods of the invention are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes.
• the present invention is drawn to methods for regulating pest resistance or tolerance in organisms, particularly plants or plant cells.
• resistance is intended that the pest (e.g., insect) is killed upon ingestion or other contact with the polypeptides of the invention.
• tolerance is intended an impairment or reduction in the movement, feeding, reproduction, or other functions of the pest.
• the methods involve transforming organisms with a nucleotide sequence encoding a pesticidal protein of the invention.
• the nucleotide sequences of the invention are useful for preparing plants and microorganisms that possess pesticidal activity.
• the methods described herein are useful for controlling or killing hemipteran pest populations and for producing compositions with pesticidal activity against hemipteran pests.
• pesticidal toxin or “pesticidal protein” is intended a toxin that has toxic activity against one or more hemipteran pests, including, but not limited to, Lygus spp., such as Western tarnished plant bug (Lygus hesperus), the tarnished plant bug (Lygus lineolaris), and green plant bug (Lygus elisus); aphids, such as the green peach aphid (Myzus persicae), cotton aphid (Aphis gossypii), cherry aphid or black cherry aphid (Myzus cerasi), soybean aphid (Aphis glycines Matsumura); brown plant hopper (Nilaparvata lugens), rice green leafhopper (Nephotettix spp.), whitebacked planthopper (Sogatella furcifera) and small brown planthopper (Laodelphax striatellus); and stink bugs, such as
• Pesticidal proteins include amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site, or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in, or in the pest after ingestion of the protein.
• the pesticidal protein comprises an AxmiOl 1 protein set forth in SEQ ID NO: 3 or 4, as well as variants and fragments thereof.
• methods for killing or controlling a hemipteran pest population comprising contacting the pest, or exposing the pest to, a composition compris the pesticidal toxin of the invention.
• nucleic acid molecules comprising nucleotide sequences encoding pesticidal proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology. Also encompassed herein are nucleotide sequences capable of hybridizing to the nucleotide sequences of the invention under stringent conditions as defined elsewhere herein.
• nucleic acid molecule is intended to include DNA molecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
• the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double- stranded DNA.
• nucleic acid sequence or DNA
• an isolated or recombinant nucleic acid sequence is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a recombinant bacterial or plant host cell.
• an isolated or recombinant nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
• isolated when used to refer to nucleic acid molecules excludes isolated chromosomes.
• the isolated delta- endotoxin encoding nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
• a delta-endotoxin protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-delta-endotoxin protein (also referred to herein as a "contaminating protein").
• Nucleotide sequences encoding the proteins of the present invention include the sequence set forth in SEQ ID NO: 1-2, and variants, fragments, and complements thereof.
• complement is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex.
• the corresponding amino acid sequences for the pesticidal proteins encoded by these nucleotide sequences are set forth in SEQ ID NO:3 or 4.
• Nucleic acid molecules that are fragments of these nucleotide sequences encoding pesticidal proteins are also encompassed by the present invention.
• fragment is intended a portion of the nucleotide sequence encoding a pesticidal protein.
• a fragment of a nucleotide sequence may encode a biologically active portion of a pesticidal protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
• Nucleic acid molecules that are fragments of a nucleotide sequence encoding a pesticidal protein comprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1350, 1400 contiguous nucleotides, or up to the number of nucleotides present in a full-length nucleotide sequence encoding a pesticidal protein disclosed herein, depending upon the intended use.
• contiguous nucleotides is intended nucleotide residues that are immediately adjacent to one another.
• Fragments of the nucleotide sequences of the present invention will encode protein fragments that retain the biological activity of the pesticidal protein and, hence, retain pesticidal activity.
• biologically-active fragments of the polypeptides disclosed herein are also encompassed.
• By "retains activity” is intended that the fragment will have at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the pesticidal activity of the pesticidal protein.
• the pesticidal activity is coleoptericidal activity.
• the pesticidal activity is lepidoptericidal activity.
• the pesticidal activity is nematocidal activity.
• the pesticidal activity is diptericidal activity. In another embodiment, the pesticidal activity is hemiptericidal activity.
• Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83 :2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Patent No. 5,743,477; and Heong et al. (2013) Research methods in toxicology and insecticide resistance monitoring of rice planthoppers, 2nd edition, Los Banos (Philippines):
• a fragment of a nucleotide sequence encoding a pesticidal protein that encodes a biologically active portion of a protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300 contiguous amino acids, or up to the total number of amino acids present in a full-length pesticidal protein of the invention.
• the fragment is a proteolytic cleavage fragment.
• the proteolytic cleavage fragment may have an N-terminal or a C-terminal truncation of at least about 30 amino acids, at least about 40 amino acids, at least about 50, at least about 100 amino acids, about 120, about 130, about 140, about 150, or about 160 amino acids relative to SEQ ID NO:3 or 4.
• the fragments encompassed herein result from the removal of the C-terminal crystallization domain, e.g., by proteolysis, or by insertion of a stop codon in the coding sequence.
• the fragments encompassed herein result from the removal of the N-terminal signal peptide (e.g., SEQ ID NO:4).
• N-terminal truncations may further comprise a methionine residue at the N-terminus.
• Preferred pesticidal proteins of the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequence of SEQ ID NO: 1-2, or the pesticidal proteins are sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 3 or 4.
• “sufficiently identical” is intended 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 greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters.
• these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
• the sequences are aligned for optimal comparison purposes.
• the two sequences are the same length.
• the percent identity is calculated across the entirety of the reference sequence (i.e., the sequence disclosed herein as any of SEQ ID NO: 1-3).
• 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, typically exact matches are counted.
• a gap i.e. a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues.
• the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• a nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.
• Gapped BLAST in BLAST 2.0
• PSI- Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
• the default parameters of the respective programs e.g., BLASTX and BLASTN
• Alignment may also be performed manually by inspection.
• ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence.
• the ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed.
• GENEDOCTM A non-limiting example of a software program useful for analysis of ClustalW alignments.
• GENEDOCTM (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins.
• Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 1 1-17. Such an algorithm is incorporated into the ALIGN program
• GAP Version 10 which uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used to determine sequence identity or similarity using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used.
• Equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
• the invention also encompasses variant nucleic acid molecules.
• "Variants" of the pesticidal protein encoding nucleotide sequences include those sequences that encode the pesticidal proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above.
• Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below.
• Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the pesticidal proteins disclosed in the present invention as discussed below.
• Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, pesticidal activity. By "retains activity” is intended 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.
• variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence 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. Such variant nucleotide sequences are also encompassed by the present invention.
• conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues.
• a “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a pesticidal protein without altering the biological activity, whereas an "essential” amino acid residue is required for biological activity.
• a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential 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).
• 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 similar or related toxins to the sequences of the invention (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins).
• residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
• residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
• residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
• one of skill in the art
• variant nucleotide sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer pesticidal activity to identify mutants that retain activity.
• the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques. Using methods such as PCR, hybridization, and the like corresponding pesticidal sequences can be identified, such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).
• hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor.
• Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known pesticidal protein-encoding nucleotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleotide sequence or encoded amino acid sequence can additionally be used.
• the probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of nucleotide sequence encoding a pesticidal protein of the invention or a fragment or variant thereof. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra herein incorporated by reference.
• an entire pesticidal sequence disclosed herein, or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding pesticidal protein-like sequences and messenger RNAs.
• probes include sequences that are unique and are preferably at least about 10 nucleotides in length, or at least about 20 nucleotides in length.
• Such probes may be used to amplify corresponding pesticidal sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism.
• Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
• the present invention encompasses probes for hybridization, as well as nucleotide sequences capable of hybridization to all or a portion of a nucleotide sequence of the invention (e.g., at least about 300 nucleotides, at least about 400, at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or up to the full length of a nucleotide sequence disclosed herein).
• Hybridization of such sequences may be carried out under stringent conditions.
• stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
• Stringent conditions are sequence-dependent and will be different in different circumstances.
• stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
• a probe is less than about 900 nucleotides in length, preferably less than 450 nucleotides in length.
• stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
• Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
• Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
• Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
• wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
• T m 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
• the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
• Pesticidal proteins are also encompassed within the present invention.
• pesticidal protein is intended a protein having the amino acid sequence set forth in SEQ ID NO:3 or 4. Fragments, biologically active portions, and variants thereof are also provided, and may be used to practice the methods of the present invention.
• recombinant protein is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
• “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 3 or 4, and that exhibit pesticidal activity.
• a biologically active portion of a pesticidal protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, or more amino acids in length.
• Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83 :2480-2485; Andrews et al. (1988) Biochem. J.
• a fragment comprises at least 8 contiguous amino acids of SEQ ID NO:3 or 4.
• the invention encompasses other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, or more amino acids in length.
• variants proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any of SEQ ID NO: 3 or 4.
• Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO: 1-2, or a complement thereof, under stringent conditions.
• variants include polypeptides that differ in amino acid sequence due to mutagenesis.
• Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining pesticidal activity. In some embodiments, the variants have improved activity relative to the native protein.
• Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83 :2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Patent No. 5,743,477; and Heong et al. (2013) Research methods in toxicology and insecticide resistance monitoring of rice planthoppers, 2nd edition, Los Banos (Philippines): International Rice Research Institute, all of which are herein incorporated by reference in their entirety.
• Bacterial genes such as the axmi genes of this invention, quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can initiate at a TTG codon, though in this event the TTG encodes a methionine.
• pesticidal proteins include amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, and amino acid sequences that are shorter than the full-length sequences due to the use of an alternate downstream start site.
• Antibodies to the polypeptides of the present invention, or to variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; U.S. Patent No. 4, 196,265).
• one aspect of the invention concerns 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 their homo logs, fusions or fragments.
• the antibody specifically binds to a protein having the amino acid sequence set forth in SEQ ID NO: 3 or 4 or a fragment thereof.
• the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO:3 or 4 or a fragment thereof.
• Antibodies of the invention may be used to quantitatively or qualitatively detect the protein or peptide molecules of the invention, or to detect post translational modifications of the proteins.
• an antibody or peptide is said to "specifically bind" to a protein or peptide molecule of the invention if such binding is not competitively inhibited by the presence of non-related molecules.
• DNA sequences of a pesticidal protein may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by a pesticidal protein of the present invention.
• This protein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of SEQ ID NO: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.
• amino acid sequence variants of a pesticidal protein can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess the desired pesticidal activity. However, it is understood that the ability of a pesticidal protein to confer pesticidal activity may be improved by the use of such techniques upon the compositions of this invention. For example, one may express a pesticidal protein in host cells that exhibit high rates of base misincorporation during DNA replication, such as XL- 1 Red (Stratagene, La Jolla, CA).
• the DNA for example by preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a vector
• culture the pesticidal protein mutations in a non-mutagenic strain and identify mutated genes with pesticidal activity, for example by performing an assay to test for pesticidal activity.
• the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293.
• Such assays can include contacting plants with one or more pests and determining the plant's ability to survive and/or cause the death of the pests. Examples of mutations that result in increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.
• alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity.
• This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification.
• the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions.
• Such fusion proteins are often used to (1) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in
• Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different pesticidal protein coding regions can be used to create a new pesticidal protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
• sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene of the invention and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity.
• Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751 ; Stemmer (1994) Nature 370:389-391 ; Crameri ei a/. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.
• Domain swapping or shuffling is another mechanism for generating altered pesticidal proteins. Domains may be swapped between pesticidal proteins, resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol. 62: 1537-1543; Ge et al. (1991) J. Biol. Chem. 266: 17954- 17958; Schnepf ei a/. (1990) J. Biol. Chem. 265:20923-20930; Rang ed/. 91999) Appl. Environ. Microbiol. 65:2918-2925).
• a pesticidal sequence of the invention may be provided in an expression cassette for expression in a plant of interest.
• plant expression cassette is intended a DNA construct that is capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically these contain a promoter and a coding sequence. Often, such constructs will also contain a 3' untranslated region. Such constructs may contain a "signal sequence” or “leader sequence” to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus.
• signal sequence is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are protolytically activated in the gut of the target pest (Chang (1987) Methods Enzymol.
• the signal sequence is located in the native sequence, or may be derived from a sequence of the invention.
• leader sequence is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts,
• plant transformation vector is intended a DNA molecule that is necessary for efficient transformation of a plant cell. Such a molecule may consist of one or more plant expression cassettes, and may be organized into more than one "vector" DNA molecule.
• binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).
• Vector refers to a nucleic acid construct designed for transfer between different host cells.
• Expression vector refers to a vector that has the ability to incorporate, integrate and express
• the cassette will include 5' and/or 3' regulatory sequences operably linked to a sequence of the invention.
• operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence
• 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.
• the cassette may additionally contain at least one additional gene to be cotransformed into the organism.
• the additional gene(s) can be provided on multiple expression cassettes.
• the nucleotide sequence of the invention is operably linked to a promoter, e.g., a plant promoter.
• a promoter e.g., a plant promoter.
• Promoter refers to a nucleic acid sequence that functions to direct transcription of a downstream coding sequence.
• the promoter together with other transcriptional and translational regulatory nucleic acid sequences are necessary for the expression of a DNA sequence of interest.
• Such an expression cassette is provided with a plurality of restriction sites for insertion of the pesticidal sequence to be under the transcriptional regulation of the regulatory regions.
• the expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the invention, and a translational and transcriptional termination region (i.e., termination region) functional in plants.
• the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "native" or "homologous" to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or “heterologous” to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
• the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with 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).
• Convenient termination regions are available from the Ti-plasmid of A. tumefacien , such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
• the gene(s) may be optimized for increased expression in the transformed host cell. That is, the genes can be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-1 1 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, U.S. Patent Publication No.
• the pesticidal protein is targeted to the chloroplast for expression.
• the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the pesticidal protein to the chloroplasts.
• transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem.
• the pesticidal gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle.
• the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by reference. Plant Transformation
• Methods of the invention involve introducing a nucleotide construct into a plant.
• introducing is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant.
• the methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to the interior of at least one cell of the plant.
• Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
• plant is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same.
• Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
• Transgenic plants or “transformed plants” or “stably transformed” plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell.
• “Heterologous” generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.
• the transgenic plants of the invention express one or more of the novel toxin sequences disclosed herein.
• the transgenic plant further comprises one or more additional genes for insect resistance (e.g., Cryl, such as members of the CrylA, Cry IB, CrylC, Cry ID, Cry IE, and Cry IF families; Cry2, such as members of the Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.).
• Cryl such as members of the CrylA, Cry IB, CrylC, Cry ID, Cry IE, and Cry IF families
• Cry2A family such as members of the Cry2A family
• Cry9 such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families
• the transgenic plant may comprise any gene imparting an agronomic trait
• Transformation of plant cells can be accomplished by one of several techniques known in the art.
• the pesticidal gene of the invention may be modified to obtain or enhance expression in plant cells.
• a construct that expresses such a protein would contain a promoter to drive transcription of the gene, as well as a 3' untranslated region to allow transcription termination and polyadenylation.
• the organization of such constructs is well known in the art.
• the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.
• This "plant expression cassette” will be inserted into a "plant transformation vector".
• This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation.
• DNA vectors needed for achieving plant transformation.
• Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
• Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "gene of interest" (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders.
• a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
• This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and
• Agrobacterium strains e.g. LBA4404, GV3101, EHA101, EHA105, etc.
• LBA4404, GV3101, EHA101, EHA105, etc. can be used for plant
• the second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
• plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
• Explants are typically transferred to a fresh supply of the same medium and cultured routinely.
• the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent.
• the shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet.
• the transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g. Hiei et al.
• Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
• Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mQdiatQd transformation), bombardment of plant cells with heterologous foreign 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. WO 91/00915; U.S. Published Application No. 2002015066), Lecl transformation, and various other non-particle direct-mediated methods to transfer DNA.
• plastid transformation can be accomplished by trans activation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
• tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 :7301- 7305.
• heterologous foreign DNA Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.
• the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having 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 expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome. Evaluation of Plant Transformation
• transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
• PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
• PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
• Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled 32 P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001 , supra).
• RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, 2001 , supra).
• Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the pesticidal protein.
• Methods described above by way of example may be utilized to generate transgenic plants, but the manner in which the transgenic plant cells are generated is not critical to this invention. Methods known or described in the art such as Agrobacterium-mQdiatQd transformation, biolistic transformation, and non-particle- mediated methods may be used at the discretion of the experimenter.
• Plants expressing a pesticidal protein may be isolated by common methods described in the art, for example by transformation of callus, selection of transformed callus, and regeneration of fertile plants from such transgenic callus. In such process, one may use any gene as a selectable marker so long as its expression in plant cells confers ability to identify or select for transformed cells.
• markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like.
• Other genes that encode a product involved in chloroplast metabolism may also be used as selectable markers.
• genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use. Such genes have been reported
• the genes disclosed herein are useful as markers to assess transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art. In one embodiment, the presence of the transgene is detected by testing for pesticidal activity.
• Fertile plants expressing a pesticidal protein may be tested for pesticidal activity, and the plants showing optimal activity selected for further breeding. Methods are available in the art to assay for pest activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293.
• the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
• plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
• Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon.
• plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape., etc.).
• crop plants for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape., etc.
• Bacillus strains containing a nucleotide sequence of the present invention, or a variant thereof, or the microorganisms that have been genetically altered to contain a pesticidal gene of the invention and protein may be used for protecting agricultural crops and products from pests.
• (pesticide)-producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).
• the pesticide is produced by introducing a pesticidal gene into a cellular host. Expression of the pesticidal gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. In one aspect of this invention, these cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest(s). The resulting product retains the toxicity of the toxin.
• These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein. Alternatively, one may formulate the cells expressing a gene of this invention such as to allow application of the resulting material as a pesticide.
• the active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds.
• These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time- release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation.
• Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
• the formulations may be prepared into edible "baits” or fashioned into pest "traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.
• Methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
• composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation,
• the polypeptide may be present in a concentration of from about 1% to about 99% by weight.
• Hemipteran pests may be killed or reduced in numbers in a given area by the methods of the invention, or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest.
• a pesticidally-effective amount is intended an amount of the pesticide that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development.
• 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 compositions described may be made by formulating either the bacterial cell, the crystal and/or the spore suspension, or the isolated protein component with the desired agriculturally-acceptable carrier.
• the compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer.
• the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application.
• Suitable agricultural carriers can be solid or liquid and are well known in the art. The term
• “agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation.
• the formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in U.S. Patent No. 6,468,523, herein incorporated by reference.
• Hemipteran pests include, but are not limited to, Lygus spp., such as Western tarnished plant bug (Lygus hesperus), the tarnished plant bug (Lygus lineolaris), and green plant bug (Lygus elisus); aphids, such as the green peach aphid (Myzus persicae), cotton aphid (Aphis gossypii), cherry aphid or black cherry aphid (Myzus cerasi), soybean aphid (Aphis glycines Matsumura); brown plant hopper (Nilaparvata lugens), rice green leafhopper (Nephotettix spp.), whitebacked planthopper (Sogatella furcifera) and small brown planthopper (Laodelphax striatellus); and stink bugs, such as green stink bug (Acrosternum hilare), brown marmorated stink bug (Halyomorpha halys), southern green
• the methods comprise providing a plant or plant cell expressing a polynucleotide encoding the pesticidal polypeptide sequence disclosed herein and growing the plant or a seed thereof in a field infested with (or susceptible to infestation by) a pest against which said polypeptide has pesticidal activity.
• the AxmiOl 1 polypeptide described herein has pesticidal activity against a hemipteran pest, and said field is infested with said hemipteran pest.
• the hemipteran pest is a planthopper pest, such as brown planthopper
• the Yield of the plant refers to the quality and/or quantity of biomass produced by the plant.
• biomass is intended any measured plant product.
• An increase in biomass production is any improvement in the yield of the measured plant product.
• Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products.
• An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the pesticidal sequence.
• plant yield is increased as a result of improved pest resistance of a plant expressing a pesticidal protein disclosed herein. Expression of the pesticidal protein results in a reduced ability of a pest to infest or feed.
• the plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides, or fungicides.
• exemplary chemical compositions include: Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides:
• Aldicarb Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Triflumuron,Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Clothianidin, Thiame
• Clodinafop-P Clopyralid, Dicamba, Diclofop-M, Diflufenican, Fenoxaprop, Florasulam, Flucarbazone-NA, Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Flurtamone, Glyphosate, Iodosulfuron, Ioxynil, Isoproturon, MCPA, Mesosulfuron, Metsulfuron, Pendimethalin, Pinoxaden, Propoxycarbazone, Prosulfocarb, Pyroxsulam, Sulfosulfuron, Thifensulfuron, Tralkoxydim, Triasulfuron, Tribenuron, Trifluralin, Tritosulfuron; Cereals Fungicides:
• Cyproconazole, Cyprodinil, Dimoxystrobin Epoxiconazole, Fenpropidin, Fenpropimorph, Fluopyram, Fluoxastrobin, Fluquinconazole, Fluxapyroxad, Isopyrazam, Kresoxim-methyl, Metconazole, Metrafenone, Penthiopyrad, Picoxystrobin, Prochloraz, Propiconazole, Proquinazid, Prothioconazole, Pyraclostrobin, Quinoxyfen, Spiroxamine, Tebuconazole, Thiophanate-methyl , Trifloxystrobin; Cereals Insecticides: Dimethoate.
• Cyazypyr Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Etofenprox, Carbofuran, Benfuracarb, Sulfoxaflor; Rice Fungicides: Azoxystrobin, Carbendazim, Carpropamid, Diclocymet, Difenoconazole, Edifenphos, Ferimzone,
• Tricyclazole Trifloxystrobin, Validamycin; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,
• Aldicarb Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma- Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid
• Trifloxystrobin Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate;
• Soybean Insecticides Lambda-cyhalothrin. Methomyl, Imidacloprid, Clothianidin,
• Cyazypyr Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, ⁇ - Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Bixafen, Boscalid,
• Carbendazim Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione, Isotianil, Mancozeb, Maneb, Metconazole, Metominostrobin, Myclobutanil, Penthiopyrad, Picoxystrobin, Propiconazole, Propineb, Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin; Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusul
• Trifloxystrobin Vinclozolin; Canola Insecticides: Carbofuran, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, ⁇ -Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cy azypyr, 4- [ [(6-Chlorpyridin-3 -y l)methy 1] (2 ,2 -difluorethy l)amino] furan-2 (5 H)- on.
• the AxmiOl 1 gene (SEQ ID NO: 1) was isolated from a Bacillus thuringiensis strain that was obtained from the ARS Culture Collection of the United States Department of
• the AXMIOl 1 amino acid sequence (SEQ ID NO:3) has 23% sequence identity to Mtx2.
• AxmOl 1 Full-length AxmOl 1, as well as a truncated version of AxmiOl 1 in which the nucleotide sequence encoding the putative signal sequence was removed and replaced with an N-terminal codon encoding methionine, was expressed and assayed for bioactivity.
• the amino acid sequence for the truncated AxmiOl 1 protein is set forth in SEQ ID NO:4.
• the genes were PCR amplified using Herculase polymerase with primers incorporating a Notl linker on the 5' end and an Ascl linker at the 3' end.
• Amplified PCR product was digested with Ascl and Notl and ligated into the pMalC4X vector to create plasmid pAX7606.
• the clones were confirmed by sequencing and pAX7606 was transformed in B121 competent cells. A single colony of each was inoculated in LB media and grown at 37°C until log phase, and induced with 1.0 mM IPTG at 16 °C for 16 hours.
• Purified AxmiOl 1 and truncated AxmiOl 1 were submitted to bioassay vs. selected insect pests according to standard protocol. Severe stunting and greater than 75% mortality was demonstrated against Nilaparvata lugens (BPH) using both AxmiO 11 and the truncated AxmiO 11.
• the coding regions of the invention are connected with appropriate promoter and terminator sequences for expression in plants. Such sequences are well known in the art. Techniques for producing and confirming promoter - gene - terminator constructs also are well known in the art.
• synthetic DNA sequences are designed and generated. These synthetic sequences have altered nucleotide sequence relative to the parent sequence, but encode proteins that are essentially identical to the parent sequence.
• the synthetic DNA sequence comprises SEQ ID NO:2.
• modified versions of the synthetic genes are designed such that the resulting peptide is targeted to a plant organelle, such as the endoplasmic reticulum or the apoplast.
• a plant organelle such as the endoplasmic reticulum or the apoplast.
• Peptide sequences known to result in targeting of fusion proteins to plant organelles are known in the art.
• the N-terminal region of the acid phosphatase gene from the White Lupin Lupinus albus (GENBANK® ID
• GI: 14276838, Miller et al. (2001) Plant Physiology 127: 594-606) is known in the art to result in endoplasmic reticulum targeting of heterologous proteins.
• the resulting fusion protein also contains an endoplasmic reticulum retention sequence comprising the peptide N- terminus-lysine-aspartic acid-glutamic acid-leucine (i.e., the "KDEL" motif, SEQ ID NO:5) at the C-terminus, the fusion protein will be targeted to the endoplasmic reticulum.
• the fusion protein lacks an endoplasmic reticulum targeting sequence at the C-terminus, the protein will be targeted to the endoplasmic reticulum, but will ultimately be sequestered in the apoplast.
• this gene encodes a fusion protein that contains the N-terminal thirty-one amino acids of the acid phosphatase gene from the White Lupin Lupinus albus (GENBANK® ID GI: 14276838 , Miller et al, 2001, supra) fused to the N-terminus of the amino acid sequence of the invention, as well as the KDEL (SEQ ID NO:5) sequence at the C-terminus.
• the resulting protein is predicted to be targeted the plant endoplasmic reticulum upon expression in a plant cell.
• the plant expression cassettes described above are combined with an appropriate plant selectable marker to aid in the selection of transformed cells and tissues, and ligated into plant transformation vectors.
• plant transformation vectors may include binary vectors from Agrobacterium- mediated transformation or simple plasmid vectors for aerosol or biolistic transformation.
• an expression cassette including a synthetic gene encoding
• AxmiOl 1 (SEQ ID NO:2) operably linked to the promoter region of the sucrose synthase 1 gene of Oryza sativa (Wang et al. (1992) Plant Molecular Biology, 19, 881-885) or the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al. (1985) Nature 313, 810-812) and the leader sequence of the chlorophyll a/b binding protein gene oi Petunia hybrid (Harpster et al. (1988) Molecular and General Genetics 212, 182-190).
• the expression cassettes further comprised 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 AxmiOl 1 sequence.
• Immature rice seeds containing embryos at the right developmental stage, were collected from donor plants grown under well controlled conditions in the greenhouse. After sterilization of the seeds, immature embryos were excised and preinduced on a solid medium for 3 days. After preinduction, embryos were immersed for several minutes in a suspension of Agrobacterium harboring the desired vectors. Then embryos were cocultivated on a solid medium containing acetosyringone and incubated in the dark for 4 days. Explants were then transferred to a first selective medium containing phosphinotricin as selective agent. After approximately 3 weeks, scutella with calli developing were cut into several smaller pieces and transferred to the same selective medium. Subsequent subcultures were performed approximately every 2 weeks.

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