USOO8541366B2

(12) United States Patent (10) Patent No.: US 8,541,366 B2 Carozzi et al. (45) Date of Patent: Sep. 24, 2013

(54) SYNTHETIC AXMI-004 DELTA-ENDOTOXIN AngSuthanasombat, C., et al., “Directed Mutagenesis of the Bacillus GENES AND METHODS FOR THEIR USE thuringiensis Cry11A Toxin Reveals a Crucial Role in Larvicidal Activity of Arginine-136 in Helix 4.” J. Biochem. Mol. Biol. Sep. 2001, pp. 402-407, vol. 34, No. 5. (75) Inventors: Nadine Carozzi, Raleigh, NC (US); Aronson, A.I., and Shai.Y., “Why Bacillus thuringiensis Insecticidal Nalini Desai, Chapel Hill, NC (US); Toxins are so Effective: Unique Features of Their Mode of Action.” Daniel J. Tomso, Bahama, NC (US); FEMS Microbiology Letters, 2001, pp. 1-8. Vadim Beilinson, Cary, NC (US); De Maagd, R.A., et al., “Identification of Bacillus thuringiensis Theodore Kahn, Cary, NC (US) Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in InsectSpecificity.” Appl. Environ. Microbiol. Oct. 1999, pp. 4369 4374, vol. 65, No. 10. (73) Assignee: Athenix Corporation, Morrisville, NC De Maagd, R.A., et al., “How Bacillus thuringiensis has Evolved (US) Specific Toxins to Colonize the Insect World.” Trends Genet., Apr. 2001, pp. 193-199, vol. 17, No. 4. (*) Notice: Subject to any disclaimer, the term of this Guo, H.H., et al., “Protein Tolerance to Random Amino Acid patent is extended or adjusted under 35 Change.” PNAS, Jun. 22, 2004, pp. 9205-9210, vol. 101, No. 25. U.S.C. 154(b) by 763 days. Hill, M.A. and Preiss, J., “Functional Analysis of Conserved Histidines in ADP-Glucose Pyrophosphorylase from Escherichia coli,' Biochem. Biophys. Res. Comm., Mar. 1998, pp. 573-577, vol. (21) Appl. No.: 12/209,354 244. Honée, G., et al., “Nucleotide Sequence of Crystal Protein Gene (22) Filed: Sep. 12, 2008 Isolated from B. thuringiensis subspecies entomocidus 60.5 Coding for a Toxin Highly Active Against Spodoptera Species.” Nucleic (65) Prior Publication Data Acids Research, May 13, 1988, p. 6240, vol. 16, No. 13. Jenkins, J.L., et al., “Binding of Bacillus thuringiensis Cry1Ac Toxin US 2009/0099,081 A1 Apr. 16, 2009 to Manduca sexta Aminopeptidase-N Receptor is Not Directly Related to Toxicity.” FEBS Letters, 1999, pp. 373-376, vol. 462. Kalman, et al., “Cloning of a Novel cryIC-type Gene from a Strain of Related U.S. Application Data Bacillus thuringiensis subsp. galleriae, "Applied and Environmental Microbiology, 1993, pp. 1131-1137, vol. 59, No. 4. (60) Provisional application No. 60/972.546, filed on Sep. Lazar, E., et al., “Transforming Growth Factor C. Mutation of 14, 2007. Aspartic Acid 47 and Leucine 48 Results in Different Biological Activities.” Mol. Cell. Biol. Mar. 1988, pp. 1247-1252, vol. 8, No. 3. Lee, M.K., et al., “Mutations at the Argine Residues in O.8 Loop of (51) Int. Cl. Bacillus thuringiensis 6-endotoxin Cry1Ac Affect Toxicity and AOIN37/18 (2006.01) Binding to Manduca sexta and Lymantria dispar Aminopeptidase C07K 14/325 (2006.01) N.” FEBS Letters, 2001, pp. 108-112, vol. 497. (52) U.S. Cl. Masson, L., et al., “Mutagenic Analysis of a Conserved Region of USPC ...... 514/45 Domain III in the Cry1Ac Toxin of Bacillus thuringiensis," Appl. Environ. Microbiol. Jan. 2002, pp. 194-200, vol. 68, No. 1. (58) Field of Classification Search Rajamohan, F., et al., “Mutations at Domain II, Loop 3, of Bacillus None thuringiensis Cry1Aa and Cry1Ab Ö-Endotoxins Suggest Loop 3 is See application file for complete search history. Involved in Initial Binding to Lepidopteran Midguts.” J. Biol. Chem. Oct. 11, 1996, pp. 25220-25226, vol. 271, No. 41. (56) References Cited Sanchis, V., et al., “Nucleotide Sequence and Analysis of the N-ter minal Coding Region of the Spodoptera-active Ö-endotoxin Gene of U.S. PATENT DOCUMENTS Bacillus thuringiensis aizawai 7.29.” Mol. Microbiol. 1989, pp. 229 238, vol. 3, No. 2. 5,380,831 A 1/1995 Adang et al. 5,866,784. A 2f1999 Van Mellaert et al. (Continued) 5,908,970 A 6, 1999 Van Mellaert et al. 6,172.281 B1 1/2001 Van Mellaert et al. Primary Examiner — Anne Kubelik 6,177,615 B1 1/2001 Baum (57) ABSTRACT 6,833,449 B1 12/2004 Barton et al. 7,355,099 B2 * 4/2008 Carozzi et al...... 800, 302 Compositions and methods for conferring pesticidal activity 2001/0003849 A1 6, 2001 Barton to bacteria, plants, plant cells, tissues and seeds are provided. 2004/O19791.6 A1* 10, 2004 Carozzi et al...... 435/468 Compositions comprising a coding sequence for a delta-en 2008/0040827 A1 2/2008 Donovan et al. dotoxin polypeptide are provided, particularly synthetically 2008/O172764 A1 7/2008 Carozzi et al. derived coding sequences. The coding sequences can be used 2008. O1768O1 A1 7/2008 Carozzi et al. in DNA constructs or expression cassettes for transformation FOREIGN PATENT DOCUMENTS and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, WO WO 2004/074462 A2 9, 2004 and seeds. In particular, isolated delta-endotoxin nucleic acid WO WO 2005,107383 * 11/2005 molecules are provided. Additionally, amino acid sequences WO WO 2005,107383 A2 11/2005 corresponding to the polynucleotides are encompassed, and OTHER PUBLICATIONS antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for de Maagdet al., 1999, Appl. Environ. Microbiol. 65:4369-4374.* isolated nucleic acid molecules comprising nucleotide Tounsi et al., 2003, J. Appl. Microbiol.95:23-28.* sequences encoding the amino acid sequence shown in SEQ Aaronson etal (2001, FEMS Microbiol. Lett. 195:1-8).* de Maagdet al (2001, Trends Genet. 17:193-199).* ID NO:9, 11, 13, 15, or 18, or the nucleotide sequence set Guo et al (2004, Proc. Natl. Acad. Sci. USA 101:9205-9210).* forth in SEQID NO:1, 2, 4, 6, 7, 8, 10, 12, 14, 16, or 17, as Partial International Search Report, Annex to Form PCT/ISA. 206, well as variants and fragments thereof. issued Dec. 12, 2008 for PCT/US/2008/076104. 8 Claims, 2 Drawing Sheets US 8,541,366 B2 Page 2

(56) References Cited NCBI Database Report for Accession No. AAF37224, Direct Sub mission on Dec. 14, 1999. OTHER PUBLICATIONS NCBI Database Report for Accession No. AAM00264. Direct Sub Schwartz, J.L., et al., “Single-Site Mutations in the Conserved Alter mission on Mar. 16, 2001. nating-Arginine Region Affect Ionic Channels Formed by Cry1Aa, a Bacillus thuringiensis Toxin.” Appl. Environ. Microbiol., Oct. 1997, NCBI Database Report for Accession No. AAN16462, Direct Sub pp. 3978-3984, vol. 63, No. 10. mission on Aug. 22, 2002. Tounsi, S., et al., “Cloning and Study of the Expression of a Novel NCBI Database Report for Accession No. CAA65457, Direct Sub cry1 Ia-type Gene from Bacillus thuringiensis subsp. Kurstaki,” J mission on Mar. 18, 1996. Appl. Microbiol. 2003, pp. 23-28, vol. 95. NCBI Database Report for Accession No. AAA22343, 1991. * cited by examiner

US 8,541,366 B2 1. 2 SYNTHETIC AXMI-OO4 DELTA-ENDOTOXIN numerals have been exchanged for Arabic numerals in the GENES AND METHODS FOR THEIR USE primary rank. Proteins with less than 45% sequence identity have different primary ranks, and the criteria for secondary CROSS-REFERENCE TO RELATED and tertiary ranks are 78% and 95%, respectively. APPLICATIONS The crystal protein does not exhibit insecticidal activity until it has been ingested and solubilized in the insect midgut. This application claims the benefit of U.S. Provisional The ingested protoxin is hydrolyzed by proteases in the insect Application No. 60/972.546, filed Sep. 14, 2007, which is digestive tract to an active toxic molecule. (Höfte and White hereby incorporated in its entirety by reference herein. ley (1989) Microbiol. Rev. 53:242-255). This toxin binds to 10 apical brush border receptors in the midgut of the target larvae REFERENCE TO SEQUENCE LISTING and inserts into the apical membrane creating ion channels or SUBMITTED ELECTRONICALLY pores, resulting in larval death. Delta-endotoxins generally have five conserved sequence The official copy of the sequence listing is Submitted elec domains, and three conserved structural domains (see, for tronically via EFS-Web as an ASCII formatted sequence list 15 example, de Maagd et al. (2001) Trends Genetics 17:193 ing with a file named "362196. SequenceListing..txt, created 199). The first conserved structural domain consists of seven on Sep. 8, 2008, and having a size of 60 kilobytes and is filed alpha helices and is involved in membrane insertion and pore concurrently with the specification. The sequence listing con formation. Domain II consists of three beta-sheets arranged in tained in this ASCII formatted document is part of the speci a Greek key configuration, and domain III consists of two fication and is herein incorporated by reference in its entirety. antiparallel beta-sheets in jelly-roll” formation (de Maagdet al., 2001, supra). Domains II and III are involved in receptor FIELD OF THE INVENTION recognition and binding, and are therefore considered deter minants of toxin specificity. This invention relates to the field of molecular biology. Because of the devastation that insects can confer there is a Provided are novel genes that encode pesticidal proteins. 25 continual need to discover new forms of Bacillus thuringien These proteins and the nucleic acid sequences that encode sis delta-endotoxins. them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants. SUMMARY OF INVENTION

BACKGROUND OF THE INVENTION 30 Compositions and methods for conferring pest resistance to bacteria, plants, plant cells, tissues and seeds are provided. Bacillus thuringiensis is a Gram-positive spore forming Compositions include nucleic acid molecules encoding soil bacterium characterized by its ability to produce crystal sequences for delta-endotoxin polypeptides, vectors com line inclusions that are specifically toxic to certain orders and prising those nucleic acid molecules, and host cells compris species of insects, but are harmless to plants and other non 35 ing the vectors. Compositions also include the polypeptide targeted organisms. For this reason, compositions including sequences of the endotoxin, and antibodies to those polypep Bacillus thuringiensis strains or their insecticidal proteins can tides. The nucleotide sequences can be used in DNA con be used as environmentally-acceptable insecticides to control structs or expression cassettes for transformation and expres agricultural insect pests or insect vectors for a variety of sion in organisms, including microorganisms and plants. The human or animal diseases. 40 nucleotide or amino acid sequences may be synthetic Crystal (Cry) proteins (delta-endotoxins) from Bacillus sequences that have been designed for expression in an organ thuringiensis have potent insecticidal activity against pre ism including, but not limited to, a microorganism or a plant. dominantly Lepidopteran, Dipteran, and Coleopteran larvae. Compositions also comprise transformed bacteria, plants, These proteins also have shown activity against plant cells, tissues, and seeds. Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and 45 In particular, isolated nucleic acid molecules correspond Acari pest orders, as well as other invertebrate orders such as ing to delta-endotoxin nucleic acid sequences are provided. Nemathelminthes, Platyhelminthes, and Sarcomastigorphora Additionally, amino acid sequences corresponding to the (Feitelson (1993) The Bacillus Thuringiensis family tree. In polynucleotides are encompassed. In particular, the present Advanced Engineered Pesticides, Marcel Dekker, Inc., New invention provides for an isolated nucleic acid molecule com York, N.Y.) These proteins were originally classified as Cry I 50 prising a nucleotide sequence encoding the amino acid to Cry V based primarily on their insecticidal activity. The sequence shown in SEQ ID NO:9, 11, 13, 15, or 18, or a major classes were Lepidoptera-specific (I), Lepidoptera nucleotide sequence set forth in SEQID NO:1, 2, 4, 6, 7, 8, and Diptera-specific (TI), Coleoptera-specific (III), Diptera 10, 12, 14, 16, or 17, as well as variants and fragments thereof. specific (IV), and nematode-specific (V) and (VI). The pro Nucleotide sequences that are complementary to a nucleotide teins were further classified into subfamilies; more highly 55 sequence of the invention, or that hybridize to a sequence of related proteins within each family were assigned divisional the invention are also encompassed. letters such as Cry1A, Cry 1B, Cry 1C, etc. Even more closely Methods are provided for producing the polypeptides of related proteins within each division were given names Such the invention, and for using those polypeptides for controlling as Cry 1C1, Cry 1C2, etc. or killing a lepidopteran or coleopteran pest. Methods and kits A new nomenclature was recently described for the Cry 60 for detecting the nucleic acids and polypeptides of the inven genes based upon amino acid sequence homology rather than tion in a sample are also included. insect target specificity (Crickmore et al. (1998) Microbiol. The compositions and methods of the invention are useful Mol. Biol. Rev. 62:807-813). In the new classification, each for the production of organisms with pesticide resistance, toxin is assigned a unique name incorporating a primary rank specifically bacteria and plants. These organisms and com (an Arabic number), a secondary rank (an uppercase letter), a 65 positions derived from them are desirable for agricultural tertiary rank (a lowercase letter), and a quaternary rank (an purposes. The compositions of the invention are also useful other Arabic number). In the new classification, Roman for generating altered or improved delta-endotoxin proteins US 8,541,366 B2 3 4 that have pesticidal activity, or for detecting the presence of identify delta-endotoxin encoding nucleic acids. As used delta-endotoxin proteins or nucleic acids in products or herein, the term “nucleic acid molecule' is intended to organisms. include DNA molecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and ana DESCRIPTION OF FIGURES logs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double FIGS. 1A and 1B show an alignment of AXMI-004 (SEQ stranded, but preferably is double-stranded DNA. ID NO:3) with AXMI-004B-2M (SEQ ID NO:5), AXMI An "isolated or “purified nucleic acid molecule or pro 004B-3M (SEQID NO:9), AXMI-004B-3M-ALT1 (SEQID tein, or biologically active portion thereof, is substantially NO:11), AXMI-004B-3M-ALT2 (SEQ ID NO:13), and 10 free of other cellular material, or culture medium when pro AXMI-004B-3M-ALT3 (SEQID NO:15). duced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically Syn DETAILED DESCRIPTION thesized. Preferably, an "isolated nucleic acid is free of sequences (preferably protein encoding sequences) that natu The present invention is drawn to compositions and meth 15 rally flank the nucleic acid (i.e., sequences located at the 5' ods for regulating pest resistance in organisms, particularly and 3' ends of the nucleic acid) in the genomic DNA of the plants or plant cells. The methods involve transforming organism from which the nucleic acid is derived. For pur organisms with a nucleotide sequence encoding a delta-en poses of the invention, “isolated when used to refer to dotoxin protein of the invention. In particular, the nucleotide nucleic acid molecules excludes isolated chromosomes. For sequences of the invention are useful for preparing plants and example, in various embodiments, the isolated delta-endot microorganisms that possess pesticidal activity. Thus, trans oxin encoding nucleic acid molecule can contain less than formed bacteria, plants, plant cells, plant tissues and seeds are about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of provided. Compositions are delta-endotoxin nucleic acids nucleotide sequences that naturally flank the nucleic acid and proteins of Bacillus thuringiensis. The sequences find use molecule in genomic DNA of the cell from which the nucleic in the construction of expression vectors for Subsequent trans 25 acid is derived. A delta-endotoxin protein that is substantially formation into organisms of interest, as probes for the isola free of cellular material includes preparations of protein hav tion of other delta-endotoxin genes, and for the generation of ing less than about 30%, 20%, 10%, or 5% (by dry weight) of altered pesticidal proteins by methods known in the art, Such non-delta-endotoxin protein (also referred to herein as a as domain Swapping or DNA shuffling. The proteins find use “contaminating protein'). in controlling or killing lepidopteran, coleopteran, and nema 30 Nucleotide sequences encoding the proteins of the present tode pest populations, and for producing compositions with invention include the sequence set forth in SEQID NO:1, 2, pesticidal activity. 4, 6, 7, 8, 10, 12, 14, 16, or 17, and variants, fragments, and By “delta-endotoxin' is intended a toxin from Bacillus complements thereof. By “complement' is intended a nucle thuringiensis that has toxic activity against one or more pests, otide sequence that is Sufficiently complementary to a given including, but not limited to, members of the Lepidoptera, 35 nucleotide sequence Such that it can hybridize to the given Diptera, and Coleoptera orders or members of the Nematoda nucleotide sequence to thereby form a stable duplex. The phylum, or a protein that has homology to Such a protein. In corresponding amino acid sequence for the delta-endotoxin Some cases, delta-endotoxin proteins have been isolated from protein encoded by this nucleotide sequence are set forth in other organisms, including Clostridium bifermentans and SEQID NO:9, 11, 13, 15, or 18. Paenibacillus popilliae. Delta-endotoxin proteins include 40 Nucleic acid molecules that are fragments of these delta amino acid sequences deduced from the full-length nucle endotoxin encoding nucleotide sequences are also encom otide sequences disclosed herein, and amino acid sequences passed by the present invention (for example, SEQID NO:6 that are shorter than the full-length sequences, either due to is a fragment of SEQ ID NO.4 and 10: SEQ ID NO:4 is a the use of an alternate downstream start site, or due to pro fragment of SEQ ID NO:2). By “fragment' is intended a cessing that produces a shorter protein having pesticidal 45 portion of the nucleotide sequence encoding a delta-endot activity. Processing may occur in the organism the protein is oxin protein. A fragment of a nucleotide sequence may expressed in, or in the pest after ingestion of the protein. encode a biologically active portion of a delta-endotoxin pro Delta-endotoxins include proteins identified as cry 1 through tein, or it may be a fragment that can be used as a hybridiza cry43, cyt1 and cyt2, and Cyt-like toxin. There are currently tion probe or PCR primer using methods disclosed below. over 250 known species of delta-endotoxins with a wide 50 Nucleic acid molecules that are fragments of a delta-endot range of specificities and toxicities. For an expansive list see oxin nucleotide sequence comprise at least about 50, 100, Crickmore et al. (1998), Microbiol. Mol. Biol. Rev. 62:807 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1050, 1100, 813, and for regular updates see Crickmore et al. (2003) 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, “Bacillus thuringiensis toxin nomenclature.” at www.bi 1650, 1700, 1750, 1800, 1850, 1860, 1870, 1880, 1885 con ols. SusX.ac.uk/Home/Neil Crickmore/Bt/index. 55 tiguous nucleotides, or up to the number of nucleotides Provided herein are novel isolated nucleotide sequences present in a full-length delta-endotoxin encoding nucleotide that confer pesticidal activity. Also provided are the amino sequence disclosed herein (for example, 1890 nucleotides for acid sequences of the delta-endotoxin proteins. The protein SEQ ID NO:1 and 2, 1806 nucleotides for SEQ ID NO:4, resulting from translation of this gene allows cells to control 1743 nucleotides for SEQID NO:6,7,8, and 16, 1809 nucle or kill pests that ingest it. 60 otides for SEQID NO:10, and 1752 nucleotides for SEQ ID Isolated Nucleic Acid Molecules, and Variants and Fragments NO:12 and 14) depending upon the intended use. By “con Thereof tiguous nucleotides is intended nucleotide residues that are One aspect of the invention pertains to isolated or recom immediately adjacent to one another. Fragments of the nucle binant nucleic acid molecules comprising nucleotide otide sequences of the present invention will encode protein sequences encoding delta-endotoxin proteins and polypep 65 fragments that retain the biological activity of the delta-en tides or biologically active portions thereof, as well as nucleic dotoxin protein and, hence, retain pesticidal activity. By acid molecules sufficient for use as hybridization probes to “retains activity” is intended that the fragment will have at US 8,541,366 B2 5 6 least about 30%, at least about 50%, at least about 70%, 80%, When utilizing BLAST, Gapped BLAST, and PSI-Blast pro 90%. 95% or higher of the pesticidal activity of the delta grams, the default parameters of the respective programs endotoxin protein. Methods for measuring pesticidal activity (e.g., BLASTX and BLASTN) can be used. Alignment may are well known in the art. See, for example, Czapla and Lang also be performed manually by inspection. (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. 5 Another non-limiting example of a mathematical algo (1988) Biochem. J. 252: 199-206; Marrone et al. (1985).J. of rithm utilized for the comparison of sequences is the Clust Economic Entomology 78:290–293; and U.S. Pat. No. 5,743, alW algorithm (Higgins et al. (1994) Nucleic Acids Res. 477, all of which are herein incorporated by reference in their 22:4673-4680). ClustalW compares sequences and aligns the entirety. entirety of the amino acid or DNA sequence, and thus can A fragment of a delta-endotoxin encoding nucleotide 10 provide data about the sequence conservation of the entire sequence that encodes a biologically active portion of a pro amino acid sequence. The ClustalW algorithm is used in tein of the invention will encode at least about 15, 25, 30, 50, several commercially available DNA/amino acid analysis 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, software packages, such as the ALIGNX module of the Vector 550,560,570, 575,580,585,590,595,600 contiguous amino NTI Program Suite (Invitrogen Corporation, Carlsbad, acids, or up to the total number of amino acids present in a 15 Calif.). After alignment of amino acid sequences with Clust full-length delta-endotoxin protein of the invention (for alW, the percent amino acid identity can be assessed. A non example, 580 amino acids for SEQID NO:9, 602 amino acids limiting example of a Software program useful for analysis of for SEQID NO:11, and 583 amino acids for SEQID NO:13 ClustalW alignments is GENEDOCTM. GENEDOCTM (Karl and 15). Nicholas) allows assessment of amino acid (or DNA) simi Preferred delta-endotoxin proteins of the present invention larity and identity between multiple proteins. Another non are encoded by a nucleotide sequence Sufficiently identical to limiting example of a mathematical algorithm utilized for the the nucleotide sequence of SEQID NO:1, 2, 4, 6, 7, 8, 10, 12, comparison of sequences is the algorithm of Myers and 14, 16, or 17. By “sufficiently identical is intended an amino Miller (1988) CABIOS 4: 11-17. Such an algorithm is incor acid or nucleotide sequence that has at least about 60% or porated into the ALIGN program (version 2.0), which is part 65% sequence identity, about 70% or 75% sequence identity, 25 of the GCG Wisconsin Genetics Software Package, Version about 80% or 85% sequence identity, about 90%, 91%, 92%, 10 (available from Accelrys, Inc., 9685 Scranton Rd., San 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence Diego, Calif., USA). When utilizing the ALIGN program for identity compared to a reference sequence using one of the comparing amino acid sequences, a PAM 120 weight residue alignment programs described herein using standard param table, a gap length penalty of 12, and a gap penalty of 4 can be eters. One of skill in the art will recognize that these values 30 used. can be appropriately adjusted to determine corresponding Unless otherwise stated, GAP Version 10, which uses the identity of proteins encoded by two nucleotide sequences by algorithm of Needleman and Wunsch (1970) J. Mol. Biol. taking into account codon degeneracy, amino acid similarity, 48(3):443-453, will be used to determine sequence identity or reading frame positioning, and the like. similarity using the following parameters: % identity and % To determine the percent identity of two amino acid 35 similarity for a nucleotide sequence using GAP Weight of 50 sequences or of two nucleic acids, the sequences are aligned and Length Weight of 3, and the nwsgapdna.cmp scoring for optimal comparison purposes. The percent identity matrix;% identity or 96 similarity for an amino acid sequence between the two sequences is a function of the number of using GAP weight of 8 and length weight of 2, and the identical positions shared by the sequences (i.e., percent BLOSUM62 scoring program. Equivalent programs may identity=number of identical positions/total number of posi 40 also be used. By "equivalent program' is intended any tions (e.g., overlapping positions)x100). In one embodiment, sequence comparison program that, for any two sequences in the two sequences are the same length. The percent identity question, generates an alignment having identical nucleotide between two sequences can be determined using techniques residue matches and an identical percent sequence identity similar to those described below, with or without allowing when compared to the corresponding alignment generated by gaps. In calculating percent identity, typically exact matches 45 GAP Version 10. are counted. The invention also encompasses variant nucleic acid mol The determination of percent identity between two ecules (for example, SEQ ID NO:2 is a variant of SEQ ID sequences can be accomplished using a mathematical algo NO:1; SEQID NO:7 and 8 are variants of SEQID NO:6; SEQ rithm. A nonlimiting example of a mathematical algorithm ID NO:10 is a variant of SEQID NO.4 and 12; and SEQID utilized for the comparison of two sequences is the algorithm 50 NO:14 is a variant of SEQ ID NO:12). “Variants” of the of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA delta-endotoxin encoding nucleotide sequences include those 87:2264, modified as in Karlin and Altschul (1993) Proc. sequences that encode the delta-endotoxin proteins disclosed Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is herein but that differ conservatively because of the degen incorporated into the BLASTN and BLASTX programs of eracy of the genetic code as well as those that are sufficiently Altschul et al. (1990).J. Mol. Biol. 215:403. BLAST nucle 55 identical as discussed above. Naturally occurring allelic vari otide searches can be performed with the BLASTN program, ants can be identified with the use of well-known molecular score=100, wordlength=12, to obtain nucleotide sequences biology techniques, such as polymerase chain reaction (PCR) homologous to delta-endotoxin-like nucleic acid molecules and hybridization techniques as outlined below. Variant of the invention. BLAST protein searches can be performed nucleotide sequences also include synthetically derived with the BLASTX program, score=50, wordlength=3, to 60 nucleotide sequences that have been generated, for example, obtain amino acid sequences homologous to delta-endotoxin by using site-directed mutagenesis but which still encode the protein molecules of the invention. To obtain gapped align delta-endotoxin proteins disclosed in the present invention as ments for comparison purposes, Gapped BLAST (in BLAST discussed below. Variant proteins encompassed by the present 2.0) can be utilized as described in Altschul et al. (1997) invention are biologically active, that is they continue to Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be 65 possess the desired biological activity of the native protein, used to perform an iterated search that detects distant rela that is, retaining pesticidal activity. By “retains activity” is tionships between molecules. See Altschuletal. (1997) supra. intended that the variant will have at least about 30%, at least US 8,541,366 B2 7 8 about 50%, at least about 70%, or at least about 80% of the tical between all proteins contained in an alignment of the pesticidal activity of the native protein. Methods for measur amino acid sequences of the present invention and known ing pesticidal activity are well known in the art. See, for delta-endotoxin sequences. Examples of residues that are example, Czapla and Lang (1990) J. Econ. Entomol. 83: conserved but that may allow conservative amino acid Sub 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; stitutions and still retain activity include, for example, resi Marrone et al. (1985) J. of Economic Entomology 78:290 dues that have only conservative substitutions between all 293; and U.S. Pat. No. 5,743,477, all of which are herein proteins contained in an alignment of the amino acid incorporated by reference in their entirety. sequences of the present invention and known delta-endot The skilled artisan will further appreciate that changes can oxin sequences. However, one of skill in the art would under be introduced by mutation of the nucleotide sequences of the 10 stand that functional variants may have minor conserved or invention thereby leading to changes in the amino acid nonconserved alterations in the conserved residues. sequence of the encoded delta-endotoxin proteins, without Alternatively, variant nucleotide sequences can be made by altering the biological activity of the proteins. Thus, variant introducing mutations randomly along all or part of the cod isolated nucleic acid molecules can be created by introducing ing sequence, Such as by Saturation mutagenesis, and the one or more nucleotide Substitutions, additions, or deletions 15 resultant mutants can be screened for ability to confer delta into the corresponding nucleotide sequence disclosed herein, endotoxin activity to identify mutants that retain activity. Such that one or more amino acid Substitutions, additions or Following mutagenesis, the encoded protein can be expressed deletions are introduced into the encoded protein. Mutations recombinantly, and the activity of the protein can be deter can be introduced by standard techniques, such as site-di mined using standard assay techniques. rected mutagenesis and PCR-mediated mutagenesis. Such Using methods such as PCR, hybridization, and the like variant nucleotide sequences are also encompassed by the corresponding delta-endotoxin sequences can be identified, present invention. Such sequences having Substantial identity to the sequences For example, conservative amino acid Substitutions may be of the invention. See, for example, Sambrook and Russell made at one or more predicted, nonessential amino acid resi (2001) Molecular Cloning. A Laboratory Manual. (Cold dues. A “nonessential amino acid residue is a residue that 25 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) can be altered from the wild-type sequence of a delta-endot and Innis, et al. (1990) PCR Protocols. A Guide to Methods oxin protein without altering the biological activity, whereas and Applications (Academic Press, NY). an “essential amino acid residue is required for biological In a hybridization method, all or part of the delta-endotoxin activity. A “conservative amino acid substitution' is one in nucleotide sequence can be used to screen cDNA or genomic which the amino acid residue is replaced with an amino acid 30 libraries. Methods for construction of such cDNA and residue having a similar side chain. Families of amino acid genomic libraries are generally known in the art and are residues having similar side chains have been defined in the disclosed in Sambrook and Russell, 2001, supra. The so art. These families includeamino acids with basic side chains called hybridization probes may be genomic DNA fragments, (e.g., lysine, arginine, histidine), acidic side chains (e.g., cDNA fragments, RNA fragments, or other oligonucleotides, aspartic acid, glutamic acid), uncharged polar side chains 35 and may be labeled with a detectable group such as P. or any (e.g., glycine, asparagine, glutamine, serine, threonine, other detectable marker, such as other radioisotopes, a fluo tyrosine, cysteine), nonpolar side chains (e.g., alanine, Valine, rescent compound, an enzyme, or an enzyme co-factor. leucine, isoleucine, proline, phenylalanine, methionine, tryp Probes for hybridization can be made by labeling synthetic tophan), beta-branched side chains (e.g., threonine, Valine, oligonucleotides based on the known delta-endotoxin-encod isoleucine) and aromatic side chains (e.g., tyrosine, phenyla 40 ing nucleotide sequence disclosed herein. Degenerate prim lanine, tryptophan, histidine). ers designed on the basis of conserved nucleotides or amino Delta-endotoxins generally have five conserved sequence acid residues in the nucleotide sequence or encoded amino domains, and three conserved structural domains (see, for acid sequence can additionally be used. The probe typically example, de Maagd et al. (2001) Trends Genetics 17:193 comprises a region of nucleotide sequence that hybridizes 199). The first conserved structural domain consists of seven 45 under stringent conditions to at least about 12, at least about alpha helices and is involved in membrane insertion and pore 25, at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, formation. Domain II consists of three beta-sheets arranged in 350, or 400 consecutive nucleotides of delta-endotoxin a Greek key configuration, and domain III consists of two encoding nucleotide sequence of the invention or a fragment antiparallel beta-sheets in jelly-roll” formation (de Maagdet or variant thereof. Methods for the preparation of probes for al., 2001, supra). Domains II and III are involved in receptor 50 hybridization are generally known in the art and are disclosed recognition and binding, and are therefore considered deter in Sambrook and Russell, 2001, supra herein incorporated by minants of toxin specificity. reference. Conserved group 1 is found from about amino acid residue For example, an entire delta-endotoxin sequence disclosed 174 to about 196 of SEQ ID NO:3. Conserved group 2 is herein, or one or more portions thereof, may be used as a found from aboutamino acid residue 250 to about 292 of SEQ 55 probe capable of specifically hybridizing to corresponding ID NO:3. Conserved group 3 is found from about amino acid delta-endotoxin-like sequences and messenger RNAS. To residue 476 to about 521 of SEQID NO:3. Conserved group achieve specific hybridization under a variety of conditions, 4 is found from about amino acid residue 542 to about 552 of Such probes include sequences that are unique and are pref SEQID NO:3. Conserved group 5 is found from about amino erably at least about 10 nucleotides in length, or at least about acid residue 618 to about 628 of SEQID NO:3. 60 20 nucleotides in length. Such probes may be used to amplify Amino acid Substitutions may be made in nonconserved corresponding delta-endotoxin sequences from a chosen regions that retain function. In general. Such substitutions organism by PCR. This technique may be used to isolate would not be made for conserved amino acid residues, or for additional coding sequences from a desired organism or as a amino acid residues residing within a conserved motif, where diagnostic assay to determine the presence of coding Such residues are essential for protein activity. Examples of 65 sequences in an organism. Hybridization techniques include residues that are conserved and that may be essential for hybridization screening of plated DNA libraries (either protein activity include, for example, residues that are iden plaques or colonies; see, for example, Sambrook et al. (1989) US 8,541,366 B2 10 Molecular Cloning: A Laboratory Manual (2d ed., Cold ently described. If the desired degree of mismatching results Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). in a T of less than 45° C. (aqueous solution) or 32° C. Hybridization of Such sequences may be carried out under (formamide solution), it is preferred to increase the SSC stringent conditions. By "stringent conditions' or 'stringent concentration so that a higher temperature can be used. An hybridization conditions” is intended conditions under which extensive guide to the hybridization of nucleic acids is found a probe will hybridize to its target sequence to a detectably in Tijssen (1993) Laboratory Techniques in Biochemistry and greater degree than to other sequences (e.g., at least 2-fold Molecular Biology—Hybridization with Nucleic Acid over background). Stringent conditions are sequence-depen Probes, Part I, Chapter 2 (Elsevier, NewYork); and Ausubelet dent and will be different in different circumstances. By con al., eds. (1995) Current Protocols in Molecular Biology, trolling the Stringency of the hybridization and/or washing 10 Chapter 2 (Greene Publishing and Wiley-Interscience, New conditions, target sequences that are 100% complementary to York). See Sambrook et al. (1989) Molecular Cloning. A the probe can be identified (homologous probing). Alterna Laboratory Manual (2d ed., Cold Spring Harbor Laboratory tively, stringency conditions can be adjusted to allow some Press, Cold Spring Harbor, N.Y.). mismatching in sequences so that lower degrees of similarity Isolated Proteins and Variants and Fragments Thereof are detected (heterologous probing). Generally, a probe is less 15 Delta-endotoxin proteins are also encompassed within the than about 1000 nucleotides in length, preferably less than present invention. By “delta-endotoxin protein' is intended a 500 nucleotides in length. protein having the amino acid sequence set forth in SEQID Typically, stringent conditions will be those in which the NO:9, 11, 13, 15, or 18. Fragments, biologically active por salt concentration is less than about 1.5 M Naion, typically tions, and variants thereofare also provided, and may be used about 0.01 to 1.0 MNaion concentration (or other salts) at pH to practice the methods of the present invention. 7.0 to 8.3 and the temperature is at least about 30°C. for short “Fragments’ or “biologically active portions' include probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for polypeptide fragments comprising amino acid sequences Suf long probes (e.g., greater than 50 nucleotides). Stringent con ficiently identical to the amino acid sequence set forth in SEQ ditions may also be achieved with the addition of destabiliz ID NO:9, 11, 13, 15, or 18, and that exhibit pesticidal activity ing agents such as formamide. Exemplary low stringency 25 (for example, SEQID NO:11, 13, and 15 are variants of SEQ conditions include hybridization with a buffer solution of 30 ID NO:9). A biologically active portion of a delta-endotoxin to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl protein can be a polypeptide that is, for example, 10, 25, 50. sulphate) at 37°C., and a wash in 1x to 2xSSC (20xSSC=3.0 100 or more amino acids in length. Such biologically active M. NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary portions can be prepared by recombinant techniques and moderate stringency conditions include hybridization in 40 to 30 evaluated for pesticidal activity. Methods for measuring pes 45% formamide, 1.0 MNaCl, 1% SDS at 37°C., and a wash ticidal activity are well known in the art. See, for example, in 0.5x to 1xSSC at 55 to 60° C. Exemplary high stringency Czapla and Lang (1990).J. Econ. Entomol. 83:2480-2485: conditions include hybridization in 50% formamide, 1 M Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et NaCl, 1% SDS at 37°C., and a wash in 0.1xSSC at 60 to 65° al. (1985).J. of Economic Entomology 78:290–293; and U.S. C. Optionally, wash buffers may comprise about 0.1% to 35 Pat. No. 5,743,477, all of which are herein incorporated by about 1% SDS. Duration of hybridization is generally less reference in their entirety. As used here, afragment comprises than about 24 hours, usually about 4 to about 12 hours. at least 8 contiguous amino acids of SEQID NO:9, 11, 13, 15, Specificity is typically the function of post-hybridization or 18. The invention encompasses other fragments, however, washes, the critical factors being the ionic strength and tem Such as any fragment in the protein greater than about 10, 20, perature of the final wash solution. For DNA-DNA hybrids, 40 30, 50, 100, 150, 200,250, 300,350, 400, 400, 450, 500,550, the T can be approximated from the equation of Meinkoth or 600 amino acids. and Wahl (1984) Anal. Biochem. 138:267-284: T-81.5° By “variants’ is intended proteins or polypeptides having C.+16.6 (log M)+0.41 (%GC)-0.61 (% form)-500/L: where an amino acid sequence that is at least about 60%. 65%, about M is the molarity of monovalent cations, 96 GC is the per 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, centage of guanosine and cytosine nucleotides in the DNA,% 45 94%. 95%, 96%, 97%, 98% or 99% identical to the amino form is the percentage of formamide in the hybridization acid sequence of SEQ ID NO:9, 11, 13, 15, or 18. Variants solution, and L is the length of the hybridin base pairs. TheT also include polypeptides encoded by a nucleic acid molecule is the temperature (under defined ionic strength and pH) at that hybridizes to the nucleic acid molecule of SEQID NO:1, which 50% of a complementary target sequence hybridizes to 2, 4, 6, 7, 8, 10, 12, 14, 16, or 17, or a complement thereof, a perfectly matched probe. T is reduced by about 1° C. for 50 understringent conditions. Variants include polypeptides that each 1% of mismatching; thus, T., hybridization, and/or differ in amino acid sequence due to mutagenesis. Variant wash conditions can be adjusted to hybridize to sequences of proteins encompassed by the present invention are biologi the desired identity. For example, if sequences with >90% cally active; that is, they continue to possess the desired identity are sought, the T can be decreased 10° C. Generally, biological activity of the native protein that is retaining pes stringent conditions are selected to be about 5° C. lower than 55 ticidal activity. Methods for measuring pesticidal activity are the thermal melting point (T) for the specific sequence and well known in the art. See, for example, Czapla and Lang its complement at a defined ionic strength and pH. However, (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. severely stringent conditions can utilize a hybridization and/ (1988) Biochem. J. 252: 199-206; Marrone et al. (1985).J. of or wash at 1, 2, 3, or 4°C. lower than the thermal melting point Economic Entomology 78:290–293; and U.S. Pat. No. 5,743, (T); moderately stringent conditions can utilize a hybridiza 60 477, all of which are herein incorporated by reference in their tion and/or wash at 6,7,8,9, or 10° C. lower than the thermal entirety. melting point (T), low stringency conditions can utilize a Bacterial genes, such as the genes of this invention, quite hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. often possess multiple methionine initiation codons in proX lower than the thermal melting point (T). Using the equa imity to the start of the open reading frame. Often, translation tion, hybridization and wash compositions, and desired T. 65 initiation at one or more of these start codons will lead to those of ordinary skill will understand that variations in the generation of a functional protein. For example, SEQ ID stringency of hybridization and/or wash Solutions are inher NO:9 (encoded by SEQID NO:6, 7, 8, and 16) represents a US 8,541,366 B2 11 12 downstream start site of SEQ ID NO:5. These start codons of inclusion of amino acid encoding sequences in the oligo can include ATG codons. However, bacteria such as Bacillus nucleotides utilized in the PCR amplification. Alternatively, sp. also recognize the codon GTG as a start codon, and pro the protein sequences added can include entire protein-cod teins that initiate translation at GTG codons contain a ing sequences, such as those used commonly in the art to methionine at the first amino acid. Furthermore, it is not often 5 generate protein fusions. Such fusion proteins are often used determined a priori which of these codons are used naturally to (1) increase expression of a protein of interest (2) introduce in the bacterium. Thus, it is understood that use of one of the a binding domain, enzymatic activity, or epitope to facilitate alternate methionine codons may also lead to generation of either protein purification, protein detection, or other experi delta-endotoxin proteins that encode pesticidal activity. mental uses known in the art (3) target secretion or translation These delta-endotoxin proteins are encompassed in the 10 of a protein to a Subcellular organelle, such as the periplasmic present invention and may be used in the methods of the space of Gram-negative bacteria, or the endoplasmic reticu present invention. lum of eukaryotic cells, the latter of which often results in Antibodies to the polypeptides of the present invention, or glycosylation of the protein. to variants or fragments thereof, are also encompassed. Meth Variant nucleotide and amino acid sequences of the present ods for producing antibodies are well known in the art (see, 15 invention also encompass sequences derived from mutagenic for example, Harlow and Lane (1988) Antibodies: A Labora and recombinogenic procedures such as DNA shuffling. With tory Manual, Cold Spring Harbor Laboratory, Cold Spring Such a procedure, one or more different delta-endotoxin pro Harbor, N.Y.; U.S. Pat. No. 4,196,265). tein coding regions can be used to create a new delta-endot Altered or Improved Variants oxin protein possessing the desired properties. In this manner, It is recognized that DNA sequences of a delta-endotoxin libraries of recombinant polynucleotides are generated from a may be altered by various methods, and that these alterations population of related sequence polynucleotides comprising may result in DNA sequences encoding proteins with amino sequence regions that have Substantial sequence identity and acid sequences different than that encoded by a delta-endot can be homologously recombined in vitro or in vivo. For oxin of the present invention. This protein may be altered in example, using this approach, sequence motifs encoding a various ways including amino acid substitutions, deletions, 25 domain of interest may be shuffled between a delta-endotoxin truncations, and insertions of one or more amino acids of SEQ gene of the invention and other known delta-endotoxin genes ID NO:9, 11, 13, 15, or 18, including up to about 2, about 3, to obtain a new gene coding for a protein with an improved about 4, about 5, about 6, about 7, about 8, about 9, about 10, property of interest, such as an increased insecticidal activity. about 15, about 20, about 25, about 30, about 35, about 40, Strategies for such DNA shuffling are known in the art. See, about 45, about 50, about 55, about 60, about 65, about 70, 30 for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA about 75, about 80, about 85, about 90, about 100, about 105, 91: 10747-10751; Stemmer (1994) Nature 370:389-391; about 110, about 115, about 120, about 125, about 130 or Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et more amino acid Substitutions, deletions or insertions. al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Methods for Such manipulations are generally known in the Proc. Natl. Acad. Sci. USA 94:45.04-4509: Crameri et al. art. For example, amino acid sequence variants of a delta 35 (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 endotoxin protein can be prepared by mutations in the DNA. and 5,837,458. This may also be accomplished by one of several forms of Domain Swapping or shuffling is another mechanism for mutagenesis and/or in directed evolution. In some aspects, the generating altered delta-endotoxin proteins. Domains II and changes encoded in the amino acid sequence will not Substan III may be swapped between delta-endotoxin proteins, result tially affect the function of the protein. Such variants will 40 ing in hybrid or chimeric toxins with improved pesticidal possess the desired pesticidal activity. However, it is under activity or target spectrum. Methods for generating recombi stood that the ability of a delta-endotoxin to confer pesticidal nant proteins and testing them for pesticidal activity are well activity may be improved by the use of such techniques upon known in the art (see, for example, Naimov et al. (2001) Appl. the compositions of this invention. For example, one may Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) express a delta-endotoxin in host cells that exhibit high rates 45 Appl. Environ. Microbiol. 62:1537-1543; Geet al. (1991).J. of base misincorporation during DNA replication, Such as Biol. Chem. 266:17954-17958; Schnepfet al. (1990).J. Biol. XL-1 Red (Stratagene). After propagation in Such strains, one Chem. 265:20923-20930; Ranget al. 91999) Appl. Environ. can isolate the delta-endotoxin DNA (for example by prepar Microbiol. 65:2918-2925). ing plasmid DNA, or by amplifying by PCR and cloning the Vectors resulting PCR fragment into a vector), culture the delta-en 50 A delta-endotoxin sequence of the invention may be pro dotoxin mutations in a non-mutagenic strain, and identify vided in an expression cassette for expression in a plant of mutated delta-endotoxin genes with pesticidal activity, for interest. By “plant expression cassette' is intended a DNA example by performing an assay to test for pesticidal activity. construct that is capable of resulting in the expression of a Generally, the protein is mixed and used in feeding assays. protein from an open reading frame in a plant cell. Typically See, for example Marrone et al. (1985).J. of Economic Ento 55 these contain a promoter and a coding sequence. Often, Such mology 78:290–293. Such assays can include contacting constructs will also contain a 3' untranslated region. Such plants with one or more pests and determining the plants constructs may contain a “signal sequence' or “leader ability to survive and/or cause the death of the pests. sequence' to facilitate co-translational or post-translational Examples of mutations that result in increased toxicity are transport of the peptide to certain intracellular structures Such found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 60 as the chloroplast (or other plastid), endoplasmic reticulum, 62:775-806. or Golgi apparatus. Alternatively, alterations may be made to the protein By "signal sequence' is intended a sequence that is known sequence of many proteins at the amino or carboxy terminus or Suspected to result in cotranslational or post-translational without substantially affecting activity. This can include peptide transport across the cell membrane. In eukaryotes, insertions, deletions, or alterations introduced by modern 65 this typically involves secretion into the Golgi apparatus, with molecular methods, such as PCR, including PCR amplifica Some resulting glycosylation. By “leader sequence' is tions that alter or extend the protein coding sequence by virtue intended any sequence that when translated, results in an US 8,541,366 B2 13 14 amino acid sequence Sufficient to trigger co-translational Mol Gen. Genet. 262:141-144; Proudfoot (1991) Cell transport of the peptide chain to a sub-cellular organelle. 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149: Thus, this includes leader sequences targeting transport and/ Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. or glycosylation by passage into the endoplasmic reticulum, (1990) Gene 91:151-158: Ballas et al. (1989) Nucleic Acids passage to vacuoles, plastids including chloroplasts, mito 5 Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. chondria, and the like. 15:9627-9639. By “plant transformation vector is intended a DNA mol Bacillus thuringiensis genes have been found to be prob ecule that is necessary for efficient transformation of a plant lematic when used for expression in higher plants. Expression cell. Such a molecule may consist of one or more plant from these native genes often leads to low accumulation of expression cassettes, and may be organized into more than 10 steady state mRNA and protein. This low accumulation has one “vector DNA molecule. For example, binary vectors are been shown to be due in part to ectopic splicing and/or poly plant transformation vectors that utilize two non-contiguous adenylation of the nascent transcript by plant mRNA process DNA vectors to encode all requisite cis- and trans-acting ing systems. Thus, several gene(s) of the invention have been functions for transformation of plant cells (Hellens and Mul optimized for increased expression in the transformed host lineaux (2000) Trends in Plant Science 5:446-451). “Vector” 15 cell. refers to a nucleic acid construct designed for transfer Any of the nucleotide sequences described herein can be between different host cells. “Expression vector” refers to a further optimized for expression in a host cell of interest. vector that has the ability to incorporate, integrate and express Thus, in one aspect of the invention, synthetic DNA heterologous DNA sequences or fragments in a foreign cell. sequences are designed for a given polypeptide, Such as the The cassette will include 5' and 3' regulatory sequences oper polypeptides of the invention. Expression of the open reading ably linked to a sequence of the invention. By “operably frame of the synthetic DNA sequence in a cell results in linked' is intended a functional linkage between a promoter production of the polypeptide of the invention. Synthetic and a second sequence, wherein the promoter sequence ini DNA sequences can be useful to simply remove unwanted tiates and mediates transcription of the DNA sequence cor restriction endonuclease sites, to facilitate DNA cloning strat responding to the second sequence. Generally, operably 25 egies, to alter or remove any potential codon bias, to alter or linked means that the nucleic acid sequences being linked are improve GC content, to remove or alter alternate reading contiguous and, where necessary to join two protein coding frames, and/or to alter or remove intron/exon splice recogni regions, contiguous and in the same reading frame. The cas tion sites, polyadenylation sites, Shine-Delgarno sequences, sette may additionally contain at least one additional gene to unwanted promoter elements and the like that may be present be cotransformed into the organism. Alternatively, the addi 30 in a native DNA sequence. It is also possible that synthetic tional gene(s) can be provided on multiple expression cas DNA sequences may be utilized to introduce other improve Settes. ments to a DNA sequence, such as introduction of an intron “Promoter refers to a nucleic acid sequence that functions sequence, creation of a DNA sequence that in expressed as a to direct transcription of a downstream coding sequence. The protein fusion to organelle targeting sequences, such as chlo promoter together with other transcriptional and translational 35 roplast transit peptides, apoplast/vacuolar targeting peptides, regulatory nucleic acid sequences (also termed "control or peptide sequences that result in retention of the resulting sequences') are necessary for the expression of a DNA peptide in the endoplasmic reticulum. Synthetic genes can sequence of interest. also be synthesized using host cell-preferred codons for Such an expression cassette is provided with a plurality of improved expression, or may be synthesized using codons at restriction sites for insertion of the delta-endotoxin sequence 40 a host-preferred codon usage frequency. See, for example, to be under the transcriptional regulation of the regulatory Campbell and Gowri (1990) Plant Physiol.92:1-11; U.S. Pat. regions. Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. The expression cassette will include in the 5'-3' direction of Published Application Nos. 2004.0005600 and 20010003849, transcription, a transcriptional and translational initiation and Murray et al. (1989) Nucleic Acids Res. 17:477-498, region (i.e., a promoter), a DNA sequence of the invention, 45 herein incorporated by reference. and a translational and transcriptional termination region The delta-endotoxin gene to be targeted to the chloroplast (i.e., termination region) functional in plants. The promoter may be optimized for expression in the chloroplast to account may be native or analogous, or foreign or heterologous, to the for differences in codon usage between the plant nucleus and plant host and/or to the DNA sequence of the invention. this organelle. In this manner, the nucleic acids of interest Additionally, the promoter may be the natural sequence or 50 may be synthesized using chloroplast-preferred codons. See, alternatively a synthetic sequence. Where the promoter is for example, U.S. Pat. No. 5,380,831, herein incorporated by “native' or “homologous' to the plant host, it is intended that reference. the promoter is found in the native plant into which the Plant Transformation promoter is introduced. Where the promoter is “foreign” or Methods of the invention involve introducing a nucleotide "heterologous' to the DNA sequence of the invention, it is 55 construct into a plant. By “introducing is intended to present intended that the promoter is not the native or naturally occur to the plant the nucleotide construct in Such a manner that the ring promoter for the operably linked DNA sequence of the construct gains access to the interior of a cell of the plant. The invention. methods of the invention do not require that a particular The termination region may be native with the transcrip method for introducing a nucleotide construct to a plant is tional initiation region, may be native with the operably 60 used, only that the nucleotide construct gains access to the linked DNA sequence of interest, may be native with the plant interior of at least one cell of the plant. Methods for introduc host, or may be derived from another source (i.e., foreign or ing nucleotide constructs into plants are known in the art heterologous to the promoter, the DNA sequence of interest, including, but not limited to, stable transformation methods, the plant host, or any combination thereof). Convenient ter transient transformation methods, and virus-mediated meth mination regions are available from the Ti-plasmid of A. 65 ods. tumefaciens, such as the octopine synthase and nopaline Syn By plant' is intended whole plants, plant organs (e.g., thase termination regions. See also Guerineau et al. (1991) leaves, stems, roots, etc.), seeds, plant cells, propagules, US 8,541,366 B2 15 16 embryos and progeny of the same. Plant cells can be differ In general, plant transformation methods involve transfer entiated or undifferentiated (e.g. callus, Suspension culture ring heterologous DNA into target plant cells (e.g. immature cells, protoplasts, leaf cells, root cells, phloem cells, pollen). or mature embryos, Suspension cultures, undifferentiated cal “Transgenic plants' or “transformed plants’ or “stably lus, protoplasts, etc.), followed by applying a maximum transformed plants or cells or tissues refers to plants that 5 threshold level of appropriate selection (depending on the have incorporated or integrated exogenous nucleic acid selectable marker gene) to recover the transformed plant cells sequences or DNA fragments into the plant cell. These from a group of untransformed cell mass. Explants are typi nucleic acid sequences include those that are exogenous, or cally transferred to a fresh Supply of the same medium and not present in the untransformed plant cell, as well as those cultured routinely. Subsequently, the transformed cells are that may be endogenous, or present in the untransformed 10 plant cell. “Heterologous' generally refers to the nucleic acid differentiated into shoots after placing on regeneration sequences that are not endogenous to the cell or part of the medium supplemented with a maximum threshold level of native genome in which they are present, and have been added selecting agent. The shoots are then transferred to a selective to the cell by infection, transfection, microinjection, elec rooting medium for recovering rooted shoot or plantlet. The troporation, microprojection, or the like. 15 transgenic plantlet then grows into a mature plant and pro Transformation of plant cells can be accomplished by one duces fertile seeds (e.g. Hiei et al. (1994) The Plant Journal of several techniques known in the art. The delta-endotoxin 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745 gene of the invention may be modified to obtain or enhance 750). Explants are typically transferred to a fresh supply of expression in plant cells. Typically a construct that expresses the same medium and cultured routinely. A general descrip Such a protein would contain a promoter to drive transcription tion of the techniques and methods for generating transgenic of the gene, as well as a 3' untranslated region to allow plants are found in Ayres and Park (1994) Critical Reviews in transcription termination and polyadenylation. The organiza Plant Science 13:219-239 and Bommineni and Jauhar (1997) tion of Such constructs is well known in the art. In some Maydica 42:107-120. Since the transformed material con instances, it may be useful to engineer the gene Such that the tains many cells; both transformed and non-transformed cells resulting peptide is secreted, or otherwise targeted within the 25 are present in any piece of Subjected target callus or tissue or plant cell. For example, the gene can be engineered to contain group of cells. The ability to kill non-transformed cells and a signal peptide to facilitate transfer of the peptide to the allow transformed cells to proliferate results in transformed endoplasmic reticulum. It may also be preferable to engineer plant cultures. Often, the ability to remove non-transformed the plant expression cassette to contain an intron, such that cells is a limitation to rapid recovery of transformed plant mRNA processing of the intron is required for expression. 30 cells and Successful generation of transgenic plants. Typically this “plant expression cassette' will be inserted Transformation protocols as well as protocols for introduc into a “plant transformation vector'. This plant transforma ing nucleotide sequences into plants may vary depending on tion vector may be comprised of one or more DNA vectors the type of plant or plant cell, i.e., monocot or dicot, targeted needed for achieving plant transformation. For example, it is for transformation. Generation of transgenic plants may be a common practice in the art to utilize plant transformation 35 performed by one of several methods, including, but not vectors that are comprised of more than one contiguous DNA limited to, microinjection, electroporation, direct gene trans segment. These vectors are often referred to in the art as fer, introduction of heterologous DNA by Agrobacterium into “binary vectors'. Binary vectors as well as vectors with plant cells (Agrobacterium-mediated transformation), bom helper plasmids are most often used for Agrobacterium-me bardment of plant cells with heterologous foreign DNA diated transformation, where the size and complexity of DNA 40 adhered to particles, ballistic particle acceleration, aerosol segments needed to achieve efficient transformation is quite beam transformation (U.S. Published Application No. large, and it is advantageous to separate functions onto sepa 2001 0026941; U.S. Pat. No. 4,945,050; International Publi rate DNA molecules. Binary vectors typically contain a plas cation No. WO 91/00915; U.S. Published Application No. mid vector that contains the cis-acting sequences required for 2002015066), Lec1 transformation, and various other non T-DNA transfer (such as left border and right border), a 45 particle direct-mediated methods to transfer DNA. selectable marker that is engineered to be capable of expres Methods for transformation of chloroplasts are known in Sionin a plant cell, and a “gene of interest' (a gene engineered the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. to be capable of expression in a plant cell for which genera Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. tion of transgenic plants is desired). Also present on this Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO plasmid vector are sequences required for bacterial replica 50 J. 12:601-606. The method relies on particle gun delivery of tion. The cis-acting sequences are arranged in a fashion to DNA containing a selectable marker and targeting of the allow efficient transfer into plant cells and expression therein. DNA to the plastid genome through homologous recombina For example, the selectable marker gene and the delta-endot tion. Additionally, plastid transformation can be accom oxin are located between the left and right borders. Often a plished by transactivation of a silent plastid-borne transgene second plasmid vector contains the trans-acting factors that 55 by tissue-preferred expression of a nuclear-encoded and plas mediate T-DNA transfer from Agrobacterium to plant cells. tid-directed RNA polymerase. Such a system has been This plasmid often contains the virulence functions (Vir reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA genes) that allow infection of plant cells by Agrobacterium, 91:7301-73.05. and transfer of DNA by cleavage at border sequences and Following integration of heterologous foreign DNA into vir-mediated DNA transfer, as is understood in the art (Hel 60 plant cells, one then applies a maximum threshold level of lens and Mullineaux (2000) Trends in Plant Science 5:446 appropriate selection in the medium to kill the untransformed 451). Several types of Agrobacterium strains (e.g. LBA4404, cells and separate and proliferate the putatively transformed GV3101, EHA101, EHA105, etc.) can be used for plant cells that survive from this selection treatment by transferring transformation. The second plasmid vector is not necessary regularly to a fresh medium. By continuous passage and for transforming the plants by other methods such as micro 65 challenge with appropriate selection, one identifies and pro projection, microinjection, electroporation, polyethylene liferates the cells that are transformed with the plasmid vec glycol, etc. tor. Molecular and biochemical methods can then be used to US 8,541,366 B2 17 18 confirm the presence of the integrated heterologous gene of transformed callus, and regeneration of fertile plants from interest into the genome of the transgenic plant. Such transgenic callus. In such process, one may use any gene The cells that have been transformed may be grown into as a selectable marker so long as its expression in plant cells plants in accordance with conventional ways. See, for confers ability to identify or select for transformed cells. example, McCormicket al. (1986) Plant Cell Reports 5:81– 5 A number of markers have been developed for use with 84. These plants may then be grown, and either pollinated plant cells, such as resistance to chloramphenicol, the ami with the same transformed strain or different strains, and the noglycoside G418, hygromycin, or the like. Other genes that resulting hybrid having constitutive expression of the desired encode a product involved in chloroplast metabolism may phenotypic characteristic identified. Two or more generations also be used as selectable markers. For example, genes that may be grown to ensure that expression of the desired phe 10 provide resistance to plant herbicides such as glyphosate, notypic characteristic is stably maintained and inherited and bromoxynil, or imidazolinone may find particular use. Such then seeds harvested to ensure expression of the desired phe genes have been reported (Stalker et al. (1985).J. Biol. Chem. notypic characteristic has been achieved. In this manner, the 263:6310-6314 (bromoxynil resistance nitrilase gene); and present invention provides transformed seed (also referred to Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS as “transgenic seed') having a nucleotide construct of the 15 imidazolinone resistance gene). Additionally, the genes dis invention, for example, an expression cassette of the inven closed herein are useful as markers to assess transformation tion, stably incorporated into their genome. of bacterial or plant cells. Methods for detecting the presence Evaluation of Plant Transformation of a transgene in a plant, plant organ (e.g., leaves, stems, roots, Following introduction of heterologous foreign DNA into etc.), seed, plant cell, propagule, embryo or progeny of the plant cells, the transformation or integration of heterologous same are well known in the art. In one embodiment, the gene in the plant genome is confirmed by various methods presence of the transgene is detected by testing for pesticidal Such as analysis of nucleic acids, proteins and metabolites activity. associated with the integrated gene. Fertile plants expressing a delta-endotoxin may be tested PCR analysis is a rapid method to screen transformed cells, for pesticidal activity, and the plants showing optimal activity tissue or shoots for the presence of incorporated gene at the 25 selected for further breeding. Methods are available in the art earlier stage before transplanting into the Soil (Sambrook and to assay for pest activity. Generally, the protein is mixed and Russell (2001) Molecular Cloning: A Laboratory Manual. used in feeding assays. See, for example Marrone etal. (1985) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, J. of Economic Entomology 78:290–293. N.Y.). PCR is carried out using oligonucleotide primers spe The present invention may be used for transformation of cific to the gene of interest or Agrobacterium vector back 30 any plant species, including, but not limited to, monocots and ground, etc. dicots. Examples of plants of interest include, but are not Plant transformation may be confirmed by Southern blot limited to, corn (maize), sorghum, wheat, sunflower, tomato, analysis of genomic DNA (Sambrook and Russell, 2001, crucifers, peppers, potato, cotton, rice, soybean, Sugarbeet, supra). In general, total DNA is extracted from the transfor Sugarcane, tobacco, barley, and oilseed rape, Brassica sp., mant, digested with appropriate restriction enzymes, frac 35 alfalfa, rye, millet, safflower, peanuts, Sweet potato, cassaya, tionated in anagarose geland transferred to a nitrocellulose or coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, nylon membrane. The membrane or “blot' is then probed avocado, fig, guava, mango, olive, papaya, cashew, macad with, for example, radiolabeled P target DNA fragment to amia, almond, oats, vegetables, ornamentals, and conifers. confirm the integration of introduced gene into the plant Vegetables include, but are not limited to, tomatoes, let genome according to standard techniques (Sambrook and 40 tuce, greenbeans, lima beans, peas, and members of the genus Russell, 2001, Supra). Curcunnis Such as cucumber, cantaloupe, and musk melon. In Northern blot analysis, RNA is isolated from specific Ornamentals include, but are not limited to, azalea, hydran tissues of transformant, fractionated in a formaldehyde aga gea, hibiscus, roses, tulips, daffodils, petunias, carnation, rose gel, and blotted onto a nylon filter according to standard poinsettia, and chrysanthemum. Preferably, plants of the procedures that are routinely used in the art (Sambrook and 45 present invention are crop plants (for example, maize, Sor Russell, 2001, supra). Expression of RNA encoded by the ghum, wheat, Sunflower, tomato, crucifers, peppers, potato, delta-endotoxin is then tested by hybridizing the filter to a cotton, rice, soybean, Sugarbeet, Sugarcane, tobacco, barley, radioactive probe derived from a delta-endotoxin, by methods oilseed rape, etc.). known in the art (Sambrook and Russell, 2001, supra). Use in Pest Control Western blot, biochemical assays and the like may be car 50 General methods for employing strains comprising a ried out on the transgenic plants to confirm the presence of nucleotide sequence of the present invention, or a variant protein encoded by the delta-endotoxin gene by standard thereof, in pesticide control or in engineering other organisms procedures (Sambrook and Russell, 2001, Supra) using anti as pesticidal agents are known in the art. See, for example bodies that bind to one or more epitopes present on the delta U.S. Pat. No. 5,039,523 and EPO480762A2. endotoxin protein. 55 The Bacillus strains containing a nucleotide sequence of Pesticidal Activity in Plants the present invention, or a variant thereof, or the microorgan In another aspect of the invention, one may generate trans isms that have been genetically altered to contain a pesticidal genic plants expressing a delta-endotoxin that has pesticidal gene and protein may be used for protecting agricultural activity. Methods described above by way of example may be crops and products from pests. In one aspect of the invention, utilized to generate transgenic plants, but the manner in which 60 whole, i.e., unlysed, cells of a toxin (pesticide)-producing the transgenic plant cells are generated is not critical to this organism are treated with reagents that prolong the activity of invention. Methods known or described in the art such as the toxin produced in the cell when the cell is applied to the Agrobacterium-mediated transformation, biolistic transfor environment of target pest(s). mation, and non-particle-mediated methods may be used at Alternatively, the pesticide is produced by introducing a the discretion of the experimenter. Plants expressing a delta 65 delta-endotoxin gene into a cellular host. Expression of the endotoxin may be isolated by common methods described in delta-endotoxin gene results, directly or indirectly, in the the art, for example by transformation of callus, selection of intracellular production and maintenance of the pesticide. In US 8,541,366 B2 19 20 one aspect of this invention, these cells are then treated under may also vary with respect to climatic conditions, environ conditions that prolong the activity of the toxin produced in mental considerations, and/or frequency of application and/ the cell when the cell is applied to the environment of target or severity of pest infestation. pest(s). The resulting product retains the toxicity of the toxin. The pesticide compositions described may be made by These naturally encapsulated pesticides may then be formu formulating either the bacterial cell, crystal and/or spore sus lated in accordance with conventional techniques for appli pension, or isolated protein component with the desired agri cation to the environment hosting a target pest, e.g., Soil, culturally-acceptable carrier. The compositions may be for water, and foliage of plants. See, for example EPA 0192319, mulated prior to administration in an appropriate means Such and the references cited therein. Alternatively, one may for as lyophilized, freeze-dried, desiccated, or in an aqueous 10 carrier, medium or Suitable diluent, such as Saline or other mulate the cells expressing a gene of this invention Such as to buffer. The formulated compositions may be in the form of a allow application of the resulting material as a pesticide. dust or granular material, or a Suspension in oil (vegetable or Pesticidal Compositions mineral), or water or oil/water emulsions, or as a wettable The active ingredients of the present invention are nor powder, or in combination with any other carrier material mally applied in the form of compositions and can be applied 15 Suitable for agricultural application. Suitable agricultural car to the crop area or plant to be treated, simultaneously or in riers can be solid or liquid and are well known in the art. The Succession, with other compounds. These compounds can be term 'agriculturally-acceptable carrier covers all adjuvants, fertilizers, weed killers, cryoprotectants, Surfactants, deter inert components, dispersants, Surfactants, tackifiers, bind gents, pesticidal Soaps, dormant oils, polymers, and/or time ers, etc. that are ordinarily used in pesticide formulation tech release or biodegradable carrier formulations that permit nology; these are well known to those skilled in pesticide long-term dosing of a target area following a single applica formulation. The formulations may be mixed with one or tion of the formulation. They can also be selective herbicides, more Solidor liquid adjuvants and prepared by various means, chemical insecticides, Virucides, microbicides, amoebicides, e.g., by homogeneously mixing, blending and/or grinding the pesticides, fungicides, bacteriocides, nematocides, mollusci pesticidal composition with Suitable adjuvants using conven cides or mixtures of several of these preparations, if desired, 25 tional formulation techniques. Suitable formulations and together with further agriculturally acceptable carriers, Sur application methods are described in U.S. Pat. No. 6,468.523, factants or application-promoting adjuvants customarily herein incorporated by reference. employed in the art of formulation. Suitable carriers and “Pest includes but is not limited to, insects, fungi, bacte adjuvants can be solid or liquid and correspond to the Sub ria, nematodes, mites, ticks, and the like. Insect pests include 30 insects selected from the orders Coleoptera, Diptera, stances ordinarily employed in formulation technology, e.g. Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemi natural or regenerated mineral Substances, solvents, dispers ptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, ants, wetting agents, tackifiers, binders or fertilizers. Like Anoplura, Siphonaptera, Trichoptera, etc., particularly wise the formulations may be prepared into edible “baits” or Coleoptera, Lepidoptera, and Diptera. fashioned into pest “traps' to permit feeding or ingestion by 35 The order Coleoptera includes the suborders Adephaga and a target pest of the pesticidal formulation. Polyphaga. Suborder Adephaga includes the Superfamilies Methods of applying an active ingredient of the present Caraboidea and Gyrinoidea, while suborder Polyphaga invention or an agrochemical composition of the present includes the superfamilies Hydrophiloidea, Staphylinoidea, invention that contains at least one of the pesticidal proteins Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dry produced by the bacterial strains of the present invention 40 opoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, include leaf application, seed coating and Soil application. Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Ceramby The number of applications and the rate of application depend coidea, Chrysomeloidea, and Curculionoidea. Superfamily on the intensity of infestation by the corresponding pest. Caraboidea includes the families Cicindelidae, Carabidae, The composition may be formulated as a powder, dust, and Dytiscidae. Superfamily Gyrinoidea includes the family pellet, granule, spray, emulsion, colloid, Solution, or Such 45 Gyrinidae. Superfamily Hydrophiloidea includes the family like, and may be prepared by Such conventional means as Hydrophilidae. Superfamily Staphylinoidea includes the desiccation, lyophilization, homogenation, extraction, filtra families Silphidae and Staphylinidae. Superfamily tion, centrifugation, sedimentation, or concentration of a cul Cantharoidea includes the families Cantharidae and Lampy ture of cells comprising the polypeptide. In all Such compo ridae. Superfamily Cleroidea includes the families Cleridae sitions that contain at least one Such pesticidal polypeptide, 50 and Dermestidae. Superfamily Elateroidea includes the fami the polypeptide may be present in a concentration of from lies Elateridae and Buprestidae. Superfamily Cucujoidea about 1% to about 99% by weight. includes the family Coccinellidae. Superfamily Meloidea Lepidopteran or coleopteran pests may be killed or reduced includes the family Meloidae. Superfamily Tenebrionoidea in numbers in a given area by the methods of the invention, or includes the family Tenebrionidae. Superfamily Scarabae may be prophylactically applied to an environmental area to 55 oidea includes the families Passalidae and Scarabaeidae. prevent infestation by a susceptible pest. Preferably the pest Superfamily Cerambycoidea includes the family Ceramby ingests, or is contacted with, a pesticidally-effective amount cidae. Superfamily Chrysomeloidea includes the family of the polypeptide. By "pesticidally-effective amount” is Chrysomelidae. Superfamily Curculionoidea includes the intended an amount of the pesticide that is able to bring about families Curculionidae and Scolytidae. death to at least one pest, or to noticeably reduce pest growth, 60 The order Diptera includes the Suborders Nematocera, feeding, or normal physiological development. This amount Brachycera, and Cyclorrhapha. Suborder Nematocera will vary depending on Such factors as, for example, the includes the families Tipulidae, Psychodidae, Culicidae, Cer specific target pests to be controlled, the specific environ atopogonidae, Chironomidae, Simuliidae, Bibionidae, and ment, location, plant, crop, or agricultural site to be treated, Cecidomyiidae. Suborder Brachycera includes the families the environmental conditions, and the method, rate, concen 65 Strationnyidae, Tabanidae. Therevidae, Asilidae, Mydidae, tration, stability, and quantity of application of the pesticid Bombyliidae, and Dolichopodidae. Suborder Cyclorrhapha ally-effective polypeptide composition. The formulations includes the Divisions Aschiza and Aschiza. Division US 8,541,366 B2 21 22 Aschiza includes the families Phoridae, Syrphidae, and aleurodes abutilonea, bandedwinged whitefly; Lygus lin Conopidae. Division Aschiza includes the Sections Acalyp eolaris, tarnished plant bug; Melanoplus femurrubrum, tratae and Calyptratae. Section Acalyptratae includes the redlegged grasshopper, Melanoplus differentialis, differen families Otitidae, Tephritidae, Agromyzidae, and Drosophil tial grasshopper; Thrips tabaci, onion thrips; Franklinkiella idae. Section Calyptratae includes the families Hippobos 5 fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spi cidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Cal der mite; Tetranychus urticae, two spotted spider mite; Rice: liphoridae, and Sarcophagidae. Diatraea saccharalis, Sugarcane borer, Spodoptera fru The order Lepidoptera includes the families Papilionidae, giperda, fall armyworm; Helicoverpa zea, corn earworm; Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Colaspis brunnea, grape colaspis; Lissorhoptrus Oryzophi Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arcti 10 lus, rice water weevil; Sitophilus Oryzae, rice weevil; Nepho idae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae. tettix nigropictus, rice leafhopper, Blissus leucopterus leu Insect pests of the invention for the major crops include: copterus, chinch bug: Acrosternum hilare, green Stink bug: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsi Soybean: Pseudoplusia includens, soybean looper, Anticar lon, black cutworm; Helicoverpa zea, corn earworm; sia gemmatalis, Velvetbean caterpillar, Plathypena scabra, Spodoptera frugiperda, fall armyworm; Diatraea grandi 15 green cloverworm; Ostrinia nubilalis, European corn borer; Osella, Southwestern corn borer, Elasmopalpus lignosellus, Agrotis ipsilon, black cutworm, Spodoptera exigua, beet lesser cornstalk borer, Diatraea saccharalis, Surgarcane armyworm; Heliothis virescens, cotton budworm; Helicov borer; Diabrotica virgifera, western corn rootworm: erpa zea, cotton bollworm; Epilachna varivestis, Mexican Diabrotica longicornis barberi, northern corn rootworm: bean beetle; Myzus persicae, green peach aphid; Empoasca Diabrotica undecimpunctata howardi, Southern corn root 20 fabae, potato leafhopper, Acrosternum hilare, green Stink worm; Melanotus spp., wireworms; Cyclocephala borealis, bug, Melanoplus femurrubrum, redlegged grasshopper, Mel northern masked chafer (white grub); Cyclocephala immacu anoplus differentialis, differential grasshopper, Hvlemya pla lata, Southern masked chafer (white grub); Popillia japonica, tura, seedcorn maggot; Sericothrips variabilis, soybean Japanese beetle; Chaetocnema pulicaria, corn flea beetle; thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, Sphenophorus maidis, maize billbug, Rhopalosiphum mai 25 Strawberry spider mite; Tetranychus urticae, two spotted spi dis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; der mite; Barley: Ostrinia nubilalis, European corn borer; Blissus leucopterus leucopterus, chinch bug; Melanoplus Agrotis ipsilon, black cutworm, Schizaphis graminum, green femurrubrum, redlegged grasshopper, Melanoplus san bug: Blissus leucopterus leucopterus, chinch bug: Acroster guinipes, migratory grasshopper, Hvlemya platura, seedcorn nun hilare, green Stink bug; Euschistus servus, brown Stink maggot, Agromyza parvicornis, cornblot leafminer, Anapho 30 bug: Delia platura, Seedcorn maggot, Mayetiola destructor, thrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Hessian fly; Petrobia latens, brown wheat mite: Oil Seed Tetranychus urticae, twospotted spidermite: Sorghum: Chilo Rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta partellus, Sorghum borer; Spodoptera frugiperda, fall army cruciferae, Flea beetle; Mamestra configurata, Bertha army worm; Helicoverpa zea, corn earworm; Elasmopalpus worm, Plutella xylostella, Diamond-back moth; Delia ssp., lignosellus, lesser cornstalk borer, Feltia subterranea, granu 35 Root maggots. late cutworm; Phyllophaga crimita, white grub; Eleodes, Nematodes include parasitic nematodes such as root-knot, Conoderus, and Aeolus spp., wireworms; Oulema melano cyst, and lesion nematodes, including Heterodera spp., pus, cereal leaf beetle; Chaetocnema pulicaria, corn flea Meloidogyne spp., and Globodera spp.; particularly members beetle; Sphenophorus maidis, maize billbug: Rhopalosiphum of the cyst nematodes, including, but not limited to, Het maidis; corn leaf aphid, Sipha flava, yellow Sugarcane aphid; 40 erodera glycines (soybean cyst nematode); Heterodera Blissus leucopterus leucopterus, chinch bug: Contarinia Schachtii (beet cyst nematode); Heterodera avenae (cereal Sorghicola, Sorghum midge; Tetranychus cinnabarinus, car cyst nematode); and Globodera rostochiensis and Globodera mine spider mite; Tetranychus urticae, twoSpotted spider pailida (potato cyst nematodes). Lesion nematodes include mite; Wheat: Pseudaletia unipunctata, army worm; Pratylenchus spp. Spodoptera frugiperda, fall armyworm; Elasmopalpus 45 Methods for Increasing Plant Yield lignosellus, lesser cornstalk borer; Agrotis Orthogonia, west Methods for increasing plant yield are provided. The meth ern cutworm; Elasmopalpus lignosellus, lesser cornstalk ods comprise introducing into a plant or plant cell a poly borer; Oulema melanopus, cereal leaf beetle: Hypera punc nucleotide comprising apesticidal sequence disclosed herein. tata, clover leaf weevil; Diabrotica undecimpunctata As defined herein, the "yield of the plant refers to the quality howardi, Southern corn rootworm, Russian wheat aphid; 50 and/or quantity of biomass produced by the plant. By “bio Schizaphis graminum, greenbug; Macrosiphum avenae, mass” is intended any measured plant product. An increase in English grain aphid; Melanoplus femurrubrum, redlegged biomass production is any improvement in the yield of the grasshopper, Melanoplus differentialis, differential grasshop measured plant product. Increasing plant yield has several per, Melanoplus sanguinipes, migratory grasshopper, May commercial applications. For example, increasing plant leaf etiola destructor, Hessian fly. Sitodiplosis mosellana, wheat 55 biomass may increase the yield of leafy vegetables for human midge; Meromyza americana, wheat stem maggot; Hylemya oranimal consumption. Additionally, increasingleafbiomass coarctata, wheat bulb fly; Frankliniella fisca, tobacco thrips: can be used to increase production of plant-derived pharma Cephus cinctus, wheat stem sawfly, Aceria tulipae, wheat ceutical or industrial products. An increase in yield can com curl mite; Sunflower: Suleima helianthana, sunflower bud prise any statistically significant increase including, but not moth; Homoeosoma electellum, Sunflower moth; Zygo 60 limited to, at least a 1% increase, at least a 3% increase, at gramma exclamationis, Sunflowerbeetle. Bothyrus gibbosus, least a 5% increase, at least a 10% increase, at least a 20% carrot beetle; Neoliasioptera murtfeldtiana, Sunflower seed increase, at least a 30%, at least a 50%, at least a 70%, at least midge: Cotton: Heliothis virescens, cotton budworm; Heli a 100% or a greater increase in yield compared to a plant not coverpa zea, cotton bollworm, Spodoptera exigua, beet expressing the pesticidal sequence. armyworm, Pectinophora gossypiella, pink bollworm; 65 In specific methods, plant yield is increased as a result of Anthonomus grandis, boll weevil: Aphis gossypii, cotton improved pest resistance of a plant expressing a pesticidal aphid; Pseudatomoscelis seriatus, cotton fleahopper; Tri protein disclosed herein. Expression of the pesticidal protein US 8,541,366 B2 23 24 results in a reduced ability of a pest to infest or feed on the TABLE 1 plant, thus improving plant yield. The following examples are offered by way of illustration Vectoring of Synthetic and variant Sequences and not by way of limitation. Nucleotide Amino acid Clone 5 Gene Name SEQ ID NO: SEQID NO: Designation EXPERIMENTAL Synaxmi-004B-2M 4 5 pAX3252 Synaxmi-004B-3M 6 9 pAX4538 Synaxmi-004C-3M 7 9 pAX3141 Synaxmi-004D-3M 8 9 pAX3142 Example 1 10 Synaxmi-004B-3m-alt1 10 11 pAX4522 pAX4515 Synaxmi-004B-3M-alt2 12 13 pAX4517 Generation of Synthetic and Variant Genes Synaxmi-004B-3M-alti 14 15 pAX4518

In one aspect of the invention, synthetic axmi-004 15 sequences were generated, for example synaxmi-004 (SEQ Example 3 ID NO:1) and synaxmi-004B (SEQ ID NO:2). These syn thetic sequences have an altered DNA sequence relative to the Assays for Pesticidal Activity axmi-004 sequence (SEQ ID NO:3) recited in U.S. patent application Ser. No. 10/782,020, herein incorporated by ref The ability of a pesticidal protein to act as a pesticide upon erence), and encode the original AXMI-004 protein. Like a pest is often assessed in a number of ways. One way well wise, synaxmi-004B-2M (SEQID NO:4) was designed and known in the art is to perform a feeding assay. In Such a encodes the axmi-004 alternate start site (herein referred to as feeding assay, one exposes the pest to a sample containing axmi-004B-2M and set forth in SEQ ID NO:5) originally either compounds to be tested, or control samples. Often this identified in U.S. patent application Ser. No. 10/782,020. 25 is performed by placing the material to be tested, or a suitable dilution of such material, onto a material that the pest will In another aspect of the invention, a third start site was ingest, Such as an artificial diet. The material to be tested may identified in the axmi-004 coding sequence. This coding be composed of a liquid, solid, or slurry. The material to be region is designated axmi-004B-3M (SEQ ID NO:16) and tested may be placed upon the Surface and then allowed to dry. encodes the AXMI-004B-3M amino acid sequence set forth 30 Alternatively, the material to be tested may be mixed with a in SEQID NO:9. Synthetic sequences encoding the AXMI molten artificial diet, then dispensed into the assay chamber. 004B-3M protein were also designed. These synthetic nucle The assay chamber may be, for example, a cup, a dish, or a otide sequences were designated Synaxmi-004B-3M, Syn well of a microtiter plate. axmi-004C-3M, and synaxmi-004D-3M and are set forth in Assays for Sucking pests (for example aphids) may involve SEQ ID NO:6, 7, and 8, respectively. 35 separating the test material from the insect by a partition, In another aspect of the invention, modified versions of the ideally a portion that can be pierced by the Sucking mouth nucleotide sequence encoding AXMI-004B-3M protein were parts of the Sucking insect, to allow ingestion of the test material. Often the test material is mixed with a feeding designed such that additional N-terminal residues are added stimulant, Such as Sucrose, to promote ingestion of the test to the encoded protein. These sequence are designated Syn 40 compound. axmi-004B-3M-alt1 (SEQ ID NO:10), synaxmi-004B-3M Other types of assays can include microinjection of the test alt2 (SEQ ID NO:12), synaxmi-004B-3M-alt3 (SEQ ID material into the mouth, or gut of the pest, as well as devel NO:14), and synaxmi-004B-3M-alta (SEQ ID NO:17). The opment of transgenic plants, followed by test of the ability of encoded proteins are designated AXMI-004B-3M-ALT1 the pest to feed upon the transgenic plant. Plant testing may (SEQID NO:11), AXMI-004B-3M-ALT2 (SEQID NO:13), 45 involve isolation of the plant parts normally consumed, for AXMI-004B-3M-ALT3 (SEQID NO:15), and AXMI-004B example, Small cages attached to a leaf, or isolation of entire 3M-ALT4 (SEQID NO:18). plants in cages containing insects. Other methods and approaches to assay pests are known in Example 2 the art, and can be found, for example in Robertson, J. L. & H. 50 K. Preisler. 1992. Pesticide bioassays with arthropods. CRC, Boca Raton, Fla. Alternatively, assays are commonly Vectoring of the Pesticidal Genes of the Invention for described in the journals “Arthropod Management Tests” and Plant Expression “Journal of Economic Entomology” or by discussion with members of the Entomological Society of America (ESA). 55 Each of the coding regions of the genes of the invention are Example 4 connected independently with appropriate promoter and ter minator sequences for expression in plants. Such sequences Transformation of the Genes of the Invention into are well known in the art and may include the rice actin Plant Cells by Agrobacterium-Mediated promoter or maize ubiquitin promoter for expression in 60 Transformation monocots, the Arabidopsis UBQ3 promoter or CaMV 35S promoter for expression indicots, and the nos or PinII termi Ears are collected 8-12 days after pollination. Embryos are nators. Techniques for producing and confirming promoter— isolated from the ears, and those embryos 0.8-1.5 mm in size gene—terminator constructs also are well known in the art. are used for transformation. Embryos are plated scutellum Select sequences (Table 1) were expressed in a bacterial 65 side-up on a suitable incubation media, and incubated over host cell and were found to have pesticidal activity against night at 25°C. in the dark. However, it is not necessary perse Lepidopteran species. to incubate the embryos overnight. Embryos are contacted US 8,541,366 B2 25 26 with an Agrobacterium strain containing the appropriate vec particular selection utilized. After the selection period, the tors for Tiplasmid mediated transfer for 5-10 min, and then resulting callus is transferred to embryo maturation media, plated onto co-cultivation media for 3 days (25° C. in the until the formation of mature somatic embryos is observed. dark). After co-cultivation, explants are transferred to recov The resulting mature Somatic embryos are then placed under ery period media for five days (at 25°C. in the dark). Explants low light, and the process of regeneration is initiated by are incubated in selection media for up to eight weeks, methods known in the art. The resulting shoots are allowed to depending on the nature and characteristics of the particular root on rooting media, and the resulting plants are transferred selection utilized. After the selection period, the resulting to nursery pots and propagated as transgenic plants. callus is transferred to embryo maturation media, until the Materials formation of mature somatic embryos is observed. The result 10 ing mature somatic embryos are then placed under low light, and the process of regeneration is initiated as known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propa DN62ASS Media gated as transgenic plants. 15 Components per liter Source Example 5 Chu SN6 Basal Salt Mixture 3.98 g/L Phytotechnology Labs (Prod. No. C 416) Transformation of Maize Cells with the Pesticidal Chu's N6 Vitamin Solution 1 mL/L (of Phytotechnology Labs Genes of the Invention (Prod. No. C 149) 1000x Stock) L-Asparagine 800 mg/L Phytotechnology Labs Maize ears are collected 8-12 days after pollination. Myo-inositol 100 mg/L Sigma Embryos are isolated from the ears, and those embryos 0.8- L-Proline 1.4 g/L Phytotechnology Labs 1.5 mm in size are used for transformation. Embryos are Casaminoacids 100 mg/L Fisher Scientific plated Scutellum side-up on a Suitable incubation media, Such 25 Sucrose 50 g/L Phytotechnology Labs as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of 1000x 2,4-D (Prod. No. D-7299) 1 mL/L (of Sigma Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L 1 mg/mL Stock) Myo-inositol: 1.4 g/L L-Proline; 100 mg/L Casaminoacids: 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D), and incubated overnight at 25°C. in the dark. 30 Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N The resulting explants are transferred to mesh squares (30 KC1, add Gelrite (Sigma) to 3 g/L, and autoclave. After cool 40 per plate), transferred onto osmotic media for 30-45min ing to 50° C., add 2 ml/L of a 5 mg/ml stock solution of Silver utes, then transferred to a beaming plate (see, for example, Nitrate (Phytotechnology Labs). Recipe yields about 20 PCT Publication No. WO/O138514 and U.S. Pat. No. 5,240, plates. 842). 35 All publications and patent applications mentioned in the DNA constructs designed to express the genes of the inven specification are indicative of the level of skill of those skilled tion in plant cells are accelerated into plant tissue using an in the art to which this invention pertains. All publications and aerosol beam accelerator, using conditions essentially as patent applications are herein incorporated by reference to the described in PCT Publication No. WO/O138514. After beam same extent as if each individual publication or patent appli ing, embryos are incubated for 30 minonosmotic media, then 40 cation was specifically and individually indicated to be incor placed onto incubation media overnight at 25°C. in the dark. To avoid unduly damaging beamed explants, they are incu porated by reference. bated for at least 24 hours prior to transfer to recovery media. Although the foregoing invention has been described in Embryos are then spread onto recovery period media, for 5 Some detail by way of illustration and example for purposes days, 25°C. in the dark, then transferred to a selection media. 45 of clarity of understanding, it will be obvious that certain Explants are incubated in selection media for up to eight changes and modifications may be practiced within the scope weeks, depending on the nature and characteristics of the of the appended claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 18

SEO ID NO 1 LENGTH: 1890 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: synthetic nucleotide sequence encoding AXMI-OO4 (synaxmi - OO4)

<4 OOs SEQUENCE: 1

atgagcgaac talagggcaa gttcaagaag to caccalacc gCacgtgctg cctic ctdaag 60

atcatcaa.ca togggggcag agg tatgaac agcaaggaac atgattacct Caaggtttgc 12O

aacgatctgt cc gatgcaaa catcaiacatg galacggttcg at aagaacga tgcact cqaa 18O

US 8,541,366 B2 31 32 - Continued

Ala Phe Met Glu His Wall Glu Glu Luell Ile Asp Thir Lys Ile Glu Gly 1OO 105 11 O

Ala Lys Asn Lys Ala Lell Ser Glu Luell Ala Gly Ile Glin Arg Asn 115 12 O 125

Lell Glu Thir Ile Glin Lell Arg Asn Glu Trp Glu Asn Asp Ile Glu 13 O 135 14 O

Asn Ser Ala Glin Gly Wall Ala Asn Tyr Glu Ser Luell Glu 145 150 155 160

Glin Ala Wall Glu Arg Ser Met Pro Glin Phe Ala Wall Glu Asn Phe Glu 1.65 17O 17s

Wall Pro Luell Luell Thir Wall Wall Glin Ala Ala Asn Lell His Luell Luell 18O 185 19 O

Lell Luell Arg Asp Wall Ser Wall Tyr Gly Trp Gly Trp Ser Glu 195

Glin Lys Ile Ile Tyr Asp Glin Ile Lys Thir His Glu 21 O 215 22O

Tyr Thir Asn His Cys Wall Asn Trp Asn Lys Gly Lell Glu Arg Luell 225 23 O 235 24 O

Asn Gly Ser Ser Glin Asp Trp Asn Asn Arg Phe 245 250 255

Arg Arg Glu Met Thir Lell Thir Wall Luell Asp Ile Wall Ala Luell Phe Pro 26 O 265 27 O

His Asp Wall Glin Thir Pro Ile Thir Thir Wall Ala Glin Luell Thir 28O 285

Arg Glu Wall Thir Asp Pro Luell Luell Asn Phe Asn Pro Luell His 29 O 295 3 OO

Ser Wall Ser Glin Lell Pro Ser Phe Ser Asp Met Glu Asn Ala Thir Ile 3. OS 310 315

Arg Thir Pro His Lell Met Glu Phe Luell Arg Met Lell Thir Ile Tyr Thir 3.25 330 335

Asp Trp Ser Wall Gly Arg Asn Tyr Trp Gly Gly His Arg Wall 34 O 345 35. O

Thir Ser Tyr His Wall Gly Gly Glu Asn Ile Arg Ser Pro Luell Gly 355 360 365

Arg Glu Ala Asn Glin Glu Wall Pro Arg Asp Phe Tyr Phe Gly Pro 37 O 375

Wall Phe Thir Lell Ser Pro Thir Luell Arg Pro Lell Glin Glin Pro 385 390 395 4 OO

Ala Pro Ala Pro Pro Phe Asn Luell Arg Ser Luell Glu Gly Wall Glu Phe 4 OS 415

His Thir Pro Thir Gly Ser Phe Met Tyr Arg Glu Arg Gly Ser Wall Asp 425 43 O

Ser Phe Asn Glu Lell Pro Pro Phe Asn Pro Wall Gly Lell Pro His 435 44 O 445

Wall Tyr Ser His Arg Lell Cys His Ala Thir Phe Wall Arg Ser Gly 450 45.5 460

Thir Pro Luell Thir Thir Gly Ala Ile Phe Ser Trp Thir His Arg Ser 465 470

Ala Glu Glu Thir Asn Thir Ile Glu Ser Asn Ile Ile Thir Glin Ile Pro 485 490 495

Lell Wall Ala Tyr Glin Ile Gly Ser Gly Thir Thir Wall Arg Gly SOO 505

Pro Gly Phe Thir Gly Gly Asp Ile Luell Arg Arg Thir Gly Pro Gly Thir 515 52O 525

US 8,541,366 B2 35 36 - Continued gtc.cgitalagg gtc.ctggittt Caccggtggit gatat cotca ggcgt accgg toctggtact 15OO titcggtgata tgaggatcaa Cat Caacgc.c c cc ct ct coc agagg tacag ggtc.cgitatic 1560 cgttacgc.ct ctaccaccga tictogcagttc gtcacct caa t caacggcac CaC catcaac 162O atcggcaact tcc ccaagac catcaacaac ctgaacaccc tgggctc.cga gggct accgc 168O accotct citt totccacccc titt cagottc tctaacgc.cc agt coat citt cc.gc.ctcggit 1740 atcCaggctt t cagcggtgt C caggaggtt tacgt.cgata agat.cgagtt catc.ccc.gtc 18OO gagtga 1806

SEO ID NO 5 LENGTH: 6O1 TYPE : PRT ORGANISM: Bacillus thuringiensis

< 4 OOs SEQUENCE: 5

Met Asn. Ser Lys Glu His Asp Tyr Luell Lys Wall Asn Asp Luell Ser 1. 5 15

Asp Asn Ile Asn Met Glu Arg Phe Asp Asn Asp Ala Lieu. Glu 2O 25

Ile Met Ser Ile Wall Ser Glu Luell Ile Gly Met Ile Pro Gly Gly 35 4 O 45

Thir Luell Glin Phe Wall Phe Asn Glin Luell Trp Ser Arg Luell Gly Asp 55 6 O

Ser Trp Asn Ala Phe Met Glu His Wall Glu Glu Lell Ile Asp Thr 65 70

Glu Gly Tyr Ala Lys Asn Ala Luell Ser Glu Luell Ala Gly 85 90 95

Ile Arg Asn Lieu. Glu Thr Tyr Ile Glin Luell Arg Asn Glu Trp. Glu 1OO 105 11 O

Asn Asp Ile Glu Asn Ser Lys Ala Glin Gly Wall Ala Asn 115 12 O 125

Glu Ser Luell Glu Glin Ala Wall Glu Arg Ser Met Pro Glin Phe Ala Wall 13 O 135 14 O

Glu Asn Phe Glu Wall Pro Leu Luell Thir Wall Tyr Wall Glin Ala Ala Asn 145 150 155 160

Lell His Luell Lieu. Luell Lieu. Arg Asp Wall Ser Wall Gly Cys Trp 1.65 17O 17s

Gly Trp Ser Glu Glin Lys Ile Llys Ile Asp Glin Ile Llys 18O 185 19 O

Thir His Glu Tyr Thir Asn His Wall ASn Trp Tyr Asn 195 2O5

Lell Glu Arg Lieu Lys Asn Lys Gly Ser Ser Glin Asp Trp Tyr Asn 21 O 215 22O

Tyr Asn Arg Phe Arg Arg Glu Met Thir Luell Thir Wall Lell Asp Ile Wall 225 23 O 235 24 O

Ala Luell Phe Pro His Tyr Asp Wall Glin Thir Pro Ile Thir Thir Wall 245 250 255

Ala Glin Luell Thr Arg Glu Val Tyr Thir Asp Pro Lell Lell Asn Phe Asn 26 O 265 27 O

Pro Luell His Ser Wall Ser Glin Luell Pro Ser Phe Ser Asp Met Glu 28O 285

Asn Ala Thir Ile Arg Thir Pro His Luell Met Glu Phe Lell Arg Met Lieu. 29 O 295 3 OO US 8,541,366 B2 38 - Continued

Thir Ile Thr Asp Trp Tyr Ser Val Gly Arg Asn Tyr Tyr Trp Gly 3. OS 310 315 32O

Gly His Arg Val Thr Ser Tyr His Val Gly Gly Glu Asn Ile Arg Ser 3.25 330 335

Pro Luell Tyr Gly Arg Glu Ala Asn Glin Glu Wall Pro Arg Asp Phe Tyr 34 O 345 35. O

Phe Gly Pro Val Phe Lys Thr Lieu. Ser Lys Pro Thir Luell Arg Pro 355 360 365

Lell Glin Glin Pro Ala Pro Ala Pro Pro Phe Asn Lell Arg Ser Lieu. Glu 37 O 375

Gly Wall Glu Phe His Thr Pro Thr Gly Ser Phe Met Arg Glu Arg 385 390 395 4 OO

Gly Ser Wall Asp Ser Phe Asn. Glu Leul Pro Pro Phe Asn Pro Val Gly 4 OS 41O 415

Lell Pro His Llys Val Tyr Ser His Arg Lieu. Cys His Ala Thir Phe Wall 425 43 O

Arg Ser Gly Thr Pro Tyr Lieu Thir Thr Gly Ala Ile Phe Ser Trp 435 44 O 445

Thir His Arg Ser Ala Glu Glu. Thir Asn. Thir Ile Glu Ser Asn Ile Ile 450 45.5 460

Thir Glin Ile Pro Lieu Val Lys Ala Tyr Glin Ile Gly Ser Gly Th Thr 465 470 47s 48O

Wall Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Lell Arg Arg Thr 485 490 495

Gly Pro Gly Thr Phe Gly Asp Met Arg Ile ASn Ile Asn Ala Pro Leu SOO 505

Ser Glin Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thir Thir Asp Lieu. 515 525

Glin Phe Wall Thir Ser Ile Asn Gly Thir Thir Ile Asn Ile Gly Asn. Phe 53 O 535 54 O

Pro Thir Ile ASn Asn Lieu. Asn Thr Lieu. Gly Ser Glu Gly Tyr Arg 5.45 550 555 560

Thir Wall Ser Phe Ser Thr Pro Phe Ser Phe Ser Asn Ala Glin Ser Ile 565 st O sts

Phe Arg Luell Gly Ile Glin Ala Phe Ser Gly Val Glin Glu Wall Tyr Val 585 59 O

Asp Ile Glu Phe Ile Pro Wall Glu 595

SEQ ID NO 6 LENGTH: 1743 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic nucleotide sequence encoding AXMI-OO4B-3M (synaxmi-OO4B-3m)

SEQUENCE: 6 atggagcgct tcgataagaa cgatgcc.ctic gagat.cggca tgtc.cat cqt citc.cgagctic 6 O atcggcatga tcc.ccggcgg caccgc.cctg cagttcgt.ct t caac cagot Ctggagcc.gc 12 O

Ctcggcgact Caggctggaa cgctitt catg gag catgtcg aggagct cat cgataccaag 18O atcgagggct acgc.caagaa Caaggcc.ctg tcc.gagctgg ccggitat coa gcgta acctg 24 O gaga cctaca tccagctgag galacgagtgg gagaacgata tcq agaactic Caaggcc.ca.g 3OO ggcaaggtog Ctalactacta cgaga.gc.ctg gag cagg.ccg tcgagcgttc aatgcct cag 360

US 8,541,366 B2 43 44 - Continued caccg.cgtga cat catatica tgttggtggit gaaaa catca gat.cgc.cgct Ctatggalaga 96.O gaagcaaatc aagaagttcC aagagatttic tacttictato gacctgtc.tt caaga cattg tcaaagccala cattgaggcc gct coagcag cgc.cgc.catt Caacttgagg agcttggaag gagttgagtt C Caca Cacca actggcagct t catgtacag agaaagagga 14 O t cagtggaca gct tcaatga gctgcc.gc.ca ttcaa.ccctg ttgggct tcc ticaca aggt c 2OO tacago cacc gcct ctogc.ca tgcaacct tc gtgaggalaga gcggcacgcc gtacct cacc 26 O accgc.gc.ca tottct catg gacccaccgc tctgctgaag aaacaaacac catcgagagc 32O aacatcatca cccagat.ccc gctggtgaag gcc taccaaa ttggat Cagg aacaa.ca.gtg aggaaaggac Ctggct tcac tggaggagac atc.ttgagaa gaactggacc tigaacattt 44 O ggagacatga ggat caac at Caacgc.gc.cg Ctgagccaaa gatacagggit gaggat Caga SOO tatgcttcaa caactgat ct t caatttgttg acaagcatca atggcaccac catcaa.catc 560 ggcaact tcc ccaagaccat Caacaacctic aac accttgg gct cagalagg Ctacaggacg gtgagcttct coacgc.catt cagcttctica aatgctcaaa gcatct tccg cct cqgcatc caagcc ttct ctdgagttca agaagttitat gtggacalaga ttgagttcat ccc.ggtggaa 74 O taa 743

<210s, SEQ ID NO 9 &211s LENGTH: 58O 212. TYPE: PRT &213s ORGANISM: Bacillus thuringiensis

<4 OOs, SEQUENCE: 9

Met Glu Arg Phe Asp Lys Asn Asp Ala Lieu. Glu Ile Gly Met Ser Ile 1. 1O 15

Wall Ser Glu Lieu. Ile Gly Met Ile Pro Gly Gly Thir Ala Leul Glin Phe 25 3O

Wall Phe Asin Glin Lieu. Trp Ser Arg Lieu. Gly Asp Ser Gly Trp Asn Ala 35 4 O 45

Phe Met Glu. His Wall Glu Glu Lieu. Ile Asp Thr Lys Ile Glu Gly Tyr SO 55 6 O

Ala Asn Lys Ala Lieu. Ser Glu Lieu Ala Gly Ile Glin Arg Asn Lieu 65 70 7s

Glu Thir Tyr Ile Glin Lieu. Arg Asn Glu Trp Glu Asn Asp Ile Glu Asn 85 90 95

Ser Ala Glin Gly Llys Val Ala Asn Tyr Tyr Glu Ser Lieu. Glu Glin 105 11 O

Ala Wall Glu Arg Ser Met Pro Glin Phe Ala Wall Glu Asn Phe Glu Wall 115 12 O 125

Pro Luell Lieu. Thr Val Tyr Val Glin Ala Ala Asn Lell His Luell Luell Luell 13 O 135 14 O

Lell Arg Asp Val Ser Val Tyr Gly Gly Trp Ser Glu Glin 145 150 155 160

Ile Lys Ile Tyr Tyr Asp Llys Glin Ile Llys Thir His Glu Tyr 1.65 17O 17s

Thir Asn His Cys Val Asn Trp Tyr Asn Lys Gly Lell Glu Arg Lieu Lys 18O 185 19 O

Asn Gly Ser Ser Tyr Glin Asp Trp Tyr Asn Tyr Asn Arg Phe Arg 195

Arg Glu Met Thir Lieu. Thir Wall Lieu. Asp Ile Val Ala Lell Phe Pro His 21 O 215 22O US 8,541,366 B2 45 46 - Continued Tyr Asp Val Glin Thr Tyr Pro Ile Thr Thr Val Ala Gln Lieu. Thir Arg 225 23 O 235 24 O Glu Val Tyr Thr Asp Pro Leu Lieu. Asn Phe ASn Pro Llys Lieu. His Ser 245 250 255 Val Ser Gln Leu Pro Ser Phe Ser Asp Met Glu Asn Ala Thr Ile Arg 26 O 265 27 O Thr Pro His Leu Met Glu Phe Lieu. Arg Met Lieu. Thir Ile Tyr Thr Asp 27s 28O 285 Trp Tyr Ser Val Gly Arg Asn Tyr Tyr Trp Gly Gly His Arg Val Thr 29 O 295 3 OO Ser Tyr His Val Gly Gly Glu Asn Ile Arg Ser Pro Leu Tyr Gly Arg 3. OS 310 315 32O Glu Ala Asn Glin Glu Val Pro Arg Asp Phe Tyr Phe Tyr Gly Pro Val 3.25 330 335 Phe Llys Thr Lieu Ser Lys Pro Thr Lieu. Arg Pro Leu Gln Glin Pro Ala 34 O 345 35. O Pro Ala Pro Pro Phe Asn Lieu. Arg Ser Leu Glu Gly Val Glu Phe His 355 360 365 Thr Pro Thr Gly Ser Phe Met Tyr Arg Glu Arg Gly Ser Val Asp Ser 37 O 375 38O Phe Asin Glu Lieu Pro Pro Phe Asin Pro Val Gly Lieu Pro His Llys Val 385 390 395 4 OO Tyr Ser His Arg Lieu. Cys His Ala Thr Phe Val Arg Lys Ser Gly Thr 4 OS 41O 415 Pro Tyr Lieu. Thir Thr Gly Ala Ile Phe Ser Trp Thr His Arg Ser Ala 42O 425 43 O

Glu Glu. Thir Asn. Thir Ile Glu Ser Asn. Ile Ile Thr Glin Ile Pro Leu 435 44 O 445 Val Lys Ala Tyr Glin Ile Gly Ser Gly Thr Thr Val Arg Lys Gly Pro 450 45.5 460 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Gly Pro Gly Thr Phe 465 470 47s 48O Gly Asp Met Arg Ile Asn. Ile Asn Ala Pro Lieu. Ser Glin Arg Tyr Arg 485 490 495 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Glin Phe Val Thr Ser SOO 505 51O Ile Asin Gly Thr Thr Ile Asn Ile Gly Asn Phe Pro Llys Thir Ile Asn 515 52O 525 Asn Lieu. Asn Thr Lieu. Gly Ser Glu Gly Tyr Arg Thr Val Ser Phe Ser 53 O 535 54 O Thr Pro Phe Ser Phe Ser Asn Ala Glin Ser Ile Phe Arg Lieu. Gly Ile 5.45 550 555 560 Glin Ala Phe Ser Gly Val Glin Glu Val Tyr Val Asp Lys Ile Glu Phe 565 st O sts

Ile Pro Wall Glu 58O

<210s, SEQ ID NO 10 &211s LENGTH: 1809 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic nucleotide sequence encoding AXMI-OO4B-3M-ALT1 (synaxmi-O 04B-3m-alt1)

<4 OOs, SEQUENCE: 10

US 8,541,366 B2 49 50 - Continued

35 4 O 45

Gly Thir Ala Luell Glin Phe Wall Phe Asn Glin Luell Trp Ser Arg Luell Gly SO 55 6 O

Asp Ser Gly Trp Asn Ala Phe Met Glu His Wall Glu Glu Luell Ile Asp 65 70

Thir Ile Glu Gly Tyr Ala Asn Lys Ala Lell Ser Luell Ala 85 90 95

Gly Ile Glin Arg Asn Lell Glu Thir Tyr Ile Glin Lell Arg Glu Trp 105

Glu Asn Asp Ile Glu Asn Ser Lys Ala Glin Gly Wall Asn Tyr 115 12 O 125

Glu Ser Luell Glu Glin Ala Wall Glu Arg Ser Met Pro Phe Ala 13 O 135 14 O

Wall Glu Asn Phe Glu Wall Pro Luell Luell Thir Wall Wall Ala Ala 145 150 155 160

Asn Luell His Luell Lell Lell Lell Arg Asp Wall Ser Wall Lys Cys 1.65 17O 17s

Trp Gly Trp Ser Glu Glin Ile Lys Ile Asp Lys Glin Ile 18O 185 19 O

Thir His Glu Tyr Thir Asn His Wall Asn Trp Asn 195

Gly Luell Glu Arg Lell Asn Gly Ser Ser Tyr Glin Asp Trp Tyr 21 O 215 22O

Asn Tyr Asn Arg Phe Arg Arg Glu Met Thir Luell Thir Wall Luell Asp Ile 225 23 O 235 24 O

Wall Ala Luell Phe Pro His Asp Wall Glin Thir Pro Ile Thir Thir 245 250 255

Wall Ala Glin Luell Thir Arg Glu Wall Tyr Thir Asp Pro Lell Luell Asn Phe 26 O 265 27 O

Asn Pro Lys Luell His Ser Wall Ser Glin Luell Pro Ser Phe Ser Asp Met 27s 28O 285

Glu Asn Ala Thir Ile Arg Thir Pro His Luell Met Glu Phe Luell Arg Met 29 O 295 3 OO

Lell Thir Ile Thir Asp Trp Ser Wall Gly Arg Asn Trp 3. OS 310 315

Gly Gly His Arg Wall Thir Ser His Wall Gly Gly Glu Asn Ile Arg 3.25 330 335

Ser Pro Luell Tyr Gly Arg Glu Ala Asn Glin Glu Wall Pro Arg Asp Phe 34 O 345 35. O

Phe Tyr Gly Pro Wall Phe Lys Thir Luell Ser Pro Thir Luell Arg 355 360 365

Pro Luell Glin Glin Pro Ala Pro Ala Pro Pro Phe Asn Lell Arg Ser Luell 37 O 375 38O

Glu Gly Wall Glu Phe His Thir Pro Thir Gly Ser Phe Met Arg Glu 385 390 395 4 OO

Arg Gly Ser Wall Asp Ser Phe Asn Glu Luell Pro Pro Phe Asn Pro Wall 4 OS 415

Gly Luell Pro His Lys Wall Ser His Arg Luell His Ala Thir Phe 425 43 O

Wall Arg Lys Ser Gly Thir Pro Tyr Luell Thir Thir Gly Ala Ile Phe Ser 435 44 O 445

Trp Thir His Arg Ser Ala Glu Glu Thir Asn Thir Ile Glu Ser Asn Ile 450 45.5 460

US 8,541,366 B2 53 - Continued tacctgacca ccggtgctat cittct cotgg acc cataggit cc.gctgagga gacta acacc 32O atcgagt caa a catcatcac ccagatcc cc Ctcgt Caagg Cctaccagat cqgcagoggc accaccgt.cc gta agggit Co tggittt cacc ggtggtgata t cct caggcg taccggit cot 44 O ggtactitt.cg gtgatatgag gat caa.catc aacgc.ccc.cc tct Cocagag gtacagggit c SOO cgitatic cqtt acgcct citac caccgatctg cagttcgt.ca cct caat caa cqgcaccacc 560 atcaa.cat cq gcaact tcc.c caagaccatc aacaacctga acaccCtggg Ctc.cgagggc taccgcaccg tot ctittct c cacco citt to agcttcticta acgcc cagtic catct tcc.gc

Ctcggt at CC aggctitt Cag cggtgtc.cag gaggtttacg tcqataagat cqagttcatc 74 O cc.cgt.cgagt ga

SEQ ID NO 13 LENGTH: 583 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: variant AXMI-OO4B-3M sequence ALT2)

SEQUENCE: 13

Met Ala Ala Met Glu Arg Phe Asp Lys Asn Asp Ala Lell Glu Ile Gly 1. 5 1O 15

Met Ser Ile Wal Ser Glu Lieu. Ile Gly Met Ile Pro Gly Gly Thr Ala 25 3O

Lell Glin Phe Wall Phe Asn. Glin Lieu. Trp Ser Arg Lell Gly Asp Ser Gly 35 4 O 45

Trp Asn Ala Phe Met Glu. His Wall Glu Glu Lieu. Ile Asp Thr Lys Ile SO 55 6 O

Glu Gly Tyr Ala Lys Asn Lys Ala Lieu. Ser Glu Lell Ala Gly Ile Glin 65 70 7s 8O

Arg Asn Lieu. Glu Thr Tyr Ile Glin Lieu. Arg Asn Glu Trp Glu Asn Asp 90 95

Ile Glu Asn Ser Lys Ala Glin Gly Llys Val Ala Asn Tyr Tyr Glu Ser 105 11 O

Lell Glu Glin Ala Val Glu Arg Ser Met Pro Glin Phe Ala Wall Glu Asn 115 12 O 125

Phe Glu Wall Pro Leu Lleu. Thir Wall Tyr Val Glin Ala Ala Asn Lieu. His 13 O 135 14 O

Lell Luell Lieu. Lieu. Arg Asp Val Ser Val Tyr Gly Lys Trp Gly Trp 145 150 155 160

Ser Glu Gln Lys Ile Lys Ile Tyr Glin Ile Lys Tyr Thr 1.65 17O 17s

His Glu Tyr Thr Asn His Cys Val Asn Trp Tyr Asn Gly Lieu. Glu 18O 185 19 O

Arg Luell Lys Asn Lys Gly Ser Ser Tyr Glin Asp Trp Tyr Asn Tyr Asn 195 2O5

Arg Phe Arg Arg Glu Met Thr Lieu. Thir Wall Lieu. Asp Ile Wall Ala Lieu. 21 O 215

Phe Pro His Tyr Asp Val Glin Thr Tyr Pro Ile Thir Thir Wall Ala Glin 225 23 O 235 24 O

Lell Thir Arg Glu Val Tyr Thr Asp Pro Luell Luell Asn Phe Asn Pro Llys 245 250 255

Lell His Ser Wal Ser Glin Leu Pro Ser Phe Ser Asp Met Glu Asn Ala 26 O 265 27 O US 8,541,366 B2 55 56 - Continued

Thir Ile Arg Thr Pro His Lieu Met Glu Phe Lieu. Arg Met Luell Thir Ile 285

Thir Asp Trp Tyr Ser Val Gly Arg Asn Tyr Tyr Trp Gly Gly His 29 O 295 3 OO

Arg Wall Thir Ser Tyr His Val Gly Gly Glu Asn Ile Arg Ser Pro Leu 3. OS 310 315

Gly Arg Glu Ala ASn Glin Glu Val Pro Arg Asp Phe Phe Tyr 3.25 330 335

Gly Pro Wall Phe Lys Thr Lieu Ser Llys Pro Thr Lell Arg Pro Lieu. Glin 34 O 345 35. O

Glin Pro Ala Pro Ala Pro Pro Phe Asn Lieu. Arg Ser Lell Glu Gly Val 355 360 365

Glu Phe His Thr Pro Thr Gly ser Phe Met Tyr Arg Glu Arg Gly Ser 37 O 375

Wall Asp Ser Phe Asn. Glu Lieu. Pro Pro Phe Asn Pro Wall Gly Leul Pro 385 390 395 4 OO

His Wall Tyr Ser His Arg Lieu Cys His Ala Thir Phe Wall Arg Llys 4 OS 41O 415

Ser Gly Thir Pro Tyr Lieu. Thir Thr Gly Ala Ile Phe Ser Trp Thir His 425 43 O

Arg Ser Ala Glu Glu Thir Asn. Thir Ile Glu Ser Asn Ile Ile Thr Gin 435 44 O 445

Ile Pro Luell Val Lys Ala Tyr Glin Ile Gly Ser Gly Thir Thir Val Arg 450 45.5 460

Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Lieu. Arg Arg Thir Gly Pro 465 470 48O

Gly Thir Phe Gly Asp Met Arg Ile ASn Ile Asn Ala Pro Luell Ser Glin 485 490 495

Arg Arg Val Arg Ile Arg Tyr Ala Ser Thr Thir Asp Luell Glin Phe SOO 505

Wall Thir Ser Ile Asn Gly Thr Thr Ile ASn Ile Gly Asn Phe Pro Llys 515 525

Thir Ile Asn Asn Lieu. Asn. Thir Lieu. Gly Ser Glu Gly Arg Thir Wall 53 O 535 54 O

Ser Phe Ser Thr Pro Phe Ser Phe Ser Asn Ala Glin Ser Ile Phe Arg 5.45 550 555 560

Lell Gly Ile Glin Ala Phe Ser Gly Wall Glin Glu Wall Wall Asp Llys 565 st O sts

Ile Glu Phe Ile Pro Wall Glu

SEQ ID NO 14 LENGTH: 1752 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic nucleotide sequence encoding AXMI-OO4B-3M-ALT3 (synaxmi-OO4B-3m-alt3)

<4 OOs, SEQUENCE: 14 atgcagat.ca tagcgctt cgataagaac gatgccCtcg agat.cggcat gtc. catcgt.c 6 O tcc.gagctica tcggcatgat CCC cqgcggc accoccctgc agttcgt citt caaccagctic 12 O

tcggcgactic aggctggaac gctitt catgg agcatgtcga ggagct catc 18O gataccalaga tcgagggcta cgc.caagaac aaggc cctgt cc.gagctggc cggitatic cag 24 O cgta acctgg agacct acat Ccagctgagg aacgagtggg agaacgatat cgagaactic C 3OO

US 8,541,366 B2 59 60 - Continued

Ile Glu Asn Ser Lys Ala Glin Gly Lys Wall Ala Asn Tyr Tyr Glu Ser 1OO 105 11 O

Lell Glu Glin Ala Wall Glu Arg Ser Met Pro Glin Phe Ala Wall Glu Asn 115 12 O 125

Phe Glu Wall Pro Lell Lell Thir Wall Wall Glin Ala Ala Asn Luell His 13 O 135 14 O

Lell Luell Luell Luell Arg Asp Wall Ser Wall Gly Trp Gly Trp 145 150 155 160

Ser Glu Glin Ile Ile Asp Glin Ile Tyr Thir 1.65 17O 17s

His Glu Thir Asn His Wall Asn Trp Asn Gly Luell Glu 18O 185 19 O

Arg Luell Lys Asn Lys Gly Ser Ser Glin Asp Trp Tyr Asn Asn 195 2O5

Arg Phe Arg Arg Glu Met Thir Luell Thir Wall Luell Asp Ile Wall Ala Luell 21 O 215

Phe Pro His Asp Wall Glin Thir Pro Ile Thir Thir Wall Ala Glin 225 23 O 235 24 O

Lell Thir Arg Glu Wall Thir Asp Pro Luell Luell Asn Phe Asn Pro 245 250 255

Lell His Ser Wall Ser Glin Lell Pro Ser Phe Ser Asp Met Glu Asn Ala 26 O 265 27 O

Thir Ile Arg Thir Pro His Lell Met Glu Phe Luell Arg Met Luell Thir Ile 285

Thir Asp Trp Tyr Ser Wall Gly Arg Asn Tyr Trp Gly Gly His 29 O 295 3 OO

Arg Wall Thir Ser Tyr His Wall Gly Gly Glu ASn Ile Arg Ser Pro Luell 3. OS 310 315

Gly Arg Glu Ala Asn Glin Glu Wall Pro Arg Asp Phe Phe 3.25 330 335

Gly Pro Wall Phe Lys Thir Lell Ser Lys Pro Thir Lell Arg Pro Luell Glin 34 O 345 35. O

Glin Pro Ala Pro Ala Pro Pro Phe Asn Luell Arg Ser Lell Glu Gly Wall 355 360 365

Glu Phe His Thir Pro Thir Gly Ser Phe Met Arg Glu Arg Gly Ser 37 O 375

Wall Asp Ser Phe Asn Glu Lell Pro Pro Phe ASn Pro Wall Gly Luell Pro 385 390 395 4 OO

His Wall Ser His Arg Luell His Ala Thir Phe Wall Arg 4 OS 415

Ser Gly Thir Pro Tyr Lell Thir Thir Gly Ala Ile Phe Ser Trp Thir His 425 43 O

Arg Ser Ala Glu Glu Thir Asn Thir Ile Glu Ser Asn Ile Ile Thir Glin 435 44 O 445

Ile Pro Luell Wall Lys Ala Tyr Glin Ile Gly Ser Gly Thir Thir Wall Arg 450 45.5 460

Lys Gly Pro Gly Phe Thir Gly Gly Asp Ile Luell Arg Arg Thir Gly Pro 465 470

Gly Thir Phe Gly Asp Met Arg Ile Asn Ile ASn Ala Pro Luell Ser Glin 485 490 495

Arg Arg Wall Arg Ile Arg Ala Ser Thir Thir Asp Luell Glin Phe SOO 505 51O

Wall Thir Ser Ile Asn Gly Thir Thir Ile Asn Ile Gly Asn Phe Pro Lys 515 52O 525 US 8,541,366 B2 61 62 - Continued

Thir Ile Asn Asn Lieu. Asn. Thir Lieu. Gly Ser Glu Gly Tyr Arg Thr Val 53 O 535 54 O

Ser Phe Ser Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Ile Phe Arg 5.45 550 555 560 Lieu. Gly Ile Glin Ala Phe Ser Gly Val Glin Glu Val Tyr Val Asp Llys 565 st O sts

Ile Glu Phe Ile Pro Wall Glu

<210s, SEQ I D NO 16 &211s LENGT H: 1743 212. TYPE : DNA <213> ORGANISM: Bacillus thuringiensis <4 OOs, SEQUENCE: 16 atggaacggit ttgataagaa tgatgcactg gaaattggta tgtc.cattgt atctgaactt 6 O attgg tatga titcCaggcgg aac agctittg caatttgttgt ttaat Caatt gtggit ct ct 12 O ttaggtgatt Ctggatggaa tgcgttcatg gaacatgtgg aggaattaat tgatact aaa 18O atagaagggit atgcaaaaaa taaagcctta tctgaattag Caggtataca aagaaac citt 24 O gaaacatata tacaattacg taatgaatgg gaaaatgata ttgaaaactic aaaggct Caa 3OO ggta agg tag Ctaattacta tgaaagttctt gag cagg.cgg ttgaaaggag tatgcct caa 360 tittgcagtgg agaattittga agtaccactt ttaactgtct atgtgcaa.gc tgctaatctt catttatt at tattaa.gaga tgttt cagtt tatggaaagt gttggggatg gtcggagcag aaaattaaaa. titt attatga taalacagatt aagtataccc atgaatacac aaatcattgt 54 O gtaaattggit ataataaagg actitgagaga ttaaaaaata aaggttcttic ttatcaagat tggtacaatt ataatcgttt cc.gtagagaa atgactic tita ctgttittaga tat cqttgct 660 ttatt cocq c actatogatgt acaaactitat CCaataacaa. cc.gttgctica gctaacaagg 72 O gaagtttata cggat.c ctitt act taattitt aat CCtaaat tacattctgt gtct caatta cctagttitta gtgacatgga aaatgcaa.ca attagaactic cacatctgat ggaatttitta 84 O agaatgctaa CaatittataC agattggitat agtgttgggala gaaact atta ttggggagga 9 OO catcgc.gtga cgt.cttacca tgtaggagga gagaatataa gat cacct ct atatggtaga 96.O gaggcaaatc aagaggitt CC tagagattitt tatttittatg gaccc.gttitt taagacgtta tcaaag.ccga ctictaagacc attacagcag cctgcaccag ctic ct cottt taatttacgt. agcttagagg gagtagaatt CCaCactCct acagg tagtt titatgtatcg tgaaagagga 14 O tcgg tagatt cittittaatga tittaatccag ttgggitt acc t catalaggta 2OO tacagt cacc gtt tatgtca tgcaacgttt gttcgtaaat Ctgggaccc.c ttatttaa.ca 26 O acaggtgc.ca tottttcttg gacacatcgt. agtgctgaag aalaccalataC aattgaatca 32O aatatt atta cgcaaatc cc gttagtaaaa gcatat caaa ttgggtcagg cactact gta aggaaaggac caggatt cac aggaggggat at actitcgaa gaacaggtoc tggaacattt 44 O ggagatatga gaataaat at taatgcacca ttatctoaaa. gat atctgt aaggatt.cgt. SOO tatgcttcta cgacagattit acaatttgtc acgagtatta atgggaccac Cattaatatt 560 ggtaact tcc cgaaaactat taataatcta aatactittag gttctgaggg ctatagaaca gtat cqttta gtactic catt tagtttctica aatgcacaaa gcatatttag attaggtata caag cattitt Ctggagttca agaagttitat gtggataaaa ttgaattitat tcc tigttgaa 74 O tag 743

US 8,541,366 B2 65 66 - Continued

<4 OOs, SEQUENCE: 18

Met Ala Ala Glu Arg Phe Asp Asn Asp Ala Lell Glu Ile Gly Met 1. 5 15

Ser Ile Wall Ser Glu Lell Ile Gly Met Ile Pro Gly Gly Thir Ala Luell 2O 25 3O

Glin Phe Wall Phe Asn Glin Lell Trp Ser Arg Luell Gly Asp Ser Gly Trp 35 4 O 45

Asn Ala Phe Met Glu His Wall Glu Glu Luell Ile Asp Thir Ile Glu SO 55 6 O

Gly Tyr Ala Asn Lys Ala Luell Ser Glu Luell Ala Gly Ile Glin Arg 65 70

Asn Luell Glu Thir Tyr Ile Glin Luell Arg Asn Glu Trp Glu Asn Asp Ile 85 90 95

Glu Asn Ser Lys Ala Glin Gly Wall Ala ASn Tyr Glu Ser Luell 105 11 O

Glu Glin Ala Wall Glu Arg Ser Met Pro Glin Phe Ala Wall Glu Asn Phe 115 12 O 125

Glu Wall Pro Luell Lell Thir Wall Wall Glin Ala Ala Asn Luell His Luell 13 O 135 14 O

Lell Luell Luell Arg Asp Wall Ser Wall Gly Lys Trp Gly Trp Ser 145 150 155 160

Glu Glin Ile Lys Ile Asp Lys Glin Ile Thir His 1.65 17s

Glu Tyr Thir Asn His Wall Asn Trp Tyr ASn Gly Luell Glu Arg 18O 185 19 O

Lell Asn Gly Ser Ser Tyr Glin Asp Trp Asn Tyr Asn Arg 195 2OO

Phe Arg Arg Glu Met Thir Lell Thir Wall Luell Asp Ile Wall Ala Luell Phe 21 O 215

Pro His Asp Wall Glin Thir Pro Ile Thir Thir Wall Ala Glin Luell 225 23 O 235 24 O

Thir Arg Glu Wall Tyr Thir Asp Pro Luell Luell ASn Phe Asn Pro Lys Luell 245 250 255

His Ser Wall Ser Glin Lell Pro Ser Phe Ser Asp Met Glu Asn Ala Thir 26 O 265 27 O

Ile Arg Thir Pro His Lell Met Glu Phe Luell Arg Met Lell Thir Ile 27s 28O 285

Thir Asp Trp Ser Wall Gly Arg Asn Tyr Trp Gly Gly His Arg 29 O 295 3 OO

Wall Thir Ser His Wall Gly Gly Glu Asn Ile Arg Ser Pro Luell Tyr 3. OS 310 315

Gly Arg Glu Ala Asn Glin Glu Wall Pro Arg Asp Phe Phe Tyr Gly 3.25 330 335

Pro Wall Phe Lys Thir Lell Ser Pro Thir Luell Arg Pro Luell Glin Glin 34 O 345 35. O

Pro Ala Pro Ala Pro Pro Phe Asn Luell Arg Ser Lell Glu Gly Wall Glu 355 360 365

Phe His Thir Pro Thir Gly Ser Phe Met Tyr Arg Glu Arg Gly Ser Wall 37 O 375

Asp Ser Phe Asn Glu Lell Pro Pro Phe Asn Pro Wall Gly Luell Pro His 385 390 395 4 OO

Wall Ser His Arg Lell His Ala Thir Phe Wall Arg Lys Ser 4 OS 41O 415 US 8,541,366 B2 67 68 - Continued

Gly Thir Pro Tyr Lell Thir Thir Gly Ala Ile Phe Ser Trp Thir His Arg 425 43 O

Ser Ala Glu Glu Thir Asn Thir Ile Glu Ser ASn Ile Ile Thir Glin Ile 435 44 O 445

Pro Luell Wall Lys Ala Glin Ile Gly Ser Gly Thir Thir Wall Arg Llys 450 45.5 460

Gly Pro Gly Phe Thir Gly Gly Asp Ile Luell Arg Arg Thir Gly Pro Gly 465 470 48O

Thir Phe Gly Asp Met Arg Ile Asn Ile Asn Ala Pro Lell Ser Glin Arg 485 490 495

Arg Wall Arg Ile Arg Ala Ser Thir Thir Asp Lell Glin Phe Wall SOO 505

Thir Ser Ile Asn Gly Thir Thir Ile Asn Ile Gly Asn Phe Pro Lys Thr 515 525

Ile Asn Asn Luell Asn Thir Lell Gly Ser Glu Gly Tyr Arg Thir Wall Ser 53 O 535 54 O

Phe Ser Thir Pro Phe Ser Phe Ser Asn Ala Glin Ser Ile Phe Arg Lieu. 5.45 550 555 560

Gly Ile Glin Ala Phe Ser Gly Wall Glin Glu Wall Wall Asp Lys Ile 565 st O sts

Glu Phe Ile Pro Wall Glu 58O

That which is claimed: 5. The composition of claim3, wherein said composition is 1. An isolated polypeptide with pesticidal activity, selected prepared by desiccation, lyophilization, homogenization, from the group consisting of extraction, filtration, centrifugation, sedimentation, or con a) a polypeptide comprising the amino acid sequence of 3s centration of a culture of Bacillus thuringiensis cells. SEQID NO:11, 13, 15, or 18; 6. The composition of claim 3, comprising from about 1% b) a polypeptide that is encoded by the nucleotide sequence to about 99% by weight of said polypeptide. of SEQID NO:10, 12, 14, or 17. 7. A method for controlling a lepidopteran or coleopteran 2. The polypeptide of claim 1 further comprising heterolo pest population comprising contacting said population with a gous amino acid sequences. 40 pesticidally-effective amount of the polypeptide of claim 1. 3. A composition comprising the polypeptide of claim 1. 8. A method for killing a lepidopteran or coleopteran pest, 4. The composition of claim3, wherein said composition is comprising contacting said pest with, or feeding to said pest, selected from the group consisting of a powder, dust, pellet, a pesticidally-effective amount of the polypeptide of claim 1. granule, spray, emulsion, colloid, and solution. k k k k k