USOO7772369B2 (12) United States Patent (10) Patent No.: US 7,772,369 B2 Rupar et al. (45) Date of Patent: Aug. 10, 2010 (54) COLEOPTERAN-TOXIC POLYPEPTIDE 6, 197,312 B1 3/2001 Peak et al. COMPOSITIONS AND INSECTRESISTANT 6,372.480 B1* 4/2002 Narva et al. .............. 435/2525 TRANSGENC PLANTS (75) Inventors: Mark J. Rupar, Wilmington, DE (US); FOREIGN PATENT DOCUMENTS William P. Donovan, Levittown, PA EP 0454485 A2 10, 1991 (US); Chih-Rei Chu, Exton, PA (US); WO WO 9314205 7, 1993 Elizabeth Pease, Danville, PA (US); WO WO 97/40162 10, 1997 Yuping Tan, Fremont, CA (US); Annette C. Slaney, Burlington, NJ (US); Thomas M. Malvar, Troy, MO (US); OTHER PUBLICATIONS James A. Baum, Webster Groves, MO Ely, S., The Engineering of Plants to Express Bacillus thuringiensis (US) Delta-Endotoxins, Bacillus thuringiensis, An Environmental Biopesticide: Theory and Practice, Edited by P.F. Entwistle et al., (73) Assignee: Monsanto Technology LLC, St. Louis, XP-002054693, pp. 105-124 (1993). MO (US) Donovan, W.P. et al., Characterization of Two Genes Encoding Bacillus thuringiensis Insecticidal Crystal Proteins Toxic to (*) Notice: Subject to any disclaimer, the term of this Coleoptera Species, Applied and Environmental Microbiology, patent is extended or adjusted under 35 XP-000876861, pp. 3921-3927 (1992). U.S.C. 154(b) by 24 days. Fidock et al., “Conservation of the Plasmodium falciparum Sporozoite Surface Protein Gene, STARP in Field Isolates and Dis (21) Appl. No.: 12/256,812 tinct Species of Plasmodium.” Molecular and Biochemical Parasitol ogy, 67:255-267 (1994). (22) Filed: Oct. 23, 2008 Yuan et al., GenBank Accession No. AJO00743 (1998). Salomon et al., GenBank Accession No. S42303 (1993). (65) Prior Publication Data Morio et al., GenBank Accession No. C89791 (1998). US 2009/OO94714 A1 Apr. 9, 2009 * cited by examiner Related U.S. Application Data Primary Examiner Anne Kubelik (62) Division of application No. 1 1/485,079, filed on Jul. (74) Attorney, Agent, or Firm Timothy K. Ball, Esq.; 12, 2006, now Pat. No. 7,442,540, which is a division Howrey LLP of application No. 10/408.692, filed on Apr. 7, 2003, now Pat. No. 7,078,592, which is a division of appli (57) ABSTRACT cation No. 09/563,269, filed on May 3, 2000, now Pat. No. 6,555,655. Disclosed are novel insecticidal polypeptides, and composi tions comprising these polypeptides, peptide fragments (60) Provisional application No. 60/172.240, filed on May thereof, and antibodies specific therefor. Also disclosed are 4, 1999. vectors, transformed host cells, and transgenic plants that contain nucleic acid segments that encode the disclosed Ö-en (51) Int. Cl. dotoxin polypeptides. Also disclosed are methods of identi C07K I4/325 (2006.01) fying related polypeptides and polynucleotides, methods of (52) U.S. Cl. ...................................................... 530/350 making and using transgenic cells comprising these poly (58) Field of Classification Search ....................... None nucleotide sequences, as well as methods for controlling an See application file for complete search history. insect population, Such as Colorado potato beetle, Southern (56) References Cited corn rootworm and western corn rootworm, and for confer ring to a plant resistance to a target insect species. U.S. PATENT DOCUMENTS 6,127,180 A 10, 2000 Narva et al. 10 Claims, 3 Drawing Sheets U.S. Patent Aug. 10, 2010 Sheet 1 of 3 US 7,772,369 B2 Hind g g> D 3 ECOR ECOR s r f HindIII FIG. 1 U.S. Patent Aug. 10, 2010 Sheet 2 of 3 US 7,772,369 B2 HindII Cla Ssp ECOR HindIII SSp Xmn ECOR HindII FIG. 2 U.S. Patent Aug. 10, 2010 Sheet 3 of 3 US 7,772,369 B2 O LO N. L. r CN | | | | s US 7,772,369 B2 1. 2 COLEOPTERAN-TOXC POLYPEPTOE insect, Solubilization in the insect midgut (a combination COMPOSITIONS AND INSECTRESISTANT stomach and Small intestine), resistance to digestive enzymes TRANSGENC PLANTS Sometimes with partial digestion actually “activating the toxin, binding to the midgut cells, formation of a pore in the This application is a divisional of application Ser. No. 5 insect cells and the disruption of cellular homeostasis (En 1 1/485,079, filed Jul 12, 2006 now U.S. Pat. No. 7,442,540, glish and Slatin, 1992). which is a divisional of application Ser. No. 10/408.692, filed One of the unique features of B. thuringiensis is its pro Apr. 7, 2003, now U.S. Pat. No. 7,078,592, which is a divi duction of crystal proteins during sporulation which are spe sional of application Ser. No. 09/563,269, filed May 3, 2000, cifically toxic to certain orders and species of insects. Many now U.S. Pat. No. 6,555,655, which claims the benefit of U.S. 10 different strains of B. thuringiensis have been shown to pro Provisional Application No. 60/172,240, filed May 4, 1999. duce insecticidal crystal proteins. Compositions including B. 1.O BACKGROUND OF THE INVENTION thuringiensis strains which produce proteins having insecti cidal activity against lepidopteran and dipteran insects have 1.1 Field of the Invention 15 been commercially available and used as environmentally The present invention relates generally to the fields of acceptable insecticides because they are quite toxic to the molecular biology. More particularly, certain embodiments specific target insect, but are harmless to plants and other concern methods and compositions comprising DNA seg non-targeted organisms. ments, and proteins derived from bacterial species. More The mechanism of insecticidal activity of the B. thuring particularly, it concerns novel genes from Bacillus thuring- 20 iensis crystal proteins has been studied extensively in the past iensis encoding coleopteran-toxic crystal proteins. Various decade. It has been shown that the crystal proteins are toxic to methods for making and using these DNA segments, DNA the insect only after ingestion of the protein by the insect. The segments encoding synthetically-modified Ö-endotoxin alkaline pH and proteolytic enzymes in the insect mid-gut polypeptides, and native and synthetic crystal proteins are solubilize the proteins, thereby allowing the release of com disclosed, such as, for example, the use of DNA segments as 25 diagnostic probes and templates for protein production, and ponents which are toxic to the insect. These toxic components the use of proteins, fusion protein carriers and peptides in disrupt the mid-gut cells, cause the insect to cease feeding, various immunological and diagnostic applications. Also dis and, eventually, bring about insect death. For this reason, B. closed are methods of making and using nucleic acid seg thuringiensis has proven to be an effective and environmen ments in the development of transgenic plant cells containing 30 tally safe insecticide in dealing with various insect pests. As noted by Höfte et al., (1989) the majority of insecticidal the polynucleotides disclosed herein. B. thuringiensis strains are active against insects of the order 1.2. Description of the Related Art Lepidoptera, i.e. caterpillar insects. Other B. thuringiensis Because crops of commercial interest are often the target of insect attack, environmentally-sensitive methods for control stains are insecticidally active against insects of the order 35 Diptera, i.e., flies and mosquitoes, or against both lepi ling or eradicating insect infestation are desirable in many dopteran and dipteran insects. In recent years, a few B. thur instances. This is particularly true for farmers, nurserymen, ingiensis strains have been reported as producing crystal pro growers, and commercial and residential areas which seek to control insect populations using eco-friendly compositions. teins that are toxic to insects of the order Coleoptera, i.e., The most widely used environmentally-sensitive insecticidal beetles (Krieg et al., 1983; Sicket al., 1990; Donovan et al., formulations developed in recent years have been composed 40 1992: Lambert et al., 1992a: 1992b). of microbial pesticides derived from the bacterium Bacillus 1.2.2 Genes Encoding Crystal Proteins thuringiensis. B. thuringiensis is a Gram-positive bacterium Many of the 8-endotoxins are related to various degrees by that produces crystal proteins or inclusion bodies which are similarities in their amino acid sequences. Historically, the specifically toxic to certain orders and species of insects. proteins and the genes which encode them were classified Many different strains of B. thuringiensis have been shown to 45 based largely upon their spectrum of insecticidal activity. The produce insecticidal crystal proteins. Compositions including review by Höfte and Whiteley (1989) discusses the genes and B. thuringiensis strains which produce insecticidal proteins proteins that were identified in B. thuringiensis prior to 1990, have been commercially-available and used as environmen and sets forth the nomenclature and classification scheme tally-acceptable insecticides because they are quite toxic to which has traditionally been applied to B. thuringiensis genes the specific target insect, but are harmless to plants and other 50 and proteins. cryI genes encode lepidopteran-toxic Cry I pro non-targeted organisms. teins, and cryII genes encode CryII proteins that are toxic to 1.2.1 Ö-Endotoxins both lepidopterans and dipterans. cry III genes encode Ö-endotoxins are used to controla wide range of leaf-eating coleopteran-toxic CryIII proteins, while cryIV genes encode caterpillars and beetles, as well as mosquitoes. These pro- 55 dipteran-toxic Cry IV proteins. teinaceous parasporal crystals, also referred to as insecticidal Based on the degree of sequence similarity, the proteins crystal proteins, crystal proteins, Bt inclusions, crystalline were further classified into subfamilies; more highly related inclusions, inclusion bodies, and Bt toxins, are a large collec proteins within each family were assigned divisional letters tion of insecticidal proteins produced by B. thuringiensis that such as CryIA, CryIB, CryIC, etc. Even more closely related are toxic upon ingestion by a Susceptible insect host.