USOO6555655B1 (12) United States Patent (10) Patent No.: US 6,555,655 B1 Rupar et al. 45) Date of Patent: Apr. 29,9 2003 (54) COLEOPTERAN-TOXIC POLYPEPTIDE FOREIGN PATENT DOCUMENTS COMPOSITIONS AND INSECT-RESISTANT TRANSGENC PLANTS EP O 454 485 10/1990 WO WO 93 14205 7/1993 (75) Inventors: Mark J. Rupar, Wilmington, DE (US); WO WO 97/40162 10/1997 William P. Donovan, Levittown, PA (US); Chih-Rei Chu, Exton, PA (US); OTHER PUBLICATIONS Elizabeth Pease, Danville, PA (US); Yuping Tan, Fremont, CA (US); Ely, S., The Engineering of Plants to Express Bacillus Annette C. Slaney, Burlington, NJ thuringiensis Ö-Endotoxins, Bacillus thuringiensis, An (US); Thomas M. Malvar, Troy, MO Environmental Biopesticide: Theory and Practice, Edited by (US); James A. Baum, Webster Groves, P.F. Entwistle et al., XP-002054693, pp. 105-124 (1993). MO (US (US) Donovan, W.P., et al., Characterization of Two Genes (73) Assignee: Monsanto Technology, LLC, St. Louis, Encoding Bacillus thuringiensis Insecticidal Crystal Pro MO (US) teins Toxic to Coleoptera Species, Applied and Environmen tal Microbiology, XP-000876861, pp. 3921–3927 (1992). (*) Notice: Subject to any disclaimer, the term of this pstent g is5), extended O Gy diustedJuSled under 35 Primary Examiner-Gabrielle Bugaisky a -- (74) Attorney, Agent, or Firm- Timothy K. Ball, Esq.; (21) Appl. No.: 09/563,269 Howrey Simon Arnold & White, LLP y - - - 9 (22) Filed: May 3, 2000 (57) ABSTRACT O O Disclosed are novel insecticidal polypeptides, and compo Related U.S. Application Data Sitions comprising these polypeptides, peptide fragments (60) Provisional application No. 60/172.240, filed on May 4, thereof, and antibodies Specific therefor. Also disclosed are 1999. vectors, transformed host cells, and transgenic plants that (51) Int. Cl." ....................... C07K 14/325; AO1N 37/18 contain nucleic acid Segments that encode the disclosed (52) U.S. Cl. ............................. 530/350, 514/2; 514/12; Ö-endotoxin polypeptides. Also disclosed are methods of 536/23.71 identifying related polypeptides and polynucleotides, meth (58) Field of Search ................................. 530/350, 825: Ods of making and using transgenic cells comprising these 514/2, 12; 536/23.71; 424/93.461 polynucleotide Sequences, as well as methods for controlling an insect population, Such as Colorado potato beetle, South (56) References Cited ern corn rootworm and western corn rootworm, and for conferring to a plant resistance to a target insect Species. U.S. PATENT DOCUMENTS 6,127,180 A 10/2000 Narva et al. ................ 435/419 12 Claims, 3 Drawing Sheets U.S. Patent Apr. 29, 2003 Sheet 1 of 3 US 6,555,655 B1 HindII LO N H L CD t R O CY) 2 ECOR N- L SB d ECOR O CD st CD Hind FIG. 1 U.S. Patent Apr. 29, 2003 Sheet 2 of 3 US 6,555,655 B1 Hind Cla SSp ECOR Hind SSp Xmn ECOR Hindi FIG. 2 U.S. Patent Apr. 29, 2003 Sheet 3 of 3 US 6,555,655 B1 BSA standards 1 2 3 4 5 6 7 8 M 8. F.G. 3 US 6,555,655 B1 1 2 COLEOPTERAN-TOXC POLYPEPTIDE midgut cells, formation of a pore in the insect cells and the COMPOSITIONS AND INSECTRESISTANT disruption of cellular homeostasis (English and Slatin, TRANSGENIC PLANTS 1992). One of the unique features of B. thuringiensis is its This application is based on U.S. Provisional Application production of crystal proteins during sporulation which are No. 60/172,240, filed May 4, 1999, the entire contents of Specifically toxic to certain orders and Species of insects. which are hereby incorporated by reference. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions 1.O. BACKGROUND OF THE INVENTION including B. thuringiensis Strains which produce proteins having insecticidal activity against lepidopteran and 1.1 Field of the Invention dipteran insects have been commercially available and used The present invention relates generally to the fields of as environmentally-acceptable insecticides because they are molecular biology. More particularly, certain embodiments quite toxic to the Specific target insect, but are harmless to concern methods and compositions comprising DNA plants and other non-targeted organisms. Segments, and proteins derived from bacterial Species. More 15 The mechanism of insecticidal activity of the B. thuring particularly, it concerns novel genes from Bacillus thuring iensis crystal proteins has been Studied extensively in the iensis encoding coleopteran-toxic crystal proteins. Various past decade. It has been shown that the crystal proteins are methods for making and using these DNA segments, DNA toxic to the insect only after ingestion of the protein by the Segments encoding Synthetically-modified Ö-endotoxin insect. The alkaline pH and proteolytic enzymes in the insect polypeptides, and native and Synthetic crystal proteins are mid-gut Solubilize the proteins, thereby allowing the release disclosed, Such as, for example, the use of DNA segments as of components which are toxic to the insect. These toxic diagnostic probes and templates for protein production, and components disrupt the mid-gut cells, cause the insect to the use of proteins, fusion protein carriers and peptides in cease feeding, and, eventually, bring about insect death. For various immunological and diagnostic applications. Also this reason, B. thuringiensis has proven to be an effective disclosed are methods of making and using nucleic acid 25 and environmentally Safe insecticide in dealing with various Segments in the development of transgenic plant cells con insect pests. taining the polynucleotides disclosed herein. As noted by Hofte et al., (1989) the majority of insecti 1.2 Description of the Related Art cidal B. thuringiensis Strains are active against insects of the Because crops of commercial interest are often the target order Lepidoptera, i.e. caterpillar insects. Other B. thuring of insect attack, environmentally-Sensitive methods for con iensis Strains are insecticidally active against insects of the trolling or eradicating insect infestation are desirable in order Diptera, i.e., flies and mosquitoes, or against both many instances. This is particularly true for farmers, lepidopteran and dipteran insects. In recent years, a few B. nurserymen, growers, and commercial and residential areas thuringienSiS Strains have been reported as producing crystal which Seek to control insect populations using eco-friendly proteins that are toxic to insects of the order Coleoptera, i.e., compositions. The most widely used environmentally 35 beetles (Krieg et al., 1983; Sicket al., 1990; Donovan et al., Sensitive insecticidal formulations developed in recent years 1992; Lambert et al., 1992a; 1992b). have been composed of microbial pesticides derived from 1.2.2 Genes Encoding Crystal Proteins the bacterium Bacillus thuringiensis. B. thuringiensis is a Many of the Ö-endotoxins are related to various degrees Gram-positive bacterium that produces crystal proteins or by Similarities in their amino acid Sequences. Historically, inclusion bodies which are specifically toxic to certain 40 the proteins and the genes which encode them were classi orders and species of insects. Many different strains of B. fied based largely upon their spectrum of insecticidal activ thuringiensis have been shown to produce insecticidal crys ity. The review by Hofte and Whiteley (1989) discusses the tal proteins. Compositions including B. thuringiensis Strains genes and proteins that were identified in B. thuringiensis which produce insecticidal proteins have been prior to 1990, and sets forth the nomenclature and classifi commercially-available and used as environmentally 45 cation Scheme which has traditionally been applied to B. acceptable insecticides because they are quite toxic to the thuringiensis genes and proteins. cry I genes encode Specific target insect, but are harmless to plants and other lepidopteran-toxic CryI proteins, and cryII genes encode non-targeted organisms. Cry II proteins that are toxic to both lepidopterans and 1.2.1 Ö-Endotoxins dipterans. cryIII genes encode coleopteran-toxic Cry III Ö-endotoxins are used to control a wide range of leaf 50 proteins, while cryIV genes encode dipteran-toxic CryIV eating caterpillars and beetles, as well as mosquitoes. These proteins. proteinaceous parasporal crystals, also referred to as insec Based on the degree of Sequence Similarity, the proteins ticidal crystal proteins, crystal proteins, Bt inclusions, crys were further classified into subfamilies; more highly related talline inclusions, inclusion bodies, and Bt toxins, are a large proteins within each family were assigned divisional letters collection of insecticidal proteins produced by B. thuring 55 such as CryIA, CryIB, CryIC, etc. Even more closely related iensis that are toxic upon ingestion by a Susceptible insect proteins within each division were given names Such as host. Over the past decade research on the Structure and CryIC1, CryIC2, etc. function of B. thuringiensis toxins has covered all of the Recently, a new nomenclature was developed which Sys major toxin categories, and while these toxins differ in tematically classified the Cry proteins based upon amino Specific Structure and function, general Similarities in the 60 acid Sequence homology rather than upon insect target Structure and function are assumed. Based on the accumu specificities (Crickmore et al., 1998). The classification lated knowledge of B. thuringiensis toxins, a generalized Scheme for many known toxins, not including allelic varia mode of action for B. thuringiensis toxins has been created tions in individual proteins, is Summarized in Section 4.3. and includes: ingestion by the insect, Solubilization in the 1.2.3 Crystal Proteins Toxic to Coleopteran Insects insect midgut (a combination Stomach and Small intestine), 65 The cloning and expression of the cry3Bb gene has been resistance to digestive enzymes Sometimes with partial described (Donovan et al., 1992). This gene encodes a digestion actually “activating the toxin, binding to the 74-kDa protein having insecticidal activity against US 6,555,655 B1 3 4 Coleopterans, such as Colorado potato beetle (CPB), and known insecticidal polypeptides.