US 20090087863A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0087863 A1 Mettus et al. (43) Pub. Date: Apr. 2, 2009 (54) METHOD FOR DETECTING POLYPEPTIDE now Pat. No. 7,022,897, which is a division of appli TOXC TO DIA BROTICA INSECTS cation No. 09/184,748, filed on Nov. 2, 1998, now Pat. No. 6,468,523. (76) Inventors: Anne-Marie Light Mettus, Publication Classification Feasterville, PA (US); James A. (51) Int. Cl. Baum, Doylestown, PA (US) GOIN 33/53 (2006.01) C07K 6/12 (2006.01) Correspondence Address: HOWREY LLP (52) U.S. Cl. ....................................... 435/7.2:530/389.5 C/O IP DOCKETING DEPARTMENT, 2941 (57) ABSTRACT FAIRVIEW PARK DRIVE SUTE 200 Disclosed is a novel Lepidopteran- and Coleopteran-active FALLS CHURCH, VA 22042 (US) Ö-endotoxin polypeptide, and compositions comprising the polypeptide, peptide fragments thereof, and antibodies spe (21) Appl. No.: 12/240,110 cific therefor. Also disclosed are vectors, transformed host cells, and transgenic plants that comprise nucleic acid seg (22) Filed: Sep. 29, 2008 ments encoding the polypeptide. Also disclosed are methods of identifying related polypeptides and polynucleotides, methods of making and using transgenic cells comprising the Related U.S. Application Data novel sequences of the invention, as well as methods for (62) Division of application No. 1 1/332,654, filed on Jan. controlling an insect population, Such as the Western Corn 13, 2006, now Pat. No. 7,429,454, which is a division Rootworm and Colorado potato beetle, and for conferring to of application No. 10/120.255, filed on Apr. 10, 2002, a plant population resistance to the target insect species. 1 kb xii; pEG1648 (Ssp Bal) (Hp B) (KSsp) HE H Hp & CryET70 Patent Application Publication Apr. 2, 2009 US 2009/0087863 A1 1 kb pEG1648 (Ssp Bal) (Hp B) (KSsp) HE H Hp Ori1030 ery pUC19BMb) cryET70 (Mb/B) pEG1657 pUC18 CryET70 US 2009/0087863 A1 Apr. 2, 2009 METHOD FOR DETECTING POLYPEPTIDE formation of a pore in the insect cells and the disruption of TOXCTO DIA BROTCA NSECTS cellular homeostasis (English and Slatin, 1992). 0006. One of the unique features of B. thuringiensis is its 1.O BACKGROUND OF THE INVENTION production of crystal proteins during sporulation which are 0001. 1.1 Field of the Invention specifically toxic to certain orders and species of insects. 0002 The present invention relates generally to the fields Many different strains of B. thuringiensis have been shown to of molecular biology. Provided are methods and composi produce insecticidal crystal proteins. Compositions including tions comprising DNA segments, and polypeptides derived B. thuringiensis strains which produce proteins having insec from bacterial species for use in insecticidal formulations and ticidal activity against lepidopteran and dipteran insects have the development of transgenic insect-resistant plants. More been commercially available and used as environmentally particularly, it concerns novel nucleic acids obtained from acceptable insecticides because they are quite toxic to the Bacillus thuringiensis that encode coleopteran- and lepi specific target insect, but are harmless to plants and other dopteran-toxic polypeptides. Various methods for making non-targeted organisms. and using these nucleic acids, DNA segments encoding Syn 0007. The mechanism of insecticidal activity of the B. thetically-modified CryET70 polypeptides, and native and thuringiensis crystal proteins has been studied extensively in synthetic polypeptide compositions are also disclosed. The the past decade. It has been shown that the crystal proteins are use of DNA segments as diagnostic probes and templates for toxic to the insect only after ingestion of the protein by the protein production, and the use of polypeptides, fusion pro insect. The alkaline pH and proteolytic enzymes in the insect teins, antibodies, and peptide fragments in various immuno logical and diagnostic applications are also disclosed, as are mid-gut solubilize the proteins, thereby allowing the release methods of making and using nucleic acid segments in the of components which are toxic to the insect. These toxic development of transgenic plant cells comprising the poly components disrupt the mid-gut cells, cause the insect to nucleotides disclosed herein. cease feeding, and, eventually, bring about insect death For 0003) 1.2. Description of the Related Art this reason, B. thuringiensis has proven to be an effective and 0004 Because crops of commercial interest are often the environmentally safe insecticide in dealing with various target of insect attack, environmentally-sensitive methods for insect pests. controlling or eradicating insect infestation are desirable in 0008. As noted by Höfte et al., (1989) the majority of many instances. This is particularly true for farmers, nursery insecticidal B. thuringiensis stains are active against insects men, growers, and commercial and residential areas which of the order Lepidoptera, i.e., caterpillar insects. Other B. seek to control insect populations using eco-friendly compo thuringiensis stains are insecticidally active against insects of sitions. The most widely used environmentally-sensitive the order Diptera, i.e., flies and mosquitoes, or against both insecticidal formulations developed in recent years have been lepidopteran and dipteran insects. In recent years, a few B. composed of microbial pesticides derived from the bacterium thuringiensis strains have been reported as producing crystal Bacillus thuringiensis. B. thuringiensis is a Gram-positive proteins that are toxic to insects of the order Coleoptera, i.e., bacterium that produces crystal proteins or inclusion bodies beetles (Krieg et al., 1983; Sick et al., 1990; Lambert et al., which are specifically toxic to certain orders and species of 1992a: 1992b). insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal 1.2.2 Genes Encoding Crystal Proteins proteins have been commercially-available and used as envi 0009. Many of the 8-endotoxins are related to various ronmentally-acceptable insecticides because they are quite degrees by similarities in their amino acid sequences. Histori toxic to the specific target insect, but are harmless to plants cally, the proteins and the genes which encode them were and other non-targeted organisms. classified based largely upon their spectrum of insecticidal activity. The review by Höfte and Whiteley (1989) discusses 12.1 8-ENDOTOXINS the genes and proteins that were identified in B. thuringiensis 0005 ö-endotoxins are used to control a wide range of prior to 1990, and sets forth the nomenclature and classifica leaf-eating caterpillars and beetles, as well as mosquitoes. tion scheme which has traditionally been applied to B. thur These proteinaceous parasporal crystals, also referred to as ingiensis genes and proteins. cry I genes encode lepidopteran insecticidal crystal proteins, crystal proteins, Bt inclusions, toxic CryI proteins. cryII genes encode Cry II proteins that are crystalline inclusions, inclusion bodies, and Bt toxins, are a toxic to both lepidopterans and dipterans. cryIII genes encode large collection of insecticidal proteins produced by B. thur coleopteran-toxic CryIII proteins, while cryIV genes encode ingiensis that are toxic upon ingestion by a susceptible insect dipteran-toxic Cry IV proteins. host. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the 0010 Based on the degree of sequence similarity, the pro major toxin categories, and while these toxins differ in spe teins were further classified into subfamilies; more highly cific structure and function, general similarities in the struc related proteins within each family were assigned divisional ture and function are assumed. Based on the accumulated letters such as CryIA, CryIB, Cry1C, etc. Even more closely knowledge of B. thuringiensis toxins, a generalized mode of related proteins within each division were given names Such action for B. thuringiensis toxins has been created and as CryIC1, CryIC2, etc. includes: ingestion by the insect, Solubilization in the insect 0011 Recently, a new nomenclature was developed which midgut (a combination stomach and Small intestine), resis systematically classified the Cry proteins based upon amino tance to digestive enzymes sometimes with partial digestion acid sequence homology rather than upon insect target speci actually “activating the toxin, binding to the midgut cells, ficities. The classification scheme for many known toxins, not US 2009/0087863 A1 Apr. 2, 2009 including allelic variations in individual proteins, is Summa nucleic acid segment comprising at least 45 contiguous rized in Table 2 of Section 4.3. nucleotides from SEQ ID NO:1, and in one embodiment comprise the coding region from nucleotide 92 to nucleotide 1.2.3 Identification of Crystal Proteins Toxic to WCRW 2254 of SEQID NO:1. The invention also discloses compo Insects sitions and insecticidal formulations that comprise Such a polypeptide. Such composition may be a cell extract, cell 0012. The cloning and expression of the cry3Bb gene has Suspension, cell homogenate, cell lysate, cell Supernatant, been described (Donovan et al., 1992). This gene codes for a protein of 74 kDa with activity against Coleopteran insects, cell filtrate, or cell pellet of a bacteria cell that comprises a particularly the Colorado potato beetle