USOO5840868A United States Patent (19) 11 Patent Number: 5,840,868 Warren et al. (45) Date of Patent: Nov. 24, 1998

54). PESTICIDAL PROTEINS AND STRAINS Heimpel, A.M., “The pH in the Gut and Blood of the Larch Sawfly, Pristiphora erichsonii (HTG.), and Other Insects 75 Inventors: Gregory W. Warren; Michael G. with Reference to the Pathogenicity of Bacillus cereuS FR. Koziel, both of Cary; Martha A. and FR.", Can. J. Zool, 33:99-106 (1955). Mullins, Raleigh; Brian Carr; Nalini Heimpel, A.M., “Investigations of the Mode of Action of M. Desai, both of Cary; Kristy Strains of Bacillus cereus FR. and FR. Pathogenic for the Kostichka, Durham, all of N.C. Larch Sawfly, Pristiphora erichsonii (HTG.)", Can. J. Zool, 73 Assignee: Novartis Finance Corporation, New 33:311-326 (1995). York, N.Y. Hofte, H., et al., “Insecticidal Crystal Proteins of Bacillus thuringiensis", Microbiological Reviews, 53(2):242-255 21 Appl. No.: 471,044 (1989). Koziel, M.G., et al., “Field Performance of Elite Tansgenic 22 Filed: Jun. 6, 1995 Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis”, Bio/Technology, 11:194-200 Related U.S. Application Data (1993). 60 Division of Ser. No. 463,483, Jun. 5, 1995, which is a Krieg, A., “Thuricin, a Bacteriocin Produced by Bacillus continuation-in-part of Ser. No. 314,594, Sep. 28, 1994, thuringiensis”, J. Invert. Path., 15:291 (1970). abandoned, which is a continuation-in-part of Ser. No. 218,018, Mar. 23, 1994, abandoned, which is a continuation Krieg, A., “Concerning Alpha-exotoxin Produced by Veg in-part of Ser. No. 37,057, Mar. 25, 1993, abandoned. etative Cells of Bacillus thuringiensis and Bacillus cereus', 51) Int. Cl...... C07H 21/02; CO7H 21/04; J. Invert. Path., 17:134–135 (1971). C12O 1/68; CO7K 1/00 Kushner, D.J., et al., “Lecithinase Production by Strains of 52 U.S. Cl...... 536/23.1; 536/24.1; 435/6; Bacillus cereuS FR. and FR. Pathogenic for the Larch 435/320.1; 530/350 Sawfly, Pristiphora erichsonii (HTG.)”, Can. J. Microbiol, 58 Field of Search ...... 536/23.1, 24.1; 3:547-551 (1957). 435/252.3, 320.1, 6; 530/350 Luthy, P., et al., “Bacillus thuringiensis as a Bacterial Insecticide: Basic Consideration and Application', In: 56) References Cited Microbial and Viral Pesticides, E. Kurstak, Ed., Marcel Dekker, NY 1982, pp. 37–39, 54–56. U.S. PATENT DOCUMENTS Myers, P.S., et al., “Localization of a Mosquito-Larval 3,632,747 1/1972 Satohiro et al...... 424/93 Toxin of Bacillus Sphaericus 1593”, Appl. Enviro. Micro 3,651,215 3/1972 Satohiro et al...... 424/93 biol, 39(1):1205-1211 (1980). 4,996,155 2/1991 Sicket al...... 435/252.3 5,262,323 11/1993 Baird et al...... 435/252.5 Porter, A.G., et al., “Mosquitocidal Toxins of Bacilli and Their Genetic Manipulation for Effective Biological Control FOREIGN PATENT DOCUMENTS of Mosquitoes”, Microbiological Reviews, 57(4):838–861 0498537A2 1/1992 European Pat. Off.. (1993). 0501650A2 2/1992 European Pat. Off.. Sekar, V., “The Insecticidal Crystal Protein Gene is WO88/08880 11/1988 WIPO. Expressed in Vegetative Cells of Bacillus thuringiensis var. WO90/13651 11/1990 WIPO. temebropmos”, Current Microbiology, 17:347-349. WO91/16432 10/1991 WIPO. WO91/16434 10/1991 WIPO. Shivakumar, A.G., et al., Abstract,:Cloned Crystal Protein WO 94/21795 9/1994 WIPO. Genes Express Vegetatively in Bacillus subtilis, Plasmid, OTHER PUBLICATIONS 16(3):230 (1986). Thanabalu, T., et al., “Proteolytic Processing of the Mos Arellano, A., et al., “Evidence of a New Bacillus thurieng quitocidal Toxin from Bacillus Sphaericus SSII-1, J. Bat iensis Toxin Active Against the Australian Sheep Blowfly eriol., 174(15):5051-5056 (1992). Lucilla cuprina”, Proceedings and Abstracts of the 5th International Colloquium On Invertebrate Pathology and (List continued on next page.) Microbial Control, Adelaide, Austrailia, 20–24 Aug., 1990, p. 291. Primary Examiner Nancy Degen Beecher, Douglas J., et al., “A Novel Bicomponent Hemol Assistant Examiner Andrew Wang ysin from Bacillus cereus', Inspection and Immunity, Attorney, Agent, or Firm-Gary M. Pace 58(7):2220–2227 (1990). Faust, R.M., "Bacterial Diseases”, In: Insect Diseases, G. 57 ABSTRACT Cantwell, ed., Marcel Dekker, NY 1974, pp. 90–102. Faust, R.M., et al., “Bacteria and Their Toxins as Insecti The present invention is drawn to pesticidal Strains and cides”, In: Microbial and Viral Pesticides, E. Kurstak, Ed., proteins. Bacillus Strains which are capable of producing Marcel Dekker, NY, 1982, pp. 84–89, 108-120. pesticidal proteins and auxiliary proteins during vegetative Gilmore, Michael S., et al., “A Bacillus cereus Cytolytic growth are provided. Also provided are the purified proteins, Determinant, Cereolysin AB, Which Comprises the Phos nucleotide Sequences encoding the proteins and methods for pholipase C and Sphingomyelinase Genes: Nucleotide using the Strains, proteins and genes for controlling pests. Sequence and Genetic Linkage”, Journal of Bacteriology, 171(2):744–753 (1989). 10 Claims, 1 Drawing Sheet 5,840,868 Page 2

OTHER PUBLICATIONS Thanabalu et al., “Cytotoxicity and ADP-Ribosylating Activity of the Mosquitocidal Toxin from Bacillus Sphaeri Yoshisue, H., et al., “Effects of Bacillus thuringiensis var. cus SSII-1: Possible Roles of the 27- and 70-Kilodalton israelensis 20 kDa Protein on Production of the Bti 130-kDa Peptides”, Journal of Bacteriology, 175(8):2314–2320 Crystal Protein in Escherichia coli'', BioScience, Biotech nology, and Biochemistry, 56(9): 1429-1433 (1992). (1993). Bernier et al., “Bacillus Thuringiensis Strains A20 and A29 Vaithlingam et al., “Anti-Coleopteran Toxin and Gene”, and Insecticidal Compounds. Therefrom, and Compositions Abstract No. 226442, New Zealand Patent Office Journal, Containing These Compounds”, Abstract No. 227249, New 80(7):931, (1991). Zealand Patent Office Journal, 80(6):798, (1988). Wahisaka et al., “Bacillus Thuringiensis Mutant and Bacte Jellis et al., “Bacillus Thuringiensis 8-Endotoxin Variants rial Insecticide”, Abstract No. 199725, New Zealand Patent and Insecticidal Compositions”, Abstract No. 228108, New Office Journal, (1982). Zealand Patent Office Journal, 81(3):359, (1992). Schurter et al., “Genetic Manifpulation of B.thuringiensis Walther et al., “Analysis of Mosquito Larvicidal Potential and B. cereus Vectors and Insecticidal Composition', Exhibited by Vegetative Cells of Bacillus thuringiensis Abstract No. 229191, New Zealand Patent Office Journal, subsp. israelensis”, Applied and Environmental Microbiol 81(3):363, (1992). ogy, 52(4): 650–653 (1986). Tayabali et al., “Semiautomated Quantification of Cytotoxic Ward et al., “Bacillus thuringiensis var. israelensis 8-Endot Damage Induced in Cultured Insect Cells Exposed to Com OXin Cloning and Expression of the Toxin in Sporogenic and mercial Bacillus thuringiensis Biopesticides”, Journal of ASporogenic Strains of Bacillus subtilis”,s Journal of Applied Toxicology, 15(5):365-373 (1995). Molecular Biology, 191(1):13–22 (1986). U.S. Patent Nov. 24, 1998 5,840,868

Figure 1

Characterization of pCIB6022 Activity vs. RV C WCRW 3: : pCIB 6022 ----

pCIB6203 pCIB6023 -

pCIB6206 --

pCIB6024 - Functional Complementation of VIP Clones

pCLB6203 pCB6023

pCIB6203 pCIB6206

pCIB6023 pCB6024 5,840,868 1 2 PESTICIDAL PROTEINS AND STRANS Particularly, new pesticidal proteins are disclosed which are isolatable from the vegetative growth stage of Bacillus. This is a divisional application of Ser. No. 08/463,483, Bacillus Strains, proteins, and genes encoding the proteins filed Jun. 5, 1995 which is a continuation-in-part of Ser. No. are provided. 08/314,594 filed Sep. 28, 1994, now abandoned, which is a The methods and compositions of the invention may be continuation-in-part of Ser. No. 08/218,018, filed Mar. 23, used in a variety of Systems for controlling plant and 1994, now abandoned, which is a continuation-in-part of non-plant pests. Ser. No. 08/037,057, filed Mar. 25, 1993, now abandoned. DETAILED DESCRIPTION OF THE FIELD OF THE INVENTION INVENTION The present invention is drawn to methods and compo Compositions and methods for controlling plant pests are Sitions for controlling plant and non-plant pests. provided. In particular, novel pesticidal proteins are pro BACKGROUND OF THE INVENTION Vided which are produced during vegetative growth of 15 Bacillus Strains. The proteins are useful as pesticidal agents. Insect pests are a major factor in the loSS of the World's The present invention recognizes that pesticidal proteins commercially important agricultural crops. Broad Spectrum are produced during vegetative growth of Bacillus Strains. chemical pesticides have been used extensively to control or To date, all of the identified pesticidal proteins of the eradicate pests of agricultural importance. There is, invention are secreted from the cell. Prior to the present however, Substantial interest in developing effective alter invention, there was no recognition in the art that a class or native pesticides. classes of pesticidal proteins are produced during vegetative Microbial pesticides have played an important role as growth of Bacillus. The only report was of a single mos alternatives to chemical pest control. The most extensively quitocidal toxin from Bacillus Sphaericus SSII-1 by Myers used microbial product is based on the bacterium Bacillus and Yousten in Infect. Immun., 19:1047-1053 (1978). Hav thuringiensis (Bt). Bt is a gram-positive spore forming 25 ing recognized that Such a class exists, the present invention Bacillus which produces an insecticidal crystal protein (ICP) embraces all vegetative insecticidal proteins, hereinafter during sporulation. referred to as VIPs, except for the mosquitocidal toxin from Numerous varieties of Bt are known that produce more B. Sphaericus. than 25 different but related ICP's. The majority of ICP's The present VIPs are not abundant after sporulation and made by Bt are toxic to larvae of certain insects in the orders are particularly expressed during log phase growth before Lepidoptera, Diptera and Coleoptera. In general, when an Stationary phase. For the purpose of the present invention ICP is ingested by a Susceptible insect the crystal is Solu vegetative growth is defined as that period of time before the bilized and transformed into a toxic moiety by the insect gut onset of sporulation. Genes encoding such VIPs can be proteases. None of the ICP's active against coleopteran isolated, cloned and transformed into various delivery larvae Such as Colorado potato beetle (Leptinotarsa 35 vehicles for use in pest management programs. decemlineata) or Yellow mealworm (Tenebrio molitor) have For purposes of the present invention, pests include but demonstrated Significant effects on members of the genus are not limited to insects, fungi, bacteria, nematodes, mites, Diabrotica particularly Diabrotica virgifera virgifera, the ticks, protozoan pathogens, animal-parasitic liver flukes, and western corn rootworm (WCRW) or Diabrotica longicornis the like. Insect pests include insects Selected from the orders barberi, the northern corn rootworm. 40 Coleoptera, Diptera, Hymenoptera, Lepidoptera, Bacillus cereus (Bc) is closely related to Bt. A major Mallophaga, Homoptera, Hemiptera, Orthroptera, distinguishing characteristic is the absence of a parasporal Thy Sanoptera, Dermaptera, Isoptera, Anoplura, crystal in Bc. Bc is a widely distributed bacterium that is Siphonaptera, Trichoptera, etc., particularly Coleoptera and commonly found in Soil and has been isolated from a variety Lepidoptera. 45 of foods and drugs. The organism has been implicated in the Tables 1-10 gives a list of pests associated with major Spoilage of food. crop plants and pests of human and Veterinary importance. Although Bt has been very useful in controlling insect Such pests are included within the Scope of the present pests, there is a need to expand the number of potential invention. biological control agents. 50 BRIEF DESCRIPTION OF THE FIGURE TABLE 1. FIG. 1: Characterization of pCIB6022. Boxed regions Lepidoptera (Butterflies and Moths) represent the extent of VIP1A(a) and VIP2A(a). White box Maize represents the portion of VIP1 encoding the 80 kDa peptide 55 Ostrinia nubialis, European corn borer observed in Bacillus. Dark box represents the N-terminal Agrotis ipsilon, black cutworm propeptide of VIP1A(a) predicted by DNA sequence Helicoverpa zea, corn earworm analysis. Stippled box represents the VIP2A(a) coding Spodoptera frugiperda, fall armyworm region. Large 'X' represents the location of the frameshift Diatraea grandioSella, southwestern corn borer mutation introduced into VIP1A(a). Arrows represent con 60 Elasnopalpus ignoSellus, lesser cornstalk Structs transcribed by the beta-galactosidase promoter. borer Restriction Sites: C-Cla I; X-Xba I, S-Sca I, RI- Eco RI; Diatraea Saccharais, sugarcane borer B-Bgl II; RV-Eco RV. Sorghum. SUMMARY OF THE INVENTION Chilo parteilus, Sorghurn borer 65 Spodoptera frugiperda, fall armyworm The present invention is drawn to compositions and Helicoverpa zea, corn earworm methods for controlling plant and non-plant pests. 5,840,868 3 4

TABLE 1-continued TABLE 2-continued

Lepidoptera (Butterflies and Moths) Coleoptera (Beetles) Elasnopalpus ignoSellus, lesser cornstalk borer Cotton Feltia Subterranea, granulate cutworm Wheat Anthonomus grandis, boll weevil Pseudaletia unipunctata, army worm Rice Spodoptera frugiperda, fall armyworm Elasnopalpus ignoSellus, lesser cornstalk borer Colaspis brunnea, grape colaspis Agrotis Orthogonia, pale western cutworm Lissorhoptrus Oryzophilus, rice water weevil Elasnopalpus ignoSellus, lesser cornstalk Sitophilus Oryzae, rice weevil borer Soybean Sunflower 15 Suleina helianthana, sunflower bud moth Epilachna varivestis, Mexican bean beetle Homoeosoma electeilun, sunflower moth Cotton Heliothis virescens, cotton boll worm Helicoverpa zea, cotton bollworm TABLE 3 Spodoptera exigua, beet armyworm Pectinophora gossypiella, pink bollworm Homoptera (Whiteflies, Aphids etc.) Rice Maize Diatraea Saccharalis, sugarcane borer Spodoptera frugiperda, fall armyworm 25 RhopaloSiphum maidis, corn leaf aphid Helicoverpa zea, corn earworm Anuraphis maidiradicis, corn root aphid Soybean Sorghum Pseudoplusia includens, soybean looper Rhopalosiphum maidis, corn leaf aphid Anticarsia gemmataiis, velvetbean Sipha flava, yellow Sugarcane aphid caterpillar Wheat Plathypena Scabra, green cloverworm Russian wheat aphid Ostrinia nubialis, European corn borer Schizaphis graninum, greenbug Agrotis ipsilon, black cutworm Macrosiphun avenae, English grain aphid Spodoptera exigua, beet armyworm Cotton Heliothis virescens, cotton boll worm Helicoverpa zea, cotton bollworm 35 Aphis gossypii, cotton aphid Barley Pseudatomoscelis Seriatus, cotton fleahopper Trialeurodes abutionea, bandedwinged whitefly Ostrinia nubialis, European corn borer Rice Agrotis ipsilon, black cutworm Nephotettix nigropictus, rice leafhopper 40 Soybean Myzus persicae, green peach aphid TABLE 2 Empoasca fabae, potato leafhopper Coleoptera (Beetles) Barley Maize 45 Schizaphis graninum, greenbug Oil Seed Rape Diabrotica virgifera virgifera, western corn rootworm Diabrotica longicornis barberi, northern corn rootworm Brevicoryne brassicae, cabbage aphid Diabrotica undecimpunctata howardi, southern corn rootworm Melanotus spp., wireworms Cyclocephala borealis, northern masked chafer (white grub) 50 Cyclocephala immaculata, southern masked chafer (white grub) TABLE 4 Popillia japonica, Japanese beetle Chaetocnema pulicaria, corn flea beetle Hemiptera (Bugs) Sphenophorus maidis, maize billbug Sorghum Maize 55 Phyllophaga crinita, white grub Blissus leucopterus leucopterus, chinch bug Eleodes, Conoderus, and Aeolus spp., wireworms Sorghum. Oulema melanopus, cereal leaf beetle Chaetocnema pulicaria, corn flea beetle Blissus leucopterus leucopterus, chinch bug Sphenophorus maidis, maize billbug Cotton Wheat 60 Lygus lineolaris, tarnished plant bug Oulema melanopus, cereal leaf beetle Rice Hypera punctata, clover leaf weevil Diabrotica undecimpunctata howardi, southern corn rootworm Blissus leucopterus leucopterus, chinch bug Sunflower Acrosternum hilare, green stink bug Soybean Zygogramma exclamationis, Sunflower beetle 65 Bothyrus gibbosus, carrot beetle Acrosternum hilare, green stink bug 5,840,868 S 6

TABLE 4-continued TABLE 7

Hemiptera (Bugs) Thysanoptera (Thrips) Maize Barley Anaphothrips obscurus, grass thrips Wheat Blissus leucopterus leucopterus, chinch bug Acrosternum hilare, green stink bug 1O Frankliniella fusca, tobacco thrips EuSchistus Servus, brown stink bug Cotton Thrips tabaci, onion thrips Frankliniella fusca, tobacco thrips Soybean TABLE 5 15 Sericothrips variabilis, soybean thrips Orthoptera (Grasshoppers, Crickets, and Cockroaches) Thrips tabaci, onion thrips Maize Melanoplus femurrubrum, redlegged grasshopper Melanoplus Sanguinipes, migratory grasshopper TABLE 8 Wheat Hymenoptera (Sawflies, Ants, Wasps, etc.) Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper Maize Melanoplus Sanguinipes, migratory grasshopper Cotton 25 Solenopsis milesta, thief ant Wheat Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper Cephus cinctus, wheat stem sawfly Soybean Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper TABLE 9 Structural/Household Other Orders and Representative Species Periplaneta americana, American cockroach Blatteila germanica, German cockroach Dermaptera (Earwigs) Biatta Orientalis, oriental cockroach 35 Forficula auricularia, European earwig Isoptera (Termites) Reticuliterimes flavipes, eastern subterranean termite TABLE 6 Mallophaga (Chewing Lice) Diptera (Flies and Mosquitoes) 40 Cuciotogaster heterographa, chicken head louse Maize Bovicola bovis, cattle biting louse Anoplura (Sucking Lice) Hylenya platura, seedcorn maggot Agromyza parvicornis, corn blotch leafminer Pediculus humanus, head and body louse Sorghum Siphonaptera (Fleas) 45 Contarinia Sorghicola, sorghum midge Ctenocephalides felis, cat flea Wheat Mayetiola destructor, Hessian fly Sitodiplosis moSellana, wheat midge TABLE 10 Meromyza americana, wheat stem maggot 50 Hylemya coarctata, wheat bulb fly Acari (Mites and Ticks) Sunflower Maize Neoliasioptera murtfeldtiana, sunflower seed midge Soybean Tetranychus urticae, twospotted spider mite 55 Sorghum Hylenya platura, seedcorn maggot Barley Tetranychus cinnabarinus, carmine spider mite Tetranychus urticae, twospotted spider mite Hylenya platura, seedcorn maggot Wheat Mayetiola destructor, Hessian fly Insects attacking humans and animals and disease carriers Aceria tulipae, wheat curl mite 60 Cotton Aedes aegypti, yellowfever mosquito Aedes albopictus, forest day mosquito Tetranychus cinnabarinus, carmine spider mite Phlebotomus papatasii, sand fly Tetranychus uricae, twospotted spider mite Musca domestica, house fly Soybean Tabanus atratus, black horse fly Cochliomyia hominivorax, screwworm fly 65 Tetranychus turkestani, strawberry spider mite Tetranychus urticae, twospotted spider mite 5,840,868 7 8

TABLE 10-continued TABLE 11-continued Acari (Mites and Ticks) List of Bacillus species Barley Morphological Group 3 B. psychrophilus Petrobia latens, brown wheat mite B. sphaericus* Subgroup E2 Important human and animal Acari B. pasteurii B. pSychroSaccharolyticus Demacentor variabilis, American dog tick B. macquariensis Argas persicus, fowl tick 1O Dernatophagoides farinae, American house dust mite * = Those Bacillus strains that have been previously found associated with Dermatophagoides pteronyssinus, European house dust mite insects Grouping according to Parry, J. M. et al. (1983) Color Atlas of Bacillus species, Wolfe Medical Publications, London. In accordance with the present invention, the pesticidal 15 proteins produced during vegetative growth can be isolated Now that it has been recognized that pesticidal proteins from Bacillus. In one embodiment, insecticidal proteins can be isolated from the vegetative growth phase of Bacillus, produced during vegetative growth, can be isolated. Meth ods for protein isolation are known in the art. Generally, other Strains can be isolated by Standard techniques and proteins can be purified by conventional chromatography, tested for activity against particular plant and non-plant including gel-filtration, ion-exchange, and immunoaffinity pests. Generally Bacillus Strains can be isolated from any chromatography, by high-performance liquid environmental Sample, including Soil, plant, insect, grain chromatography, Such as reversed-phase high-performance elevator dust, and other Sample material, etc., by methods liquid chromatography, ion-exchange high-performance liq known in the art. See, for example, Travers et al. (1987) uid chromatography, size-exclusion high-performance liq Appl. Environ. Microbiol. 53:1263–1266; Saleh et al. 25 uid chromatography, high-performance chromatofocusing (1969) Can J. Microbiol. 15:1101-1104; DeLucca et al. and hydrophobic interaction chromatography, etc., by elec (1981) Can. J. Microbiol. 27:865-870; and Norris, et al. trophoretic Separation, Such as one-dimensional gel (1981) “The genera Bacillus and Sporolactobacillus.” In electrophoresis, two-dimensional gel electrophoresis, etc. Starret al; (eds.), The Prokaryotes: A Handbook on Habitats, Such methods are known in the art. See for example Current Isolation, and Identification of Bacteria, Vol. II, Springer Protocols in Molecular Biology, Vols. 1 and 2, Ausubel et al. Verlog Berlin Heidelberg. After isolation, strains can be (eds.), John Wiley & Sons, NY (1988). Additionally, anti tested for pesticidal activity during vegetative growth. In bodies can be prepared against Substantially pure prepara this manner, new pesticidal proteins and Strains can be tions of the protein. See, for example, Radka et al. (1983).J. identified. Immunol. 128:2804; and Radka et al. (1984) Immunogenet 35 ics 19:63. Any combination of methods may be utilized to purify protein having pesticidal properties. AS-the protocol Such Bacillus microorganisms which find use in the is being formulated, pesticidal activity is determined after invention include Bacillus cereus and Bacillus thuringiensis, each purification Step. as well as those Bacillus species listed in Table 11. Such purification StepS will result in a Substantially puri 40 fied protein fraction. By “substantially purified” or “sub Stantially pure' is intended protein which is Substantially TABLE 11 free of any compound normally associated with the protein List of Bacillus species in its natural State. "Substantially pure' preparations of Morphological Group 1 Unassigned Strains protein can be assessed by the absence of other detectable 45 protein bands following SDS-PAGE as determined visually B. megaterium Subgroup A or by densitometry Scanning. Alternatively, the absence of other amino-terminal Sequences or N-terminal residues in a B. cerets B. apiarus B. cereus var. mycoides B. filicolonicus purified preparation can indicate the level of purity. Purity B. thuringiensis B. thiaminolyticus can be verified by rechromatography of "pure' preparations B. licheniformis B. alcalophilus 50 showing the absence of other peaks by ion exchange, reverse B. Subtilis Subgroup B phase or capillary electrophoresis. The terms "Substantially B. punius B. cirroflagellosus pure” or “substantially purified” are not meant to exclude B. firmus B. chitinosporus artificial or synthetic mixtures of the proteins with other B. coagulans B. lentus compounds. The terms are also not meant to exclude the Morphological Group 2 Subgroup C 55 presence of minor impurities which do not interfere with the B. polymyxa B. badius biological activity of the protein, and which may be present, B. macerans B. aneurinolyticus for example, due to incomplete purification. B. circulians B. macroides Once purified protein is isolated, the protein, or the B. Stearothermophilus B. freundenreichi B. alvei Subgroup D polypeptides of which it is comprised, can be characterized 60 and Sequenced by Standard methods known in the art. For B. laterosporus B. pantothenticus example, the purified protein, or the polypeptides of which B. brevis B. epiphytus it is comprised, may be fragmented as with cyanogen B. pulvifaciens Subgroup E1 bromide, or with proteases Such as papain, chymotrypsin, B. popilliae B. anninovorans trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. B. lentimorbus B. globisporus 65 Biol. Chem. 257:9751–9758; Liu et al. (1983) Int. J. Pept. B. larvae * B. insolitus Protein Res. 21:209-215). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and 5,840,868 9 10 electroblotting onto PVDF membranes, and subjected to The pesticidal proteins of the invention can be used in amino acid Sequencing. To accomplish this task, the peptides combination with Bt endotoxins or other insecticidal pro are preferably analyzed by automated Sequenators. It is teins to increase insect target range. Furthermore, the use of recognized that N-terminal, C-terminal, or internal amino the VIPs of the present invention in combination with Bt acid Sequences can be determined. From the amino acid Ö-endotoxins or other insecticidal principles of a distinct Sequence of the purified protein, a nucleotide Sequence can nature has particular utility for the prevention and/or man be Synthesized which can be used as a probe to aid in the agement of insect resistance. Other insecticidal principles isolation of the gene encoding the pesticidal protein. include protease inhibitors (both Serine and cysteine types), It is recognized that the pesticidal proteins may be oligo lectins, C.-amylase and peroxidase. In one preferred meric and will vary in molecular weight, number of embodiment, expression of VIPs in a transgenic plant is protomers, component peptides, activity against particular 1O accompanied by the expression of one or more Bt pests, and in other characteristics. However, by the methods Ö-endotoxins. This co-expression of more than one insecti Set forth herein, proteins active against a variety of pests cidal principle in the same transgenic plant can be achieved may be isolated and characterized. by genetically engineering a plant to contain and express all Once the purified protein has been isolated and charac the genes necessary. Alternatively, a plant, Parent 1, can be terized it is recognized that it may be altered in various ways 15 genetically engineered for the expression of VIPs. A Second including amino acid Substitutions, deletions, truncations, plant, Parent 2, can be genetically engineered for the expres and insertions. Methods for Such manipulations are gener sion of Bt Ö-endotoxin. By crossing Parent 1 with Parent 2, ally known in the art. For example, amino acid Sequence progeny plants are obtained which express all the genes variants of the pesticidal proteins can be prepared by muta introduced into Parents 1 and 2. Particularly preferred Bt tions in the DNA. Such variants will possess the desired Ö-endotoxins are those disclosed in U.S. Pat. No. 5,625,136, pesticidal activity. Obviously, the mutations that will be herein incorporated by reference. made in the DNA encoding the variant must not place the A Substantial number of cytotoxic proteins, though not all, Sequence out of reading frame and preferably will not create are binary in action. Binary toxins typically consist of two complementary regions that could produce Secondary 25 protein domains, one called the A domain and the other mRNA structure. See, EP Patent Application Publication No. called the B domain (see Sourcebook of Bacterial Protein 75,444. Toxins, J. E. Alouf and J. H. Freer eds.(1991) Academic In this manner, the present invention encompasses the Press). The A domain possesses a potent cytotoxic activity. pesticidal proteins as well as components and fragments The B domain binds an external cell Surface receptor before thereof. That is, it is recognized that component protomers, being internalized. Typically, the cytotoxic A domain must polypeptides or fragments of the proteins may be produced be escorted to the cytoplasm by a translocation domain. which retain pesticidal activity. These fragments include Often the A and B domains are Separate polypeptides or truncated Sequences, as well as N-terminal, C-terminal, protomers, which are associated by a protein-protein inter internal and internally deleted amino acid Sequences of the action or a di-sulfide bond. However, the toxin can be a proteins. 35 Single polypeptide which is proteolytically processed within Most deletions, insertions, and Substitutions of the protein the cell into two domains as in the case for Pseudomonas Sequence are not expected to produce radical changes in the eXotoxin A. In Summary binary toxins typically have three characteristics of the pesticidal protein. However, when it is important domains, a cytotoxic Adomain, a receptor binding difficult to predict the exact effect of the Substitution, B domain and a translocation domain. The A and B domain deletion, or insertion in advance of doing So, one skilled in 40 are often associated by protein-protein interacting domains. the art will appreciate that the effect will be evaluated by The receptor binding domains of the present invention are routine Screening assayS. useful for delivering any protein, toxin, enzyme, transcrip The proteins or other component polypeptides described tion factor, nucleic acid, chemical or any other factor into herein may be used alone or in combination. That is, Several target insects having a receptor recognized by the receptor proteins may be used to control different insect pests. 45 binding domain of the binary toxins described in this patent. Some proteins are Single polypeptide chains while many Similarly, Since binary toxins have translocation domains proteins consist of more than one polypeptide chain, i.e., which penetrate phoSopholipid bilayer membranes and they are oligomeric. Additionally, Some VIPs are pesticid escort cytotoxins across those membranes, Such transloca ally active as oligomers. In these instances, additional pro tion domains may be useful in escorting any protein, toxin, tomers are utilized to enhance the pesticidal activity or to 50 enzyme, transcription factor, nucleic acid, chemical or any activate pesticidal proteins. Those protomers which enhance other factor acroSS a phospholipid bilayer Such as the plasma or activate are referred to as auxiliary proteins. Auxiliary membrane or a vesicle membrane. The translocation domain proteins activate or enhance a pesticidal protein by interact may itself perforate membranes, thus having toxic or insec ing with the pesticidal protein to form an oligomeric protein ticidal properties. Further, all binary toxins have cytotoxic having increased pesticidal activity compared to that 55 domains, Such a cytotoxic domain may be useful as a lethal observed in the absence of the auxiliary protein. protein, either alone or when delivered into any target cell(s) Auxiliary proteins activate or increase the activity of by any means. pesticidal proteins such as the VIP1 protein from AB78. Finally, Since binary toxins comprised of two polypep Such auxiliary proteins are exemplified by, but not limited tides often form a complex, it is likely that there are to, the VIP2 protein from AB78. As demonstrated in the 60 protein-protein interacting regions within the components of Experimental Section of the application, auxiliary proteins the binary toxins of the invention. These protein-protein can activate a number of pesticidal proteins. Thus, in one interacting domains may be useful in forming associations embodiment of the invention, a plant, Parent 1, can be between any combination of toxins, enzymes, transcription transformed with an auxiliary protein. This Parent 1 can be factors, nucleic acids, antibodies, cell binding moieties, or crossed with a number of Parent 2 plants transformed with 65 any other chemicals, factors, proteins or protein domains. one or more pesticidal proteins whose pesticidal activities Toxins, enzymes, transcription factors, antibodies, cell are activated by the auxiliary protein. binding moieties or other protein domains can be fused to 5,840,868 11 12 pesticidal or auxiliary proteins by producing in frame tides may vary in molecular weight, having at least a genetic fusions which, when translated by ribosomes, would molecular weight of 50 kDa up to at least 200 kDa, prefer produce a fusion protein with the combined attributes of the ably about 100 kDa to 150 kDa. VIP and the other component used in the fusion. An auxiliary protein may be used in combination with the Furthermore, if the protein domain fused to the VIP has an pesticidal proteins of the invention to enhance activity or to affinity for another protein, nucleic acid, carbohydrate, lipid, activate the pesticidal protein. To determine whether the or other chemical or factor, then a three-component complex auxiliary protein will affect activity, the pesticidal protein can be formed. This complex will have the attributes of all can be expressed alone and in combination with the auxil of its components. A similar rationale can be used for iary protein and the respective activities compared in feed producing four or more component complexes. These com ing assays for pesticidal activity. plexes are useful as insecticidal toxins, pharmaceuticals, It may be beneficial to Screen Strains for potential pesti laboratory reagents, and diagnostic reagents, etc. Examples cidal activity by testing activity of the Strain alone and in where Such complexes are currently used are fusion toxins combination with the auxiliary protein. In Some instances an for potential cancer therapies, reagents in ELISA assays and auxiliary protein in combination with the native proteins of immunoblot analysis. 15 the Strains yields pesticidal activity where none is seen in the One Strategy of altering pesticidal or auxiliary proteins is absence of an auxiliary protein. to fuse a 15-amino-acid “S-tag to the protein without The auxiliary protein can be modified, as described above, destroying the insect cell binding domain(s), translocation by various methods known in the art. Therefore, for pur domains or protein-protein interacting domains of the pro poses of the invention, the term “Vegetative Insecticidal teins. The S-tag has a high affinity (K=10M) for a Protein” (VIP) encompasses those proteins produced during ribonuclease S-protein, which, when bound to the S-tag, vegetative growth which alone or in combination can be forms an active ribonuclease (See F. M. Richards and H. W. used for pesticidal activity. This includes pesticidal proteins, Wyckoff(1971) in “The Enzymes”, Vol. IV (Boyer, P. D. auxiliary proteins and those proteins which demonstrate ed.). pp. 647-806. Academic Press, New York). The fusion activity only in the presence of the auxiliary protein or the can be made in Such a way as to destroy or remove the 25 polypeptide components of these proteins. cytotoxic activity of the pesticidal or auxiliary protein, It is recognized that there are alternative methods avail thereby replacing the VIP cytotoxic activity with a new able to obtain the nucleotide and amino acid Sequences of cytotoxic ribonuclease activity. The final toxin would be the present proteins. For example, to obtain the nucleotide comprised of the S-protein, a pesticidal protein and an Sequence encoding the pesticidal protein, coSmid clones, auxiliary protein, where either the pesticidal protein or the which express the pesticidal protein, can be isolated from a auxiliary protein is produced as translational fusions with genomic library. From larger active coSmid clones, Smaller the S-tag. Similar Strategies can be used to fuse other Subclones can be made and tested for activity. In this potential cytotoxins to pesticidal or auxiliary proteins manner, clones which express an active pesticidal protein including (but not limited to) ribosome inactivating proteins, can be Sequenced to determine the nucleotide Sequence of insect hormones, hormone receptors, transcription factors, 35 the gene. Then, an amino acid Sequence can be deduced for proteases, phosphatases, Pseudomonas eXotoxin A, or any the protein. For general molecular methods, See, for other protein or chemical factor that is lethal when delivered example, Molecular Cloning, A Laboratory Manual, Second into cells. Similarly, proteins can be delivered into cells which are not lethal, but might alter cellular biochemistry or Edition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring 40 Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), physiology. and the references cited therein. The Spectrum of toxicity toward different Species can be The present invention also encompasses nucleotide altered by fusing domains to pesticidal or auxiliary proteins Sequences from organisms other than Bacillus, where the which recognize cell Surface receptors from other Species. nucleotide Sequences are isolatable by hybridization with the Such domains might include (but are not limited to) 45 Bacillus nucleotide Sequences of the invention. Proteins antibodies, transferrin, hormones, or peptide Sequences iso encoded by Such nucleotide Sequences can be tested for lated from phage displayed affinity Selectable libraries. Also, pesticidal activity. The invention also encompasses the pro peptide Sequences which are bound to nutrients, Vitamins, teins encoded by the nucleotide Sequences. Furthermore, the hormones, or other chemicals that are transported into cells invention encompasses proteins obtained from organisms could be used to alter the Spectrum of toxicity. Similarly, any 50 other than Bacillus wherein the protein cross-reacts with other protein or chemical which binds a cell Surface receptor antibodies raised against the proteins of the invention. Again or the membrane and could be internalized might be used to the isolated proteins can be assayed for pesticidal activity by alter the spectrum of activity of VIP1 and VIP2. the methods disclosed herein or others well-known in the art. The pesticidal proteins of the present invention are those Once the nucleotide Sequences encoding the pesticidal proteins which confer a Specific pesticidal property. Such 55 proteins of the invention have been isolated, they can be proteins may vary in molecular weight, having component manipulated and used to express the protein in a variety of polypeptides at least a molecular weight of 30 kDa or hosts including other organisms, including microorganisms greater, preferably about 50 kDa or greater. and plants. The auxiliary proteins of the invention may vary in The pesticidal genes of the invention can be optimized for molecular weight, having at least a molecular weight of 60 enhanced expression in plants. See, for example U.S. Pat. about 15 kDa or greater, preferably about 20 kDa or greater; No. 5,625,136; EPA0359472; EPA 0385962; WO 91/16432; more preferably, about 30 kDa or greater. The auxiliary Perlak et al. (1991) Proc. Natl. Acad. Sci. USA proteins themselves may have component polypeptides. 88:3324–3328; and Murray et al. (1989) Nucleic Acids It is possible that the pesticidal protein and the auxiliary Research 17:477-498. In this manner, the genes can be protein may be components of a multimeric, insecticidal 65 Synthesized utilizing plant preferred codons. That is the protein. Such a insecticidal protein which includes the preferred codon for a particular host is the Single codon auxiliary proteins as one or more of its component polypep which most frequently encodes that amino acid in that host. 5,840,868 13 14 The maize preferred codon, for example, for a particular Picornavirus leaders, for example, EMCV leader amino acid may be derived from known gene Sequences (encephalomyocarditis 5' noncoding region) (Elroy from maize. Maize codon usage for 28 genes from maize Stein, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA plants is found in Murray et al. (1989), Nucleic Acids 86:6126–6130); Research 17:477-498, the disclosure of which is incorpo Potyvirus leaders, for example, TEV leader (Tobacco Etch rated herein by reference. Synthetic genes can also be made Virus) (Allison et al., (1986); MDMV leader (Maize based on the distribution of codons a particular host uses for Dwarf Mosaic Virus); Virology, 154:9-20), and a particular amino acid. Human immunoglobulin heavy-chain binding protein In this manner, the nucleotide Sequences can be optimized (BiP), (Macejak, D. G., and Sarnow, P., (1991), Nature, for expression in any plant. It is recognized that all or any 1O 353:90-94; part of the gene Sequence may be optimized or Synthetic. Untranslated leader from the coat protein mRNA of That is, Synthetic or partially optimized Sequences may also alfalfa mosaic virus (AMV RNA4), (Jobling, S.A., and be used. Gehrke, L., (1987), Nature, 325:622-625; In like manner, the nucleotide Sequences can be optimized 15 Tobacco mosaic virus leader (TMV), (Gallie, D. R. et al., for expression in any microorganism. For Bacillus preferred (1989), Molecular Biology of RNA, pages 237-256; codon usage, see, for example U.S. Pat. No. 5,024,837 and and Johansen et al. (1988) Gene 65:293–304. Methodologies for the construction of plant expression Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, cassettes as well as the introduction of foreign DNA into S. A. et al., (1991), Virology, 81:382–385. See also, plants are described in the art. Such expression cassettes Della-Cioppa et al., (1987), Plant Physiology, may include promoters, terminators, enhancers, leader 84:965-968. Sequences, introns and other regulatory Sequences operably A plant terminator may be utilized in the expression linked to the pesticidal protein coding Sequence. It is further cassette. See, Rosenberg et al., (1987), Gene, 56: 125; recognized that promoters or terminators of the VIP genes Guerineau et al., (1991), Mol. Gen. Genet., 226:141-144; 25 Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., can be used in expression cassettes. (1991), Genes Dev, 5:141–149; Mogen et al., (1990), Plant Generally, for the introduction of foreign DNA into plants Cell, 2:1261–1272; Munroe et al., (1990), Gene, Ti plasmid vectors have been utilized for the delivery of 91:151-158; Ballas et al., (1989), Nucleic Acids Res., foreign DNA as well as direct DNA uptake, liposomes, 17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., electroporation, micro-injection, and the use of microprojec 15:9627-9639. tiles. Such methods had been published in the art. See, for For tissue specific expression, the nucleotide Sequences of example, Guerche et al., (1987) Plant Science 52:111-116; the invention can be operably linked to tissue specific Neuhause et al., (1987) Theor. Appl. Genet. 75:30–36; Klein promoters. See, for example, U.S. Pat. No. 5,625,136 herein et al., (1987) Nature 327:70–73; Howell et al., (1980) incorporated by reference. Science 208: 1265; Horsch et al., (1985) Science 35 It is recognized that the genes encoding the pesticidal 227: 1229–1231; DeBlock et al., (1989) Plant Physiology proteins can be used to transform insect pathogenic organ 91:694-701; Methods for Plant Molecular Biology isms. Such organisms include Baculoviruses, fungi, (Weissbach and Weissbach, eds.) Academic Press, Inc. protozoa, bacteria and nematodes. (1988); and Methods in Plant Molecular Biology (Schuler The Bacillus strains of the invention may be used for and Zielinski, eds.) Academic Press, Inc. (1989). See also 40 protecting agricultural crops and products from pests. U.S. patent application Ser. No. 08/008,374 herein incorpo Alternatively, a gene encoding the pesticide may be intro rated by reference. See also, EPA 0193259 and EPA duced via a Suitable vector into a microbial host, and Said 0451878A1. It is understood that the method of transforma host applied to the environment or plants or animals. Micro tion will depend upon the plant cell to be transformed. organism hosts may be Selected which are known to occupy It is further recognized that the components of the expres 45 the “phytosphere” (phylloplane, phyllosphere, rhizosphere, Sion cassette may be modified to increase expression. For and/or rhizoplana) of one or more crops of interest. These example, truncated Sequences, nucleotide Substitutions or microorganisms are Selected So as to be capable of Success other modifications may be employed. See, for example fully competing in the particular environment with the Perlak et al. (1991) Proc. Natl. Acad. Sci. USA wild-type microorganisms, provide for Stable maintenance 88:3324–3328; Murray et al., (1989) Nucleic Acids Research 50 and expression of the gene expressing the polypeptide 17:477-498; and WO 91/16432. pesticide, and, desirably, provide for improved protection of The construct may also include any other necessary the pesticide from environmental degradation and inactiva regulators Such as terminators, (Guerineau et al., (1991), tion. Mol. Gen. Genet., 226:141–144; Proudfoot, (1991), Cell, Such microorganisms include bacteria, algae, and fungi. 64:671-674; Sanfacon et al., (1991), Genes Dev, 55 Of particular interest are microorganisms, Such as bacteria, 5:141–149; Mogen et al., (1990), Plant Cell, 2:1261–1272; e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Munroe et al., (1990), Gene, 91:151-158; Ballas et all et al., Xanthom on a S, Stre p to my c e S, Rhizobium, (1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., Rhodopseudomonas, Methylius, Agrobacterium, (1987), Nucleic Acid Res., 15:9627–9639); plant transla Acetobacter, Lactobacillus, Arthrobacter AZotobacter, tional consensus sequences (Joshi, C. P., (1987), Nucleic 60 Leuconostoc, and Alcaligenes, fungi, particularly yeast, e.g., Acids Research, 15:6643-6653), introns (Luehrsen and Saccharomyces, Cryptococcus, Kluyveromyce S, Walbot, (1991), Mol. Gen. Genet., 225:81–93) and the like, Sporobolomyces, Rhodotorula, and Aureobasidium. Of par operably linked to the nucleotide Sequence. It may be ticular interest are Such phytosphere bacterial Species as beneficial to include 5' leader Sequences in the expression Pseudomonas Syringae, Pseudomonas fluorescens, Serratia cassette construct. Such leader Sequences can act to enhance 65 marceScens, Acetobacter xylinum, Agrobacteria, translation. Translational leaders are known in the art and Rhodopseudomonas Spheroides, Xanthomonas campestris, include: Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli 5,840,868 15 16 and Azotobacter Vinlandii; and phytosphere yeast Species Host organisms of particular interest include yeast, Such such as Rhodotorula rubra, R. glutinis, R. marina, R. as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., aurantiaca, CryptococcuS albidus, C. diffluens, C. laurentii, and Sporobolomyces sp., phylloplane organisms. Such as Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Pseudomonas sp., Erwinia Sp. and Flavobacterium sp., or Sporobolomyces roSues, S. Odorus, Kluyveromyces veronae, Such other organisms as Escherichia, LactoRacillus sp., and Aureobasidium pollulans. Of particular interest are the Bacillus sp., and the like. Specific organisms include pigmented microorganisms. Pseudomonas aelurginosa, Pseudomonas fluorescens, Sac A number of ways are available for introducing a gene charomyces cerevisiae, Bacillus thuringiensis, Escherichia expressing the pesticidal protein into the microorganism coli, Bacillus Subtilis, and the like. host under conditions which allow for Stable maintenance VIP genes can be introduced into micro-organisms that and expression of the gene. For example, expression cas multiply on plants (epiphytes) to deliver VIP proteins to settes can be constructed which include the DNA constructs potential target pests. Epiphytes can be gram-positive or of interest operably linked with the transcriptional and gram-negative bacteria for example. translational regulatory Signals for expression of the DNA Root colonizing bacteria, for example, can be isolated constructs, and a DNA sequence homologous with a 15 from the plant of interest by methods known in the art. Sequence in the host organism, whereby integration will Specifically, a Bacillus cereus Strain which colonizes roots occur, and/or a replication System which is functional in the could be isolated from roots of a plant (for example see J. host, whereby integration or Stable maintenance will occur. Handelsman, S. Raffel, E. Mester, L. Wunderlich and C. Transcriptional and translational regulatory Signals Grau, Appl. Environ. Microbiol. 56:713–718, (1990)). VIP1 include but are not limited to promoter, transcriptional and/or VIP2 could be introduced into a root colonizing initiation Start Site, operators, activators, enhancers, other Bacillus cereus by standard methods known in the art. regulatory elements, ribosomal binding sites, an initiation Specifically, VIP1 and/or VIP2 derived from Bacillus codon, termination signals, and the like. See, for example, cereus Strain AB78 can be introduced into a root colonizing U.S. Pat. No. 5,039,523; U.S. Pat. No. 4,853,331; EPO Bacillus cereuS by means of conjugation using Standard 0480762A2; Sambrook et al. Supra; Molecular Cloning, a 25 methods (J. Gonzalez, B. Brown and B. Carlton, Proc. Natl. Laboratory Manual, Maniatis et al. (eds) Cold Spring Harbor Acad. Sci. 79:6951-6955, (1982)). Laboratory, Cold Spring Harbor, N.Y. (1982); Advanced Also, VIP1 and/or VIP2 or other VIPs of the invention can Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor be introduced into the root colonizing Bacillus by means of Laboratory, Cold Spring Harbor, N.Y. (1980); and the ref electro-transformation. Specifically, VIPs can be cloned into erences cited therein. a shuttle vector, for example, pHT3101 (D. Lereclus et al., Suitable host cells, where the pesticide-containing cells FEMS Microbiol. Letts, 60:211-218 (1989)) as described in will be treated to prolong the activity of the toxin in the cell Example 10. The shuttle vector pHT3101 containing the when the then treated cell is applied to the environment of coding sequence for the particular VIP can then be trans the target pest(s), may include either prokaryotes or formed into the root colonizing Bacillus by means of elec eukaryotes, normally being limited to those cells which do 35 troporation (D.Lereclus et al. 1989, FEMS Microbiol. Letts. not produce Substances toxic to higher organisms, Such as 60:211-218). mammals. However, organisms which produce Substances Expression Systems can be designed So that VIP proteins toxic to higher organisms could be used, where the toxin is are Secreted outside the cytoplasm of gram negative bacteria, unstable or the level of application sufficiently low as to E. coli, for example. Advantages of having VIP proteins avoid any possibility of toxicity to a mammalian host. AS 40 secreted are (1) it avoids potential toxic effects of VIP hosts, of particular interest will be the prokaryotes and the proteins expressed within the cytoplasm and (2) it can lower eukaryotes, Such as fungi. Illustrative prokaryotes, increase the level of VIP protein expressed and (3) can aid both Gram-negative and -positive, include in efficient purification of VIP protein. Enterobacteriaceae, Such as Escherichia, Erwinia, Shigella, VIP proteins can be made to be secreted in E. coli, for Salmonella, and Proteus, Bacillaceae, Rhizobiceae, Such as 45 example, by fusing an appropriate E. coli Signal peptide to Rhizobium, Spirillaceae, Such as photobacterium, the amino-terminal end of the VIP Signal peptide or replac Zymomonas, Serratia, Aeromonas, Vibrio, DeSulfoVibrio, ing the VIP Signal peptide with the E. coli Signal peptide. Spirillum, Lactobacillaceae, Pseudomonadaceae, Such as Signal peptides recognized by E. coli can be found in Pseudomonas and Acetobacter, Azotobacteraceae and Nitro proteins already known to be Secreted in E. coli, for example bacteraceae. Among eukaryotes are fungi, Such as Phyco 50 the Omp A protein (J. Ghrayeb, H. Kimura, M. Takahara, Y. mycetes and AScomycetes, which includes yeast, Such a Masui and M. Inouye, EMBO J., 3:2437–2442 (1984)). Saccharomyces and Schizosaccharromyces, and Basidi OmpA is a major protein of the E. coli outer membrane and omycetes yeast, Such as Rhodotorula, Aureobasidium, thus its signal peptide is thought to be efficient in the Sporobolomyces, and the like. translocation process. Also, the Omp A Signal peptide does Characteristics of particular interest in Selecting a host 55 not need to be modified before processing as may be the case cell for purposes of production include ease of introducing for other Signal peptides, for example lipoprotein Signal the protein gene into the host, availability of expression peptide (G. Duffaud, P. March and M. Inouye, Methods in Systems, efficiency of expression, Stability of the protein in Enzymology, 153:492 (1987)). the host, and the presence of auxiliary genetic capabilities. Specifically, unique BamHI restriction sites can be intro Characteristics of interest for use as a pesticide microcapsule 60 duced at the amino-terminal and carboxy-terminal ends of include protective qualities for the pesticide, Such as thick the VIP coding Sequences using Standard methods known in cell walls, pigmentation, and intracellular packaging or the art. These BamHI fragments can be cloned, in frame, into formation of inclusion bodies, leaf affinity; lack of mam the vector plN-III-ompa1, A2 or A3 (J. Ghrayeb, H. malian toxicity; attractiveness to pests for ingestion; ease of Kimura, M. Takahara, H. Hsiung, Y. Masui and M. Inouye, killing and fixing without damage to the toxin; and the like. 65 EMBO.J., 3:2437–2442 (1984)) thereby creating ompA:VIP Other considerations include ease of formulation and fusion gene which is Secreted into the periplasmic Space. handling, economics, Storage Stability, and the like. The other restriction sites in the polylinker of plN-III-ompA 5,840,868 17 18 can be eliminated by Standard methods known in the art So potential target pests. Epiphytes can be gram-positive or that the VIP amino-terminal amino acid coding Sequence is gram-negative bacteria for example. directly after the omp A Signal peptide cleavage Site. Thus, The Bacillus Strains of the invention or the microorgan the secreted VIP sequence in E. coli would then be identical isms which have been genetically altered to contain the to the native VIP sequence. pesticidal gene and protein may be used for protecting When the VIP native signal peptide is not needed for agricultural crops and products from pests. In one aspect of proper folding of the mature protein, Such signal Sequences the invention, whole, i.e., unlysed, cells of a toxin (pesticide) can be removed and replaced with the omp A Signal -producing organism are treated with reagents that prolong Sequence. Unique BamHI restriction sites can be introduced the activity of the toxin produced in the cell when the cell is at the amino-termini of the proprotein coding Sequences applied to the environment of target pest(s). directly after the Signal peptide coding Sequences of VIP and Alternatively, the pesticides are produced by introducing at the carboxy-termini of VIP coding sequence. These a heterologous gene into a cellular host. Expression of the BamHI fragments can then be cloned into the plN-III-ompA heterologous gene results, directly or indirectly, in the vectors as described above. intracellular production and maintenance of the pesticide. General methods for employing the Strains of the inven These cells are then treated under conditions that prolong the tion in pesticide control or in engineering other organisms as 15 activity of the toxin produced in the cell when the cell is pesticidal agents are known in the art. See, for example U.S. applied to the environment of target pest(s). The resulting Pat. No. 5,039,523 and EPO480762A2. product retains the toxicity of the toxin. These naturally VIPs can be fermented in a bacterial host and the resulting encapsulated pesticides may then be formulated in accor bacteria processed and used as a microbial Spray in the same dance with conventional techniques for application to the manner that Bacillus thuringiensis Strains have been used as environment hosting a target pest, e.g., Soil, Water, and insecticidal sprays. In the case of a VIP(s) which is secreted foliage of plants. See, for example EPA 0192319, and the from Bacillus, the Secretion signal is removed or mutated references cited therein. using procedures known in the art. Such mutations and/or The active ingredients of the present invention are nor deletions prevent secretion of the VIP protein(s) into the mally applied in the form of compositions and can be growth medium during the fermentation process. The VIPs 25 applied to the crop area or plant to be treated, Simultaneously are retained within the cell and the cells are then processed or in Succession, with other compounds. These compounds to yield the encapsulated VIPs. Any Suitable microorganism can be both fertilizers or micronutrient donors or other can be used for this purpose. PSuedomonas has been used to preparations that influence plant-growth. They can also be express Bacillus thuringiensis endotoxins as encapsulated Selective herbicides, insecticides, fungicides, bactericides, proteins and the resulting cells processed and Sprayed as an nematicides, mollusicides or mixtures of Several of these insecticide. (H. Gaertner et al. 1993, In Advanced Engi preparations, if desired, together with further agriculturally neered Pesticides, L. Kim ed.) acceptable carriers, Surfactants or application-promoting Various Strains of Bacillus thuringiensis are used in this adjuvants customarily employed in the art of formulation. manner. Such Bt Strains produce endotoxin protein(s) as Suitable carriers and adjuvants can be Solid or liquid and well as VIPs. Alternatively, such strains can produce only 35 correspond to the Substances ordinarily employed in formu VIPs. A sporulation deficient strain of Bacillus Subtilis has lation technology, e.g. natural or regenerated mineral been shown to produce high levels of the CryIIIA endotoxin Substances, Solvents, dispersants, wetting agents, tackifiers, from Bacillus thuringiensis (Agaisse, H. and Lereclus, D., binders or fertilizers. “Expression in Bacillus Subtilis of the Bacillus thuringiensis Preferred methods of applying an active ingredient of the CryIIIA toxin gene is not dependent on a sporulation 40 present invention or an agrochemical composition of the Specific Sigma factor and is increased in a SpoOA mutant', present invention which contains at least one of the pesti J. Bacteriol. 176:4734–4741 (1994)). A similar spoCA cidal proteins produced by the bacterial Strains of the present mutant can be prepared in Bacillus thuringiensis and used to invention are leaf application, Seed coating and Soil appli produce encapsulated VIPs which are not secreted into the cation. The number of applications and the rate of applica medium but are retained within the cell. 45 tion depend on the intensity of infestation by the correspond To have VIPs maintained within the Bacillus cell the ing pest. Signal peptide can be disarmed So that it no longer functions In one embodiment of the invention a Bacillus cereuS as a Secretion signal. Specifically, the putative signal peptide microorganism has been isolated which is capable of killing for VIP1 encompasses the first 31 amino acids of the protein Diabrotica virgifera virgifera, and Diabrotica longicornis with the putative consensus cleavage site, Ala-X-Ala, at the 50 barberi. The novel B. cereus strain AB78 has been deposited C-terminal portion of this sequence (G. von Heijne, J. Mol. in the Agricultural Research Service, Patent Culture Collec Biol. 184:99-105 (1989)) and the putative signal peptide for tion (NRRL), Northern Regional Research Center, 1815 VIP2 encompasses the first 40 amino acids of the protein North University Street, Peoria, Ill. 61604, USA and given with the putative cleavage Site after Ala-10. The cleavage Accession No. NRRL B-21058. sites in either VIP1 or VIP2 can be mutated with methods 55 A fraction protein has been substantially purified from the known in the art to replace the cleavage Site consensus B. cereus strain. This purification of the protein has been Sequence with alternative amino acids that are not recog verified by SDS-PAGE and biological activity. The protein nized by the Signal peptidases. has a molecular weight of about 60 to about 100 kDa, Alternatively, the signal peptides of VIP1, VIP2 and/or particularly about 70 to about 90 kDa, more particularly other VIPs of the invention can be eliminated from the 60 about 80 kDa, hereinafter VIP. Sequence thereby making them unrecognizable as Secretion Amino-terminal Sequencing has revealed the N-terminal proteins in Bacillus. Specifically, a methionine Start Site can amino-acid Sequence to be: NH-Lys-Arg-Glu-Ile-Asp-Glu be engineered in front of the proprotein Sequence in VIP1, Asp-Thr-Asp-Thr-ASX-Gly-Asp-Ser-Ile-Pro- (SEQ ID Starting at ASp32, or the proprotein Sequence in VIP2, NO:8) where ASX represents either Asp or Asn. The entire Starting at Glu41 using methods known in the art. 65 amino acid sequence is given in SEQ ID NO:7. The DNA VIP genes can be introduced into micro-organisms that Sequence which encodes the amino acid Sequence of SEQID multiply on plants (epiphytes) to deliver VIP proteins to NO:7 is disclosed in SEO ID NO:6. 5,840,868 19 20 An oligonuleotide probe for the region of the gene encod ing amino acids 3–9 of the NH2-terminus has been gener TABLE 12-continued ated. The probe was Synthesized based on the codon usage of a Bacillus thuringiensis (Bt) Ö-endotoxin gene. The Antibiotic activity of AB78 culture Supernatant nucleotide Sequence of the oligonucleotide probe used for 5 Zone of inhibition (cm Southern hybridizations was as follows: 5'-GAA ATT GAT CAA GAT ACN GAT-3' (SEQ ID Bacteria tested ABF8 Streptomycin NO:9) where N represents any base. B. mycoides 1.3 2.1 In addition, the DNA probe for the Bc AB78 VIP1 gene B. cereus CB 1.O 2.O described herein, permits the Screening of any Bacillus B. cereus 11950 1.3 2.1 B. cereus 14579 1.O 2.4 strain or other organisms to determine whether the VIP1 B. cerets AB78 O.O 2.2 gene (or related gene) is naturally present or whether a Bt var. israelensis 1.1 2.2 particular transformed organism includes the VIP1 gene. Bt var. tenebrionis O.9 2.3 The invention now being generally described, the same will be better understood by reference to the following 15 detailed examples that are provided for the purpose of Morphological characteristics of AB78 are as follows: illustration and are not to be considered limiting of the Vegetative rods straight, 3.1-5.0 mm long and 0.5-2.0 invention unless So Specified. mm wide. Cells with rounded ends, Single in Short chains. A Standard nomenclature has been developed based on the Single Subterminal, cylindrical-oval, endospore formed per Sequence identity of the proteins encompassed by the cell. No parasporal crystal formed. Colonies opaque, erose, present invention. The gene and protein names for the lobate and flat. No pigments produced. Cells motile. Flagella detailed examples which follow and their relationship to the present. names used in the parent application are shown below. Growth characteristics of AB78 are as follows: Facultative anaerobe with optimum growth temperature 25 of 21°–30° C. Will grow at 15, 20°, 25°,30° and 37° C. Will Gene/Protein Name under Standard Gene/protein not grow above 40° C. Grows in 5–7% NaCl. Nomenclature Name in Parent Description of Protein Table 13 provides the biochemical profile of AB78. VIP1A(a) VIP1 VIP1 from strain AB78 as TABLE 13 disclosed in SEO ID NO: 5. VIP2A(a) VIP2 VIP2 from strain AB78 as Biochemical characteristics of B. cereus strain AB78. disclosed in SEO ID NO: 2. VIP1A(b) VIP1 homolog VIP1 from Bacillus thuringiensis Acid from L-arabinose - Methylene blue reoxidized -- var. tenebrionis as disclosed in Gas from L-arabinose - Nitrate reduced -- SEO ID NO: 21. Acid from D-xylose - NO reduced to NO, -- VIP2A(b) VIP2 homolog VIP2 from Bacillus thuringiensis 35 Gas from D-xylose - VP -- var. tenebrionis as disclosed in Acid from D-glucose HO, decomposed -- SEO ID NO: 2O. Gas from D-glucose - Indole VIP3A(a) VIP from strain AB88 as Acid from lactose - Tyrosine decomposed -- disclosed in SEO ID NO: 28 of the Gas from lactose - Dihydroxiacetone present application Acid from sucrose - Litmus milk acid VIP3A(b) VIP from strain AB424 as 40 Gas from Sucrose - Litmus milk coagulated disclosed in SEO ID NO:31 of the Acid from D-mannitol - Litmus milk alkaline present application Gas from D-mannitol - Litmus milk peptionized Proprionate utilization + Litmus milk reduced Citrate utilization + Casein hydrolyzed -- Hippurate hydrolysis w Starch hydrolyzed -- EXPERIMENTAL Methylene blue reduced + Gelatin liquidified -- 45 Lecithinase produced w EXAMPLE 1. w = weak reaction AB78 ISOLATION AND CHARACTERIZATION EXAMPLE 2 Bacillus cereus strain AB78 was isolated as a plate 50 contaminant in the laboratory on T3 media (per liter: 3 g BACTERIAL CULTURE tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05M Sodium A Subculture of Bc strain AB78 was used to inoculate the phosphate (pH 6.8), and 0.005 g MnOl; Travers, R. S. following medium, known as TB broth: 1983). During log phase growth, AB78 gave significant activity against Western corn rootworm. Antibiotic activity 55 Tryptone 12 g/l against gram-positive Bacillus spp. was also demonstrated Yeast Extract 24 g/l (Table 12). Glycerol 4 mlf KHPO 2.1 g/l TABLE 12 KHPO, 14.7 g/l pH 7.4 60 Antibiotic activity of AB78 culture Supernatant Zone of inhibition (cm The potassium phosphate was added to the autoclaved broth after cooling. Flasks were incubated at 30° C. on a Bacteria tested ABF8 Streptomycin rotary shaker at 250 rpm for 24 h.-36 h, which represents an E. coli O.O 3.0 65 early to mid-log growth phase. B. megaterium 1.1 2.2 The above procedure can be readily Scaled up to large fermentors by procedures well known in the art. 5,840,868 21 22 During vegetative growth, usually 24-36 h. after Starting the culture, which represents an early to mid-log growth TABLE 14-continued phase, AB78 bacteria were centrifuged from the culture Supernatant. The culture Supernatant containing the active Activity of AB78 culture Supernatant against various insect species protein was used in bioassayS. Insect species tested to date Order Activity EXAMPLE 3 (Diabrotica undecimpunctata INSECT BIOASSAYS howardi) Colorado potato beetle Col (Leptinotarsa decenlineata) B. cereuS Strain AB78 was tested against various insects Yellow mealworm Col as described below. (Tenebrio molitor) Western, Northern and Southern corn rootworm, European corn borer Lep (Ostrinia nubialis) Diabrotica virgifera virgifera, D. longcornis barberi and D. Tobacco budworm Lep undecempunctata howardi, respectively: dilutions were 15 (Heliothis virescens) made of AB78 culture Supernatant grown 24-36 h., mixed Tobacco hornworm Lep with molten artificial diet (Marrone et al. (1985) J. of (Manduca Sexta) Economic Entomology 78:290–293) and allowed to solidify. Beet armyworm Lep Solidified diet was cut and placed in dishes. Neonate larvae (Spodoptera exigua) Black cutworm Lep were placed on the diet and held at 30° C. Mortality was (Agrotis ipsilon) recorded after 6 dayS. Northern house mosquito Dip E. coli clone bioassay: E. coli cells were grown overnight Culex plppipiens in broth containing 100 ug/ml ampicillin at 37 C. Ten ml culture was Sonicated 3x for 20 sec each. 500 ul of Sonicated culture was added to molten western corn rootworm diet. Colorado potato beetle, Leptinotarsa decemlineata: dilu 25 tions in Triton X-100 (to give final concentration of 0.1% TX-100) were made of AB78 culture Supernatant grown The newly discovered B. cereus strain AB78 showed a 24–36 h. Five cm potato leaf pieces were dipped into these Significantly different Spectrum of insecticidal activity as dilutions, air dried, and placed on moistened filter paper in compared to known coleopteran active Ö-endotoxins from plastic dishes. Neonate larvae were placed on the leaf pieces Bt. In particular, AB78 showed more selective activity and held at 30° C. Mortality was recorded after 3-5 days. against beetles than known coleopteran-active Bt Strains in Yellow mealworm, Tenebrio molitor: dilutions were made that it was Specifically active against Diabrotica spp. More of AB78 culture Supernatant grown 24-36 h., mixed with specifically, it was most active against D. virgifera virgifera molten artificial diet (BioServ #F9240) and allowed to and D. longicornis barberi but not D. undecimpunctata Solidify. Solidified diet was cut and placed in plastic dishes. 35 Neonate larvae were placed on the diet and held at 30° C. howardi. Mortality was recorded after 6-8 days. European corn borer, black cutworm, tobacco budworm, tobacco hornworm and beet armyworm; Ostrinia nubilalis, A number of Bacillus strains were bioassayed for activity Agrotis ipsilon, Heliothis virescens, Manduca Sexta and 40 during vegetative growth (Table 15) against western corn Spodoptera exigua, respectively: dilutions, in TX-100 (to give final concentration of 0.1% TX-100), were made of rootworm. The results demonstrate that AB78 is unique in AB78 culture Supernatant grown 24-36 hrs. 100 ul was that activity against Western corn rootworm is not a general pipetted onto the surface of 18 cm° of solidified artificial diet phenomenon. (BioServ #F9240) and allowed to air dry. Neonate larvae 45 were then placed onto the surface of the diet and held at 30 TABLE 1.5 C. Mortality was recorded after 3–6 days. Activity of culture supernatants from various Northern house mosquito, Culex pipiens:-dilutions were Bacillus spp. against Western corn rootworm made of AB78 culture Supernatant grown 24-36 h. 100 ul was pipetted into 10 ml water in a 30 ml plastic cup. Third 50 Percent instar larvae were added to the water and held at room Bacillus strain WCRW mortality temperature. Mortality was recorded after 24-48 hours. The B. cereus AB78 (Bat.1) 1OO spectrum of entomocidal activity of AB78 is given in Table B. cereus AB78 (Bat.2) 1OO 14. B. cereus (Carolina Bio.) 12 B. cereuS ATCC 11950 12 55 B. cereuS ATCC 14579 8 TABLE 1.4 B. mycoides (Carolina Bio.) 3O B. popilliae 28 Activity of AB78 culture Supernatant against various insect species B. thuringiensis HD135 41 B. thuringiensis HD.191 9 Insect species B. thuringiensis GC91 4 tested to date Order Activity 60 B. thuringiensis is realensis 24 Water Control 4 Western corn rootworm Col ------(Diabrotica virgifera virgifera) Northern corn rootworm Col ------(Diabrotica longicornis barberi) 65 Southern corn rootworm Col Specific activity of AB78 against western corn rootworm is provided in Table 16. 5,840,868 23 24 Transfer was performed at 70 V constant voltage for 1 TABLE 16 hour. Activity of AB78 culture Supernatant against Following transfer, the membrane was rinsed with water neonate Western corn rootworm and stained for two minutes with 0.25% Coomassie Blue R-250 in 50% MeOH. Culture supernatant Percent Destaining was done with several rinses with 50% MeOH concentration (ul/ml) WCRW mortality 40% water 10% acetic acid. 1OO 1OO Following destaining the membrane was air dried prior to 25 87 1O 8O excision of the bands for Sequence analysis. A BlottCar 5 40 tridge and appropriate cycles were utilized to achieve maxi 2.5 2O mum efficiency and yield. Data analysis was performed 1. 6 using model 610 Sequence Analysis Software for identifying O O and quantifying the PTH-amino acid derivatives for each 15 Sequential cycle. The LCso was calculated to be 6.2 ul of culture Superna The N-terminal sequence was determined to be: NH2 tant per ml of Western corn rootworm diet. Lys-Arg-Glu-Ile-Asp-Glu-Asp-Thr-Asp-Thr-ASX-Gly-Asp The cell pellet was also bioassayed and had no activity Ser-Ile-Pro- (SEQ ID NO:8) where ASX represents Asp or against WCRW. Thus, the presence of activity only in the ASn. The complete amino acid Sequence for the 80 kDa Supernatant indicates that this VIP is an exotoxin. component is disclosed in SEQ ID NO:7. The DNA sequence which encodes SEQ ID NO:7 is disclosed in SEQ EXAMPLE 4 ID NO:6.

ISOLATION AND PURIFICATION OF CORN EXAMPLE 6 ROOTWORMACTIVE PROTEINS FROM AB78 25 Culture media free of cells and debris was made to 70% CONSTRUCTION OF DNA PROBE saturation by the addition of solid ammonium sulfate (472 An oligonucleotide probe for the region of the gene g/L). Dissolution was at room temperature followed by encoding amino acids 3–9 of the N-terminal Sequence cooling in an ice bath and centrifugation at 10,000xg for (Example 5) was generated. The probe was Synthesized thirty minutes to pellet the precipitated proteins. The Super based on the codon usage of a Bacillus thuringiensis (Bt) natant was discarded and the pellet was dissolved in /10 the Ö-endotoxin gene. The nucleotide sequence 5'-GAA ATT original volume of 20 mM TRIS-HCl at pH 7.5. The GAT CAA GAT ACN GAT-3' (SEQ ID NO:9) was used as dissolved pellet was desalted either by dialysis in 20 mM a probe in Southern hybridizations. The oligonucleotide was TRIS-HCl pH 7.5, or passing through a desalting column. Synthesized using Standard procedures and equipment. The desalted material was titrated to pH 3.5 using 20 mM 35 Sodium citrate pH 2.5. Following a thirty minute room EXAMPLE 7 temperature incubation the Solution was centrifuged at 3000xg for ten minutes. The Supernatant at this stage ISOELECTRIC POINT DETERMINATION OF contained the greatest amount of active protein. THE CORN ROOTWORMACTIVE PROTEIN Following neutralization of the pH to 7.0 the Supernatant 40 Purified protein from step 5 of the purification process was applied to a Mono-Q, anion exchange, column equili was analyzed on a 3-9 p. isoelectric focusing gel using the brated with 20 mM TRIS pH 7.5 at a flow rate of 300 Phastgel electrophoresis System (Pharmacia). Standard oper mL/min. The column was developed with a stepwise and ating procedures for the unit were followed for both the linear gradient employing 400 mM NaCl in 20 mM TRIS pH Separation and Silver Staining development procedures. The 7.5. 45 pI was approximated at about 4.9. Bioassay of the column fractions and SDS-PAGE analysis were used to confirm the active fractions. SDS-PAGE analy EXAMPLE 8 sis identified the biologically active protein as having com ponents of a molecular weight in the range of about 80 kDa PCR DATA ON AB78 and 50 kDa. 50 PCR analysis (See, for example U.S. patent application EXAMPLE 5 Ser. No. 08/008,006; and, Carozzi et al. (1991) Appl. Envi SEQUENCE ANALYSIS OF THE CORN ron. Microbiol. 57(11):3057-3061, herein incorporated by ROOTWORMACTIVE PROTEIN reference.) was used to verify that the B. cereus strain AB78 55 did not contain any insecticidal crystal protein genes of B. The 80 kDa component isolated by SDS-PAGE was thuringiensis or B. Sphaericus (Table 17). transferred to PVDF membrane and was subjected to amino terminal Sequencing as performed by repetitive Edman TABLE 1.7 cycles on an ABI 470 pulsed-liquid Sequencer. Transfer was carried out in 10 mM CAPS buffer with 10% methanol pH 60 Bacillus insecticidal crystal protein gene primers 11.0 as follows: tested by PCR against AB78 DNA. Incubation of the gel following electrophoresis was done Primers Tested Product Produced in transfer buffer for five minutes. ProBlott PVDF mem 2 sets specific for CryIIIA Negative brane was wetted with 100% MeOH briefly then equili CryIIIB Negative brated in transfer buffer. The sandwich was arranged 65 2 sets specific for CryIA Negative between foam Sponges and filter paper Squares with the CryIA(a) Negative configuration of cathode-gel-membrane-anode. 5,840,868 25 26 3. Add together: TABLE 17-continued 100 ulcells 100 ul diluted packaging mixture Bacillus insecticidal crystal protein gene primers 100 ul 10 mM MgSO tested by PCR against AB78 DNA. 30 ul TB Primers Tested Product Produced 4. Adsorb at room temperature for 30 minutes with no Shaking. CryIA(b) specific Negative CryIB Negative 5. Add 1 ml TB and mix gently. Incubate 30 minutes at CryIC specific Negative 37o C. CryIE specific Negative 6. Plate 200 ul onto L-amp plates. Incubate at 37 C. 2 sets specific for B. Sphaericus Negative overnight. 2 sets specific for CryIV Negative At least 400 coSmid clones were Selected at random and Bacillus control (PI-PLC) Positive Screened for activity against Western corn rootworm as described in Example 3. DNA from 5 active clones and 5 15 non-active clones were used in Southern hybridizations. EXAMPLE 9 Results demonstrated that hybridization using the above described oligonucleotide probe correlated with western COSMID CLONING OF TOTAL DNA FROM B. corn rootworm activity (Table 18). CEREUSSTRAIN AB78 Cosmid clones P3-12 and P5-4 have been deposited with The VIP1A(a) gene was cloned from total DNA prepared the Agricultural Research Service Patent Culture Collection from strain AB78 as follows: (NRRL) and given Accession Nos. NRRL B-21061 and Isolation of AB78 DNA was as follows: NRRL B-21059 respectively. 1. Grow bacteria in 10 ml L-broth overnight. (Use 50 ml Sterile centrifuge tube) TABLE 1.8 2. Add 25 ml of fresh L-broth and amplicillin (30 tug/ml). 25 3. Grow cells 2-6 h. at 30° C. with shaking. Activity of AB78 COSmid clones against Western corn rootworm. 4. Spin cells in a 50 ml polypropylene orange cap tube in Mean IEC benchtop clinical centrifuge at % Speed. Clone percent mortality (N = 4) 5. Resuspend cell pellet in 10 ml TES (TES=50 mM TRIS Clones which hybridize with probe pH 8.0, 100 mM EDTA, 15 mM NaCl). 6. Add 30 mg lysozyme and incubate 2 hrs at 37 C. P1-73 47 P1-83 64 7. Add 200 ul 20% SDS and 400 ul Proteinase K stock (20 P2-2 69 mg/ml). Incubate at 37° C. P3-12 85 8. Add 200 ul fresh Proteinase K. Incubate 1 hr. at 55° C. P5-4 97 Add 5 ml TES to make 15 ml final volume. 35 Clones which do not hybridize with probe 9. Phenol extract twice (10 ml phenol, spin at room tem P1-2 5 perature at % Speed in an IEC benchtop clinical P3-8 4 centrifuge). Transfer Supernatant (upper phase) to a clean P3-9 12 tube using a wide bore pipette. P3-18 O 10. Extract once with 1:1 vol. phenol:chloroform/isoamyl 40 P4-6 9 alcohol (24:1 ratio). 11. Precipitate DNA with an equal volume of cold isopro panol; Centrifuge to pellet DNA. EXAMPLE 10 12. Resuspend pellet in 5 ml TE. 13. Precipitate DNA with 0.5 ml 3M NaOAc pH 5.2 and 11 45 IDENTIFICATION OF A 6 KB REGION ACTIVE ml 95% ethanol. Place at -20° C. for 2 h. AGAINST WESTERN CORN ROOTWORM 14. “Hook' DNA from tube with a plastic loop, transfer to a microfuge tube, Spin, pipette off exceSS ethanol, dry in DNA from P3-12 was partially digested with restriction WCUO. enzyme Sau 3A, and ligated into the E. coli vector puC19 15. Resuspend in 0.5 ml TE. Incubate 90 min. at 65° C. to 50 and transformed into E. coli. A DNA probe specific for the help get DNA back into solution. 80 kDa VIP1A(a) protein was synthesized by PCR ampli 16. Determine concentration using Standard procedures. fication of a portion of P3-12 DNA. Oligonucleotides Cosmid Cloning of AB78 MK113 and MK117, which hybridize to portions of VIP1A All procedures, unless indicated otherwise, were per (a), were Synthesized using the partial amino acid sequence 55 of the 80 kDa protein. Plasmid subclones were identified by formed according to Stratagene Protocol, SupercoS 1 colony hybridization to the PCR-generated probe, and tested Instruction Manual, Cat. No. 251301. for activity against Western corn rootworm. One Such clone, Generally, the Steps were as follows: PL2, hybridized to the PCR-generated fragment, and was A. Sau 3A partial digestion of the AB78 DNA. active against Western corn rootworm in the assay previ B. Preparation of vector DNA 60 ously described. C. Ligation and packaging of DNA A 6 kb Cla I restriction fragment from pl. 2 was cloned D. Tittering the cosmid library into the Sma I site of the E. coli-Bacillus shuttle vector pHT 1. Start a culture of HB101 cells by placing 50 ml of an 3101 (Lereclus, D. et al., FEMS Microbiology Letters overnight culture in 5 mls of TB with 0.2% maltose. 60:211-218 (1989)) to yield pCIB6201. This construct con Incubate 3.5 hrs. at 37 C. 65 fers anti-western corn rootworm activity upon both Bacillus 2. Spin out cells and resuspend in 0.5 ml 10 mM and E.coli strains, in either orientation. pCIB6022 contains MgSO. this same 6 kb Cla I fragment in pBluescript SK(+) 5,840,868 27 28 (Stratagene), produces equivalent VIP1A(a) protein (by lacks most of the VIP1A(a) coding region. pCIB6024 was western blot), and is also active against western corn root constructed by gel purifying the 2.2 kb Cla I-Sca I restriction WO. fragment from pCIB6022, filling in the single-stranded ends The nucleotide sequence of pCIB6022 was determined by with DNA polymerase (Klenow fragment) and dNTPs, and the dideoxy termination method of Sanger et al., Proc. Natl. ligating this fragment into pBlueScript SK(+) vector Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready (Stratagene) digested with the enzyme Eco RV. Cells con Reaction Dye Deoxy Terminator Cycle Sequencing Kits and taining pCIB6024 exhibit no activity against western corn PRISM Sequenase(R) Terminator Double-Stranded DNA rootworm. However, a mixture of cells containing Sequencing Kit and analyzed on an ABI 373 automatic pCIB6024 and cells containing pCIB6023 shows high activ sequencer. The sequence is given in SEQ ID NO:1. The 6 kb ity against western corn rootworm. (See FIG. 1). fragment encodes both VIP1A(a) and VIP2A(a), as indi Thus, pCIB6023 and pCIB6206 must produce a func cated by the open reading frames described in SEQ ID tional VIP1A(a) gene product, while pCIB6203 and NO:1. The sequence encoding VIP1A(a) is further disclosed pCIB6024 must produce a functional VIP2A(a) gene prod in SEQ ID NO:4. The relationship between VIP1A(a) and uct. These results Suggest a requirement for a gene product VIP2A(a) within the 6 kb fragment found in pCIB6022 is 15 (s) from the VIP2A(a) region, in combination with VIP1A depicted in FIG. 1. pCIB6022 was deposited with the (a), to confer maximal western corn rootworm activity. (See Agricultural Research Service, Patent Culture Collection, FIG. 1) (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA, and given the EXAMPLE 12 Accession No. NRRL B-21222. AB78 ANTIBODY PRODUCTION EXAMPLE 11 Antibody production was initiated in 2 Lewis rats to allow for both the possibility of moving to production of hybri FUNCTIONAL DISSECTION OF THE VIP1A(a) doma cell lines and also to produce enough Serum for limited DNA REGION 25 Screening of genomic DNA library. Another factor was the To confirm that the VIP1A(a) open reading frame (ORF) very limited amount of antigen available and the fact that it is necessary for insecticidal activity a translational frame could only be produced to purity by PAGE and Subsequent shift mutation was created in the gene. The restriction electrotransfer to nitrocellulose. enzyme Bgl II recognizes a unique site located 857 bp into Due to the limited availability of antigen on the coding region of VIP1A(a). pCIB6201 was digested with nitrocellulose, the nitrocellulose was emulsified in DMSO Bgl II, and the single-stranded ends filled-in with DNA and injected into the hind footpads of the animals to elicit polymerase (Klenow fragment) and dNTPS. The plasmid B-cell production in the popliteal lymph nodes just was re-ligated and transformed into E. coli. The resulting upstream. A strong reacting Serum was produced as judged plasmid, pCIB6203, contains a four nucleotide insertion in by western blot analysis with the first production bleed. the coding region of VIP1A(a). pCIB6203 does not confer 35 Several Subsequent injections and bleeds produced enough WCRW insecticidal activity, confirming that VIP1A(a) is an Serum to accomplish all of the Screening required. essential component of Western corn rootworm activity. Hybridoma production with one of the rats was then To further define the region necessary to encode VIP1A initiated. The popliteal lymph node was excised, macerated, (a), Subclones of the VIP1A(a) and VIP2A(a) (auxiliary and the resulting cells fused with mouse myeloma protein) region were constructed and tested for their ability 40 P3x63Ag8.653. Subsequent cell screening was accom to complement the mutation in pCIB6203. pCIB6023 con plished as described below. Four initial wells were selected tains the 3.7 kb Xba I-EcoRV fragment in pBluescript SK(+) which gave the highest emulsified antigen reaction to be (Stratagene). Western blot analysis indicates that pCIB6023 moved to limited dilution cloning. An additional 10 wells produces VIP1A(a) protein of equal size and quantity as were chosen for expansion and cryoperServation. clones PL2 and pCIB6022. pCIB6023 contains the entire 45 gene encoding the 80 kD protein. pCIB6023 was deposited Procedure to Emulsify AB78 on nitrocellulose in with the Agricultural Research Service, Patent Culture DMSO for ELISA screening Collection, (NRRL), Northern Regional Research Center, After electrotransfer of AB78 samples run on PAGE to 1815 North University Street, Peoria, Ill. 61604, USA, and 50 nitrocellulose, the reversible strain Ponceau S is used to given the Accession No. NRRL B-21223N. pCIB6206 con Visualize all protein transferred. The band corresponding to tains the 4.3 kb Xba I-Cla I fragment from pCIB6022 in AB78 toxin, previously identified and N-terminal pBluescript SK(+) (Stratagene). pCIB6206 was also depos Sequenced, was identified and excised from nitrocellulose. ited with the Agricultural Research Service, Patent Culture Each band is approximately 1 mmx5 mm in Size to minimize Collection, (NRRL), Northern Regional Research Center, 55 the amount of nitrocellulose emulsified. A Single band is 1815 North University Street, Peoria, Ill. 61604, USA, and placed in a microfuge tube with 250 ul of DMSO and given the Accession No. NRRL B-21321. macerated using a plastic pestle (Kontes, Vineland, N.J.). To pCIB6023, pCIB6206, and pCIB6203 do not produce aid in emulsification, the DMSO mixture is heated for 2-3 detectable western corn rootworm activity when tested indi minutes at 37 C-45 C. Some further maceration might be vidually. However, a mixture of cells containing pCIB6203 60 necessary following heating; however, all of the nitrocellu (VIP1A(a)-mutated, plus VIP2A(a)) and cells containing lose should be emulsified. Once the AB78 sample is pCIB6023 (only VIP1A(a)) shows high activity against emulsified, it is placed on ice. In preparation for microtiter western corn rootworm. Similarly, a mixture of cells con plate coating with the emulsified antigen, the Sample must be taining pCIB6206 and cells containing pCIB6203 shows diluted in borate buffered Saline as follows: 1:5, 1:10, 1:15, high activity against Western corn rootworm. 65 1:20, 1:30, 1:50, 1:100, and 0. The coating antigen must be To further define the limits of VIP2A(a), we constructed prepared fresh immediately prior to use. pCIB6024, which contains the entirety of VIP2A(a), but ELISA protocol: 5,840,868 29 30 1. Coat with AB78/DMSO in BBS. Incubate overnight at 4 C. ... Wash plate 3x with 1xELISA wash buffer. lnsect species Percent Block (1% BSA & 0.05% Tween 20 in PBS) for 30 tested Mortality minutes at Room Temperature. 5 Ostrinia nubialis O Agrotis ipsilon O ... Wash plate 3x with 1xELISA wash buffer. Diabrotica virgifera virgifera 55 Add rat serum. Incubate 1.5 hours at 37 C. ... Wash plate 3x with 1xELISA wash buffer. . Add goat anti-rat at a concentration of 2 ug/ml in ELISA EXAMPLE 1.5 diluent. Incubate 1 hr. at 37 C. ISOLATION AND BIOLOGICAL ACTIVITY OF 8 ... Wash plate 3x with 1xELISA wash buffer. B. THURINGIENSIS AB6 9. Add rabbit anti-goat alkaline phosphatase at 2 tug/ml in ELISA diluent. Incubate 1 hr. at 37° C. A B. thuringiensis Strain, designated AB6, was isolated 10. Wash 3X with 1xELISA wash buffer. from grain bin dust Samples by Standard methods known in 15 the art. A Subculture of AB6 was grown and prepared for 11. Add Substrate. Incubate 30 minutes at room temperature. bioassay as described in Example 2. Half of the Sample was 12. Stop with 3N NaOH after 30 minutes. autoclaved 15 minutes to test for the presence of B-eXotoxin. Preparation of VIP2A(a) Antisera Biological activity was evaluated as described in Example A partially purified AB78 culture Supernatant was sepa 3. The results are as follows: rated by discontinuous SDS PAGE (Novex) following manufacturers instructions. Separated proteins were elec trophoresed to nitrocellulose (S&S #21640) as described by Insect species Percent Towbin et al., (1979). The nitrocellulose was stained with tested Mortality Ponceau S and the VIP2A(a) band identified. The VIP2A(a) Ostrinia nubialis O 25 Agrotis ipsilon 1OO band was excised and emulsified in DMSO immediately Agrotis ipsilon (autoclaved sample) O prior to injection. A rabbit was initially immunized with Diabrotica virgifera virgifera O emulsified VIP2A(a) mixed approximately 1:1 with Fre und's Complete adjuvant by intramuscular injection at four different Sites. Subsequent immunizations occurred at four The reduction of insecticidal activity of the culture Super week intervals and were identical to the first, except for the natant to insignificant levels by autoclaving indicates that use of Freund Incomplete adjuvant. The first serum har the active principle is not f-eXotoxin. vested following immunization reacted with VIP2A(a) pro Strain AB6 has been deposited in the Agricultural tein. Western blot analysis of AB78 culture Supernatant Research Service, Patent Culture Collection (NRRL), using this antisera identifies predominately full length Northern Regional Research Center, 1815 North University 35 Street, Peoria, Ill. 61604, USA, and given Accession No. VIP2A(a) protein. NRRL B-21060. EXAMPLE 16 EXAMPLE 13 ISOLATION AND BIOLOGICAL CHARACTERIZATION OF B. THURINGIENSIS ACTIVATION OF INSECTICIDAL ACTIVITY OF 40 NON-ACTIVE BT STRAINS WITH AB78 VIP AB88 CLONES ABt Strain, designated AB88, was isolated from grain bin dust Samples by Standard methodologies. A Subculture of Adding pCIB6203 together with a 24 h culture (early to AB88 was grown and prepared for bioassay as described in mid-log phase) Supernatant from Bt Strain GC91 produces Example 2. Half of the sample was autoclaved 15 minutes 100% mortality in Diabrotica virgifera virgifera. Neither 45 to test for the presence of B-eXotoxin. Biological activity pCIB6203 nor GC91 is active on Diabrotica virgifera vir was evaluated against a number of insect Species as gifera by itself Data are shown below: described in Example 3. The results are as follows:

50 Percent mortality of Test material Percent Diabrotica mortality culture Supernatant pCIB6203 O GC91 16 Insect species tested Order Non-autoclaved Autoclaved pCIB6203 + GC91 1OO Control O Agrotis ipsilon Lepidoptera 1OO 5 55 Ostrinia nubialis Lepidoptera 1OO O Spodoptera frugiperda Lepidoptera 1OO 4 Helicoverpa zea Lepidoptera 1OO 12 Heliothis virescens Lepidoptera 1OO 12 EXAMPLE 1.4 Leptinotarsa decenlineata Coleoptera O O Diabrotica virgifera Coleoptera O 5 virgifera ISOLATION AND BIOLOGICAL ACTIVITY OF 60 B. CEREUS AB81 The reduction of insecticidal activity of the culture Super A Second B. cereuS Strain, designated AB81, was isolated natant to insignificant levels by autoclaving indicates that from grain bin dust Samples by Standard methodologies. A the active principle is not f-eXotoxin. Subculture of AB81 was grown and prepared for bioassay as 65 Delta-endotoxin crystals were purified from strain AB88 described in Example 2. Biological activity was evaluated as by Standard methodologies. No activity from pure crystals described in Example 3. The results are as follows: was observed when bioassayed against AgrOtis ipsilon. 5,840,868 31 32 EXAMPLE 1.7

PURIFICATION OF VIPS FROM STRAIN AB88 Agrotis VIP N-terminal sequence of major N-terminal sequences Ö-endotoxin proteins Bacterial liquid culture was grown overnight at 30° C. in 130 kDa TB media. Cells were spun out and the Supernatant retained. MDNNPNINE Proteins were precipitated with ammonium sulfate (70% (SEQ ID NO: 14) Saturation), centrifuged and the pellet retained. The pellet 80 kDa 80 kDa was resuspended in the original volume of 20 mM Tris pH MNKNNTKLPTRALP MDNNPNINE (SEQ ID NO: 12) (SEQ ID NO: 15) 7.5 and dialyzed against the same buffer. AB88 dialysate was 1O 60 kDa more turbid than comparable material from AB78. AB88 MNVLNSGRTTI proteins have been Separated by Several different methods 35 kDa (SEQ ID NO: 16) following clarification including isoelectric focusing ALSENTGKDGGYIVP (Rotofor, BioRad, Hercules, Calif.), precipitation at pH 4.5, (SEQ ID NO: 13) ion-exchange chromotography, Size exclusion chromatogra 15 phy and ultrafiltration. The Ostrinia nubilalis activity is due to a 60 kDa VIP and European corn borer (ECB)-active protein remained in the the Spodoptera frugiperda activity is due to a VIP of pellet obtained by pH 4.5 precipitation of dialysate. When unknown size. preparative IEF was done on the dialysate using pH 3-10 Bacillus thuringiensis strain AB88 has been deposited in ampholytes, ECB insecticidal activity was found in all the Agricultural Research Service, Patent Culture Collection fractions with pH of 7 or greater. SDS-PAGE analysis of (NRRL), Northern Regional Research Center, 1815 North these fractions showed protein bands of MW ~60 kDa and University Street, Peoria, Ill. 61604, USA and given the ~80 kDa. The 60 kDa and 80 kDa bands were separated by Accession No. NRRL B-21225. anion exchange HPLC on a Poros-Q column (PerSeptive BioSystems, Cambridge, Mass.). N-terminal Sequence was 25 EXAMPLE 18A obtained from two fractions containing proteins of Slightly differing MW, but both of approximately 60 kDa in size. The ISOLATION AND BIOLOGICAL ACTIVITY OF Sequences obtained were similar to each other and to Some B. THURINGIENSIS AB424 Ö-endotoxins. A B. thuringiensis Strain, designated AB424, was isolated from a moSS covered pine cone sample by Standard methods anion exchange fraction 23 (smaller): XEPFWSAXXXOXXX known in the art. A subculture of AB424 was grown and (SEQ ID NO: 10) prepared for bioassay as described in Example 2. anion exchange fraction 28 (larger): XEYENVEPFWSAX Biological activity was evaluated as described in Example (SEQ ID NO: 11) 3. The results are as follows: 35 When the ECB-active pH 4.5 pellet was further separated by anion eXchange on a Poros-Q column, activity was found Insect species tested Percent mortality only in fractions containing a major band of ~60 kDa. Ostrinia nubialis 1OO Agrotis ipsilon 1OO Black cutworm-active protein also remained in the pellet 40 Diabrotica virgifera O when AB88 dialysate was brought down to pH 4.5. In virgifera preparative IEF using pH 3-10 ampholytes, activity was not found in the ECB-active IEF fractions; instead, it was highest in a fraction of pH 4.5-5.0. Its major components Strain AB424 has been deposited in the Agricultural have molecular weights of ~35 and ~80 kDa. 45 Research Service, Patent Culture Collection (NRRL), The pH 4.5 pellet was separated by anion exchange HPLC Northern Regional Research Center, 1815 North University to yield fractions containing only the 35 kDa material and Street, Peoria, Ill. 61604, USA, and given Accession No. fractions containing both 35 kDa and 80 kDa bands. NRRL B-21439. EXAMPLE 1.8B EXAMPLE 1.8 50 CLONING OF THE VIP3A(a) and VIP3A(b) CHARACTERIZATION OF AB88 VIP GENES WHICH ENCODE PROTEINS ACTIVE Fractions containing the various lepidopteran active veg AGAINST BLACK CUTWORM etative proteins were generated as described in Example 17. DNA from isolates AB88 and AB424 was digested with Biological analysis of fractions demonstrated that different 55 the restriction enzymes Xbaland EcoRI respectively, ligated VIPs were responsible for the different lepidopteran species into pBlueScript vector previously linearized with the same activity. enzymes and dephosphorylated, and transformed into E. coli The Agrotis ipsilon activity is due to an 80 kDa and/or a DH5C. Strain. Recombinant clones were blotted onto nitro 35 kDa protein, either delivered singly or in combination. cellulose filters which were subsequently probed with a These proteins are not related to any 8-endotoxins from Bt 60 33-bases long oligonucleotide corresponding to the 11-N as evidenced by the lack of Sequence homology of known Bt terminal amino acids of the 80 kDa protein active against Ö-endotoxin Sequences. Also, these proteins are not found in Agrotis epsilon (black cutworm). Four out of 400 recombi the AB88 Ö-endotoxin crystal. N-terminal sequences of the nant clones were positive. Insect bioassays of the positive major Ö-endotoxin proteins were compared with the recombinants exhibited toxicity to black cutworm larvae N-terminal sequences of the 80 kDa and 35 kDa VIP and 65 comparable to that of AB88 or AB424 Supernantants. revealed no Sequence homology. A Summary of the results The nucleotide sequence of pCIB7104, a positive recom follows: binant clone from AB88, and of pCIB7107, a positive 5,840,868 33 34 recombinant clone from AB424, was determined by the These Strains were isolated from environmental Samples by dideoxy termination method of Sanger et al., Proc. Natl. Standard methodologies. Isolates were prepared for bioassay Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready and assayed as described in Examples 2 and 3 respectively. Reaction Dye Deoxy Terminator Cycle Sequencing Kits and Isolates which produced insecticidal proteins during vegeta PRISM Sequenase(R) Terminator Double-Stranded DNA 5 tive growth with activity against Agrotis ipsilon in the Sequencing Kit and analysed on an ABI 373 automatic bioassay are tabulated below. No correlation was observed Sequencer. between the presence of a 6-endotoxin crystal and vegetative The clone pCIB7104 contains the VIP3A(a) gene whose insecticidal protein production. coding region is disclosed in SEQ ID NO:28 and the encoded protein sequence is disclosed in SEQ ID NO:29. A Synthetic version of the coding region designed to be highly Presence of 8-endotoxin expressed in maize is given in SEQ ID NO:30. Any number Bacillus isolate crystal Percent mortality of Synthetic genes can be designed based on the amino acid AB6 -- 1OO sequence given in SEQ ID NO:29. AB53 8O AB88 -- 1OO The clone pCIB7107 contains the VIP3A(b) gene whose 15 AB195 60 coding region is disclosed in SEQ ID NO:31 and the AB211 70 encoded protein is disclosed in SEQ ID NO:32. Both AB217 83 pCIB7104 and pCIB7107 have been deposited with the AB272 8O AB279 70 Agricultural Research Service Patent Culture Collection AB289 -- 1OO (NRRL) and given Accession Nos. NRRL B-21422 and AB292 -- 8O B-21423, respectively. AB294 1OO AB3OO 8O EXAMPLE 1.8C AB359 1OO IDENTIFICATION OF NOVEL VIP3-LIKE GENES BY HYBRIDIZATION 25 Isolates AB289, AB294 and AB359 have been deposited To identify Bacillus containing genes related to the in the Agricultural Research Service, Patent Culture Collec VIP3A(a) from isolate AB88, a collection of Bacillus iso tion (NRRL), Northern Regional Research Center, 1815 lates was screened by hybridization. Cultures of 463 Bacil North University Street, Peoria Ill. 61604, USA and given lus Strains were grown in microtiter Wells until Sporulation. the Accession Numbers NRRL B-21227, NRRL B-21229, A 96-pin colony Stampel was used to transfer the cultures to and NRRL B-21226 respectively. 150 mm plates containing L-agar. Inoculated plates were Bacillus isolates which produce insecticidal proteins dur kept at 30° C. for 10 hours, then at 4 C. overnight. Colonies ing vegetative growth with activity against Diabrotica vir were blotted onto nylon filters and probed with a 1.2 Kb gifera virgifera are tabulated below. HindIII VIP3A(a) derived fragment. Hybridization was per formed overnight at 62 C. using hybridization conditions of 35 Maniatis et al. Molecular Cloning. A Laboratory Manual Presence of 8-endotoxin (1982). Filters were washed with 2xSSC/0.1% SDS at 62° Bacillus isolate crystal Percent mortality C. and exposed to X-ray film. AB52 50 Of the 463 Bacillus strains Screened, 60 contain VIP3-like AB59 71 genes that could detected by hybridization. AB68 -- 60 40 ABA8 1OO EXAMPLE 1.8D AB122 57 AB218 64 CHARACTERIZATION OF A B. thuringiensis AB256 64 STRAIN M21.94 CONTAINING A CRYPTIC VIP3-LIKE GENE A B. thuringiensis strain, designated M2194, was shown 45 Isolates AB59 and AB256 have been deposited in the to contain VIP3-like gene(s) by colony hybridization as Agricultural Research Service, Patent Culture Collection described in Example 18C. The M2194 VIP3 like gene is (NRRL), Northern Regional Research Center, 1815 North considered cryptic Since no expression can be detected University Street, Peoria Ill. 61604, USA, and given the throughout the bacterial growth phases either by immuno Accession Numbers NRRL B-21228 and NRRL B-21230, blot analysis using polyclonal antibodies raised against the 50 respectively. VIP3A(a) protein isolated from AB88 or by bioassay as described in Example 3. EXAMPLE 2.0 The M2194 VIP3-like gene was cloned into pKS by IDENTIFICATION OF NOVEL VIP1/VIP2 LIKE following the protocol described in Example 9, which cre GENES BY HYBRIDIZATION ated pCIB7108. E. coli containing pCIB7108 which com 55 prises the M2194 VIP3 gene were active against black To identify Strains containing genes related to those found cutworm demonstrating that the gene encodes a functional in the VIP1A(a)/VIP2A(a) region of AB78, a collection of protein with insecticidal activity. The plasmid pCIB7108 has Bacillus Strains was Screened by hybridization. Independent been deposited with the Agricultural Research Service cultures of 463 Bacillus strains were grown in wells of 96 Patent Culture Collection (NRRL) and given Accession No. 60 well microtiter dishes (five plates total) until the cultures NRRL B-21438. Sporulated. Of the Strains tested, 288 were categorized as EXAMPLE 1.9 Bacillus thuringiensis, and 175 were categorized as other Bacillus species based on the presence or absence of ISOLATION AND BIOLOGICAL ACTIVITY OF Ö-endotoxin crystals. For each microtiter dish, a 96-pin OTHER BACILLUS SP. 65 colony Stamper was used to transfer approximately 10 ul of Other Bacillus species have been isolated which produce Spore culture to two 150 mm plates containing L-agar. proteins with insecticidal activity during vegetative growth. Inoculated plates were grown 4-8 hours at 30° C., then 5,840,868 35 36 chilled to 4 C. Colonies were transferred to nylon filters, tified. All twenty eight clones are identical, and contain and the cells lysed by standard methods known in the art. VIP1A(a)VIP2A(a) homologs. Clone pCIB7100 has been The filters were hybridized to a DNA probe generated from deposited in the Agricultural Research Service, Patent Cul DNA fragments containing both VIP1A(a) and VIP2A(a) ture Collection (NRRL), Northern Regional Research DNA sequences. Hybridization was performed overnight at Center, 1815 North University Street, Peoria Ill. 61604, 65 C. using the hybridization conditions of Church and USA, and given the Accession Number B-21322. Several Gilbert (Church, G. M., and W. Gilbert, PNAS, subclones were constructed from pCIB7100. A 3.8 kb Xba 81:1991–1995 (1984)). Filters were washed with 2XSSC I fragment from pCIB7100 was cloned into pBluescript containing 0.1% SDS at 65° C. and exposed to X-Ray film. SK(+) to yield pCIB7101. A 1.8 kb Hind III fragment and a Of the 463 Bacillus strains Screened, 55 strains were 1.4 kb Hind III fragment from pCIB7100 were cloned into identified that hybridized to the VIP1A(a)/VIP2A(a) probe. pBluescript SK(+) to yield pCIB7102 and pCIB7103, DNA was isolated from 22 of these strains, and analyzed respectively. Subclones pCIB7101, pCIB7102 and using a Southern blot with VIP1A(a)/VIP2A(a) DNA as pCIB7103 have been deposited in the Agricultural Research probes. These Strains were grouped into 8 classes based on Service, Patent Culture Collection (NRRL), Northern their Southern blot pattern. Each class differed in Southern 15 Regional Research Center, 1815 North University Street, blot pattern from AB78. One class had a pattern identical to Peoria Ill. 61604, USA, and given the Accession Numbers that of the VIP1A(a)/VIP2A(a) homologs from Bacillus B-21323, B-21324 and B-21325 respectively. thuringiensis war tenebrionis (see below). Each of the 22 The DNA sequence of the region of pCIB7100 containing Strains was tested for activity against Western corn rootworm the VIP2A(a)/VIP1A(a) homologs was determined by the (WCRW). Three strains, AB433, AB434, and AB435 were dideoxy chain termination method (Sanger et al., 1977, Proc. found to be active on WCRW. Western blot analysis using Natl. Acad. Sci. USA 74:5463-5467). Reactions were per VIP2A(a) antisera revealed that strains AB6, AB433, formed using PRISM Ready Reaction Dye Deoxy Termina AB434, AB435, AB444, and AB445 produce a protein(s) of tor Cycle Sequencing Kits and PRISM Sequenase(R) Termi equivalent size to VIP2A(a). nator Double-Stranded DNA Sequencing Kits, and analyzed Notable among the strains identified was Bacillus thur 25 on an ABI model 373 automated Sequencer. Custom oligo ingiensis strain AB6, (NRRL B-21060) which produced a nucleotides were used as primers to determine the DNA VIP active against black cutworm (Agrotis ipsilon) as Sequence in certain regions. The DNA sequence of this described in Example 15. Western blot analysis with poly region is shown in SEQ ID NO:19. clonal antisera to VIP2A(a) and polyclonal antisera to The 4 kb region shown in SEQ ID NO:19 contains two VIP1A(a) suggests that AB6 produces proteins similar to open readings frames (ORFs), which encode proteins with a VIP2A(a) and VIP1A(a). Thus, AB6 may contain VIPs high degree of similarity to VIP1A(a) and VIP2A(a) proteins similar to VIP1A(a) and VIP2A(a), but with a different from Strain AB78. The amino acid sequence of the VIP2A(a) spectrum of insecticidal activity. homolog, designated as VIP2A(b) using the Standardized EXAMPLE 21 35 nomenclature, is found at SEQID NO:20 and the amino acid sequence of the VIP1A(a) homolog, designated as VIP1A(b) CLONING OF A VIP1A(a)/VIP2A(a) HOMOLOG using the Standardized nomenclature, is disclosed at SEQID FROM BACILLUS THURINGIENSIS WAR. NO:21. The VIP2A(b) protein exhibits 91% amino acid TENEBRIONIS identity to VIP2A(a) from AB78. An alignment of the amino 40 acid sequences of the two VIP2 proteins is provided in Table Several previously characterized Bacillus Strains were 19. The VIP1A(b) protein exhibits 77% amino acid identity tested for presence of DNA similar to VIP1A(a)/VIP2A(a) to VIP1A(a) from AB78. An alignment of these two VIP1 by Southern blot analysis. DNA from Bacillus strains AB78, proteins is provided in Table 20. The alignment shown in AB88, GC91, HD-1 and ATCC 10876 was analyzed for Table 21 discloses the similarity between VIP1A(b) and presence of VIP1A(a)/VIP2A(a) like sequences. DNA from 45 VIP1A(a) from AB78. This alignment reveals that the amino Bt strains GC91 and HD-1, and the Bc strain ATCC 10876 terminal regions of the two VIP1 proteins share higher did not hybridize to VIP2A(a)/VIP1A(a) DNA, indicating amino acid identity in the amino-terminal region than in the they lack DNA sequences similar to VIP1A(a)/VIP2A(a) carboxy terminal region. In fact, the amino terminal two genes. Similarly, DNA from the insecticidal strain AB88 thirds (up to aa 618 of the VIP1A(b) sequence shown in (Example 16) did not hybridize to VIP1A(a)/VIP2A(a) DNA 50 Table 20) of the two proteins exhibit 91% identity, while the region, Suggesting that the VIP activity produced by this carboxy-terminal third (from aa 619–833 of VIP1 A(b)) strain does not result from VIP1A(a)/VIP2A(a) homologs. exhibit only 35% identity. In contrast, Bacillus thuringiensis var. tenebrionis (Btt) contained sequences that hybridized to the VIP1A(a)/VIP2A Western blot analysis indicated that Bacillus thuringiensis (a) region. Further analysis confirmed that Btt contains 55 var. tenebrionis (Btt) produces both VIP1A(a) like and VIP1A(a)/VIP2A(a) like sequences. VIP2A(a) like proteins. However, these proteins do not To characterize the Btt homologs of VIP2A(a) and VIP1A appear to have activity against Western corn rootworm. (a), the genes encoding these proteins were cloned. Southern Bioassay for activity against Western corn rootworm was blot analysis identified a 9.5 kb Eco RI restriction fragment performed using either a 24 h culture Supernatant from Btt likely to contain the coding regions for the homologs. 60 or E. coli clone pCIB7100 (which contains the entire region Genomic DNA was digested with EcoRI, and DNA frag of the VIP1A(a)/VIP2A(a) homologs). No activity against ments of approximately 9.5 kb in length were gel-purified. western corn rootworm was detected in either case. This DNA was ligated into pBluescript SK(+) digested with Given the similarity between the VIP2 proteins from Btt EcoRI, and transformed into E. coli to generate a plasmid and AB78, the ability of VIP2A(b) from Btt to substitute for library. Approximately 10,000 colonies were screened by 65 VIP2A(a) from AB78 was tested. Cells containing colony hybridization for the presence of VIP2A(a) homolo pCIB6206 (which produces AB78 VIP1A(a) but not VIP2A gous Sequences. Twenty eight positive colonies were iden (a) protein) were mixed with Btt culture Supernatant, and 5,840,868 37 38 tested for activity against western corn rootworm. While Thus, the ability to identify new strains with insecticidal neither Btt culture Supernatant nor cells containing activity by using VIP DNA as hybridization probes has been pCIB6206 had activity on WCRW, the mixture of Btt and demonstrated. Furthermore, Bacillus Strains that contain pCIB6206 gave high activity against WCRW. Furthermore, VIP1A(a)/VIP2A(a) like sequences, produce VIP1A(a)/ additional bioassay showed that the Btt clone pCIB7100, VIP2A(a) like protein, yet demonstrate toxicity toward which contains the Btt VIP1A(b)/VIP2A(b) genes in E. coli, different insect pests. Similar methods can identify many also confers activity against WCRW when mixed with pCIB6206. Thus, the VIP2A(b) protein produced by Btt is more members of the VIP1/VIP2 family. Furthermore, use functionally equivalent to the VIP2A(a) protein produced by of Similar methods can identify homologs of other varieties AB78. of VIPs (for example, the VIPs from AB88).

TABLE 19 Alignment of VIP2 Amino Acid Sequences from Bacillus thuringienis var. tenebrionis (VIP2A(b)) vs. AB78 (VIP2A(a)) Btt 1 MOR ME G. KLF V V S KTL OVW T R T V L L S T V Y S I T L L NNV VI KADOL NI NS O S K 50 SEQID NO: 20 Abis Mk Midki is kk. Viki villvi is le Vikki list k so sold No.2 51 YTNL ONLKI PDNAE D F KEDK GKAKE WGKEKG E E WRP PATE KGEMN NFL DN 100 5 y-liki if k vibhkhikikiki w ki ki khwki i Al Ekk M. B. 1OO 101 KND I KTNY KEI TFS MAGS CE DE I KD L E E I DKI F D KAN LS S S I TY KNVE P 150 1O kbi x kit Mkh f : bhik bikiksbki is hyky, 150 151 AT I GF NK S L T E GNT I NSDAMA OF KE OF L G KDMKF D S YLD THL T AO Q V S S K 200 15 if k + 1 NBAMA, kh) i? bik by bhikkh) is sk 2OO 2O1 KR VI L. KVTV P S G. K. G. S TT PT KAG WI LNN N E YKML DNG Y V L HW D KVS KVVK 250 2O E v. kyivk, TT PT KAG VI is kill by viviky KVVK 250 251 KGME CLOVE GT L KKS LD F KN DI NAE AHS WG MKI YED WAKN LT AS ORE AL D 300 251 K G VE CLOI E GT L KKS LD F KNDI Alki w knyi iwikilip ORE AL D 300 301 GY AR ODY KEI NNY LR NO G GS GNE KL DAOL KNI SDAL GKKP I PEN I TVY R W 350 3O by Abbykh NNY LR NO GGS hikib), KNI bikki TVY RW 350 351 C GMP E F GYO I SDP LP S L KDF E E OF LNT I. KEDKGYMS T S L S S E R L AAF G. S R 400 35 day, blk by 1. khbky Miss AA 400 401 KI I L R L OVP KG ST GAYL SAI G G F A S E KEI LL D KDS KY HID KATE VI I KGV 450 401 K k + k is ki did Akhill bkhsky hibkvily I KGV 450

451 KRY VVD A T L L T N 462 45 kiy Vykhill 462

TABLE 20 Alignment of VIP1 Amino Acid Sequenes from Bacillus thuringiensis var. tenebrionis (VIP1A(b)) vs. AB78(VIP1A(a)) Btt 1 MKNMKKKLAS V VT CMLL AP MF LN G. N. V N AVN ADS KI NOI S T TO E N OOKE MD 5 O SEQID NO: 21 Asis MkMkkki. A vidth| A Mid NVAVyk biki ) + , khykh is so sold No. 51 R K G L L G Y YF KGKDFN NL TMF AP T R D N T L MY DO O. T A NALL CKKO OE YOS I R 100 s, kill khk) is lish Ahlbs lit by Akilikky Ey, to 101 WI GL I ORKET GDF TF NLS KDE QAI I E I D G KI IS NKG KEKO V V H L E KEK L V 150 to will skill billi bhikshk khkh by hikák is 151 P I KI E YOS DT KF NI DS KTF K E L KLF KIDS ON OS O OVO . . . LR N P E F NKKE 197 is kily, bikh Nikhkhikki by yodel Nikki so 5,840,868 39 40

TABLE 20-continued Alignment of VIP1 Amino Acid Sequenes from Bacillus thuringiensis var. tenebrionis (VIP1A(b)) vs. AB78(VIP1A(a)) 198 SOE FLAKAS KTNL FK OKMKR DI DE DTDTD G D S I P DL WE E N G YTI ONKVAV 247 2O Akiki likikki Bibhbhbhbb why y if A 250 248 KWD D S LASKGYT KF V S N P LDS HTV GDP YTDYE KAARD LD L S NAKETF NPL 297 25 kwbb Akhykh lishly by by k.k. i bib Akhil 3OO 298 WAAF P S W NWS ME KWI L S P N E NL S N S W E S H S S T NWS YTNTE G AS E A G G G P 347 3O VAA VV Milky his vish hw, y} + k, vikhi GP 350 348 L GLS F G V S VT Y O H S E TV AOEWG T S T GNTS OF NT AS A GYLN ANVR YNN V GT 397 35 KGI SF GVS vs. ht. A will | Nth his All Aviv- 400 398 GAI YD WKP TTS F W L NNNTI ATI TAKS NST AL R S P G D S YP E GENA AIT 447 40 GAI y bykhiv Nik Ah Ak Al Sidi khoal A T 450 448 S MDD F NS HP I T L N KO OV NOL I N NKP I MLET DOT D G V Y KI RD THG NI VT G G 497 45 S. MDD F NS Hilkkvi Nikh stills, by ki kbih, VT G G 500 498 EWN GVT OOI KAKT ASI I V DD GKO VAE K R V AA KDY GHP E D KTP P L T L KDTL 547 50 EWN G VI O OI kikh A Vibhi Alki Akby is bk} is likbal 550 548 KL SYP DE I KET NG LLYY DDKP I YES S VMT YLDEN TAKE V K KOI NDT T G KF 597 55 kly biki idly ksky is shy bhikkhvi khi bhikkh 6OO 598. KD VNHLYD WKLTP KMNFT I KMAS LYD GAE NNHNS L. GT WYLTYNVAG GNT G 647 60 kbyshly by kill kivi kis hybsk is bid kwinks vskish 650 648 KROY R S A H S CAHVAL S S E AKKKLN ON ANYYL S MY MKAD S T TEPT I EVA GE 697 is keys NSF disil Nikolk kNR by is is liksikshoe Eli ibi to 698 KS AI T S K K V K L N N ONY OR VDI LVKN S E R N P MDKI Y I R GN GTT NVY GD DVT 747 to y hikits v?ki kibli Ai Niks his sinki i Eii if wbbis is 748 I P E V S AI N P A S L S DE EI QEI F KD STI EY GNP SF VAD A VT F K. 788 7s by Ask Esli is Elko vs Ryoki bat if Dkkadiivae f : NE as so so789 F.. NI EhlbyvikkviKP LONY V KEYE I YH Ysse K . . Laon votifsS HRYE KKTVF okiykdoi DI MG iVHY kibi EY Si Ikusks ARE O so830 83 KKA 833 851 Eog ss

50 EXAMPLE 22 polypeptide domains. Such linkers are known in the art. This linker can optionally be designed to contain protease cleav FUSION OF VIP PROTEINS TO MAKE A age Sites Such that once the Single fused polypeptide is SINGLE POLYPEPTIDE ingested by the target insect it is cleaved in the linker region VIP proteins may occur in nature as Single polypeptides, 55 to liberate the two polypeptide components of the active VIP or as two or more interacting polypeptides. When an active molecule. VIP is comprised of two or more interacting protein chains, VIP1A(a) and VIP2A(a) from B. cereus strain AB78 are these protein chains can be produced as a single polypeptide fused to make a Single polypeptide by fusing their coding chain from a gene resulting from the fusion of the two (or regions. The resulting DNA has the Sequence given in SEQ more) VIP coding regions. The genes encoding the two 60 ID NO:22 with the encoded protein given in SEQID NO:23. chains are fused by merging the coding regions of the genes In like manner, other fusion proteins may be produced. to produce a single open reading frame encoding both VIP The fusion of the genes encoding VIP1A(a) and VIP2A(a) polypeptides. The composite polypeptides can be fused to is accomplished using Standard techniques of molecular produce the Smaller polypeptide as the NH terminus of the biology. The nucleotides deleted between the VIP1A(a) and fusion protein, or they can be fused to produce the larger of 65 VIP2A(a) coding regions are deleted using known mutagen the polypeptides as the NH2 terminus of the fusion protein. esis techniqueS or, alternatively, the coding regions are fused A linker region can optionally be used between the two using PCR techniques. 5,840,868 41 42 The fused VIP polypeptides can be expressed in other functioning of these mechanisms have been characterized in organisms using a Synthetic gene, or partially Synthetic gene, Some detail. For example, the targeting of gene products to optimized for expression in the alternative host. For the chloroplast is controlled by a Signal Sequence found at instance, to express the fused VIP polypeptide from above in the amino-terminal end of various proteins. This signal is maize, one makes a Synthetic gene using the maize preferred cleaved during chloroplast import, yielding the mature pro codons for each amino acid, see for example U.S. Pat. No. 5 5,625,136 herein incorporated by reference. Synthetic DNA tein (e.g. Comai et al. J. Biol. Chem. 263:15104-15109 Sequences created according to these methods are disclosed (1988)). These signal Sequences can be fused to heterolo in SEQ ID NO:17 (maize optimized version of the 100 kDa gous gene products Such as VIP2 to effect the import of those VIP1A(a) coding sequence), SEQ ID NO:18 (maize opti products into the chloroplast (van den Broeck et al. Nature mized version of the 80 kDa VIP1A(a) coding sequence) and 313:358-363 (1985)). DNA encoding for appropriate signal SEQ ID NO:24 (maize optimized version of the VIP2A(a) sequences can be isolated from the 5' end of the cDNAS coding Sequence). encoding the RUBISCO protein, the CAB protein, the EPSP Synthetic VIP1 and VIP2 genes optimized for expression Synthase enzyme, the GS2 protein and many other proteins in maize can be fused using PCR techniques, or the Synthetic which are known to be chloroplast localized. genes can be designed to be fused at a common restriction Other gene products are localized to other organelles Such Site. Alternatively, the Synthetic fusion gene can be designed 15 as the mitochondrion and the peroxisome (e.g. Unger et al. to encode a single polypeptide comprised of both VIP1 and Plant Molec. Biol. 13:411–418 (1989)). The cDNAs encod VIP2 domains. ing these products can also be manipulated to effect the Addition of a peptide linker between the VIP1 and VIP2 domains of the fusion protein can be accomplished by PCR targeting of heterologous gene products Such as VIP2 to mutagenesis, use of a Synthetic DNA linker encoding the these organelles. Examples of Such Sequences are the linker peptide, or other methods known in the art. nuclear-encoded ATPases and Specific aspartate amino trans The fused VIP polypeptides can be comprised of one or ferase isoforms for mitochondria. Similarly, targeting to more binding domains. If more than one binding domain is cellular protein bodies has been described by Rogers et al. used in the fusion, multiple target pests are controlled using (Proc. Natl. Acad. Sci. USA 82:6512–6516 (1985)). such a fusion. The other binding domains can be obtained by 25 By the fusion of the appropriate targeting Sequences using all or part of other VIPs; Bacillus thuringiensis described above to coding Sequences of interest Such as endotoxins, or parts thereof, or other proteins capable of binding to the target pest or appropriate biding domains VIP2 it is possible to direct the transgene product to any derived from Such binding proteins. organelle or cell compartment. For chloroplast targeting, for One example of a fusion construction comprising a maize example, the chloroplast Signal Sequence from the optimized DNA sequence encoding a Single polypeptide RUBISCO gene, the CAB gene, the EPSPsynthase gene, or chain fusion having VIP2A(a) at the N-terminal end and the GS2 gene is fused in frame to the amino-terminal ATG VIP1A(a) at the C-terminal end is provided by pCIB5531. A of the transgene. The Signal Sequence Selected should DNA sequence encoding a linker with the peptide sequence include the known cleavage site and the fusion constructed PSTPPTPSPSTPPTPS (SEQ ID NO:47) has been inserted should take into account any amino acids after the cleavage between the two coding regions. The Sequence encoding this 35 Site which are required for cleavage. In Some cases this linker and relevant cloning sites is 5'-CCCGGGCCTTCT requirement may be fulfilled by the addition of a small ACT CCC CCAACT CCCTCTCCTAGC ACG CCT CCG ACACCTAGCGAT ATCGGATC C-3' (SEQ ID NO:48). number of amino acids between the cleavage Site and the Oligonucleotides were Synthesized to represent both the Start codon ATG, or alternatively replacement of Some upper and lower Strands and cloned into a pUC vector amino acids within the coding Sequence. Fusions con following hybridization and phosphorylation using Standard 40 structed for chloroplast import can be tested for efficacy of procedures. The stop codon in VIP2A(a) was removed using chloroplast uptake by in vitro translation of in vitro tran PCR and replaced by the BglII restriction site with a Smal scribed constructions followed by in vitro chloroplast uptake Site. A translation fusion was made by ligating the Bam using techniques described by (Bartlett et al. In: Edelmann HI/PstI fragment of the VIP2A(a) gene from pCIB5522 (see et al. (Eds.) Methods in Chloroplast Molecular Biology, Example 24), a PCR fragment containing the PstI-end 45 Elsevier. pp 1081–1091 (1982); Wasmann et al. Mol. Gen. fragment of the VIP2A(a) gene (identical to that used to Genet. 205:446-453 (1986)). These construction techniques construct pCIB5522), a synthetic linker having ends that are well known in the art and are equally applicable to would ligate with a blunt site at the 5' end and with BamHI mitochondria and peroxisomes. at the 3' end and the modified synthetic VIP1A(a) gene from The above described mechanisms for cellular targeting pCIB5526 described below (See SEQ ID NO:35). The 50 can be utilized not only in conjunction with their cognate fusion was obtained by a four way ligation that resulted in promoters, but also in conjunction with heterologous pro a plasmid containing the VIP2A(a) gene without a transla tion stop codon, with a linker and the VIP1A(a) coding moters So as to effect a specific cell targeting goal under the region without the Bacillus secretion signal. The DNA transcriptional regulation of a promoter which has an sequence for this construction is disclosed in SEQ ID expression pattern different to that of the promoter from NO:49, which encodes the fusion protein disclosed in SEQ 55 which the targeting Signal derives. ID NO:50. A single polypeptide fusion where VIP1A(a) is at A DNA sequence encoding a Secretion signal is present in the N-terminal end and VIP2A(a) is at the C-terminal end the native Bacillus VIP2 gene. This signal is not present in can be made in a similar fashion. Furthermore, either one or the mature protein which has the N-terminal Sequence of both genes can be linked in a translation fusion with or LKITDKVEDF (amino acid residues 57 to 66 of SEQ ID without a linker at either the 5' or the 3' end to other 60 NO:2). It is possible to engineer VIP2 to be secreted out of molecules like toxin encoding genes or reporter genes. the plant cell or to be targeted to Subcellular organelles Such as the endoplasmic reticulum, vacuole, mitochondria or EXAMPLE 23 plastids including chloroplasts. Hybrid proteins made by TARGETING OF VIP2 TO PLANT fusion of a Secretion signal peptide to a marker gene have ORGANELLES 65 been Successfully targeted into the Secretion pathway. Various mechanisms for targeting gene products are (Itirriaga G. et al., The Plant Cell, 1:381-390 (1989), known to exist in plants and the Sequences controlling the Denecke et al., The Plant Cell, 2:51–59 (1990). Amino 5,840,868 43 44 terminal Sequences have been identified that are responsible described in Example 23A) which had been digested with for targeting to the ER, the apoplast, and extracellular BamHI/Pst using standard procedures. The resulting maize secretion from aleurone cells (Koehler & Ho, Plant Cell optimized coding sequence is disclosed in SEQ ID NO:42 2:769–783 (1990)). which encodes the protein disclosed in SEQID NO:43. This The presence of additional signals are required for the encoded protein comprises the eukaryotic Secretion signal in protein to be retained in the endoplasmic reticulum or the place of the Bacillus Secretion Signal. vacuole. The peptide sequence KDEL/HDEL at the carboxy One example of a construction which incorporates a terminal of a protein is required for its retention in the vacuolar targetting Signal fused to a coding Sequence for a endoplasmic reticulum (reviewed by Pelham, Annual VIP is provided by pCIB5533. Oligonucleotides corre Review Cell Biol., 5:1–23 (1989). The signals for retention sponding to both the upper and lower Strand of Sequences of proteins in the vacuole have also been characterized. encoding the vacuolar targetting peptide of SEQ ID NO:3 Vacuolar targeting Signals may be present either at the was Synthesized and has the Sequence 5'-CCG CGG GCG amino-terminal portion, (Holwerda et al., The Plant Cell, TGC ACT GCC TCA GCA GCA GCA GCT TCG CCG 4:307-318 (1992), Nakamura et al., Plant Physiol., 101:1-5 ACA GCA ACC CCA TCC GCG TGA CCG ACC GCG (1993)), carboxy-terminal portion, or in the internal 15 CCG CCA GCACCC TGC AG-3' (SEQ ID NO:44). When Sequence of the targeted protein. (Tague et al., The Plant hybridized, the 5' end of the vacuolar targetting Signal Cell, 4:307–318 (1992), Saalbach et al., The Plant Cell, resembled "Sticky-ends' corresponding to restriction Sites 3:695–708 (1991)). Additionally, amino-terminal sequences SacII and Pst. The oligonucleotide was hybridized and in conjunction with carboxy-terminal Sequences are respon phosphorylated and ligated into pCIB5528 (construction Sible for vacuolar targeting of gene products (Shinshi et al. described above) which had been digested with SacII/PstI Plant Molec. Biol. 14:357–368 (1990)). Similarly, proteins using Standard procedures. The resulting maize optimized may be targeted to the mitochondria or plastids using coding sequence is disclosed in SEQ ID NO:45 which Specific carboxy terminal signal peptide fusions (Heijne et encodes the protein disclosed in SEQ ID NO:46. This al., Eur, J. Biochem., 180:535-545 (1989), Archer and encoded protein comprises the vacuolar targetting peptide in Keegstra, Plant Molecular Biology, 23:1105–1115 (1993)). 25 addition to the eukaryotic Secretion Signal. In order to target VIP2, either for secretion or to the various Subcellular organelles, a maize optimized DNA The VIP1 gene can also be designed to be secreted or Sequence encoding a known signal peptide(s) may be targeted to Subcellular organelles by Similar procedures. designed to be at the 5' or the 3' end of the gene as required. EXAMPLE 23A To secrete VIP2 out of the cell, a DNA sequence encoding the eukaryotic secretion signal peptide MGWSWIFLFLLS REMOVAL OF BACILLUS SECRETION GAAGVHCL (SEQID NO:25) from U.S. patent application SIGNAL FROM VIP1A(a) AND VIP2A(a) Ser. No. 08/267,641 or any other described in the literature (Itirriaga et al., The Plant Cell, 1:381–390 (1989), Denecke, VIP1A(a) and VIP2A(a) are secreted during the growth of et al., The Plant Cell, 2:51–59 (1990)) may be added to the 35 Strain AB78. The nature of peptide Sequences that act as 5' end of either the complete VIP2 gene sequence or to the Secretion signals has been described in the literature Sequence truncated to encode the mature protein or the gene (Simonen and Palva, Microbiological reviews, pg. 109-137 truncated to nucleotide 286 or encoding a protein to start at (1993)). Following the information in the above publication, amino acid residue 94 (methionine). To target VIP2 to be the putative Secretion Signal was identified in both genes. In retained in the endoplasmic reticulum, a DNA sequence 40 VIP1A(a) this signal is composed of amino acids 1-33 (See encoding the ER signal peptide KDEL/HDEL, in addition to SEQ ID NO:5). Processing of the secretion signal probably the Secretion signal, can be added to the 3' end of the gene. occurs after the Serine at amino acid 33. The Secretion Signal For vacuolar targeting a DNA sequence encoding the Signal in VIP2A(a) was identified as amino acids 1-49 (See SEQ peptide SSSSFADSNPIRVTDRAAST (SEQ ID NO:3; Hol ID NO:2). N-terminal peptide analysis of the secreted werda et al., The Plant Cell, 4:307–318 (1992)) can be 45 mature VIP2A(a) protein revealed the N-terminal sequence designed to be adjacent to the Secretion signal or a sequence LKITDKVEDFKEDK. This sequence is found beginning at encoding a carboxyl Signal peptide as described by Dom amino acid 57 in SEQ ID NO:2. The genes encoding these browski et al., The Plant Cell, 5:587–596 (1993) or a proteins have been modified by removal of the Bacillus functional variation may be inserted at the 3' end of the gene. Secretion signals. Similarly, VIP2 can be designed to be targeted to either the 50 A maize optimized VIP1A(a) coding region was con mitochondria or the plastids, including the chloroplasts, by Structed which had the Sequences encoding the first 33 inserting Sequences in the VIP2 Sequence described that amino acids, i.e., the Secretion Signal, removed from its 5' would encode the required targeting Signals. The bacterial end. This modification was obtained by PCR using an Secretion Signal present in VIP2 may be retained or removed forward primer that contained the sequence 5'-GGA TCC from the final construction. 55 ACC ATG AAG ACC AAC CAG ATC AGC-3' (SEQ ID One example of a construction which incorporates a NO:33), which hybridizes with the maize optimized gene eukaryotic Secretion signal fused to a coding Sequence for a (SEQ ID NO:26) at nucleotide position 100, and added a VIP is provided by pCIB5528. Oligonucleotides corre BamHI restriction site and a eukaryotic translation Start Site sponding to both the upper and lower Strand of Sequences consensus including a start codon. The reverse primer that encoding the secretion signal peptide of SEQID NO:25 was 60 contained the sequence 5'-AAG CTT CAG CTC CTTG-3' Synthesized and has the Sequence 5'-GGATCCACC ATG (SEQ ID NO:34) hybridizes on the complementary strand at GGCTGGAGCTGG ATC TTC CTG TTC CTG CTGAGC nucelotide position 507. A527 bp amplification product was GGC GCC GCG GGC GTG CAC TGCCTGCAG-3' (SEQ obtained containing the restriction sites BamHI at the 5' end ID NO:41). When hybridized, the 5' end of the secretion and HindIII site at the 3' end. The amplification product was Signal resembled "Sticky-ends' corresponding to restriction 65 cloned into a T-vector (described in Example 24, below) and sites BamHI and Pst. The oligonucleotide was hybridized sequenced to ensure the correct DNA sequence. The BamHI/ and phosphorylated and ligated into pCIB5527 (construction HindIII fragment was then obtained by restriction digest and 5,840,868 45 46 used to replace the BamHI/HindIII fragment of the maize Cloning optimized VIP1A(a) gene cloned in the root-preferred pro Oligos were synthesized by IDT Inc., and were supplied moter cassette. The construct obtained was designated as lyophilized powders. They were resuspended at a con pCIB5526. The maize optimized coding region for VIP1A centration of 200 uM. To 30 ul of each oligo formamide was (a) with the Bacillus Secretion signal removed is disclosed as added a final concentration of 25-50% and the sample was SEQ ID NO:35 and the encoded protein is disclosed as SEQ boiled for two minutes before separation on a premade 10% ID NO:36. polyacryamide/urea gel obtained from NoveX. After The gene encoding the processed form of VIP2A(a), i.e., electrophoresis, the oligo was detected by UV shadowing by a coding region with the Secretion Signal removed, was placing the gel on a TLC plate containing a fluorescent constructed by a procedure Similar to that described for that indicator and exposing it to UV light. The region containing used to construct the processed form of VIP1A(a), above. DNA of the correct size was excised and extracted from the The modification was obtained by PCR using the forward polyacryamide by an overnight incubation of the minced gel primer 5'-GGATCC ACC ATG CTG CAG AAC CTGAAG fragment in a buffer containing 0.4M LiCl, 0.1 mM EDTA. ATC AC-3' (SEQ ID NO:37). This primer hybridizes at The DNA was separated from the gel residue by centrifu nucleotide position 150 of the maize optimized VIP2A(a) 15 gation through a Millipore UFMC filter. The extracted DNA gene (SEQ ID NO:27). A silent mutation has been inserted was ethanol precipitated by the addition of 2 volumes of at nucleotide position 15 of this primer to obtain a Pst absolute alcohol. After centrifugation, the precipitate was restriction site. The reverse primer has the Sequence 5'-AAG resuspended in dI2O at a concentration of 2.5 uM. Frag CTT CCA CTC CTT CTC-3' (SEQ ID NO:38). A 259 bp ments were cloned either by hybridization of the oligos and product was obtained with HindIII restriction site at the 3' ligation with the appropriate vector or by amplification of end. The amplification product was cloned into a T-vector, the hybridized fragment using a equimolar mixture of all the sequenced and ligated to a BamHI/HindIII digested root oligos for a particular fragment as a template and end preferred promoter cassette containing the maize optimized specific PCR primers. VIP2A(a). The construct obtained was designated Cloning by hybridization and ligation pCIB5527. The maize optimized coding region for VIP2A 25 Homologous double Stranded oligo pairs were obtained (a) with the Bacillus Secretion signal removed is disclosed as by mixing 5ul of the upper and of the lower oligo for each SEQ ID NO:39 and the encoded protein is disclosed as SEQ oligo pair with buffer containing 1xpolynucleotide kinase ID NO:40. (PNK) buffer (70 mM Tris-HCl (pH 7.6), 10 mM MgCl, 5 mM dithiothreitol (DTT)), 50 mM KC1, and 5% formamide EXAMPLE 24 in a final volume of 50 ul. The oligos were boiled for 10 minutes and slow cooled to 37 C. or room temperature. 10 CONSTRUCTION AND CLONING OF THE All was removed for analysis on a 4% agarose in a TAE buffer VIP1A(a) AND VIP2A(a) MAIZE OPTIMIZED system (Metaphore(E, FMC). Each hybridized oligo pair GENES was kinased by the addition of ATP at a final concentration Design 35 of 1 mM, BSA at a final concentration of 100 ug per ml and The maize optimized genes were designed by reverse 200 units of polynucleotide kinase and 1 til of 10xPNK translation of the native VIP1A(a) and VIP2A(a) protein buffer in a volume of 10 ul. Following hybridization and Sequences using codons that are used most often in maize phosphorylation, the reaction was incubated at 37 C. for 2 (Murray et al., Nucleic Acid Research, 17:477-498 (1989)). hours to overnight. 10 ul of each of the oligo pairs for a To facilitate cloning, the DNA sequence was further modi 40 particular fragment, were mixed in a final Volume of 50 ul. fied to incorporate unique restriction sites at intervals of The oligo pairs were hybridized by heating at 80° C. for 10 every 200-360 nucleotides. VIP1A(a) was designed to be minutes and slow cooling to 37 C. 2 ul of oligos was mixed cloned in 11 Such fragments and VIP2A(a) was cloned in 5 with about 100 ng an appropriate vector and ligated using a fragments. Following cloning of the individual fragments, buffer containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCl, adjacent fragments were joined using the restriction Sites 45 10 mM DTT, 1 mM ATP. The reaction was incubated at common to both fragments, to obtain the complete gene. To room temp. for 2 hours to overnight and transformed into clone each fragment, oligonucleotides (50-85 nucleotides) DH5C. Strain of E.coli, plated on L-plates containing ampi were designed to represent both the upper and the lower cillin at a concentration of 100 tug/ml using Standard proce strand of the DNA. The upper oligo of the first oligo pair was dures. Positive clones were further characterized and con designed to have a 15bp Single Stranded region at the 3' end 50 firmed by PCR miniscreen described in detail in U.S. Pat. which was homologous to a similar Single Stranded region of No. 5,625,136 using the universal primers “Reverse” and the lower Strand of the next oligo pair to direct the orien M13 “-20” as primers. Positive clones were identified by tation and Sequence of the various oligo pairs within a given digestion of DNA with appropriate enzymes followed by fragment. The oligos are also designed Such that when the all Sequencing. Recombinants that had the expected DNA the oligos representing a fragment are hybridized, the ends 55 Sequence were then Selected for further work. have single Stranded regions corresponding to the particular PCR Amplification and cloning into T-vector: restriction site to be formed. The Structure of each oligomer PCR amplification was carried out by using a mixture of was examined for Stable Secondary Structures Such as hairpin all the oligomers that represented the upper and the lower loops using the OLIGO program from NBI Inc. Whenever Strand of a particular fragment (final concentration 5 mM necessary, nucleotides were changed to decrease the Stability 60 each) as template, specific end primers for the particular of the Secondary Structure without changing the amino acid fragment (final concentration 2 uM) 200 uM of each dATP, Sequence of the protein. A plant ribosomal binding site dTTP, dCTP and dGTP, 10 mM Tris-HCl (pH 8.3), 50 mM consensus sequence, TAAACAATG (Joshi et al., Nucleic KCl, 1.5 mM MgCl,0.01% gelatin and 5 units of Taq Acid Res., 15:6643–6653 (1987)) or eukaryotic ribosomal polymerase in a final reaction Volume of 50 ul. The ampli binding site concensus sequence CCACCATG (Kozak, 65 fication reaction was carried out in a Perkin Elmer ther Nucleic Acid Research, 12:857-872 (1984)) was inserted at mocycler 9600 by incubation at 95 C. for 1 min (1 cycle), the translational Start codon of the gene. followed by 20 cycles of 95° C. for 45 sec., 50° C. for 45 5,840,868 47 48 Sec., 72 C. for 30 sec. Finally the reaction was incubated for EXAMPLE 26 5 min at 72 C. before analyzing the product. 10 ul of the reaction was analyzed on a 2.5% Nusieve (FMC) agarose gel EXPRESSION OF MAIZE OPTIMIZED VIP1A(a) in a TAE buffer System. The correct size fragment was gel AND VIP2A(a) purified and used for cloning into a PCR cloning vector or T-vector. T-vector construction was as described by Mar E. coli Strains containing different plasmids comprising chuk et al., Nucleic Acid Research, 19:1154 (1991). VIP genes were assayed for expression of VIPs. E. coli pBlueScriptsk+ (Stratagene(E), Ca.) was used as the parent Strains harboring the individual plasmids were grown over vector. Transformation and identification of the correct clone night in L-broth and expressed protein was extracted from was carried out as described above. the culture as described in Example 3, above. Protein Fragments 1, 3, 4, 5, 6, 8, and 9 of VIP1A(a) and expression was assayed by Western Blot analysis using fragments 2 and 4 of VIP2A(a) were obtained by cloning of antibodies developed using Standard methods known in the PCR amplification products; whereas, fragments 2, 7, 10 and art, Similar to those described in Example 12, above. Also, 11 of VIP1A(a) and fragments 1, 3, and 5 of VIP2A(a) were insecticidal activity of the expressed proteins were tested obtained by hybridization/ligation. 15 against Western corn rootworm according to the method in Once fragments with the desired Sequence were obtained, Example 3, above. The results of the E. coli expression the complete gene was assembled by cloning together adja assays are described below. cent fragments. The complete gene was resequenced and tested for activity against WCRW before moving it into plant expression vectors containing the root preferred promoter Expression of VIPs in E. coli (disclosed in U.S. Pat. No. 5,466,785, herein incorporated Assay Assay by reference) and the rice actin promoter. Extract of E. coli Strain Harboring No. 1 No. 2 Protein One such plant expression vector is pCIB5521. The maize Indicated Plasmid %. Mortality Detected optimized VIP1A(a) coding region (SEQ ID NO:26) was Control O O O cloned in a plant expression vector containing the root 25 pCIB5521 (maize optimized VIP1A(a)) 47 27 yes preferred promoter at the 5' of the gene with the PEP pCIB5522 (maize optimized VIP2A(a)) 7 7 yes pCIB6024 (native VIP2A(a)) 13 13 yes Carboxylase intron #9 followed by the 35S terminator at the pCIB6206 (native VIP1A(a)) 27 40 yes 3' end. The plasmid also contains Sequences for amplicillin Extracts p0IB5521 + pCIB5522 combined 87 47 resistance from the plasmid pUC19. Another plant expres Extracts p0IB5521 + pCIB6024 combined 93 1OO sion vector is pCIB5522, which contains the maize opti Extracts p0IB5522 + pCIB6206 combined 100 1OO mized VIP2A(a) coding region (SEQID NO:27) fused to the Extracts p0IB6024 + pCIB6206 combined 100 1OO root preferred promoter at the 5' of the gene with the PEP Carboxylase intron #9 followed by the 35S terminator at the 3' end. The DNA from these plasmids was used to transiently 35 express the VIPs in a maize protoplast expression System. EXAMPLE 25 Protoplasts were isolated from maize 2717 Line 6 suspen Sion cultures by digestion of the cell walls using Cellulase NAD AFFINITY CHROMATOGRAPHY RS and Macerase R10 in appropriate buffer. Protoplasts A purification Strategy was used based on the affinity of were recovered by Sieving and centrifugation. Protoplasts VIP2 for the substrate NAD. The Supernatant from the pH 40 were transformed by a Standard direct gene transfer method 3.5 Sodium citrate buffer treatment described in Example 4 using approximately 75 lug plasmid DNA and PEG-40. was dialyzed in 20 mM TRIS pH 7.5 overnight. The Treated protoplasts were incubated overnight in the dark at neutralized Supernatant was added to an equal Volume of room temperature. Analysis of VIP expression was accom washed NAD agarose and incubated with gentle rocking at plished on protoplast explants by Western blot analysis and 4 C. overnight. The resin and protein solution were added 45 insecticidal activity against Western corn rootworm as to a 10 ml disposable polypropylene column and the protein described above for the expression in E. coli. The results of Solution allowed to flow out. The column was washed with the maize protoplast expression assays are described below. 5 column volumes of 20 mM TRIS pH 7.5 then washed with 2–5 column volumes of 20 mMTRIS pH 7.5, 100 mM NaCl, Expression of VIPs in Plant Protoplasts followed by 2-5 column volumes of 20 mM TRIS 7.5. The 50 VIP proteins were eluted in 20 mM TRIS pH 7.5 supple Assay No. 1 Assay No. 2 Protein mented with 5 mM NAD. Approximately 3 column volumes Extract Tested %. Mortality Detected of the effluent were collected and concentrated in a No DNA Control 27 1O O Centricon-10. Yield is typically about 7-15 lug of protein per pCIB5521 (p) 20 (O) 3O yes ml of resin. 55 (maize optimized VIP1A(a)) pCIB5522 (p) 20 (O) 2O yes When the purified proteins were analyzed by SDS-PAGE (maize optimizied VIP2A(a)) followed by Silver Staining, two polypeptides were visible, Extracts p0IB5521 (p) + 87 (82) 90 one with Mr of approximately 80,000 and one with Mr of pCIB5522 (p) combined approximately 45,000. N-terminal Sequencing revealed that Extracts p0IB5521 (p) + 1OO pCIB5522 (e) combined the Mr 80,000 protein corresponded to a proteolytically 60 Extracts p0IB5522 (p) + 53 (36) processed form of VIP1A(A) and the Mr 45,000 form PCIB5521 (e) combined corresponded to a proteolytically processed form of VIP2A Extracts p0IB5521 (p) + 1OO pCIB6024 (e) combined (a). The co-purification of VIP1A(a) with VIP2A(a) indi Extracts p0IB5522 (p) + 1OO cates that the two proteins probably form a complex and pCIB6206 (e) combined have protein-protein interacting regions. VIP1A(a) and 65 pCIB6024 (e) (native VIP2A(a)) O yes VIP2A(a) proteins purified in this manner were biologically pCIB6206 (e) (native VIPIA(a)) 2O yes active against Western corn rootworm. 5,840,868 49 SO -continued Expression of VIPs in Plant Protoplasts 1. E. coli PL2 Accession No. NRRL B-21221N 2. E. coli pCIB6022 Accession No. NRRL B-21222 Assay No. 1 Assay No. 2 Protein 3. E. coli pCIB6023 Accession No. NRRL B-21223N Extract Tested %. Mortality Detected 4. Bacilius thuringiensis Accession No. NRRL B-21224 HD73-78VIP pCIB5521 + pCIB5522 1OO 1OO yes 5. Bacilius thuringiensis AB88 Accession No. NRRL B-21225 (plasmids delivered by 6. Bacillus thuringiensis AB359 Accession No. NRRL B-21226 cotransformation) 7. Bacilius thuringiensis AB289 Accession No. NRRL B-21227 8. Bacillus sp. AB59 Accession No. NRRL B-21228 (p) = extract of protoplast culture transformed with indicated plasmid 1O 9. Bacillus sp. AB294 Accession No. NRRL B-21229 (e) = extract of E. coli strain harboring indicated plasmid 10. Bacillus sp. AB256 Accession No. NRRL B-21230 11. E. coli P5-4 Accession No. NRRL B-21059 The expression data obtained with both E. coli and maize 12. E. coli P3-12 Accession No. NRRL B-21061 13. Bacilius cereus AB78 Accession No. NRRL B-21058 protoplasts show that the maize optimized VIP1A(a) and 14. Bacillus thuringiensis AB6 Accession No. NRRL B-21060 VIP2A(a) genes make the same protein as the native VIP1A 15 15. E. coli pCIB6202 Accession No. NRRL B-21321 (a) and VIP2A(a) genes, respectively, and that the proteins 16. E. coli pCIB7100 Accession No. NRRL B-21322 encoded by the maize optimized genes are functionally 17. E. coli pCIB7101 Accession No. NRRL B-21323 18. E. coli pCIB7102 Accession No. NRRL B-21324 equivalent to the proteins encoded by the native genes. 19. E. coli pCIB7102 Accession No. NRRL B-21325 All publications and patent applications mentioned in this 20. E. coli pCIB7104 Accession No. NRRL B-21422 specification are indicative of the level of skill of those 21. E. coli pCIB7107 Accession No. NRRL B-21423 skilled in the art to which this invention pertains. All 22. E. coli pCIB7108 Accession No. NRRL B-21438 publications and patent applications are herein incorporated 23. Bacillus thuringiensis AB424. Accession No. NRRL B-21439 by reference to the same extent as if each individual publi cation or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in The following deposits have been made at Agricultural 25 Some detail by way of illustration and example for purposes Research Service, Patent Culture Collection (NRRL), of clarity of understanding, it will be obvious that certain Northern Regional Research Center, 1815 North University changes and modifications may be practiced within the Street, Peoria, Ill. 61604, USA: Scope of the appended claims.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 50

( 2) INFORMATION FOR SEQ ID NO:1: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 6049 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus cereus (B) STRAIN: AB78 ( C ) INDIVIDUALISOLATE: NRRL B-21058 ( i x ) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1082.2467 ( D ) OTHER INFORMATION: product=*VIP2A(a) ( i x ) FEATURE: (A) NAMEKEY: misc feature (B) LOCATION: 2475.5126 ( D.) OTHER INFORMATION: note= “Coding sequence for the 100 d VIP1A(a) protein. This coding sequence is repeated in SEQID NO:4 and translated separately. ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:1:

A T C GATA CAA T G T T G T TTT A CTTAGA C C G G TA GT C T C T G T AATTT GTTTA A T G CTATATT 6 O

CTTT ACTTT G ATA CATTTTA ATA G C CATTT CAA C CTTAT C A GT AT GTTTT T G T G GT CTTC 1 2 O

C T C CTTTTTT T C C A C G A G C T CTA G CT GC GT TT AAT C C T G T TTT G G T AC GT T C GCTAATAA 18 O

5,840,868 59 60 -continued Le u As n As n Ser G 1 u Ty r Lys Me t Le u I le A s p As n G 1 y Ty r Me t V a 1 2 25 23 O 23 5 24 O His V a 1 A s p Lys V a 1 Ser Lys V a V a l Lys Lys G 1 y V a 1 G 1 u Cy s Le u 2 4 5 25 O 25 5

G l n I le G 1 u G 1 y Thr Le u Lys Ly s Ser Le u A s p Ph e Lys As in A s p I e 2 6 O 2 65 2 7 O

As in A a G 1 u A 1 a His Ser T r p G 1 y Me t Lys As in Ty r G 1 u G 1 u T r p A 1 a 2 7 5 28 O 28 5

G l n A s p Ty r Lys G 1 u I le. As n As n Ty r Le u A rig As in G 1 in G 1 y G 1 y Se r 3 O 5 3 1 O 3 15 3 2 O

G 1 y As n G 1 u Lys Le u A s p A 1 a G 1 in I le L y S A. S i. I e S e A. S p A l a Le u 3 2 5 3 3 O 3 3 5

Me t Pro G 1 u P he G 1 y Tyr G 1 in I 1 e S e A. S p Pro Le u Pro Ser Le u Lys 3 5 5 3 6 O 3 65

Le u S e r A a I e G ly G 1 y Ph e A a Ser G 1 u Lys G 1 u I le Le l Le u As p

Lys A s p Ser Lys Ty r H is I e A s p Lys V a 1 Thr G 1 u V a 1 I le I le Lys 43 5 4 4 O 4 4 5

G l y V a l Lys Arg Ty r V a l V a 1 A s p A a Thr Le u Le u Thir As in 4 5 O 4 55 4 6 O

( 2) INFORMATION FOR SEQ ID NO:3: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: ( A ) NAME/KEY: Peptide (B) LOCATION: 1.20 ( D.) OTHER INFORMATION: note= “Signal peptide for vacuolar targetting ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:3: S e r S e r S e r S e r Ph e A a A s p Ser As n Pro I e A rig V a 1 T h r A s p Arg 1. 5 1 O 1 5

A a A a Ser Thr 2 O

( 2) INFORMATION FOR SEQ ID NO:4: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 2655 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic)

5,840,868 67 68 -continued

AGA TAT AAT AAA TAG 2 6 5 5 A rig Ty r As i. Ly S 1. 3 4 5

( 2) INFORMATION FOR SEQ ID NO:5: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 884 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear i) MOLECULETYPE: protein i ) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Me t As i. Me t Ly is Ly S L y s Le A W a Th r Le u 1. O

Le u Me t P he Le Gl A Wa 2 O

S e r G in I S e r Th G u

Me t G 1 y Le Ph P he 5 5 5 o

Le Th Me t Th Le o 7

G Th Le Le Gl 9

G in I I G 1 y Le I Th 1 O 1 1

P he Th Ph G u G A I Gl 11 1, 2

G 1 y I I L y S Gl Wa W a Le u o 1 4

G u G Gl G in S e Th r 1 4 5 1 5 1 6 O

I A. S Ph Gl Le Le 1. y

I e S e Gl G G Gl Gl G 18 18 1 9

G Ph l S e G Ph P ro 1 9

S e r I Le u Ph Th r Me G u I G u 2 1. o 2 15

Th G S e I T r p G 2 25 2 3 24 O

G 1 y Th I G in I Le u 2 4 5

A 1 a Gl Th Ph Gl 2 6 2 7

Th r Gl P ro Th r A 1 a Le u 2 7 28 28 5

l S e A 1 a G u Th Ph l Wa P he 2 2 9 5 3 O

W a Wa Me t W a I S e r P r G u 3 1. 5 3 2 O

S e G u S e S e Th T r 3 2 5

Th r Th Gl G 1 y A S e r W a Gl I e G 1 y G 1 y

5,840,868 77 78 -continued

S e Le u G u Th r Gl y As O Th r

A 1 a A rig A G u Th r 7 o

Le Wa P he P ro W a Me t

W a I e Le G u Se Wa G u 1. O 1 1 O

S e Th T r p S e Th r Th Gl G A 1 a S e r Wa G 1 1 1 2 O 1 2 5

A G I G 1 y Gl I e Ph Gl S e r Wa 13 13 1 4

Gl S e G u Th r A 1 a G in Gl Th Th r G 1 y 1 4 1 5

Th Gl P he A 1 a S e r Gl Le A 1 a 1 65 1 7

A r Wa G Th r G 1 y A I Wa L y S Th 18 O 18

Th Ph Wa Le u Th I A Th 1 9 2 O O

S e S e Th r Le I e S e P r G 2 2

G G 1 y e I e Se P he 2 3

I Th r As As 2 4 5 25

Me Le u G Th r Th Gl Wa T y I

Th G 1 y I e Gl Gl G 1 y W a 2 7 28 5

I G in Gl I e L y S Th r Se I Wa G 29 O

Gl W a A 1 a G u S Wa G u 3 O 3 2

Gl Th r Le u Th r Le A Le u S e 3 2 5 o

G u I e G u I e G Le Le T y 3 4 O 3 4

e G u S e S e r Me Th Le Th 3 6 O

A L y S G Wa Th r G in Le u Th Th P he 3 75 38

S e Le u Wa L e Th P r Me t 38 39

Th I e Le u I Le u A G S e r 4 O 5 4 1

S e I e Gl T r p Th Th r I W a G 1 y

Gl S e r A As in Th r Le s 4 4 O 4 4 5

Th r A 1 a G in l Ly is Le u I 4 5 O 4 55 4 6 5,840,868 79 80 -continued Ser Le u Ty r Me t Lys Ser G 1 u Lys As n Thr G 1 n Cy s G 1 u I le Th I e 4 6 5 4 7 4 75 4 8 O

A s p G 1 y G 1 u I le Ty r Pro I le Thr Thr Lys Thr V a l As n V a l As 4 8 4 9 O 49

A s p As in Ty r Lys Arg Le u A s p I le I l e A la His As n I le L y S S e 5 O O 5 O 5 5 1 O

Pro I le S e r S e r Le u H is I e Lys Thr As n A s p G 1 u I le Thr Le P he 5 15 5 2 O 5 25

T r p A s p A s p I le S e r I le Th r A s p V a 1 A a Se r I 1 e Lys Pro G 1 53 O 5 35 5 4 O

Le u Th r A s p Ser G 1 u I le L y s G 1 n I le Ty r S e r A rig Ty r G 1 y I 1 L y S 5 4 5 5 5 O 55 5

Le u G 1 u A s p G 1 y I le Le u I e A s p Lys Lys G 1 y G 1 y I le H is Ty 5 6 5 5 7 O 5 7 5

G u Ph e I e A s in G u. A a S e r Ph e As n I I e G 1 u Pro Le u Pro As 58 O 5 85 5 9 O

V a 1 T h r Lys Tyr G 1 u V a 1 T hr Ty r S e r S e r G 1 u Le u G 1 y Pro As Wa 5 9 5 6 O O 6 O 5

S e r A s p Thr Le u G 1 u Se r A s p Lys I le Ty r Lys A s p G 1 y Thr I 1 6 1 O 6 15 6 2 O

Ph e A s p Phe Thr Lys Ty r S e r Lys As n G 1 u G | n G 1 y Le u Ph e Ty 6 25 6 3 O 6 3 5

Ser G 1 y Le u As n Tr p A s p P he Lys I le As n A a I le Thr Ty r As 6 4.5 6 5 O 65

Ly s G 1 u Me t As n V a 1 P h e H is Arg Ty r A s in Lys 6 6 O 6 65

( 2) INFORMATION FOR SEQ ID NO:8: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( v ) FRAGMENT TYPE: N-terminal ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus cereus (B) STRAIN: AB78 ( C ) INDIVIDUALISOLATE: NRRL B-21058 ( i x ) FEATURE: ( A ) NAME/KEY: Peptide (B) LOCATION: 1.16 ( D.) OTHER INFORMATION: note= “N-terminal sequence of rotein purified from strain AB78 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:8: Lys Arg G 1 u I le A s p G 1 u A s p Th r A s p Th r A six G 1 y A s p S e r 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO:9: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) 5,840,868 81 82 -continued

( i i i ) HYPOTHETICAL: NO ( i v ). ANTI-SENSE: NO ( i x ) FEATUR E: (A) NAMEKEY: misc feature (B) LOCATION: 1.21 ( D.) OTHER INFORMATION: note= “Oligonucleotide probe based on amino acids 3 to 9 of SEQID NO:8, using codon usage of Bacillus thuringiensis’ ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GAAA TT GAT C A A GATA CNGA T

( 2) INFORMATION FOR SEQ ID NO:10: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( v ) FRAGMENT TYPE: N-terminal ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus thuringiensis (B) STRAIN: AB88 ( i x ) FEATURE: ( A ) NAME/KEY: Peptide (B) LOCATION: 1.14 (D) OTHER INFORMATION: note= “N-terminal amino acid sequence of protein known as anion exchange fraction 23 ( s m a l l e r ) ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Xa a G 1 u Pro Phi e V a S e r A a Xa a Xa a Xa a G in Xa a Xa a Xa a 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO:11: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus thuringiensis ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:11: Xa a G 1 u Tyr G 1 u As n V a 1 G 1 u Pro P he V a 1 Se r A a Xa a 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO:12: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus thurigiensis ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:12: Me t As n Lys As n As n Th r Lys Le u Pro Th r A rig A a Le u Pro 1. 5 1 O 5,840,868 83 84 -continued

( 2) INFORMATION FOR SEQ ID NO:13: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( v ) FRAGMENT TYPE: N-terminal ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus thuringiensis (B) STRAIN: AB88 ( i x ) FEATURE: ( A ) NAME/KEY: Peptide (B) LOCATION: 1.15 ( D.) OTHER INFORMATION: note= “N-terminal amino acid sequence of 35kDa VIP active against Agrotis ipsilon ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:13: A a Le u Se r G 1 u As in Th r G 1 y Ly is As p G 1 y G 1 y Ty r I e Wa P ro 1. 5 1 O 1 5

( 2) INFORMATION FOR SEQ ID NO:14: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Bacillus thuringiensis ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Me t A s p As in As n Pro As n I e As in G u 1. 5

( 2) INFORMATION FOR SEQ ID NO:15: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( v ) FRAGMENT TYPE: N-terminal ( i x ) FEATUR ( A ) NAME/KEY: Peptide (B) LOCATION: 1.9 ( D.) OTHER INFORMATION: note= “N-terminal sequence of 80 Da delta- endotoxin ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Me t A s p As in As n Pro As n I e As in G u 1. 5

( 2) INFORMATION FOR SEQ ID NO:16: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid

5,840,868 101 102 -continued

W a o S e r G 1 y Th Th Th r Ly is A 1 a G 1 y Wa 2 1 O 22 O

As in As in G u Me Le As in G 1 y Wa 2 4 O

W a 1 A sp W a W a L y S G 1 y Me t G u C y S 2 4 5

Gl W a G u Gl Th r P he Ly s 2 6 2 6

a G 1 u A 1 a Gl Me I e Ty r G 1 u T r p 2 7 5 28 28 5

in L e u Th r A 1 a A r G Le u A s p G 1 y A 1 a O 3 O O

Gl p Ty r G u L e u As in G in G 1 y 3 O

Gl in G 1 u L y S I e S e r A 1 a 3 2 5 3 3 5

Gl s L y s P O I e Wa Ty r A rig

Me o G 1 u P he G 1 y e P ro Le u Pro Le u 3 5 5 3 65

e G 1 u G u G in G 1 u A sp G 1 y O 3 7 3 8 O

r Thr S e r L e u A 1 a A a Ph e 39 5

e I e Le u A. Gl G 1 y S e r Thr A 1 a 4 15

Le r A 1 a I e Ph G u I e Le u 4 3 O

p S e r I Th r G u Wa I e 43 5 R 4 4 5 Gl 1 L y s W a A Th r Le u Le u Thr O 4 5 4 6 O

( 2 ) INFORMATION FOR SEQ ID NO:21: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 834 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein i ) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Me s As in Me t Ly is Ly is Ly S Le A S e r Wa Thr Le u 5 1 O

Le a P ro Me t P he Le Gl Wa A a Wa 2 O

S e s I e G in I S e Th G m G u As in G in G in G u 3 5 4 5

Me p A rig G 1 y Le Gl P he Ly s G 1 y P he O 5 6

in L e u Th r Me t P r Th As in Th r Me t

G in G in Th r A 1 a As Le Le Lys G 1 n G in 8 5

Gl r I e A rig T r p I Gl Le I e Lys G 1 u Th r As p 5,840,868 103 104 -continued

1 O O 1 O 5 1 1 O

Ph Th Ph As p G l A 1 a I G u I e 1 1 1 2 O Gl o I I e s G 1 y Gl i. Wa Le

Gl G I e I G in Th 1 4 1 6

Ph I e Th r Ph S P he 6 1 7 5

I S e Gl i. G S e G in Gl 18 O 18

Ph G G u Ph L e A 1 a Th s 2 O O 2 O 5

A r A. G u Th r 2 R

Th G I Le Th 2 25 2 3 2 4

I W a A W a T r Le u 2 4 5

Gl Th L y S Ph W a S e Th Wa G 2 6 2 7 O

Gl A Le u 2 o

A G u Th Ph Le A 1 a a P he S e r 29 29

W a Me t W a G u As in 3 15

W a G u S e Th T r p Th r 3 2

Gl G I A G 1 y S e r 3 4 O 3 4 3 5 O

Gl W a Wa Th Gl Gl Th r A 1 a G u 3 65

Gl Th Th r Th G Ph A 1 a 3 7 3 7

Le A 1 a Th r e

Th Ph Le u Th r 4 15

A Th Th S e A 1 a 4 2 O 4 2

Gl G I l A 1 a A 1 a 4 4 5

Me P he I e Th Le u W a

I e I e Me Th r Th 4 75 4 8

G W a I Th G 1 y I e Wa Gl 49

Gl G W a Th G in Gl i. I A Th r S e 5 O 5 5 1 O

I I G G in W a G u Wa A 5 2 O 5,840,868 105 106 -continued Lys A s p Ty r G 1 y H is Pro G 1 u A s p Lys Thr Pro Pro Le u Thir Le u Lys 53 O 5 35 5 4 O

A. S Th r Le u Lys Le u Se r Ty r Pro A s p G 1 u I l e L y S G u. Th r A is in Gl

Le u Le u T y r Ty r A s p A s p Lys Pro I le Ty r G 1 u Se r S e r V a l Me t Th r 5 6 5 5 7 O 5 7 5

58 O 5 85 5 9 O

Thr Pro Lys Me t As n Phe Thr I 1 e Lys Me t A a Ser Le u Ty r A s p G 1 y

A a G 1 u As in As n H is As n Ser Le u G 1 y Thr T r p Ty r Le u Thr Ty r A s in 6 25 6 3 O 6 3 5 6 4 O

V a 1 A 1 a G 1 y G 1 y As n Thr G 1 y Lys Arg G 1 i. T y A. g S e A. a H S S e r

G 1 u Pro Thr I le G 1 u V a 1 A a G 1 y G 1 u Lys S e r A la I le Th r S e r Lys 6 9 O 6 9 5 7 O O

G l y Th r Thr As n V a 1 Ty r G 1 y A s p A S W a Th r I e Pro G l Wa S e r 7 4 O 7 4 7 5 O

A la I le. As n Pro A a Se r Le u Se r A s p G 1 u G 1 u I le G 1 in G 1 u I le P he

7 7 O 7 7 5 7 8 O

V a 1 T h r P he Lys As n I le Lys Pro Le u G 1 in As in Ty r V a l Lys G 1 u Ty r 7 85 7 9 O 7 9 5 8 O O

G 1 u I le Ty r H is Lys Ser H is Arg Ty r G 1 u Lys Lys Thr V a 1 Phe As p 8 O 5 8 1 O 8 15

I le Me t G 1 y V a l His Ty r G 1 u Ty r S e r I 1 e A la Arg G 1 u G 1 in Lys Lys 8 2 O 8 25 8 3 O

A la A 1 a

( 2) INFORMATION FOR SEQ ID NO:22: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4041 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i x ) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1.4038 ( D.) OTHER INFORMATION: product="VIP1A(a) VIP2A(a) fusion roduct ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:22:

A T G AAA AGA A T G GAG G GA AA G T T G T TT A T G G T G T CA. AAA AAA TTA CAA Me t Lys Arg Me t G 1 u G 1 y Lly s Le u Ph e Me t V a 1 Ser Lys Lys Le u G 1 n

5,840,868 115 116 -continued

2 115 2 1 2 O 2 1 2 5 2 13 O

TAC AAG GAT GGG ACA ATT AAA TTT GAT TTT ACC AAA. TAT A GT AAA AAT 39 3 6 Ty r Lys A s p G 1 y Thr I 1 e Lys Ph e A s p Phe Thr Lys Ty r S e r Lys As in 2 13 5 21 4 O 2 1 4 5

GAA CAA. GGA TTA TTT TAT GA C A G T G G A TTA AAT T G G GAC TTT AAA ATT 39 8 4 G 1 u G | n G 1 y Le u Ph e Ty r A s p Ser G 1 y Le u As in T r p A s p P he Lys I le 2 15 O 2 15 5 21 6 O

AA T G CT ATT ACT TAT GAT G G T AAA GAG AT G AAT G TT TTT CAT AGA TAT 4 O 3 2 As in A a I le Thr Ty r A s p G 1 y Lly s G 1 u Me t As n V a 1 P h e H is Arg Ty r 21 65 2 1 7 O 2. 1 7 5

AAT AAA TAG 4 O 4 1 As in Lys 2 18 O

( 2) INFORMATION FOR SEQ ID NO:23: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1346 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:23: Me t Lys Arg Me t G 1 u G 1 y Lly s Le u Ph e Me t V a 1 Ser Lys Lys Le u G 1 n 1. 5 1 O 1 5

V a l V a 1 T hr Ly S Th r Wa Le u Le u S e r Thr Wa Ph e S e r I e S e Le u 2 O 25 3 O

Le u As n As n G 1 u V a 1 I e Lys Al a G u G in Le u As n I e A s in S e r G in 5 4 O 4 5

Ser Lys Ty r Thr As n Le u G | n As n Le u Lys I le Th r A s p Lys V a 1 G 1 u 5 O 5 5 6 A s p P he Lys G 1 u A s p Lys G 1 u Lys A 1 a Lys G 1 u T r p G 1 y Lly s G 1 u L y s 5 7 O 7 5 8 G 1 u Lys G 1 u Trip Lys Le u Th r A la Thr G 1 u Lys G 1 y Lys Me t As in As in

P he Le u A s p As n Lys As n A s p I e Lys Thr As n Ty r Lys G 1 l I e Thr

Ph e S e r Me t A a G 1 y Ser P he G 1 u A s p G 1 u I e Lys A s p Le u Lys G 1 u

y L y S A. S i. V a 1 G 1 u Pro T hir Thr I I e G 1 y P h e A s in Lys Ser Le u Thr

P he Le u A s p A rig A s p I e Lys Phe A S S e T y Le u A s p T H S Le u 18 O 18 o Th r A 1 a G 1 n G 1 n V a l S e r S e r Lys G 1 u A rig V a 1 I le Le u Lys V a 1 T h r 1 9 5 2 O O 2 O 5

V a 1 Pro Ser G 1 y Lys G 1 y Ser T hr T hr Pro T hr Ly S A. a G ly Wa I e

2 25 23 O 23 5 24 O

His V a 1 A s p Lys V a 1 Ser Lys V a V a l L S L y S G 1 y V a 1 G 1 u Cy Le u 2 4 5 2 s

G n I I e G 1 u Gl Th r Le u Lys Ly s Ser Le u A s p Ph e Lys As in A s p I e 2 6 O 2 65 2 7 O

5,840,868 135 136 -continued

l I e Le G in L e u I e

G y W a Le u I e G As in 8 O

Th Gl G u I e G in

G W a W a A 1 a e Th r 1 O l

Me Le W a L y S Th r Me t Le u Wa 1 1 1 2 5

Me Gl G u Le S e r o 1 4 O

Gl G Gl I I e W a 1 4

Le I S e Th Th G u Thr Gl I e 1 6 1 7 O

W a Gl Ph G u L e u Th P he A 1 a Th Th r 18 1 9

S e W a Gl A Le G u s 2 O

Le Th Le Th G A 1 a W a Th r Wa 2 1 22 O

Ph Gl Ph Th P he Wa Me Wa Gl 2 3 2 4

Le Gl A 1 a Le L y S Th A 1 a G I e 2 4 25 5

Th G i. W a S e r G Gl Wa G 1 y Wa 2 6 2 7

Ph W a A 1 a Le A G in A 1 a Ph Le u Th r 2 7 28 O 28 5

Le Th Th Le u G L e u A I e Th r 29 3 O O I Me G s G u G u Ph

I Le Th Th P he T y 3 2 3 3 O

W a S e A Me I e Wa G 3 4 3 5

P r G Le I Gl P he I e S e S e I e Th r 3 6 O

W a G A Le G G Wa 3 7

S e W a I e G 1 y Le u 39 39

G Gl I e T y Th 4 O 4 15 P r G o W a I Th Ph Th r o Me t Th Le G W a Th A 1 a P he Th r G 1 y 4 4 O Gl e s W a S e G u

A r Le Gl Me t Le G 1 y Wa 5,840,868 137 138 -continued

4 6 5 4 7 4 7 4 8 O

I e S e r G 1 u Thir Ph Le Th I i. Ph Gl Le u G i. A 4 8 49 49 5

Le I Th T y Le 5 O O 5 O

G u Le u Le u Le ul Al Th L e l Gl Le I 5 1. 5

Wa P r o Pro Se r G Ph I I G G 1 y I 53 O 5 3 5 4

G 1 u G 1 u A s p As n Le Gl l 5 4 5 5 5 o V a 1 A s p H is Thr G 1 G W a Th A 5 6 5 7 O

I Gl Gl L y S P r 58

G I 5

Le u L y S A. S p G u As Th Gl l Th r 6 1. 6 2

i. Th Ph Th Th r Gl Th 63 3 6 4

Gl G 1 y Gl o 65

A la T r p G 1 y A s p As Ph S e G 6 6

Th As T r Th Th G 68

S e r Thr As n I e S e Gl Th r Le Th G 1 y Gl 6 9 O o

Gl I le Le u Lys G 1 i. G in Le Th r 7 O 7 1.

V a 1 Ty r P h e Sier V a S e i. W a I 7 2

A rig G 1 u V a 1 Le u Ph G S e Gl W a 7 4 O

S e r G 1 u Me t Phi e Th Th P he Ph G 7 55 7 6 O 7 6

Le u S e r G in G 1 y As e Ty r Gl W a 7 7 O 7

A s p V a 1 Se r I 1 e Ly 7 85

( 2) INFORMATION FOR SEQ ID NO:30: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 2403 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( A ) DESCRIPTION: desc = “Synthetic DNA” ( i i i ) HYPOTHETICAL: NO ( i x ) FEATUR E: (A) NAMEKEY: misc feature (B) LOCATION: 11.2389 ( D.) OTHER INFORMATION: note= “maize optimized DNA

5,840,868 147 148 -continued (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Me As Ly S As in As i. Th L y s Le S e A 1 a Le u Ph 5

I Ty P he As i. I e Ph Th r G 1 y e 2 O

I Me Me t I Ph Th G 1 y Th r Le

l I Le u Gl L e G 1 y

G Wa Le I e G G 1 y

Th G u Gl I e G 5

G W a Wa A 1 a e Th 1 O O l

Me Le Wa Th Me t Le u W a 1 1 o 1 2 5

Me Gl G u Le S e r o 1 4 O

Gl G G u I I e W a 1 4

Le I S e r Th Th r G Th Gl I 1 6 1 7

W a Gl P he Gl L e Th r P he A 1 a Th Th 18 O 1 9

S e Wa Gl A 1 a s 2 O

Le Th Le u Th A Wa Th r W a 2 1 22 O

Ph G u Ph Th Ph Wa Me Wa Gl o 23 5 2 4

Le P he Gl A Le Th r A 1 a G I 2 4 o 25 5

Th G W a S e G G u Wa G 1 y Wa 2 6 O 2 6 2 7

Ph I e W a A Le A 1 a A 1 a Ph Le u Th 28 28 5

Le Th Le G L e A 1 a I e Th 29

I Me G L y S G u Ph

I Le P ro Th Th Ph 3 2 3 3

W a S e G u A Me t I e Wa G 3 4 3 5

P r G Le I G 1 y Ph G I S e r S e I e Th 3 6

W a Wa G A 1 a Le G in G Wa 3 75

Le u S e G Wa I e Gl As p Le u Le

5,840,868 151 152 -continued ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH:30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid (A) DESCRIPTION: idesc = "forward primer used to make

( i i i ) HYPOTHETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:33:

G GAT C CA C C A T GAA GA C CAA C CAGAT CAG C 3 O

( 2) INFORMATION FOR SEQ ID NO:34: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid (A) DESCRIPTION: idesc = “reverse primer used to make pCIB5526” ( i i i ) HYPOTHETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:34:

AAG CTT CAG C T C C TT 1 5

( 2) INFORMATION FOR SEQ ID NO:35: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 2576 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( A ) DESCRIPTION: desc = “Synthetic DNA” ( i i i ) HYPOTHETICAL: NO ( i x ) FEATUR (A) NAME/KEY: CDS (B) LOCATION: 9.2564 ( D.) OTHER INFORMATION: note= “Maize optimized sequence encoding VIP1A(a) with the Bacillus secretion signal removed as contained in pCIB5526 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:35:

GAT C CA C C A T G AAG ACC AA C CAG AT C A G C A C C A C C CAG AAG AA C C A G CAG 5 O Me t Lys Thr As n G | n I I e Ser Thr Thr G 1 n Lys As n G | n G 1 n 8 25 8 3 O 8 35

AAG GAG AT G GA C C GC AAG G G C C T G C T G G G C T A C T AC TT C AAG G GC AAG 9 8 Ly s G 1 u Me t A s p A rig L y s G 1 y Le u Le u G 1 y Ty r Ty r Ph e Lys G 1 y Lys 8 4 O 8 45 8 5 O

GAC TT C A G C AAC CT G ACC AT G T T C G C C C C C A C G C G T G A C A G C A C C C T G 1 4 6 A s p Ph e S e r A s in Le u Thr Me t Ph e A a Pro Th r A rig A s p Ser Thr Le u 8 55 8 6 O 8 65

A T C T A C G A C C A G CAG A C C GCC AAC AA G C T G C T G GAC AAG AA G CAG CAG 19 4 I le Ty r A s p G 1 in G 1 in Th r A 1 a. As n Lys Le u Le u A s p Lys Lys G 1 n G 1 in 8 7 O 8 7 5 8 8 O

GAG TAC CAG A G C AT C C G C T G G AT C G G C C T G AT C C A G A G C AAG GAG ACC 2 4 2 G 1 u Ty r G 1 n Sle r I 1 e Arg Tr p I le G 1 y Le u I le G 1 in Ser Lys G 1 u Thr 88 5 8 9 O 8 9 5

GGC GAC TT C A C C T T C AA C C T G A G C GAG GA C G A G CAG GCC ATC AT C G A G 29 O G 1 y A s p Phe Thr Phe As n Le u Se r G 1 u A s p G 1 u G 1 in A a I I e I le G 1 u

5,840,868 159 160 -continued

18 18 5 1 9

Th G I e P ro Le u G Gl l 1 9 2 O O Gl : o Th I G in I e A a Wa S e Le

A S e Gl Th P he Wa S e r Gl 2 2 2 3 2 4

Th Gl Th Ty r G 1 u A 1 a Le 25 O

S e A 1 a Th r Ph e As in Le u Wa A Ph 2 65 2 7

P r Wa S e Me G u Lys V a 1 Le u P r 2 7 28 O

l S e S e r W a Gl S e r H is S e r Th r T r 2 9 29 3 O O

Th Th Gl G 1 y A S e Wa G u. A a I e G 1 y G 3 O 3 1. 3 2

I Ph G S e W a Ty r G 1 in G u Th 3 2 5 3 3 O

Gl G Gl Th r Th G 1 y As in Th r G in P he Th A 3 4 3 4 5 3 5

S e Gl Le u A V a l Arg W a Gl Th 3 5 3 6 O 3 65

Gl A I W a P ro Th r Thr P he Wa Le 3 7 3 8 O

Th I Al Th r e A 1 a Ly s Ser Th r Al 38

I S e P r G G u L y s Ly s G in Gl e 4 O 5 4 1

I S e Ph S e r H is I e Th r l 4 2 4 3

Le Le As n L y s Me t G Th 4 3

Th W a I e Lys Th r I 4 5 4 6 O

Gl Wa I e G in I e A 4 7 o

Th S e I W a G 1 y G 1 u 4 9 O s

W a G Pro G 1 u Th r O Le o 5 O 5

Th Le Le L e l Ser Ty r G u Gl s 5 25 I G Le As n L y s I e S e 5 3 5 4 O

Me Th Th r A 1 a G u Wa Gl 5 4 t 5 6 Le Th P he L y s As p S e r 5 7 O

W a Le Th Me Wa Thr I e Le u I 58 O 5 85 o

A As n S e r I e G 1 y Th 59 6 O 5 5,840,868 162 -continued

Th r As n I e V a Gl Gl y As i. As i. Gl Ly L y S G in S e 6 1 O 6 1

As in Pro As p A 1 a Le Th Le Th A 1 a G G 6 25 6 3 5 6 i. o

Le u As in Lys As in I Le Me t

As in Th r G in G I Th e Gl G u I e 6 6 O y o

Th r Thir Lys Thr Wa W a 6 7 5

I e I e A a His I 6 9 O

Th r A s in As p G u e Ph I e 7 1. o

W a A a S e r P r l Th

G in I le Ty r S e r Gl Gl G 1 y e 7 4 O

Lys Ly s G 1 y G 1 y I Ph 7 5 7

P he As n I I e G u Le Gl i. Th L y S W a 7 7 O 7 7 5

S e r S e r G 1 u Le u P r W a S e A. 7 9 O R

I le Ty r Lys G Th e P he Th r 8 O 5 8 1. s

As in G u G in G 1 y Le Ph Le u 8 2 O

I l e A s in A 1 a I e Th Gl Me t W a Ph 8 35 R A rig Ty r A s in Lys 8 5 O

( 2) INFORMATION FOR SEQ ID NO:37: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid (A) DESCRIPTION: idesc = "forward primer used to make pCIB5527

( i i i ) HYPOTH ETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:37:

G GAT C CA C C A T G C T G CA GAA C C T GAA GAT C A C 3 2

( 2) INFORMATION FOR SEQ ID NO:38: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid (A) DESCRIPTION: idesc = “reverse primer used to make pCIB5527

5,840,868

-continued

Ph e A s p Lys Thr As in Le u Se r A s in Se I e I Th r Wa 9 O

Gl Se Le Th r G u Gl Th r 1 O O 1 O 5 1 1

I Gl G in P he Le u A rig 1 2 5

I e L y S Ph e A s p Ser Ty Le u As Th Th r A 1 a Gl G in 1 4 O

Th r Wa P ro G 1 6

G 1 y Ser Thr Thr Pro T hr Lys A 1 Gl I e Le u 1 65 1 7

Gl Ty r Lys Me t Le u I le A s p As n G 1 Me Wa 18 O 18

W a Ser Lys V a l V a l Lys Ly s G 1 y V a Le u G in Gl G 1 y 1 9 2 O O 2 O 5

Th I e Gl A 1 a 2 1. O 2 1. 5

Le Th r 24 O

G in 2 4 5

Gl G S e r G 1 y

Le A Le u G P ro 2 7 5 28 O 28

I G Me t G P he 3 O

Gl Se Gl G u 3 O 3 1 O 3 2 O

Gl P he Le u As n Th r I 1 e Ly s G 1 u As G Me t Th 3 2 5 o 3 3 Le S e r S e r G 1 u A rig Le u A a A a Ph S e I Le u 3 4 O 3 4

A r A A I e 3 5 5 3 6 O

Gl G Ph e A a Ser G 1 u L y S G u I L e Le S e 3 7

I I Ly is G 1 y Wa Ly 3 9 O 39

V a l V a 1 A s p A a Thr Le u Le u Th 4 O 5 4 1

( 2) INFORMATION FOR SEQ ID NO:41: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid (A) DESCRIPTION: idesc = "oligonucleotide encoding eukaryotic secretion signal used to construct pCIB5527 ( i i i ) HYPOTHETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:41:

5,840,868 173 174 -continued

8 5 9 O 9 5

G 1 u Pro T hir Thr I I e G 1 y P h e A s in Lys Ser Le u Thr G 1 u G 1 y As in Thr 1 O O 1 O 5 1 1 O

I e As n Sle r A s p A 1 a Me t A a G 1 in P he L y s G 1 u G 1 in P he Le u A s p Arg

Wa I e Le u As in As n S e r 1 65 1 7

18 O 18

V a 1 G 1 u Cy s Le u G | n I I e G 1 u G 1 y 1. 2 O 2 O 5

Th r Le u Lys Lys Ser Le u A s p Ph e Lys As in A s p I e A s in A a G 1 u A 1 a 2 1. O 2 1. 5 22 O

2 4 5 25 O 25 5

G 1 u I le. As n As n Ty r Le u A rig As n G. l n G 1 y G 1 y Ser G 1 y As in G 1 u Lys 2 6 O 2 65 2 7 O

Le u A s p A a G 1 n I le Lys As n I le S e r A s p A 1 a Le u G 1 y Lys Lys Pro 2 7 5 28 O 28 5 I e Pro G 1 u As n I I e Thr V a 1 Ty r A rig Tr p Cy s G 1 y Me t Pro G 1 u Ph e 29 O 2 9 5 3 O O

3 O 3 1 O 3 15 3 2 O

G l n Ph e Le u As n Th r I 1 e Lys G 1 u A s p L y S G 1 y Tyr Me t Ser T h r S e r 3 2 5 3 3 O 3 3 5

Le u Se r S e r G 1 u A rig Le u A a A 1 a Ph e G 1 y Ser Arg Lys I 1 e I e Le u 3 4 O 3 4 5 3 5 O

A rig L e u G 1 in V a 1 Pro Lys G 1 y Ser Thr G 1 y Al a Ty r Le u Se r A la I e 3 5 5 3 6 O 3 65

G 1 y G 1 Ph e A a Ser G 1 u Lys G 1 u I le Le u Le u A s p Lys A s p Ser Lys 3 7

38 5 3 9 O 39 5 4 O O

4 O 5 4 1 O

( 2) INFORMATION FOR SEQ ID NO:44: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid (A) DESCRIPTION: idesc = "oligonucleotide encoding vacuolar targetting peptide used to construct pCIB5533' ( i i i ) HYPOTHETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:44:

C C GC G G G C G T G CA C T G C C T C A G CAG CAGC A G CTT C GC CGA CAG CAA C C C C AT C C GC GT GA

5,840,868 179 18O -continued

W a Th A 1 a A a S e r Le u G in As in Le u Th 3

G P he Ly s G 1 u L y s T r

G u l Le u Thr G u G

Me P he As in A sp I e Th r 8 5 9 O

Gl Th P he I A 1 a S e r Phe G u G u e 1 O O 1 O 5 l Le I e Me t Ph A s p L y s Th r S e r 1 1 1, 2 1 2 5

I e Th P r o Thr Th r G 1 y Ph 1 35

Th G u G 1 y Th r I As n Sle r Me t A Gl P he 1 5 5 1 6 O

P he I e Lys P he S e r 1 7 O 1 7

Th Le Th r G G in W a S e r S e r G u I Le u 18 O 18 5

Th Wa P ro G 1 y G 1 y Ser Th r Th r 1 9

Gl I Le u Ty r Lys Me t G 1 y 22 O

W a Wa W a Ser Lys Wa Gl

G in G Th Le u L y s S e r Le u Ph 25 O 25

I G Ser T r p G 1 y Me t G u 2 6

Gl T r A Le S e r G 1 in G u A 1 a G 1 y 2 7 R 28 5

A G in Gl I e As in Le u G in 29 2

Gl G S e G 1 y G Le A s p A 1 a I e 3 O 3 1.

G 1 y L y S I P r o G 1 u I e Th r W a 3 3 O

T r G Gl Ty r G 1 in 3 4 5

S e Le P he G Gl i. Ph e Le u Th r I e Gl s 3 6 3 65

G Me t Th Le S e r S e r Le u A P he 3 7

Gl e Le u G in Gl S e Th r 38 4 O O

Gl Le u G G 1 y P he G u Gl I e 4 1 O 4 1

Le Le His I e Wa Th Gl Wa 4 2 4 3

I I e Ly Wa Wa Th r Le Le Th r s 4 4 5 5,840,868 181 182 -continued

( 2) INFORMATION FOR SEQ ID NO:47: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( i x ) FEATUR E: ( A ) NAME/KEY: Peptide (B) LOCATION: 1.16 ( D.) OTHER INFORMATION: note= “linker peptide for fusion of VIP1A(a) and VIP2A(a) used to construct pCIB5533” ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:47:

P r o S e r Thr Pro P r o Thr Pro S e r P r o S e r Thr Pro P r o Thr Pro S e r 1. 5 1 O 1 5

( 2) INFORMATION FOR SEQ ID NO:48: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( A ) DESCRIPTION: desc = “DNA encoding linker peptide used to construct pCIB5533' ( i i i ) HYPOTHETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:48:

C C C G G G CCTT CTA C T C C C C C AAC T C C C T C T C CTA G CAC G C C T C C GACA C C T A G C GAT ATC 6 O

G GAT C C 6 6

( 2) INFORMATION FOR SEQ ID NO:49: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 4031 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( A ) DESCRIPTION: desc = “Synthetic DNA” ( i i i ) HYPOTHETICAL: NO ( i x ) FEATUR (A) NAME/KEY: CDS (B) LOCATION: 6.4019 ( D.) OTHER INFORMATION: note= “Maize optimized DNA sequence encoding a VIP2A(a) - VIP1A(a) fusion protein as contained in pCIB5531 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GAT C C A T G A A G C GC AT G GAG G G C A A G C T G T T C A T G G T G A G C AAG AAG 4 7 Me t Lys Arg Me t G 1 u G 1 y Lly s Le u Ph e Me t V a 1 Ser Lys Lys 4 5 O 4 55 4 6 O

C T C CAG G T G G T G A C C AAG A C C G T G C T G C T G A G C A C C G T G T T C A GC ATC 9 5 Le u G 1 in V a l V a 1 T hr Lys Thr V a 1 Le u Le u Ser Thr V a 1 P h e Sier I 1 e 4 6 5 4 7 O 4 75

A G C C T G C T G AAC AA C G A G G T G ATC AAG GCC GAG CAG C T G AAC AT C AAC 1 4 3 Ser Le u Le u As n As n G 1 u V a 1 I e Lys A a G 1 u G | n Le u As n I I e A s in 4 8 O 48 5 4 9 O 4 9 5

5,840,868 191 192 -continued

GGC AAG GAG AT G AA C G T G T T C CAC C GC TAC AAC AAG TAGAT CT GAG 4 O 29 G 1 y Ly is G u Me t As in Wa P he Hi S A rig Ty r A s in Lys 1 7 8 O 1 7 8 5

CT 4 O 3 1

( 2) INFORMATION FOR SEQ ID NO:50: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 1338 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:50: Me t Lys Arg Me t G 1 u G 1 y Lys Le P h e Me t V a 1 Ser Lys Lys Le 1. 5 1 O 1. 5

V a l V a 1 T hr Lys Thr V a 1 Le u Le S e r Thr Wa Ph e S e r I e S e Le 2 O 25 3 O

Le u As n As n G 1 u V a 1 I e Lys a G u G in Le u As n I e A s in S e Gl 5 4 5

Ser Lys Ty r Thr As n Le u G 1 n Le u Lys I le Th r A s p Lys V a Gl 5 5 5 6 A s p P he Lys G 1 u A s p Lys G 1 u A 1 a Lys G 1 u T r p G 1 y Lly s G 1 65 7 O 7 5

G 1 u Lys G 1 u Trip Lys Le u Thr A Thr G 1 u Lys G 1 y Lys Me t As 8 5 9 O

Ph e Le u A s p As in Lys As in As p I Lys Thr As n Ty r Lys G 1 u I Th 1 O O 1 O 5 1 1 O

P h e S e r I 1 e A 1 a G 1 y Ser P he A s p G 1 u I l e Ly s A s p Le u L y 1 15 1, 2 1 2 5

I e A s p Lys Me t Ph e A s p Lys Th As in L e ul S e r As n S e r I I e I Th 13 O 13 1 4 O

Ty r Lys As n V a 1 G 1 u Pro Thr Th I le G 1 y Phe As in Lys Ser Le Th 1 4 1 5 O 1 5 5 1 6

G 1 u G 1 y As n Th r I 1 e As n Sle r A 1 a Me t Al a G | n Ph e Ly s G 1 Gl 1 65 1 7 O 1 7

Ph e Le u A s p A rig A s p I l e Lys Ph A s p Ser Ty r Le u A s p Thr Hi Le 18 O 18 5 1 9

Th r A a G 1 in G 1 m Wa S e r S e r S G 1 u A rig V a 1 I le Le u Lys V a Th 1 9 5 2 O 5

V a 1 Pro Ser G 1 y Thr Pro T hr Lys A a G 1 y V a 21 O 2 1 22 O

Le u As n As n Ser G 1 u Ty r Lys Me Le u I le A s p As n G 1 y Ty r Me 2 25 2 3 23 5

His V a 1 A s p Lys V a 1 Ser Lys W a V a l Lys Lys G 1 y V a 1 G 1 u Cy 2 4 5 25 O 25 s G l n I le G 1 u G 1 y Thr Le u Lys Ser Le u A s p Ph e Lys As n As 2 6 O 2 65 2 7 O

As in A a G 1 u A 1 a His Ser T r p Gl Me t Lys As n Ty r G 1 u G 1 u T r 2 7 5 28 28 5

Lys A s p Le u Th r A s p Ser G 1 n G 1 u A 1 a Le u A s p G 1 y Ty r A 1 29 O 2 9 5 3 O O

G l n A s p Ty r Lys G 1 u I le. As n Ty r Le u A rig As in G 1 in G 1 y G 1 3 O 5 3 1 O 3 15 o

G l y As in G 1 u L y S L e u A s p A 1 a Gl I e Lys As n I le S e r A s p A 1 3 2 5 3 3 O 3 3 5,840,868 193 194 -continued

Gl I G u Ty G 1 y 3 4

Me G u Ph G G in I Le Le u 3 5 5 3 6

G u Gl Gl Ph Th I e Gl G 1 y 38

Th r S e L e u A P he 39 39

I e Le Le G in W a Gl Th r A 1 a 4 15

Le A 1 a e Gl P he I e Le u 4 3 O

I e Th I e 4 4 5

Gl W a A Th r Le l Th r S e r A rig 4 55

Gl Th Th r S e P ro P ro Th r 4 6 4 7 4 7 4 8 O

S e Gl Th Me t Th Gl S e r Th r G in 4 9 5

Gl Me t G Le u 5 O 5 O

Ph Ph S e r Le Th r Me A 1 a Th r 5 25

Le I G Th r A. i. Le u o 53 5

Gl Gl S e I G 1 y I e G in o 5 5 6 O

Th Th Ph G u G u G in 5 6 5 7 5

A I I I G u 58 58

G W a Le Gl W a I e 5 9 5 o

Gl G in S e Th I e P he o 6 2

Gl Le Ph Gl G in 6 2 o

Gl G Gl e Gl P he G in 6 6 5 O 6 55

Gl Le u A S e r I Le Ph Th r G in Me t 6 6 6 6

G u I Th Th r Gl I e P ro 6 7 5 68 68 5

l Gl G Th I e Gl A rig I e A 1 a Wa 6 9 69 7 O

S e Th P he Wa 7 2 O

Le u Gl Th r W a Th r G u

Le Le G u Th r 7 4 7 4 7 5 O

P r Wa A Ph S e W a W a Me t G u Ly is Wa