USOO577O696A United States Patent (19) 11 Patent Number: 5,770,696 Warren et al. (45) Date of Patent: Jun. 23, 1998

54 AUXILIARY PROTEINS FOR ENHANCING Porter, A.G., et al., “Mosquitocidal Toxins of Bacilli and THE INSECTICIDAL ACTIVITY OF Their Genetic Manipulation for Effective Biological Control PESTICIDAL PROTEINS of Mosquitoes”, Microbiological Reviews, 57(4):838–861 (1993). 75 Inventors: Gregory W. Warren; Michael G. Sekar, V., “The Insecticidal Crystal Protein Gene is Koziel, both of Cary; Martha A. Expressed in Vegetative Cells of Bacillus thuringiensis var. Mullins, Raleigh; Gordon J. Nye, temebropmos”, Current Microbiology, 17:347-349. Apex; Brian Carr; Nalini M. Desai, both of Cary; Kristy Kostichka, Shivakumar, A.G., et al., Abstract,:Cloned Crystal Protein Durham; Nicholas B. Duck, Cary; Juan Genes Express Vegetatively in Bacillus subtilis, Plasmid, J. Estruch, Durham, all of N.C. 16(3):230 (1986). Thanabalu, T., et al., “Proteolytic Processing of the Mos 73 Assignee: Novartis Corporation quitocidal Toxin from Bacillus Sphaericus SSII-1, J. Bac teriol., 174(15):5051-5056 (1992). 21 Appl. No.: 471,033 Yoshisue, H., et al., “Effects of Bacillus thuringiensis var. israelensis 20 kDa Protein on Production of the Bti 130-kDa 22 Filed: Jun. 6, 1995 Crystal Protein in Escherichia coli'', BioScience, Biotech Related U.S. Application Data nology, and Biochemistry, 56(9): 1429-1433 (1992). Arellano, A., et al., “Evidence of a New Bacillus thuring 60 Division of Ser. No. 463,483, Jun. 5, 1995, which is a iensis Toxin Active Against the Australian Sheep Blowfly continuation-in-part of Ser. No. 314,594, Sep. 28, 1994, which is a continuation-in-part of Ser. No. 218,018, Mar. 23, Lucilla cuprina', Proceedings and Abstracts of the 5th 1994, abandoned, which is a continuation-in-part of Ser. No. International Colloquium On Invertebrate Pathology and 37,057, Mar. 25, 1993, abandoned. Microbial Control, Adelaide, Austrailia, 20–24 Aug., 1990, (51) Int. Cl." ...... C07K 14/32 p. 291. 52 U.S. Cl...... 530/350; 536/23.1; 536/23.7; Beecher, Douglas J., et al., “A Novel Bicomponent Hemol 536/23.71; 530/825 ysin from Bacillus cereus', Inspection and Immunity, 58 Field of Search ...... 530/350; 536/23.1, 58(7):2220–2227 (1990). 536/23.7, 23.71, 825 Faust, R.M., "Bacterial Diseases”, In: Insect Diseases, G. Cantwell, ed., Marcel Dekker, NY 1974, pp. 84-89 and 108-120. 56) References Cited Gilmore, Michael S., et al., “A Bacillus cereus Cytolytic Determinant, Cereolysin AB, Which Comprises the Phos U.S. PATENT DOCUMENTS pholipase C and Sphingomyelinase Genes: Nucleotide 3,632,747 1/1972 Satohiro et al...... 424/93 Sequence and Genetic Linkage”, Journal of Bacteriology, 3,651,215 3/1972 Satohiro et al...... 424/93 171(2):744–753 (1989). 4,996,155 2/1991 Sick et al...... 435/252.3 Heimpel, A.M., “The pH in the Gut and Blood of the Larch 5,262,323 11/1993 Baird et al...... 435/252.5 Sawfly, Pristiphora erichsonii (HTG), and Other Insects with Reference to the Pathogenicity of Bacillus cereuS FR. FOREIGN PATENT DOCUMENTS and FR.", Can. J. Zool, 33:99-106 (1955). O498537A2 1/1992 European Pat. Off.. Heimpel, A.M., “Investigations of the Mode of Action of O501650A2 2/1992 European Pat. Off.. Strains of Bacillus cereus FR. and FR. Pathogenic for the WO88/08880 11/1988 WIPO. Larch Sawfly, Pristiphora erichsonii (HTG.)", Can. J. Zool., WO90/13651 11/1990 WIPO. WO91/16432 10/1991 WIPO. 33:311-326 (1995). WO91/16434 10/1991 WIPO. Krieg, A., “Thuricin, a Bacteriocin Produced by Bacillus US94/03131 7/1994 WIPO. thuringiensis”, J. Invert. Path., 15:291 (1970). WO 94/21795 9/1994 WIPO. (List continued on next page.) OTHER PUBLICATIONS Primary Examiner Robert A. Wax Hofte, H., et al., “Insecticidal Crystal Proteins of Bacillus ASSistant Examiner Nashaat T. Nashed thuringiensis", Microbiological Reviews, 53(2):242-255 Attorney, Agent, or Firm-Gary M. Pace (1989). 57 ABSTRACT Koziel, M.G., et al., “Field Performance of Elite Tansgenic The present invention is drawn to pesticidal Strains and Maize Plants Expressing an Insecticidal Protein Derived proteins. Bacillus Strains which are capable of producing from Bacillus thuringiensis', Bio/Technology, 11:194-200 pesticidal proteins and auxiliary proteins during vegetative (1993). growth are provided. “The auxiliary proteins enhance the Krieg, A., “Concerning Alpha-exotoxin Produced by Veg insecticidal activity of pesticidal proteins.” Also provided etative Cells of Bacillus thuringiensis and Bacillus cereus', are the purified proteins, nucleotide Sequences encoding the J. Invert. Path., 17:134–135 (1971). proteins and methods for using the Strains, proteins and Myers, P.S., et al., “Localization of a Mosquito-Larval genes for controlling pests. Toxin of Bacillus Sphaericus 1593”, Appl. Environ. Micro biol, 39(1):1205-1211 (1980). 6 Claims, 1 Drawing Sheet 5,770,696 Page 2

OTHER PUBLICATIONS Thanabalu et al., “Cytotoxicity and ADP-Ribosylating Kushner, D.J., et al., “Lecithinase Production by Strains of Activity of the Mosquitocidal Toxin from Bacillus Sphaeri Bacillus cereuS FR. and FR. Pathogenic for the Larch cus SSII-1: Possible Roles of the 27-and 70-Kilodaron Sawfly, Pristiphora erichsonii (HTG.)", Can. J. Microbiol., Peptides”, Journal of Bacteriology, 175(8):2314–2320 3:547-551 (1957). Luthy, P., et al., “Bacillus thuringiensis as a Bacterial (1993). Insecticide: Basic Consideration and Application', In: Vaithlingam et al., “Anti-Coleopteran Toxin and Gene”, Microbial and Viral Pesticides, E. Kurstak, Ed., Marcel Dekker, NY 1982, pp. 37–39, 54–56. Abstract No. 226442, New Zealand Patent Office Journal, European International Search Report dated May 3, 1996. 80(7):931, (1991). Bernier et al., “Bacillus thuringiensis Strains A20 and A29 and Insecticidal Compounds therefrom, and Compositions Wahisaka et al., “Bacillus Thuringiensis Mutant and Bacte Containing These Compounds”, Abstract No. 227249, New rial Insecticide”, Abstract No. 199725, New Zealand Patent Zealand Patent Office Journal, 80(6):798, (1988). Office Journal, (1982). Jellis et al., “Bacillus Thuringiensis 8-Endotoxin Variants and Insecticidal Compositions”, Abstract No. 228108, New Walther et al., “Analysis of Mosquito Larvicidal Potential Zealand Patent Office Journal, 81(3):359, (1992). Exhibited by Vegetative Cells of Bacillus thuringiensis Schurter et al., “Genetic Manifpulation of B. thuringiensis B. cereus Vectors and Insecticidal Composition”, Abstract No. subsp. israelensis”, Applied and Environmental Microbiol 229191, New Zealand Patent Office Journal, 81(3):363, ogy, 52(4):650–653 (1986). (1992). 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”, Journal of Applied Toxicology, 15(5):365-373 (1995). Molecular Biology, 191(1): 13–22 (1986). U.S. Patent Jun. 23, 1998 5,770,696

Figure 1 Characterization of pCIB6022 Activity vs. WCRW S: pCIB6022 ---- WTP2A(a VIP1A(a)

pCB6203 pCIB6023

pCIB6206 -

pCLB6024 Functional Complementation of VIP Clones

pCIB6203 H pCDB6023

pCIB6203 -H pCB6206

pCIB6023 cro pCIB6024 5,770,696 1 2 AUXLIARY PROTEINS FOR ENHANCING The methods and compositions of the invention may be THE INSECTICIDAL ACTIVITY OF used in a variety of Systems for controlling plant and PESTICIDAL PROTEINS non-plant pests. This is a divisional application of Ser. No. 08/463,483, BRIEF DESCRIPTION OF THE FIGURE filed Jun. 5, 1995 which is a continuation-in-part of Ser. No. 08/314,594 filed Sep. 28, 1994, now pending, which is a FIG. 1: Characterization of pCIB6022. Boxed regions continuation-in-part of Ser. No. 08/218,018, filed Mar. 23, represent the extent of VIP1A(a) and VIP2A(a). White box 1994, now abandoned, which is a continuation-in-part of represents the portion of VIP1 encoding the 80 kDa peptide Ser. No. 08/037,057, filed Mar. 25, 1993, now abandoned. observed in Bacillus. Dark box represents the N-terminal propeptide of VIP1A(a) predicted by DNA sequence FIELD OF THE INVENTION analysis. Stippled box represents the VIP2A(a) coding The present invention is drawn to methods and compo region. Large 'X' represents the location of the frameshift Sitions for controlling plant and non-plant pests. mutation introduced into VIP1A(a). Arrows represent con 15 Structs transcribed by the beta-galactosidase promoter. BACKGROUND OF THE INVENTION Restriction Sites: C-Cla I; X-Xba I, S-Sca I, RI-Eco RI; B-Bgl II; RV-Eco RV. Insect pests are a major factor in the loSS of the World's commercially important agricultural crops. Broad Spectrum chemical pesticides have been used extensively to control or DETAILED DESCRIPTION OF THE eradicate pests of agricultural importance. There is, INVENTION however, Substantial interest in developing effective alter Compositions and methods for controlling plant pests are native pesticides. provided. In particular, novel pesticidal proteins are pro Microbial pesticides have played an important role as Vided which are produced during vegetative growth of alternatives to chemical pest control. The most extensively 25 Bacillus Strains. The proteins are useful as pesticidal agents. used microbial product is based on the bacterium Bacillus The present invention recognizes that pesticidal proteins thuringiensis (Bt). Bt is a gram-positive spore forming are produced during vegetative growth of Bacillus Strains. Bacillus which produces an insecticidal crystal protein (ICP) To date, all of the identified pesticidal proteins of the during sporulation. invention are secreted from the cell. Prior to the present Numerous varieties of Bt are known that produce more invention, there was no recognition in the art that a class or than 25 different but related ICP's. The majority of ICP's classes of pesticidal proteins are produced during vegetative made by Bt are toxic to larvae of certain insects in the orders growth of Bacillus. The only report was of a single mos Lepidoptera, Diptera and Coleoptera. In general, when an quitocidal toxin from Bacillus Sphaericus SSII-1 by Myers ICP is ingested by a Susceptible insect the crystal is Solu and Yousten in Infect. Immun., 19:1047-1053 (1978). Hav bilized and transformed into a toxic moiety by the insect gut 35 ing recognized that Such a class exists, the present invention proteases. None of the ICP's active against coleopteran embraces all vegetative insecticidal proteins, hereinafter larvae Such as Colorado potato beetle (Leptinotarsa referred to as VIPs, except for the mosquitocidal toxin from decemlineata) or Yellow mealworm (Tenebrio molitor) have B. Sphaericus. demonstrated Significant effects on members of the genus Diabrotica particularly Diabrotica virgifera virgifera, the 40 The present VIPs are not abundant after sporulation and western corn rootworm (WCRW) or Diabrotica longicornis are particularly expressed during log phase growth before barberi, the northern corn rootworm. Stationary phase. For the purpose of the present invention Bacillus cereus (Bc) is closely related to Bt. A major vegetative growth is defined as that period of time before the distinguishing characteristic is the absence of a parasporal onset of Sporulation. Genes encoding Such VIPS can be crystal in Bc. Bc is a widely distributed bacterium that is 45 isolated, cloned and transformed into various delivery commonly found in Soil and has been isolated from a variety vehicles for use in pest management programs. of foods and drugs. The organism has been implicated in the For purposes of the present invention, pests include but Spoilage of food. are not limited to insects, fungi, bacteria, nematodes, mites, Although Bt has been very useful in controlling insect ticks, protozoan pathogens, animal-parasitic liver flukes, and pests, there is a need to expand the number of potential 50 the like. Insect pests include insects Selected from the orders biological control agents. Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, SUMMARY OF THE INVENTION Thy Sanoptera, Dermaptera, Isoptera, Anoplura, The present invention is drawn to compositions and Siphonaptera, Trichoptera, etc., particularly Coleoptera and methods for controlling plant and non-plant pests. 55 Lepidoptera. Particularly, new pesticidal proteins are disclosed which are Tables 1-10 gives a list of pests associated with major isolatable from the vegetative growth stage of Bacillus. crop plants and pests of human and Veterinary importance. Bacillus Strains, proteins, and genes encoding the proteins Such pests are included within the Scope of the present are provided. invention.

TABLE 1. Lepidoptera (Butterflies and Moths) Maize Sunflower Ostrinia nubilalis, European corn borer Suleina helianthana, sunflower bud moth 5,770,696 3

TABLE 1-continued Lepidoptera (Butterflies and Moths) Agrotis ipsilon, black cutworm Homoeosoma electeilun, sunflower moth Helicoverpa zea, corn earworm Cotton Spodoptera frugiperda, fall armyworm Heliothis virescens, cotton boll worm Diatraea grandioSella, southwestern corn Helicoverpa zea, cotton bollworm borer Spodoptera exigua, beet armyworm Elasnopalpus ignoSelius, lesser cornstalk Pectinophora gossypiella, pink bollworm borer Rice Diatraea Saccharais, sugarcane borer Diatraea Saccharalis, sugarcane borer Sorghum Spodopterafrugiperda, fall armyworm Chilo partellus, sorghum borer Helicoverpa zea, corn earworm Spodoptera frugiperda, fall armyworm Soybean Helicoverpa zea, corn earworm Pseudoplusia includens, soybean looper Elasnopalpus ignoSelius, lesser cornstalk Anticarsia gemmataiis, velvetbean borer caterpillar Feltia Subterranea, granulate cutworm Plathypena Scabra, green cloverworm Wheat Ostrinia nubialis, European corn borer Pseudaletia unipunctata, army worm Agrotis ipsilon, black cutworm Spodoptera frugiperda, fall armyworm Spodoptera exigua, beet armyworm Elasnopalpus ignoSelius, lesser cornstalk Heliothis virescens, cotton boll worm borer Helicoverpa zea, cotton bollworm Agrotis Orthogonia, pale western cutworm Barley Elasnopalpus ignoSelius, lesser cornstalk Ostrinia nubialis, European corn borer borer Agrotis ipsilon, black cutworm

25

TABLE 2 TABLE 3-continued

Coleoptera (Beetles) Homoptera (Whiteflies, Aphids etc..) Maize Diabrotica virgifera virgifera, western corn rootworm Russian wheat aphid Diabrotica longicornis barberi, northern corn rootworm Diabrotica undecimpunctata howardi, southern corn rootworm Schizaphis graninum, greenbug Melanotus spp., wireworms Macrosiphun avenae, English grain aphid Cyclocephala borealis, northern masked chafer (white grub) Cotton Cyclocephala immaculata, southern masked chafer (white grub) 35 Popillia japonica, Japanese beetle Aphis gossypii, cotton aphid Chaetocnema pulicaria, corn flea beetle Pseudatomoscelis Seriatus, cotton fleahopper Sphenophorus maidis, maize billbug Trialeurodes abutionea, bandedwinged whitefly Sorghum Phyllophaga crinita, white grub Rice Eleodes, Conoderus, and Aeolus spp., wireworms 40 Nephotettix nigropictus, rice leafhopper Oulema melanopus, cereal leaf beetle Soybean Chaetocnema pulicaria, corn flea beetle Myzus persicae, green peach aphid Sphenophorus maidis, maize billbug Wheat Empoasca fabae, potato leafhopper Oulema melanopus, cereal leaf beetle Barley Hypera punctata, clover leaf weevil 45 Schizaphis graninum, greenbug Diabrotica undecimpunctata howardi, southern corn rootworm Sunflower Oil Seed Rape Zygogramma exclamationis, Sunflower beetle Brevicoryne brassicae, cabbage aphid Bothyrus gibbosus, carrot beetle Cotton Anthonomus grandis, boll weevil Rice 50 Colaspis brunnea, grape colaspis TABLE 4 Lissorhoptrus Oryzophilus, rice water weevil Sitophilus Oryzae, rice weevil Hemiptera (Bugs) Soybean Maize Epilachna varivestis, Mexican bean beetle 55 Blissus leucopterus leucopterus, chinch bug Sorghum. Blissus leucopterus leucopterus, chinch bug Cotton TABLE 3 Lygus lineolaris, tarnished plant bug Rice Homoptera (Whiteflies, Aphids etc..) Blissus leucopterus leucopterus, chinch bug 60 Acrosternum hilare, green stink bug Maize Soybean Rhopalosiphum maidis, corn leaf aphid Acrosternum hilare, green stink bug Anuraphis maidiradicis, corn root aphid Barley Sorghum. Blissus leucopterus leucopterus, chinch bug Rhopalosiphum maidis, corn leaf aphid Acrosternum hilare, green stink bug Sipha flava, yellow Sugarcane aphid 65 EuSchistus Servus, brown stink bug Wheat 5,770,696 S 6

TABLE 5 TABLE 9 Orthoptera (Grasshoppers, Crickets, and Cockroaches) Other Orders and Representative Species Maize Dermaptera (Earwigs) Forficula auricularia, European earwig Melanoplus femurrubrum, redlegged grasshopper Isoptera (Termites) Melanoplus Sanguinipes, migratory grasshopper Reticuliterimes flavipes, eastern subterranean termite Wheat Mallophaga (Chewing Lice) Melanoplus femurrubrum, redlegged grasshopper Cuciotogaster heterographa, chicken head louse Melanoplus differentialis, differential grasshopper 1O Bovicola bovis, cattle biting louse Melanoplus Sanguinipes, migratory grasshopper Anoplura (Sucking Lice) Cotton Pediculus humanus, head and body louse Melanoplus femurrubrum, redlegged grasshopper Siphonaptera (Fleas) Melanoplus differentialis, differential grasshopper Ctenocephalides felis, cat flea Soybean Melanoplus femurrubrum, redlegged grasshopper 15 Melanoplus differentialis, differential grasshopper TABLE 10 Structural/Household Periplaneta annericana, American cockroach Acari (Mites and Ticks) Blatteila germanica, German cockroach Biatta Orientalis, oriental cockroach Maize Tetranychus urticae, wOspotted spider mite Sorghum Tetranychus cinnabarinus, carmine spider mite TABLE 6 Tetranychus urticae, twospotted spider mite Wheat Diptera (Flies and Mosquitoes) curl mite 25 Aceria tulipae, whea Maize Cotton Hylenya platura, seedcorn maggot Tetranychus cinnabarinus, carmine spider mite Agromyza parvicornis, corn blotch leafminer Tetranychus urticae, twospotted spider mite Sorghum. Soybean Contarinia Sorghicola, sorghum midge Tetranychus turkestani, strawberry spider mite Wheat Tetranychus urticae, twospotted spider mite Mayetiola destructor, Hessian fly Barley Sitodiplosis mosellana, wheat midge Petrobia latens, brown wheat mite Meromyza americana, wheat stem maggot Important human and animal Acari Hylemya coarctata, wheat bulb fly Demacentor variabilis, American dog tick Sunflower Argas persicus, fowl tick Neoliasioptera murtfeldtiana, Sunflower seed midge Dernatophagoides farinae, American house dust mite Soybean 35 Dermatophagoides pteronyssinus, European house dust mite Hylenya platura, seedcorn maggot Barley Hylenya platura, seedcorn maggot Now that it has been recognized that pesticidal proteins Mayetiola destructor, Hessian fly Insects attacking humans and animals and disease carriers can be isolated from the vegetative growth phase of Bacillus, Aedes aegypti, yellowfever mosquito 40 other Strains can be isolated by Standard techniques and Aedes albopictus, forest day mosquito tested for activity against particular plant and non-plant Phlebotomus papatasii, sand fly pests. Generally Bacillus Strains can be isolated from any Musca domestica, house fly Tabanus atratus, black horse fly environmental Sample, including Soil, plant, insect, grain Cochliomyia hominivorax, screwworm fly elevator dust, and other Sample material, etc., by methods 45 known in the art. See, for example, Travers et al. (1987) Appl. Environ. Microbiol. 53:1263–1266; Saleh et al. TABLE 7 (1969) Can J. Microbiol. 15:1101-1104; DeLucca et al. (1981) Can. J. Microbiol. 27:865-870; and Norris, et al. Thysanoptera (Thrips) (1981) “The genera Bacillus and Sporolactobacillus.” In Maize 50 Starret al. (eds.), The Prokaryotes: A Handbook on Habitats, Anaphothrips obscurus, grass thrips Wheat Isolation, and Identification of Bacteria, Vol. II, Springer Frankliniella fusca, tobacco thrips Verlog Berlin Heidelberg. After isolation, strains can be Cotton tested for pesticidal activity during vegetative growth. In Thrips tabaci, onion thrips this manner, new pesticidal proteins and Strains can be Frankliniella fusca, tobacco thrips identified. Soybean 55 Sericothrips variabilis, soybean thrips Such Bacillus microorganisms which find use in the Thrips tabaci, onion thrips invention include Bacillus cereus and Bacillus thuringiensis, as well as those Bacillus species listed in Table 11. TABLE 8 60 TABLE 11 Hymenoptera (Sawflies, Ants, Wasps, etc.) List of Bacillus species Maize Solenopsis milesta, thief ant Morphologica1 Group 1 Unassigned Strains Wheat B. megaterium Subgroup A Cephus cinctus, wheat stem sawfly 65 B. cereus B. apiarus B. cereus var. mycoides B. filicolonicus 5,770,696 7 8 presence of minor impurities which do not interfere with the TABLE 11-continued biological activity of the protein, and which may be present, for example, due to incomplete purification. List of Bacillus species Once purified protein is isolated, the protein, or the B. thuringiensis B. thiaminolyticus polypeptides of which it is comprised, can be characterized B. licheniformis B. alcalophilus and Sequenced by Standard methods known in the art. For B. Subtilis Subgroup B B. punius B. cirroflagellosus example, the purified protein, or the polypeptides of which B. firmus B. chitinosporus it is comprised, may be fragmented as with cyanogen B. coagulans B. lentus bromide, or with proteases Such as papain, chymotrypsin, Morphological Group 2 Subgroup C trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. B. polymyxa B. badius B. macerans B. aneurinolyticus Biol. Chem. 257:9751–9758; Liu et al. (1983) Int. J. Pept. B. circulians B. macroides Protein Res. 21:209-215). The resulting peptides are B. Stearothermophilus B. freundenreichi separated, preferably by HPLC, or by resolution of gels and B. alvei Subgroup D electroblotting onto PVDF membranes, and subjected to B. laterosporus B. pantothenticus 15 B. brevis B. epiphytus amino acid Sequencing. To accomplish this task, the peptides B. pulvifaciens Subgroup E1 are preferably analyzed by automated Sequenators. It is B. popilliae B. anninovorans recognized that N-terminal, C-terminal, or internal amino B. lentimorbus B. globisporus acid Sequences can be determined. From the amino acid B. larvae B. insolitus Morphological Group 3 B. psychrophilus Sequence of the purified protein, a nucleotide Sequence can B. sphaericus* Subgroup E2 be Synthesized which can be used as a probe to aid in the B. pasteurii B. pSychroSaccharolyticus isolation of the gene encoding the pesticidal protein. B. macquariensis It is recognized that the pesticidal proteins may be oligo * = Those Bacillus strains that have been previously found associated with meric and will vary in molecular weight, number of insects Grouping according to Parry, J. M. et al. (1983) Color Atlas of Bacillus protomers, component peptides, activity against particular species, Wolfe Medical Publications, London. 25 pests, and in other characteristics. However, by the methods In accordance with the present invention, the pesticidal Set forth herein, proteins active against a variety of pests proteins produced during vegetative growth can be isolated may be isolated and characterized. from Bacillus. In one embodiment, insecticidal proteins Once the purified protein has been isolated and charac produced during vegetative growth, can be isolated. Meth terized it is recognized that it may be altered in various ways ods for protein isolation are known in the art. Generally, including amino acid Substitutions, deletions, truncations, proteins can be purified by conventional chromatography, and insertions. Methods for Such manipulations are gener including gel-filtration, ion-exchange, and immunoaffinity ally known in the art. For example, amino acid Sequence chromatography, by high-performance liquid variants of the pesticidal proteins can be prepared by muta chromatography, Such as reversed-phase high-performance 35 tions in the DNA. Such variants will possess the desired liquid chromatography, ion-exchange high-performance liq pesticidal activity. Obviously, the mutations that will be uid chromatography, size-exclusion high-performance liq made in the DNA encoding the variant must not place the uid chromatography, high-performance chromatofocusing Sequence out of reading frame and preferably will not create and hydrophobic interaction chromatography, etc., by elec complementary regions that could produce Secondary trophoretic Separation, Such as one-dimensional gel 40 mRNAstructure. See, EP Patent Application Publication No. electrophoresis, two-dimensional gel electrophoresis, etc. 75,444. Such methods are known in the art. See for example Current In this manner, the present invention encompasses the Protocols in Molecular Biology, Vols. 1 and 2, Ausubel et al. pesticidal proteins as well as components and fragments (eds.), John Wiley & Sons, N.Y. (1988). Additionally, anti thereof. That is, it is recognized that component protomers, bodies can be prepared against Substantially pure prepara 45 polypeptides or fragments of the proteins may be produced tions of the protein. See, for example, Radka et al. (1983).J. which retain pesticidal activity. These fragments include Immunol. 128:2804; and Radka et al. (1984) Immunogenet truncated Sequences, as well as N-terminal, C-terminal, ics 19:63. Any combination of methods may be utilized to internal and internally deleted amino acid Sequences of the purify protein having pesticidal properties. AS the protocol proteins. is being formulated, pesticidal activity is determined after 50 Most deletions, insertions, and Substitutions of the protein each purification Step. Sequence are not expected to produce radical changes in the Such purification Steps will result in a Substantially puri characteristics of the pesticidal protein. However, when it is fied protein fraction. By “substantially purified” or “sub difficult to predict the exact effect of the substitution, Stantially pure' is intended protein which is Substantially deletion, or insertion in advance of doing So, one skilled in free of any compound normally associated with the protein 55 the art will appreciate that the effect will be evaluated by in its natural State. "Substantially pure' preparations of routine Screening assayS. protein can be assessed by the absence of other detectable The proteins or other component polypeptides described protein bands following SDS-PAGE as determined visually herein may be used alone or in combination. That is, Several or by densitometry Scanning. Alternatively, the absence of proteins may be used to control different insect pests. other amino-terminal Sequences or N-terminal residues in a 60 Some proteins are Single polypeptide chains while many purified preparation can indicate the level of purity. Purity proteins consist of more than one polypeptide chain, i.e., can be verified by rechromatography of "pure' preparations they are oligomeric. Additionally, Some VIPs are pesticid showing the absence of other peaks by ion exchange, reverse ally active as oligomers. In these instances, additional pro phase or capillary electrophoresis. The terms "Substantially tomers are utilized to enhance the pesticidal activity or to pure” or “substantially purified” are not meant to exclude 65 activate pesticidal proteins. Those protomers which enhance artificial or synthetic mixtures of the proteins with other or activate are referred to as auxiliary proteins. Auxiliary compounds. The terms are also not meant to exclude the proteins activate or enhance a pesticidal protein by interact 5,770,696 9 10 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 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 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 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 pesticidal or auxiliary proteins by producing in frame The pesticidal proteins of the invention can be used in 15 genetic fusions which, when translated by ribosomes, would combination with Bt endotoxins or other insecticidal pro produce a fusion protein with the combined attributes of the teins to increase insect target range. Furthermore, the use of VIP and the other component used in the fusion. the VIPs of the present invention in combination with Bt Furthermore, if the protein domain fused to the VIP has an Ö-endotoxins or other insecticidal principles of a distinct affinity for another protein, nucleic acid, carbohydrate, lipid, nature has particular utility for the prevention and/or man or other chemical or factor, then a three-component complex agement of insect resistance. Other insecticidal principles can be formed. This complex will have the attributes of all include protease inhibitors (both Serine and cysteine types), of its components. A similar rationale can be used for lectins, C.-amylase and peroxidase. In one preferred producing four or more component complexes. These com embodiment, expression of VIPs in a transgenic plant is plexes are useful as insecticidal toxins, pharmaceuticals, accompanied by the expression of one or more Bt 25 laboratory reagents, and diagnostic reagents, etc. Examples Ö-endotoxins. This co-expression of more than one insecti where Such complexes are currently used are fusion toxins cidal principle in the same transgenic plant can be achieved for potential cancer therapies, reagents in ELISA assays and by genetically engineering a plant to contain and express all immunoblot analysis. the genes necessary. Alternatively, a plant, Parent 1, can be One Strategy of altering pesticidal or auxiliary proteins is genetically engineered for the expression of VIPs. A Second to fuse a 15-amino-acid “S-tag to the protein without plant, Parent 2, can be genetically engineered for the expres destroying the insect cell binding domain(s), translocation Sion of Bt Ö-endotoxin. By crossing Parent 1 with Parent 2, domains or protein-protein interacting domains of the pro progeny plants are obtained which express all the genes teins. The S-tag has a high affinity (K=10M) for a introduced into Parents 1 and 2. Particularly preferred Bt ribonuclease S-protein, which, when bound to the S-tag, Ö-endotoxins are those disclosed in U.S. Pat. No. 5,625,136, 35 forms an active ribonuclease (See F. M. Richards and H. W. herein incorporated by reference. Wyckoff (1971) in “The Enzymes”, Vol. IV (Boyer, P. D. A Substantial number of cytotoxic proteins, though not all, ed.). pp. 647-806. Academic Press, New York). The fusion are binary in action. Binary toxins typically consist of two can be made in Such a way as to destroy or remove the protein domains, one called the A domain and the other cytotoxic activity of the pesticidal or auxiliary protein, called the B domain (see Sourcebook of Bacterial Protein 40 thereby replacing the VIP cytotoxic activity with a new Toxins, J. E. Alouf and J. H. Freer eds.(1991) Academic cytotoxic ribonuclease activity. The final toxin would be Press). The A domain possesses a potent cytotoxic activity. comprised of the S-protein, a pesticidal protein and an The B domain binds an external cell surface receptor before auxiliary protein, where either the pesticidal protein or the being internalized. Typically, the cytotoxic A domain must auxiliary protein is produced as translational fusions with be escorted to the cytoplasm by a translocation domain. 45 the S-tag. Similar Strategies can be used to fuse other Often the A and B domains are Separate polypeptides or potential cytotoxins to pesticidal or auxiliary proteins protomers, which are associated by a protein-protein inter including (but not limited to) ribosome inactivating proteins, action or a di-sulfide bond. However, the toxin can be a insect hormones, hormone receptors, transcription factors, Single polypeptide which is proteolytically processed within proteases, phosphatases, Pseudomonas eXotoxin A, or any the cell into two domains as in the case for Pseudomonas 50 other protein or chemical factor that is lethal when delivered eXotoxin A. In Summary binary toxins typically have three into cells. Similarly, proteins can be delivered into cells important domains, a cytotoxic Adomain, a receptor binding which are not lethal, but might alter cellular biochemistry or B domain and a translocation domain. The A and B domain physiology. are often associated by protein-protein interacting domains. The Spectrum of toxicity toward different Species can be The receptor binding domains of the present invention are 55 altered by fusing domains to pesticidal or auxiliary proteins useful for delivering any protein, toxin, enzyme, transcrip which recognize cell Surface receptors from other species. tion factor, nucleic acid, chemical or any other factor into Such domains might include (but are not limited to) target insects having a receptor recognized by the receptor antibodies, transferrin, hormones, or peptide Sequences iso binding domain of the binary toxins described in this patent. lated from phage displayed affinity Selectable libraries. Also, Similarly, Since binary toxins have translocation domains 60 peptide Sequences which are bound to nutrients, Vitamins, which penetrate phoSopholipid bilayer membranes and hormones, or other chemicals that are transported into cells escort cytotoxins acroSS those membranes, Such transloca could be used to alter the Spectrum of toxicity. Similarly, any tion domains may be useful in escorting any protein, toxin, other protein or chemical which binds a cell Surface receptor enzyme, transcription factor, nucleic acid, chemical or any or the membrane and could be internalized might be used to other factor acroSS a phospholipid bilayer Such as the plasma 65 alter the spectrum of activity of VIP1 and VIP2. membrane or a vesicle membrane. The translocation domain The pesticidal proteins of the present invention are those may itself perforate membranes, thus having toxic or insec proteins which confer a Specific pesticidal property. Such 5,770,696 11 12 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 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, pesticidal Synthesized utilizing plant preferred codons. That is the protein. Such a pesticidal protein which includes the auxil preferred codon for a particular host is the Single codon iary proteins as one or more of its component polypeptides which most frequently encodes that amino acid in that host. may vary in molecular weight, having at least a molecular The maize preferred codon, for example, for a particular weight of 50 kDa up to at least 200 kDa, preferably about amino acid may be derived from known gene Sequences 100 kDa to 150 kDa. 15 from maize. Maize codon usage for 28 genes from maize An auxiliary protein may be used in combination with the plants is found in Murray et al. (1989), Nucleic Acids pesticidal proteins of the invention to enhance activity or to Research 17:477-498, the disclosure of which is incorpo activate the pesticidal protein. To determine whether the rated herein by reference. Synthetic genes can also be made auxiliary protein will affect activity, the pesticidal protein based on the distribution of codons a particular host uses for can be expressed alone and in combination with the auxil a particular amino acid. iary protein and the respective activities compared in feed In this manner, the nucleotide Sequences can be optimized ing assays for pesticidal activity. for expression in any plant. It is recognized that all or any It may be beneficial to Screen Strains for potential pesti part of the gene Sequence may be optimized or Synthetic. cidal activity by testing activity of the Strain alone and in That is, Synthetic or partially optimized Sequences may also combination with the auxiliary protein. In Some instances an 25 be used. auxiliary protein in combination with the native proteins of In like manner, the nucleotide Sequences can be optimized the Strains yields pesticidal activity where none is seen in the for expression in any microorganism. For Bacillus preferred absence of an auxiliary protein. codon usage, see, for example U.S. Pat. No. 5,024,837 and The auxiliary protein can be modified, as described above, Johansen et al. (1988) Gene 65:293–304. by various methods known in the art. Therefore, for pur Methodologies for the construction of plant expression poses of the invention, the term “Vegetative Insecticidal cassettes as well as the introduction of foreign DNA into Protein” (VIP) encompasses those proteins produced during plants are described in the art. Such expression cassettes vegetative growth which alone or in combination can be may include promoters, terminators, enhancers, leader used for pesticidal activity. This includes pesticidal proteins, 35 Sequences, introns and other regulatory Sequences operably auxiliary proteins and those proteins which demonstrate linked to the pesticidal protein coding Sequence. It is further activity only in the presence of the auxiliary protein or the recognized that promoters or terminators of the VIP genes polypeptide components of these proteins. can be used in expression cassettes. It is recognized that there are alternative methods avail Generally, for the introduction of foreign DNA into plants able to obtain the nucleotide and amino acid Sequences of 40 Ti plasmid vectors have been utilized for the delivery of the present proteins. For example, to obtain the nucleotide foreign DNA as well as direct DNA uptake, liposomes, Sequence encoding the pesticidal protein, coSmid clones, electroporation, micro-injection, and the use of microprojec which express the pesticidal protein, can be isolated from a tiles. Such methods had been published in the art. See, for genomic library. From larger active coSmid clones, Smaller example, Guerche et al., (1987) Plant Science 52:111-116; Subclones can be made and tested for activity. In this 45 Neuhause et al., (1987) Theor. App. Genet. 75:30–36; Klein manner, clones which express an active pesticidal protein et al., (1987) Nature 327: 70–73; Howell et al., (1980) can be sequenced to determine the nucleotide Sequence of Science 208:1265; Horsch et al., (1985) Science 227: the gene. Then, an amino acid Sequence can be deduced for 1229–1231; DeBlock et al., (1989) Plant Physiology the protein. For general molecular methods, See, for 91:694-701, Methods for Plant Molecular Biology example, Molecular Cloning, A Laboratory Manual, Second 50 (Weissbach and Weissbach, eds.) Academic Press, Inc. Edition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring (1988); and Methods in Plant Molecular Biology (Schuler Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and Zielinski, eds.) Academic Press, Inc. (1989). See also and the references cited therein. U.S. patent application Ser. No. 08/008,374 herein incorpo The present invention also encompasses nucleotide rated by reference. See also, EPA 0193259 and EPA Sequences from organisms other than Bacillus, where the 55 0451878A1. It is understood that the method of transforma nucleotide Sequences are isolatable by hybridization with the tion will depend upon the plant cell to be transformed. Bacillus nucleotide Sequences of the invention. Proteins It is further recognized that the components of the expres encoded by Such nucleotide Sequences can be tested for Sion cassette may be modified to increase expression. For pesticidal activity. The invention also encompasses the pro example, truncated Sequences, nucleotide Substitutions or teins encoded by the nucleotide Sequences. Furthermore, the 60 other modifications may be employed. See, for example invention encompasses proteins obtained from organisms Perlak et al. (1991) Proc. Natl. Acad. Sci. USA other than Bacillus wherein the protein cross-reacts with 88:3324–3328; Murray et al., (1989) Nucleic Acids Research antibodies raised against the proteins of the invention. Again 17:477-498; and WO 91/16432. the isolated proteins can be assayed for pesticidal activity by The construct may also include any other necessary the methods disclosed herein or others well-known in the art. 65 regulators Such as terminators, (Guerineau et al., (1991), Once the nucleotide Sequences encoding the pesticidal Mol. Gen. Genet., 226:141–144; Proudfoot, (1991), Cell, proteins of the invention have been isolated, they can be 64:671-674; Sanfacon et al., (1991), Genes Dev., 5,770,696 13 14 5:141–149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Xantho monas, Stre p to my c e S, Rhizobium, Munroe et al., (1990), Gene, 91:151-158; Ballas et al et al., Rhodopseudomonas, Methylius, Agrobacterium, (1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, (1987), Nucleic Acid Res., 15:9627-9639); plant transla Leuconostoc, and Alcaligenes, fungi, particularly yeast, e.g., tional consensus sequences (Joshi, C. P., (1987), Nucleic Saccharomyces, Cryptococcus, Kluyveromyce S, Acids Research, 15:6643-6653), introns (Luehrsen and Sporobolomyces, Rhodotorula, and Aureobasidium. Of par Walbot, (1991), Mol. Gen. Genet., 225:81–93) and the like, ticular interest are such phytosphere bacterial Species as operably linked to the nucleotide sequence. It may be Pseudomonas Syringae, Pseudomonasfluorescens, Serratia beneficial to include 5' leader Sequences in the expression marce scens, Acetobacter xylinum, Agrobacteria, cassette construct. Such leader Sequences can act to enhance Rhodopseudomonas Spheroides, Xanthomonas Campestris, translation. Translational leaders are known in the art and Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli include: and Azotobacter vinlandii; and phytosphere yeast Species such as Rhodotorula rubra, R. glutinis, R. marina, R. Picornavirus leaders, for example, EMCV leader aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, (encephalomyocarditis 5' noncoding region) (Elroy-Stein, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA 15 Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, 86:6126–6130); and Aureobasidium pollulans. Of particular interest are the Potyvirus leaders, for example, TEV leader (Tobacco Etch pigmented microorganisms. Virus) (Allison et al., (1986); MDMV leader (Maize Dwarf A number of ways are available for introducing a gene Mosaic Virus); Virology, 154:9-20), and expressing the pesticidal protein into the microorganism Human inmunoglobulin heavy-chain binding protein host under conditions which allow for stable maintenance (BiP), (Macejak, D. G., and Sarnow, P., (1991), Nature, and expression of the gene. For example, expression cas 353:90-94; settes can be constructed which include the DNA constructs Untranslated leader from the coat protein MRNA of of interest operably linked with the transcriptional and alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and 25 translational regulatory signals for expression of the DNA Gehrke, L., (1987), Nature, 325:622-625; constructs, and a DNA sequence homologous with a Tobacco mosaic virus leader (TMV), (Gallie, D. R. et al., sequence in the host organism, whereby integration will (1989), Molecular Biology of RNA pages 237-256; and occur, and/or a replication System which is finctional in the host, whereby integration or stable maintenance will occur. Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, Transcriptional and translational regulatory signals S. A. et al., (1991), Virology, 81:382–385. See also, Della include but are not limited to promoter, transcriptional Cioppa et al., (1987), Plant Physiology, 84:965–968. initiation start site, operators, activators, enhancers, other A plant terminator may be utilized in the expression regulatory elements, ribosomal binding sites, an initiation cassette. See, Rosenberg et al., (1987), Gene, 56:125; codon, termination signals, and the like. See, for example, Guerineau et al., (1991), Mol. Gen. Genet., 226:141-144, 35 U.S. Pat. No. 5,039,523; U.S. Pat. No. 4,853,331; EPO Proudfoot, (1991), Cell, 64.671-674; Sanfacon et al., 048.0762A2; Sambrook et al. Supra; Molecular Cloning, a (1991), Genes Dev., 5:141–149: Mogen et al., (1990), Plant Laboratory Manual, Maniatis et al. (eds) Cold Spring Harbor Cell, 2:1261–1272; Munroe et al., (1990), Gene, Laboratory, Cold Spring Harbor, N.Y. (1982); Advanced 91:151-158: Ballas et al., (1989), Nucleic Acids Res., Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor 17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 40 Laboratory, Cold Spring Harbor, N.Y. (1980); and the ref 15:9627-9639. erences cited therein. For tissue specific expression, the nucleotide Sequences of Suitable host cells, where the pesticide-containing cells the invention can be operably linked to tissue specific will be treated to prolong the activity of the toxin in the cell promoters. See, for example, U.S. Pat. No. 5,625,136 herein when the then treated cell is applied to the environment of incorporated by reference. 45 the target pest(s), may include either prokaryotes or It is recognized that the genes encoding the pesticidal eukaryotes, normally being limited to those cells which do proteins can be used to transform insect pathogenic organ not produce substances toxic to higher organisms, Such as isms. Such organisms include Baculoviruses, fingi, mammals. However, organisms which produce Substances protozoa, bacteria and nematodes. toxic to higher organisms could be used, where the toxin is The Bacillus strains of the invention may be used for 50 unstable or the level of application sufficiently low as to protecting agricultural crops and products from pests. avoid any possibility of toxicity to a mammalian host. AS Alternatively, a gene encoding the pesticide may be intro hosts, of particular interest will be the prokaryotes and the duced via a suitable vector into a microbial host, and Said lower eukaryotes, such as fungi. Illustrative prokaryotes, host applied to the environment or plants or animals. Micro both Gram-negative and -positive, include organism hosts may be selected which are known to occupy 55 Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, the “phytosphere” (phylloplane, phyllosphere, rhizosphere, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, Such as and/or rhizoplana) of one or more crops of interest. These Rhizobium; Spirillaceae, such as photobacterium, microorganisms are selected So as to be capable of Success Zymomonas, Serratia, Aeromonas, Vibrio, DeSulfoVibrio, fully competing in the particular environment with the Spirillum; Lactobacillaceae; Pseudomonadaceae, Such as wild-type microorganisms, provide for stable maintenance 60 Pseudomonas and Acetobacter, Azotobacteraceae and Nitro and expression of the gene expressing the polypeptide bacteraceae. Among eukaryotes are fungi, Such as Phyco pesticide, and, desirably, provide for improved protection of mycetes and Ascomycetes, which includes yeast, Such a the pesticide from environmental degradation and Saccharomyces and Schizosaccharromyces, and Basidi inactivation. omycetes yeast, such as Rhodotorula, Aureobasidium, Such microorganisms include bacteria, algae, and fungi. 65 Sporobolomyces, and the like. Of particular interest are microorganisms, such as bacteria, Characteristics of particular interest in Selecting a host e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, cell for purposes of production include ease of introducing 5,770,696 15 16 the protein gene into the host, availability of expression for other Signal peptides, for example lipoprotein Signal Systems, efficiency of expression, Stability of the protein in peptide (G. Duffaud, P. March and M. Inouye, Methods in the host, and the presence of auxiliary genetic capabilities. Enzymology. 153:492 (1987)). Characteristics of interest for use as a pesticide microcapsule Specifically, unique BamHI restriction sites can be intro include protective qualities for the pesticide, Such as thick 5 duced at the amino-terminal and carboxy-terminal ends of cell walls, pigmentation, and intracellular packaging or the VIP coding Sequences using Standard methods known in formation of inclusion bodies, leaf affinity; lack of mam the art. These BamHI fragments can be cloned, in frame, into malian toxicity; attractiveness to pests for ingestion; ease of the vector plN-III-ompa1, A2 or A3 (J. Ghrayeb, H. killing and fixing without damage to the toxin; and the like. Kimura, M. Takahara, H. Hsiung, Y. Masui and M. Inouye, Other considerations include ease of formulation and EMBO.J., 3:2437–2442 (1984)) thereby creating ompA:VIP handling, economics, Storage Stability, and the like. fusion gene which is Secreted into the periplasmic Space. Host organisms of particular interest include yeast, Such The other restriction sites in the polylinker of plN-III-ompA as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., can be eliminated by Standard methods known in the art So and Sporobolomyces sp., phylloplane organisms. Such as that the VIP amino-terminal amino acid coding Sequence is Pseudomonas sp., Erwinia Sp. and Flavobacterium sp., or 15 directly after the omp A Signal peptide cleavage site. Thus, Such other organisms as Escherichia, LactoBacillus sp., the secreted VIP sequence in E. coli would then be identical Bacillus sp., and the like. Specific organisms include to the native VIP sequence. Pseudomonas aelurginosa, Pseudomonasfluorescens, Sac When the VIP native signal peptide is not needed for charomyces cerevisiae, Bacillus thuringiensis, Escherichia proper folding of the mature protein, Such signal Sequences coli, Bacillus Subtilis, and the like. can be removed and replaced with the omp A Signal VIP genes can be introduced into micro-organisms that Sequence. Unique BamHI restriction sites can be introduced multiply on plants (epiphytes) to deliver VIP proteins to at the amino-termini of the proprotein coding Sequences potential target pests. Epiphytes can be gram-positive or directly after the Signal peptide coding Sequences of VIP and gram-negative bacteria for example. at the carboxy-termini of VIP coding sequence. These Root colonizing bacteria, for example, can be isolated 25 BamHI fragments can then be cloned into the plN-III-ompa from the plant of interest by methods known in the art. vectors as described above. Specifically, a Bacillus cereuS Strain which colonizes roots General methods for employing the Strains of the inven could be isolated from roots of a plant (for example see J. tion in pesticide control or in engineering other organisms as Handelsman, S. Raffel, E. Mester, L. Wunderlich and C. pesticidal agents are known in the art. See, for example U.S. Grau, Appl. Environ. Microbiol. 56:713–718, (1990)). VIP1 Pat. No. 5,039,523 and EPO480762A2. and/or VIP2 could be introduced into a root colonizing VIPs can be fermented in a bacterial host and the resulting Bacillus cereus by standard methods known in the art. bacteria processed and used as a microbial Spray in the same Specifically, VIP1 and/or VIP2 derived from Bacillus manner that Bacillus thuringiensis Strains have been used as cereus Strain AB78 can be introduced into a root colonizing 35 insecticidal sprays. In the case of a VIP(s) which is secreted Bacillus cereuS by means of conjugation using Standard from Bacillus, the Secretion signal is removed or mutated methods (J. Gonzalez, B. Brown and B. Carlton, Proc. Natl. using procedures known in the art. Such mutations and/or Acad. Sci. 79:6951-6955, (1982)). deletions prevent secretion of the VIP protein(s) into the Also, VIP1 and/or VIP2 or other VIPs of the invention can growth medium during the fermentation process. The VIPs be introduced into the root colonizing Bacillus by means of 40 are retained within the cell and the cells are then processed electro-transformation. Specifically, VIPs can be cloned into to yield the encapsulated VIPs. Any Suitable microorganism a shuttle vector, for example, pHT3101 (D. Lereclus et al., can be used for this purpose. PSuedomonas has been used to FEMS Microbiol. Letts., 60:211-218 (1989)) as described in express Bacillus thuringiensis endotoxins as encapsulated Example 10. The shuttle vector pHT3101 containing the proteins and the resulting cells processed and Sprayed as an coding Sequence for the particular VIP can then be trans 45 insecticide. (H. Gaertner et al. 1993, In Advanced Engi formed into the root colonizing Bacillus by means of elec neered Pesticides, L. Kim ed.) troporation (D. Lereclus et al. 1989, FEMS Microbiol. Letts. Various strains of Bacillus thuringiensis are used in this 60:211-218). manner. Such Bt Strains produce endotoxin protein(s) as well Expression Systems can be designed So that VIP proteins as VIPs. Alternatively, such strains can produce only VIPs. are Secreted outside the cytoplasm of gram negative bacteria, 50 A sporulation deficient strain of Bacillus Subtilis has been E. coli, for example. Advantages of having VIP proteins shown to produce high levels of the CryIIIA endotoxin from secreted are (1) it avoids potential toxic effects of VIP Bacillus thuringiensis (Agaisse, H. and Lereclus, D., proteins expressed within the cytoplasm and (2) it can “Expression in Bacillus Subtilis of the Bacillus thuringiensis increase the level of VIP protein expressed and (3) can aid Cry IIIA toxin gene is not dependent on a sporulation in efficient purification of VIP protein. 55 Specific Sigma factor and is increased in a SpoOA mutant', VIP proteins can be made to be secreted in E. coli, for J. Bacteriol., 176:4734–4741 (1994)). A similar spoCA example, by fusing an appropriate E. coli Signal peptide to mutant can be prepared in Bacillus thuringiensis and used to the amino-terminal end of the VIP Signal peptide or replac produce encapsulated VIPs which are not secreted into the ing the VIP Signal peptide with the E. coli Signal peptide. medium but are retained within the cell. Signal peptides recognized by E. coli can be found in 60 To have VIPs maintained within the Bacillus cell the proteins already known to be Secreted in E. coli, for example Signal peptide can be disarmed So that it no longer functions the Omp A protein (J. Ghrayeb, H. Kimura, M. Takahara, Y. as a Secretion signal. Specifically, the putative signal peptide Masui and M. Inouye, EMBO J., 3:2437–2442 (1984)). for VIP1 encompasses the first 31 amino acids of the protein OmpA is a major protein of the E. coli outer membrane and with the putative consensus cleavage site, Ala-X-Ala, at the thus its signal peptide is thought to be efficient in the 65 C-terminal portion of this sequence (G. von Heijne, J. Mol. translocation process. Also, the Omp A Signal peptide does Biol. 184:99-105 (1989)) and the putative signal peptide for not need to be modified before processing as may be the case VIP2 encompasses the first 40 amino acids of the protein 5,770,696 17 18 with the putative cleavage Site after Ala-10. The cleavage tion (NRRL), Northern Regional Research Center, 1815 sites in either VIP1 or VIP2 can be mutated with methods North University Street, Peoria, Ill. 61604, USA and given known in the art to replace the cleavage Site consensus Accession No. NRRL B-21058. Sequence with alternative amino acids that are not recog A fraction protein has been substantially purified from the nized by the Signal peptidases. 5 B. cereus Strain. This purification of the protein has been Alternatively, the signal peptides of VIP1, VIP2 and/or verified by SDS-PAGE and biological activity. The protein other VIPs of the invention can be eliminated from the has a molecular weight of about 60 to about 100 kDa, Sequence thereby making them unrecognizable as Secretion particularly about 70 to about 90 kDa, more particularly proteins in Bacillus. Specifically, a methionine Start Site can about 80 kDa, hereinafter VIP. be engineered in front of the proprotein Sequence in VIP1, Amino-terminal Sequencing has revealed the N-terminal Starting at ASp32, or the proprotein Sequence in VIP2, amino-acid Sequence to be: Starting at Glu41 using methods known in the art. NH-Lys-Arg-Glu-Ile-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp VIP genes can be introduced into micro-organisms that Ser-Ile-Pro- (SEQ ID NO:8) multiply on plants (epiphytes) to deliver VIP proteins to where ASX represents either Asp or ASn. The entire amino potential target pests. Epiphytes can be gram-positive or 15 acid sequence is given in SEQID NO:7. The DNA sequence gram-negative bacteria for example. which encodes the amino acid sequence of SEQ ID NO:7 is The Bacillus Strains of the invention or the microorgan disclosed in SEO ID NO:6. isms which have been genetically altered to contain the An oligonuleotide probe for the region of the gene encod pesticidal gene and protein may be used for protecting ing amino acids 3–9 of the NH2-terminus has been gener agricultural crops and products from pests. In one aspect of ated. The probe was Synthesized based on the codon usage the invention, whole, i.e., unlysed, cells of a toxin (pesticide) of a Bacillus thuringiensis (Bt) Ö-endotoxin gene. The -producing organism are treated with reagents that prolong nucleotide Sequence of the oligonucleotide probe used for the activity of the toxin produced in the cell when the cell is Southern hybridizations was as follows: applied to the environment of target pest(s). 25 5'-GAAATT GAT CAA GAT ACN GAT-3' (SEQ ID NO:9) Alternatively, the pesticides are produced by introducing where N represents any base. a heterologous gene into a cellular host. Expression of the In addition, the DNA probe for the Bc AB78 VIP1 gene heterologous gene results, directly or indirectly, in the described herein, permits the Screening of any Bacillus intracellular production and maintenance of the pesticide. strain or other organisms to determine whether the VIP1 These cells are then treated under conditions that prolong the gene (or related gene) is naturally present or whether a activity of the toxin produced in the cell when the cell is particular transformed organism includes the VIP1 gene. applied to the environment of target pest(s). The resulting The invention now being generally described, the same product retains the toxicity of the toxin. These naturally will be better understood by reference to the following encapsulated pesticides may then be formulated in accor detailed examples that are provided for the purpose of dance with conventional techniques for application to the 35 illustration and are not to be considered limiting of the environment hosting a target pest, e.g., Soil, Water, and invention unless So Specified. foliage of plants. See, for example EPA 0192319, and the A Standard nomenclature has been developed based on the references cited therein. Sequence identity of the proteins encompassed by the The active ingredients of the present invention are nor present invention. The gene and protein names for the mally applied in the form of compositions and can be 40 detailed examples which follow and their relationship to the applied to the crop area or plant to be treated, Simultaneously names used in the parent application are shown below. or in Succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be Gene/Protein Name Selective herbicides, insecticides, fungicides, bactericides, 45 under Standard Gene/Protein nematicides, mollusicides or mixtures of Several of these Nomenclature Name in Parent Description of Protein preparations, if desired, together with further agriculturally VIP1A(a) VIP1 VIP1 from strain AB78 as acceptable carriers, Surfactants or application-promoting disclosed in SEO ID NO: 5. adjuvants customarily employed in the art of formulation. VIP2A(a) VIP2 VIP2 from strain AB78 as disclosed in SEO ID NO: 2. Suitable carriers and adjuvants can be Solid or liquid and 50 VIP1A(b) VIP1 homolog VIP1 from Bacillus thuringiensis correspond to the Substances ordinarily employed in formu var. tenebrionis as disclosed lation technology, e.g. natural or regenerated mineral in SEO ID NO: 21. Substances, Solvents, dispersants, wetting agents, tackifiers, VIP2A(b) VIP2 homolog VIP2 from Bacillus thuringiensis var. tenebrionis as disclosed binders or fertilizers. in SEO ID NO: 2O. Preferred methods of applying an active ingredient of the 55 VIP3A(a) VIP from strain AB88 as present invention or an agrochemical composition of the disclosed in SEO ID NO: 28 of the present application present invention which contains at least one of the pesti VIP3A(b) VIP from strain AB424 as cidal proteins produced by the bacterial Strains of the present disclosed in SEO ID NO:31 of invention are leaf application, Seed coating and Soil appli the present application cation. The number of applications and the rate of applica 60 tion depend on the intensity of infestation by the correspond ing pest. EXPERIMENTAL In one embodiment of the invention a Bacillus cereuS Example 1 microorganism has been isolated which is capable of killing Diabrotica virgifera virgifera, and Diabrotica longicornis 65 AB78 Isolation and Characterization barberi. The novel B. cereus strain AB78 has been deposited Bacillus cereus strain AB78 was isolated as a plate in the Agricultural Research Service, Patent Culture Collec contaminant in the laboratory on T3 media (per liter: 3 g 5,770,696 19 tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05M Sodium -continued phosphate (pH 6.8), and 0.005 g MnOl; Travers, R. S. 1983). During log phase growth, AB78 gave significant KHPO, 2.1 g/l KHPO 14.7 g/l activity against Western corn rootworm. Antibiotic activity pH 7.4 against gram-positive Bacillus spp. was also demonstrated 5 (Table 12). The potassium phosphate was added to the autoclaved TABLE 12 broth after cooling. Flasks were incubated at 30° C. on a rotary shaker at 250 rpm for 24 h.-36 h, which represents an Antibiotic activity of AB78 culture Supernatant early to mid-log growth phase. Zone of inhibition (cm The above procedure can be readily Scaled up to large fermentors by procedures well known in the art. Bacteria tested ABA8 Streptomycin During vegetative growth, usually 24-36 h. after Starting E. coli O.O 3.0 the culture, which represents an early to mid-log growth B. megaterium 1.1 2.2 15 phase, AB78 bacteria were centrifuged from the culture B. mycoides 1.3 2.1 B. cereus CB 1.O 2.O Supernatant. The culture Supernatant containing the active B. cereus 11950 1.3 2.1 protein was used in bioassayS. B. cereus 14579 1.O 2.4 B. cerets AB78 O.O 2.2 Example 3 Bt var. israelensis 1.1 2.2 Bt var. tenebrionis O.9 2.3 Insect Bioassays B. cereuS Strain AB78 was tested against various insects Morphological characteristics of AB78 are as follows: as described below. Western, Northern and Southern corn rootworm, Vegetative rods straight, 3.1-5.0 mm long and 0.5-2.0 25 mm wide. Cells with rounded ends, Single in Short Diabrotica virgifera virgifera, D. longcornis barberi and D. chains. Single Subterminal, cylindrical-oval, endospore undecempunctata howardi, respectively: dilutions were formed per cell. No parasporal crystal formed. Colonies made of AB78 culture Supernatant grown 24-36 h., mixed opaque, erose, lobate and flat. No pigments produced. with molten artificial diet (Marrone et al. (1985) J. of Cells motile. Flagella present. Economic Entomology 78:290–293) and allowed to solidify. Solidified diet was cut and placed in dishes. Neonate larvae Growth characteristics of AB78 are as follows: were placed on the diet and held at 30° C. Mortality was Facultative anaerobe with optimum growth temperature recorded after 6 dayS. of 21°–30° C. Will grow at 15, 20°, 25°, 30° and 37° E. coli clone bioaSSay. E. coli cells were grown overnight C. Will not grow above 40° C. Grows in 5–7% NaCl. in broth containing 100 ug/ml ampicillin at 37 C. Ten ml Table 13 provides the biochemical profile of AB78. 35 culture was Sonicated 3x for 20 sec each. 500 ul of Sonicated culture was added to molten western corn rootworm diet. TABLE 13 Colorado potato beetle, Leptinotarsa decemlineata: dilu Biochemical characteristics of B. cereus strain AB78. tions in Triton X-100 (to give final concentration of 0.1% Acid from L-arabinose - Methylene blue reoxidized -- 40 TX-100) were made of AB78 culture Supernatant grown Gas from L-arabinose - Nitrate reduced -- 24-36 h. Five cm potato leaf pieces were dipped into these Acid from D-xylose - NO reduced to NO, -- dilutions, air dried, and placed on moistened filter paper in Gas from D-xylose - VP -- plastic dishes. Neonate larvae were placed on the leaf pieces Acid from D-glucose + HO decomposed -- Gas from D-glucose - Indole and held at 30° C. Mortality was recorded after 3-5 days. Acid from lactose - Tyrosine decomposed -- 45 Yellow mealworm, Tenebrio molitor: dilutions were made Gas from lactose - Dihydroxiacetone of AB78 culture Supernatant grown 24-36 h., mixed with Acid from Sucrose - Litmus milk acid Gas from sucrose - Litmus milk coagulated molten artificial diet (BioServ #F9240) and allowed to Acid from D-mannitol - Litmus milk alkaline Solidify. Solidified diet was cut and placed in plastic dishes. Gas from D-mannitol - Litmus milk peptionized Neonate larvae were placed on the diet and held at 30° C. Proprionate utilization + Litmus milk reduced 50 Mortality was recorded after 6-8 days. Citrate utilization + Casein hydrolyzed -- Hippurate hydrolysis w Starch hydrolyzed -- European corn borer, black cutworm, tobacco budworm, Methylene blue reduced + Gelatin liquidified -- tobacco hornworm and beet armyworm; Ostrinia nubilalis, Lecithinase produced w Agrotis ipsilon, Heliothis virescens, Manduca Sexta and Spodoptera exigua, respectively: dilutions, in TX-100 (to w = weak reaction 55 give final concentration of 0.1% TX-100), were made of AB78 culture Supernatant grown 24-36 hrs. 100 ul was Example 2 pipetted onto the surface of 18 cm of solidified artificial diet (BioServ #F9240) and allowed to air dry. Neonate larvae Bacterial Culture were then placed onto the surface of the diet and held at 30 60 C. Mortality was recorded after 3–6 days. A Subculture of Bc strain AB78 was used to inoculate the Northern house mosquito, Culex.pipiens:-dilutions were following medium, known as TB broth: made of AB78 culture Supernatant grown 24-36 h. 100 ul was pipetted into 10 ml water in a 30 ml plastic cup. Third Tryptone 12 g/l instar larvae were added to the water and held at room Yeast Extract 24 g/l 65 temperature. Mortality was recorded after 24-48 hours. The Glycerol 4 ml/ spectrum of entomocidal activity of AB78 is given in Table 14. 5,770,696 21 22

TABLE 1.4 TABLE 16 Activity of AB78 culture Supernatant against various insect species Activity of AB78 culture Supernatant against neonate western corn rootworm Insect species tested to date Order Activity Culture supernatant Percent concentration (ul/ml) WCRW mortality Western corn rootworm Col ------(Diabrotica virgifera 1OO 1OO virgifera) 25 87 Northern corn rootworm Col ------1O 8O (Diabrotica longicornis 5 40 barberi) 2.5 2O Southern corn rootworm Col 1. 6 (Diabrotica undecimpunctata O O howardi) Colorado potato beetle Col 15 (Leptinotarsa decenlineata) Yellow mealworm Col The LCso was calculated to be 6.2 ul of culture Superna (Tenebrio molitor) tant per ml of Western corn rootworm diet. European corn borer Lep The cell pellet was also bioassayed and had no activity (Ostrinia nubilalis) against WCRW. Thus, the presence of activity only in the Tobacco budworm Lep (Heliothis virescens) Supernatant indicates that this VIP is an exotoxin. Tobacco hornworm Lep (Manduca Sexta) Example 4 Beet armyworm Lep (Spodoptera exigua) Isolation and Purification of Corn Rootworm Active Black cutworm Lep Proteins from AB78. (Agrotis ipsilon) 25 Northern house mosquito Dip Culture media free of cells and debris was made to 70% Culex pipiensplp saturation by the addition of solid ammonium sulfate (472 g/L). Dissolution was at room temperature followed by cooling in an ice bath and centrifugation at 10,000xg for The newly discovered B. cereus strain AB78 showed a thirty minutes to pellet the precipitated proteins. The Super Significantly different spectrum of insecticidal activity as natant was discarded and the pellet was dissolved in /10 the compared to known coleopteran active Ö-endotoxins from original volume of 20 mM TRIS-HC1 at pH 7.5. The Bt. In particular, AB78 showed more selective activity dissolved pellet was desalted either by dialysis in 20 mM against beetles than known coleopteran-active Bt Strains in TRIS-HCl pH 7.5, or passing through a desalting column. that it was Specifically active against Diabrotica spp. More 35 The desalted material was titrated to pH 3.5 using 20 mM Specifically, it was most active against D. Virgifera virgifera Sodium citrate pH 2.5. Following a thirty minute room and D. longicornis barberi but not D. undecimpunctata temperature incubation the Solution was centrifuged at howardi. 3000xg for ten minutes. The Supernatant at this stage contained the greatest amount of active protein. 40 A number of Bacillus strains were bioassayed for activity Following neutralization of the pH to 7.0 the Supernatant during vegetative growth (Table 15) against western corn was applied to a Mono-Q, anion exchange, column equili rootworm. The results demonstrate that AB78 is unique in brated with 20 mM TRIS pH 7.5 at a flow rate of 300 that activity against Western corn rootworm is not a general mL/min. The column was developed with a stepwise and phenomenon. 45 linear gradient employing 400 mM NaCl in 20 mMTRIS pH 7.5. TABLE 1.5 Bioassay of the column fractions and SDS-PAGE analysis were used to confirm the active fractions. SDS-PAGE analy Activity of culture Supernatants from various Bacillus spp. against western sis identified the biologically active protein as having com corn rootWOrm 50 ponents of a molecular weight in the range of about 80 kDa Percent and 50 kDa. Bacillus strain WCRW mortality Example 5 B. cereus AB78 (Bat. 1) 1OO B. cereus AB78 (Bat. 2) 1OO Sequence Analysis of the Corn Rootworm Active Protein B. cereus (Carolina Bio.) 12 55 B. cerets ATCC 11950 12 The 80 kDa component isolated by SDS-PAGE was B. cerets ATCC 14579 8 B. mycoides (Carolina Bio.) 3O transferred to PVDF membrane and was subjected to amino B. popilliae 28 terminal Sequencing as performed by repetitive Edman B. thuringiensis HD135 41 cycles on an ABI 470 pulsed-liquid Sequencer. Transfer was B. thuringiensis HD.191 9 60 carried out in 10 mM CAPS buffer with 10% methanol pH B. thuringiensis GC91 4 11.0 as follows: B. thuringiensis is realensis 24 Water Control 4 Incubation of the gel following electrophoresis was done in transfer buffer for five minutes. ProBlott PVDF mem brane was wetted with 100% MeOH briefly then equili 65 brated in transfer buffer. The sandwich was arranged Specific activity of AB78 against western corn rootworm between foam Sponges and filter paper Squares with the is provided in Table 16. configuration of cathode-gel-membrane-anode. 5,770,696 23 24 Transfer was performed at 70 V constant voltage for 1 hour. TABLE 1.7 Following transfer, the membrane was rinsed with water Bacillus insecticidal crystal protein gene primers tested by PCR against and stained for two minutes with 0.25% Coomassie Blue ABF8 DNA. R-250 in 50% MeOH Primers Tested Product Produced Destaining was done with several rinses with 50% MeOH 2 sets specific for CryIIIA Negative 40% water 10% acetic acid. CryIIIB Negative 2 sets specific for CryIA Negative CryIA(a) Negative Following destaining the membrane was air dried prior to CryIA(b) specific Negative excision of the bands for Sequence analysis. A BlottCar CryIB Negative tridge and appropriate cycles were utilized to achieve maxi CryIC specific Negative CryIE specific Negative mum efficiency and yield. Data analysis was performed 2 sets specific for B. Sphaericus Negative using model 610 Sequence Analysis Software for identifying 15 2 sets specific for CryIV Negative and quantifying the PTH-amino acid derivatives for each Bacillus control (PI-PLC) Positive Sequential cycle. The N-terminal sequence was determined to be: NH2 Example 9 Lys-Arg-Glu-Ile-Asp-Glu-Asp-Thr-Asp-Thr-ASX-Gly-Asp Ser-Ile-Pro-(SEQ ID NO:8) where ASX represents Asp or Cosmid Cloning of Total DNA from B. cereus ASn. The complete amino acid Sequence for the 80 kDa Strain AB78 component is disclosed in SEQ ID NO:7. The DNA The VIP1A(a) gene was cloned from total DNA prepared sequence which encodes SEQ ID NO:7 is disclosed in SEQ from strain AB78 as follows: ID NO:6. 25 Isolation of AB78 DNA was as follows: 1. Grow bacteria in 10 ml L-broth overnight. (Use 50 ml Example 6 Sterile centrifuge tube) 2. Add 25 ml of fresh L-broth and amplicillin (30 tug/ml). 3. Grow cells 2-6 h. at 30° C. with shaking. Construction of DNA Probe 4. Spin cells in a 50 ml polypropylene orange cap tube in IEC benchtop clinical centrifuge at % Speed. An oligonucleotide probe for the region of the gene 5. Resuspend cell pellet in 10 ml TES (TES=50 mM TRIS encoding amino acids 3–9 of the N-terminal Sequence pH 8.0, 100 mM EDTA, 15 mM NaCl). (Example 5) was generated. The probe was Synthesized . Add 30 mg lysozyme and incubate 2 hrs at 37 C. based on the codon usage of a Bacillus thuringiensis (Bt) 35 g . Add 200ul 20% SDS and 400 ul Proteinase K stock (20 Ö-endotoxin gene. The nucleotide Sequence mg/ml). Incubate at 37 C. 8. Add 200 ul fresh Proteinase K. Incubate 1 hr. at 55° C. Add 5 ml TES to make 15 ml final volume. 5'-GAAATT GAT CAAGAT ACN GAT-3' (SEQ ID NO:9) 9. Phenol extract twice (10 ml phenol, spin at room tem 40 perature at % Speed in an IEC benchtop clinical was used as a probe in Southern hybridizations. The oligo centrifuge). Transfer Supernatant (upper phase) to a clean nucleotide was Synthesized using Standard procedures and tube using a wide bore pipette. equipment. 10. Extract once with 1:1 vol. phenol: chloroform/isoamyl alcohol (24:1 ratio). Example 7 45 11. Precipitate DNA with an equal volume of cold isopro panol; Centrifuge to pellet DNA. Isoelectric Point Determination of the Corn 12. Resuspend pellet in 5 ml TE. Rootworm Active Protein 13. Precipitate DNA with 0.5 ml 3M NaOAc pH 5.2 and 11 ml 95% ethanol. Place at -20° C. for 2 h. Purified protein from step 5 of the purification process 50 14. “Hook' DNA from tube with a plastic loop, transfer to was analyzed on a 3–9 p isoelectric focusing gel using the a microfuge tube, Spin, pipette off exceSS ethanol, dry in Phastgel electrophoresis System (Pharmacia). Standard oper WCUO. ating procedures for the unit were followed for both the 15. Resuspend in 0.5 ml TE. Incubate 90 min. at 65° C. to Separation and Silver Staining development procedures. The help get DNA back into Solution. 55 16. Determine concentration using Standard procedures. pI was approximated at about 4.9. Cosmid Cloning of AB78 All procedures, unless indicated otherwise, were per Example 8 formed according to Stratagene Protocol, SupercoS 1 Instruction Manual, Cat. No. 251301. PCR Data on AB78 60 Generally, the Steps were as follows: A. Sau 3A partial digestion of the AB78 DNA. PCR analysis (See, for example U.S. patent application B. Preparation of vector DNA Ser. No. 08/008,006; and, Carozzi et al. (1991) Appl. Envi C. Ligation and packaging of DNA ron. Microbiol. 57(11):3057-3061, herein incorporated by D. Tittering the cosmid library reference.) was used to verify that the B. cereus strain AB78 65 1. Start a culture of HB101 cells by placing 50 ml of an did not contain any insecticidal crystal protein genes of B. overnight culture in 5 mls of TB with 0.2% maltose. thuringiensis or B. Sphaericus (Table 17). Incubate 3.5 hrs. at 37° C. 5,770,696 25 26 2. Spin out cells and resuspend in 0.5 ml 10 mM MgSO. this same 6 kb Cla I fragment in pBluescript SK(+) 3. Add together: (Stratagene), produces equivalent VIP1A(a) protein (by 100 ulcells western blot), and is also active against western corn 100 ul diluted packaging mixture rOOtWOrm. 100 ul 10 mM MgSO The nucleotide sequence of pCIB6022 was determined by 30 ul TB the dideoxy termination method of Sanger et al., Proc. Natl. 4. Adsorb at room temperature for 30 minutes with no Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready Shaking. Reaction Dye Deoxy Terminator Cycle Sequencing Kits and 5. Add 1 ml TB and mix gently. Incubate 30 minutes at PRISM Sequenase(R) Terminator Double-Stranded DNA 37o C. Sequencing Kit and analyzed on an ABI 373 automatic 6. Plate 200 ul onto L-amp plates. Incubate at 37 C. sequencer. The sequence is given in SEQ ID NO: 1. The 6 overnight. kb fragment encodes both VIP1A(a) and VIP2A(a), as indicated by the open reading frames described in SEQ ID At least 400 coSmid clones were Selected at random and NO:1. The sequence encoding VIP1A(a) is further disclosed Screened for activity against Western corn rootworm as 15 described in Example 3. DNA from 5 active clones and 5 in SEQ ID NO:4. The relationship between VIP1A(a) and non-active clones were used in Southern hybridizations. VIP2A(a) within the 6 kb fragment found in pCIB6022 is Results demonstrated that hybridization using the above depicted in FIG. 1 pCIB6022 was deposited with the Agri described oligonucleotide probe correlated with western cultural Research Service, Patent Culture Collection, corn rootworm activity (Table 18). (NRRL), Northern Regional Research Center, 1815 North Cosmid clones P3-12 and P5-4 have been deposited with University Street, Peoria, Ill. 61604,USA, and given the the Agricultural Research Service Patent Culture Collection Accession No. NRRL B-21222. (NRRL) and given Accession Nos. NRRL B-21061 and Example 11 NRRL B-21059 respectively. Functional Dissection of the VIP1A(a) DNA 25 TABLE 1.8 Region. To confirm that the VIP1A(a) open reading frame (ORF) Activity of AB78 cosmid clones against western corn rootworm. is necessary for insecticidal activity a translational frame Mean shift mutation was created in the gene. The restriction Clone percent mortality (N = 4) enzyme Bgl II recognizes a unique site located 857 bp into the coding region of VIP1A(a). pCIB6201 was digested with Clones which hybridize with probe Bgl II, and the single-stranded ends filled-in with DNA P1-73 47 polymerase (Klenow fragment) and dNTPS. The plasmid P1-83 64 was re-ligated and transformed into E. coli. The resulting P2-2 69 plasmid, pCIB6203, contains a four nucleotide insertion in P3-12 85 35 P5-4 97 the coding region of VIP1A(a). ppCIB6203 does not confer Clones which do not hybridize with probe WCRW insecticidal activity, confirming that VIP1A(a) is an essential component of Western corn rootworm activity. P1-2 5 P3-8 4 To further define the region necessary to encode VIP1A P3-9 12 40 (a), subclones of the VIP1A(a) and VIP2A(a) (auxiliary P3-18 O protein) region were constructed and tested for their ability P4-6 9 to complement the mutation in pCIB6203. pCIB6023 con tains the 3.7 kb Xba I-EcoRV fragment in pBluescript SK(+) (Stratagene). Western blot analysis indicates that pCIB6023 Example 10 45 produces VIP1A(a) protein of equal size and quantity as clones PL2 and pCIB6022. pCIB6023 contains the entire Identification of a 6 KB Region Active Against gene encoding the 80 kD protein. pCIB6023 was deposited Western Corn Rootworm. with the Agricultural Research Service, Patent Culture DNA from P3-12 was partially digested with restriction Collection, (NRRL), Northern Regional Research Center, enzyme Sau 3A, and ligated into the E. coli vector puC19 50 1815 North University Street, Peoria, Ill. 61604, USA, and and transformed into E. coli. A DNA probe specific for the given the Accession No. NRRL B-21223N. pCIB6206 con 80 kDa VIP1A(a) protein was synthesized by PCR ampli tains the 4.3 kb Xba I-Cla I fragment from pCIB6022 in fication of a portion of P3-12 DNA. Oligonucleotides pBluescript SK(+) (Stratagene). pCIB6206 was also depos MK113 and MK117, which hybridize to portions of VIP1A ited with the Agricultural Research Service, Patent Culture (a), were Synthesized using the partial amino acid sequence 55 Collection, (NRRL), Northern Regional Research Center, of the 80 kDa protein. Plasmid subdlones were identified by 1815 North University Street, Peoria, Ill. 61604, USA, and colony hybridization to the PCR-generated probe, and tested given the Accession No. NRRL B-21321. for activity against Western corn rootworm. One Such clone, pCIB6023, pCIB6206, and pCIB6203 do not produce PL2, hybridized to the PCR-generated fragment, and was detectable western corn rootworm activity when tested indi active against Western corn rootworm in the assay previ 60 vidually. However, a mixture of cells containing pCIB6203 ously described. (VIP1A(a)-mutated, plus VIP2A(a)) and cells containing A 6 kb Cla I restriction fragment from p 2 was cloned pCIB6023 (only VIP1A(a)) shows high activity against into the Sma I site of the E. coli-Bacillus shuttle vector pHT western corn rootworm. Similarly, a mixture of cells con 3101 (Lereclus, D. et al., FEMS Microbiology Letters taining pCIB6206 and cells containing pCIB6203 shows 60:211-218 (1989)) to yield pCIB6201. This construct con 65 high activity against Western corn rootworm. fers anti-western corn rootworm activity upon both Bacillus To further define the limits of VIP2A(a), we constructed and E.coli strains, in either orientation. pCIB6022 contains pCIB6024, which contains the entirety of VIP2A(a), but 5,770,696 27 28 lacks most of the VIP1A(a) coding region. pCIB6024 was ELISA protocol: constructed by gel purifying the 2.2 kb Cla I-Sca I restriction 1. Coat with AB78/DMSO in BBS. Incubate overnight at 4 fragment from pCIB6022, filling in the single-stranded ends C. with DNA polymerase (Klenow fragment) and dNTPs, and ... Wash plate 3x with 1x ELISA wash buffer. ligating this fragment into pBlueScript SK(+) vector Block (1% BSA & 0.05% Tween 20 in PBS) for 30 (Stratagene) digested with the enzyme Eco RV. Cells con minutes at Room Temperature. taining pCIB6024 exhibit no activity against western corn ... Wash plate 3x with 1x ELISA wash buffer. rootworm. However, a mixture of cells containing Add rat serum. Incubate 1.5 hours at 37 C. pCIB6024 and cells containing pCIB6023 shows high activ ... Wash plate 3x with 1x ELISA wash buffer. ity against western corn rootworm.(See FIG. 1). . Add goat anti-rat at a concentration of 2 ug/ml in ELISA Thus, pCIB6023 and pCIB6206 must produce a func diluent. Incubate 1 hr.at 37° C. tional VIP1A(a) gene product, while pCIB6203 and ... Wash plate 3x with 1x ELISA wash buffer. pCIB6024 must produce a functional VIP2A(a) gene prod . Add rabbit anti-goat alkaline phosphatase at 2 tug/ml in uct. These results Suggest a requirement for a gene product ELISA diluent. Incubate 1 hr.at37 C. (s) from the VIP2A(a) region, in combination with VIP1A 15 10. Wash 3X with 1x ELISA wash buffer. (a), to confer maximal western corn rootworm activity. (See 11. Add Substrate. Incubate 30 minutes at room temperature. FIG. 1.) 12. Stop with 3N NaOH after 30 minutes. Example 12 Preparation of VIP2A(a) Antisera AB78 Antibody Production A partially purified AB78 culture Supematant was sepa rated by discontinuous SDS PAGE (Novex) following Antibody production was initiated in 2 Lewis rats to allow manufacturers instructions. Separated proteins were elec for both the possibility of moving to production of hybri trophoresed to nitrocellulose (S&S #21640) as described by doma cell lines and also to produce enough Serum for limited 25 Towbin et al., (1979). The nitrocellulose was stained with Screening of genomic DNA library. Another factor was the Ponceau S and the VIP2A(a) band identified. The VIP2A(a) very limited amount of antigen available and the fact that it band was excised and emulsified in DMSO immediately could only be produced to purity by PAGE and subsequent prior to injection. A rabbit was initially immunized with electrotransfer to nitrocellulose. emulsified VIP2A(a) mixed approximately 1:1 with Fre Due to the limited availability of antigen on und's Complete adjuvant by intramuscular injection at four nitrocellulose, the nitrocellulose was emulsified in DMSO different Sites. Subsequent immunizations occurred at four and injected into the hind footpads of the animals to elicit week intervals and were identical to the first, except for the B-cell production in the popliteal lymph nodes just use of Freund Incomplete adjuvant. The first serum har upstream. A Strong reacting Serum was produced as judged vested following immunization reacted with VIP2A(a) pro by western blot analysis with the first production bleed. 35 tein. Western blot analysis of AB78 culture Supernatant Several Subsequent injections and bleeds produced enough using this antisera identifies predominately full length Serum to accomplish all of the Screening required. VIP2A(a) protein. Hybridoma production with one of the rats was then initiated. The popliteal lymph node was excised, macerated, Example 13 and the resulting cells fused with mouse myeloma 40 P3x63Ag8.653. Subsequent cell screening was accom Activation of Insecticidal Activity of Non-Active plished as described below. Four initial wells were selected BT Strains with AB78 VIP Clones. which gave the highest emulsified antigen reaction to be moved to limited dilution cloning. An additional 10 wells Adding pCIB6203 together with a 24 h culture (early to were chosen for expansion and cryoperServation. 45 mid-log phase) Supernatant from Bt Strain GC91 produces 100% mortality in Diabrotica virgifera virgifera. Neither Procedure to Emulsify AB78 on Nitrocellulose in pCIB6203 nor GC91 is active on Diabrotica virgifera vir DMSO for ELISA Screening gifera by itself. Data are shown below: After electrotransfer of AB78 samples run on PAGE to 50 nitrocellulose, the reversible strain Ponceau S is used to Visualize all protein transferred. The band corresponding to Test material Percent Diabrotica mortality AB78 toxin, previously identified and N-terminal pCIB6203 O GC91 16 Sequenced, was identified and excised from nitrocellulose. pCIB6203 + GC91 1OO Each band is approximately 1 mmx5 mm in Size to minimize 55 Control O the amount of nitrocellulose emulsified. A Single band is placed in a microfuge tube with 250 ul of DMSO and macerated using a plastic pestle (Kontes, Vineland, N.J.). To aid in emulsification, the DMSO mixture is heated for 2-3 Example 14 minutes at 37 C-45 C. Some further maceration might be 60 necessary following heating; however, all of the nitrocellu Isolation and Biological Activity of B. cereus AB81. lose should be emulsified. Once the AB78 sample is emulsified, it is placed on ice. In preparation for microtiter A Second B. cereuS Strain, designated AB81,was isolated plate coating with the emulsified antigen, the Sample must be from grain bin dust Samples by Standard methodologies. A diluted in borate buffered Saline as follows: 1:5, 1:10, 1:15, 65 Subculture of AB81 was grown and prepared for bioassay as 1:20, 1:30, 1:50, 1:100, and 0. The coating antigen must be described in Example 2. Biological activity was evaluated as prepared fresh immediately prior to use. described in Example 3. The results are as follows: 5,770,696 30 Delta-endotoxin crystals were purified from strain AB88 by Standard methodologies. No activity from pure crystals Insect species Percent was observed when bioassayed against AgrOtis ipsilon. tested Mortality Ostrinia nubialis O Example 17 Agrotis ipsilon O Diabrotica virgifera virgifera 55 Purification of VIPs from Strain AB88 Bacterial liquid culture was grown overnight at 30° C. in Example 15 TB media. Cells were Spun out and the Supernatant retained. 1O Proteins were precipitated with ammonium sulfate (70% Isolation and Biological Activity of B. thuringiensis Saturation), centrifuiged and the pellet retained. The pellet AB6. was resuspended in the original volume of 20 mM Tris pH A B. thuringiensis Strain, designated AB6, was isolated 7.5 and dialyzed against the same buffer. AB88 dialysate was from grain bin dust Samples by Standard methods known in more turbid than comparable material from AB78. AB88 the art. A Subculture of AB6 was grown and prepared for 15 proteins have been Separated by Several different methods bioassay as described in Example 2. Half of the Sample was following clarification including isoelectric focusing autoclaved 15 minutes to test for the presence of B-eXotoxin. (Rotofor, BioRad, Hercules, Calif.), precipitation at pH 4.5, Biological activity was evaluated as described in Example ion-exchange chromotography, Size exclusion chromatogra 3. The results are as follows: phy and ultrafiltration. European corn borer (ECB)-active protein remained in the pellet obtained by pH 4.5 precipitation of dialysate. When Insect species Percent preparative IEF was done on the dialysate using pH 3-10 tested Mortality ampholytes, ECB insecticidal activity was found in all Ostrinia nubialis O fractions with pH of 7 or greater. SDS-PAGE analysis of Agrotis ipsilon 1OO 25 these fractions showed protein bands of MW ~60 kDa and Agrotis ipsilon (autoclaved sample) O ~80 kDa. The 60 kDa and 80 kDa bands were separated by Diabrotica virgifera virgifera O anion exchange HPLC on a Poros-Q column (PerSeptive BioSystems, Cambridge, Mass.). N-terminal Sequence was The reduction of insecticidal acitivity of the culture obtained from two fractions containing proteins of slightly Supernatant to insignificant levels by autoclaving indicates differing MW, but both of approximately 60 kDa in size. The that the active principle is not B-exotoxin. Sequences obtained were similar to each other and to Some Strain AB6 has been deposited in the Agricultural Ö-endotoxins. anion exchange fraction 23 (Smaller): Research Service, Patent Culture Collection (NRRL), XEPFVSAXXXQXXX (SEQ ID NO:10) anion exchange frac Northern Regional Research Center, 1815 North University 35 tion 28 (larger): xEYENVEPFVSAX (SEQ ID NO:11) Street, Peoria, Ill. 61604, USA, and given Accession No. When the ECB-active pH 4.5 pellet was further separated NRRL B-21060. by anion eXchange on a Poros-Q column, activity was found Example 16 only in fractions containing a major band of ~60 kDa. Isolation and Biological Characterization of B. Black cutworm-active protein also remained in the pellet 40 when AB88 dialysate was brought down to pH 4.5. In thuringiensis AB88. preparative IEF using pH 3-10 ampholytes, activity was not ABt Strain, designated AB88, was isolated from grain bin found in the ECB-active IEF fractions; instead, it was dust Samples by Standard methodologies. A Subculture of highest in a fraction of pH 4.5-5.0. Its major components AB88 was grown and prepared for bioassay as described in have molecular weights of ~35 and ~80 kDa. Example 2. Half of the sample was autoclaved 15 minutes 45 to test for the presence of B-eXotoxin. Biological activity The pH 4.5 pellet was separated by anion exchange HPLC was evaluated against a number of insect Species as to yield fractions containing only the 35 kDa material and described in Example 3. The results are as follows: fractions containing both 35 kDa and 80 kDa bands. Example 18 50 Insect Percent mortality of culture Supernatant Characterization of AB88 VIP. species tested Order Non-autoclaved Autoclaved Fractions containing the various lepidopteran active Veg Agrotis ipsilon Lepidoptera 1OO 5 etative proteins were generated as described in Example 17. Ostrinia nubilalis Lepidoptera 1OO O Spodoptera Lepidoptera 1OO 4 55 Biological analysis of fractions demonstrated that different frugiperda VIPs were responsible for the different lepidopteran species Helicoverpa zea Lepidoptera 1OO 12 activity. Heliothis Lepidoptera 1OO 12 wireScens The Agrotis ipsilon activity is due to an 80 kDa and/or a Leptinotarsa Coleoptera O O 35 kDa protein, either delivered singly or in combination. decenlineata 60 These proteins are not related to any 8-endotoxins from Bt Diabrotica Coleoptera O 5 as evidenced by the lack of Sequence homology of known Bt virgifera Ö-endotoxin Sequences. Also, these proteins are not found in virgifera the AB88 Ö-endotoxin crystal. N-terminal sequences of the major Ö-endotoxin proteins were compared with the The reduction of insecticidal acitivity of the culture 65 N-terminal sequences of the 80 kDa and 35 kDa VIP and Supernatant to insignificant levels by autoclaving indicates revealed no Sequence homology. A Summary of the results that the active principle is not f-exotoxin. follows: 5,770,696 31 32 dideoxy termination method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready N-terminal sequence of major 8 Reaction Dye Deoxy Terminator Cycle Sequencing Kits and Agrotis VIP N-terminal sequences endotoxin proteins PRISM Sequenase(R) Terminator Double-Stranded DNA 130 kDa Sequencing Kit and analysed on an ABI 373 automatic MDNNPNINE (SEQ ID NO: 14) Sequencer. 80 kDa 80 kDa MNKNNTKLPTRALP MDNNPNINE (SEQ ID NO: 15) The clone pCIB7104 contains the VIP3A(a) gene whose (SEQ ID NO: 12) coding region is disclosed in SEQ ID NO:28 and the 60 kDa encoded protein sequence is disclosed in SEQ ID NO:29. A MNVLNSGRTTI (SEQ ID NO: 16) Synthetic version of the coding region designed to be highly 35 kDa ALSENTGKDGGYIVP expressed in maize is given in SEQ ID NO:30. Any number (SEQ ID NO: 13) of Synthetic genes can be designed based on the amino acid sequence given in SEQ ID NO:29. The clone pCIB7107 contains the VIP3A(b) gene whose The Ostrinia nubilalis activity is due to a 60 kDa VIP and 15 coding region is disclosed in SEQ ID NO:31 and the the Spodoptera frugiperda activity is due to a VIP of encoded protein is disclosed in SEQ ID NO:32. Both unknown size. pCIB7104 and pCIB7107 have been deposited with the Bacillus thuringiensis strain AB88 has been deposited in Agricultural Research Service Patent Culture Collection the Agricultural Research Service, Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21422 and (NRRL), Northern Regional Research Center, 1815 North B-21423, respectively. University Street, Peoria, Ill. 61604, USA and given the Accession No. NRRL B-21225. Example 18C Identification of Novel VIP3-like Genes by Example 18 Hybridization 25 Isolation and Biological Activity of B. To identify Bacillus containing genes related to the Thuringiensis AB424 VIP3A(a) from isolate AB88, a collection of Bacillus iso A.B. thuringiensis Strain, designated AB424, was isolated lates was screened by hybridization. Cultures of 463 Bacil from a moSS covered pine cone sample by Standard methods lus Strains were grown in microtiter Wells until Sporulation. known in the art. A subculture of AB424 was grown and A 96-pin colony Stampel was used to transfer the cultures to prepared for bioassay as described in Example 2. 150 mm plates containing L-agar. Inoculated plates were kept at 30° C. for 10 hours, then at 4 C. overnight. Colonies Biological activity was evaluated as described in Example were blotted onto nylon filters and probed with a 1.2 Kb 3. The results are as follows: HindIII VIP3A(a) derived fragment. Hybridization was per 35 formed overnight at 62 C. using hybridization conditions of Insect species tested Percent mortality Maniatis et al. Molecular Cloning. A Laboratory Manual (1982). Filters were washed with 2xSSC/0.1% SDS at 62 Ostrinia nubialis 1OO C. and exposed to X-ray film. Agrotis ipsilon 1OO Diabrotica virgifera O Of the 463 Bacillus strains Screened, 60 contain VIP3-like virgifera 40 genes that could detected by hybridization. Example 18D Strain AB424 has been deposited in the Agricultural Characterization of a B. thuringiensis Strain M2.194 Research Service, Patent Culture Collection (NRRL), Containing a Cryptic VIP3-Like Gene Northern Regional Research Center, 1815 North University 45 Street, Peoria, Ill. 61604, USA, and given Accession No. A B. thuringiensis strain, designated M2194, was shown NRRL B-21439. to contain VIP3-like gene(s) by colony hybridization as described in Example 18C. The M2194 VIP3 like gene is Example 18B considered cryptic Since no expression can be detected throughout the bacterial growth phases either by immuno Cloning of the VIP3A(a) and VIP3A(b) Genes which 50 blot analysis using polyclonal antibodies raised against the Encode Proteins Active Against Black Cutworm. VIP3A(a) protein isolated from AB88 or by bioassay as DNA from isolates AB88 and AB424 was digested with described in Example 3. the restriction enzymes Xbaland EcoRI respectively, ligated The M2194 VIP3-like gene was cloned into pKS by into pBlueScript vector previously linearized with the same 55 following the protocol described in Example 9, which cre enzymes and dephosphorylated, and transformed into E. coli ated pCIB7108. E. coli containing pCIB7108 which com DHC. Strain. Recombinant clones were blotted onto nitro prises the M2194 VIP3 gene were active against black cellulose filters which were subsequently probed with a cutworm demonstrating that the gene encodes a functional 33-bases long oligonucleotide corresponding to the 11-N protein with insecticidal activity. The plasmid pCIB7108 has terminal amino acids of the 80 kDa protein active against 60 been deposited with the Agricultural Research Service Agrotis ipsilon (black cutworm). Four out of 400 recombi Patent Culture Collection (NRRL) and given Accession No. nant clones were positive. Insect bioassays of the positive NRRL B-21438. recombinants exhibited toxicity to black cutworm larvae Example 19 comparable to that of AB88 or AB424 Supernantants. The nucleotide sequence of pCIB7104, a positive recom 65 Isolation and Biological Activity of Other Bacillus sp. binant clone from AB88, and of pCIB7107, a positive Other Bacillus species have been isolated which produce recombinant clone from AB424, was determined by the proteins with insecticidal activity during vegetative growth. 5,770,696 33 34 These Strains were isolated from environmental Samples by Inoculated plates were grown 4-8 hours at 30° C., then Standard methodologies. Isolates were prepared for bioassay chilled to 4 C. Colonies were transferred to nylon filters, and assayed as described in Examples 2 and 3 respectively. and the cells lysed by standard methods known in the art. Isolates which produced insecticidal proteins during vegeta The filters were hybridized to a DNA probe generated from tive growth with activity against Agrotis ipsilon in the DNA fragments containing both VIP1A(a) and VIP2A(a) bioassay are tabulated below. No correlation was observed DNA sequences. Hybridization was performed overnight at between the presence of a 6-endotoxin crystal and vegetative 65 C. using the hybridization conditions of Church and insecticidal protein production. Gilbert (Church, G. M., and W. Gilbert, PNAS, 81:1991–1995 (1984)). Filters were washed with 2x SSC containing 0.1% SDS at 65° C. and exposed to X-Ray film. Presence of 8-endotoxin Of the 463 Bacillus strains Screened, 55 strains were Bacillus isolate crystal Percent mortality identified that hybridized to the VIP1A(a)/VIP2A(a) probe. AB6 -- 1OO DNA was isolated from 22 of these strains, and analyzed AB53 8O using a Southern blot with VIP1A(a)/VIP2A(a) DNA as AB88 -- 1OO 15 AB195 60 probes. These Strains were grouped into 8 classes based on AB211 70 their Southern blot pattern. Each class differed in Southern AB217 83 blot pattern from AB78. One class had a pattern identical to AB272 8O AB279 70 that of the VIP1A(a)/VIP2A(a) homologs from Bacillus AB289 -- 1OO thuringiensis war tenebrionis (see below). Each of the 22 AB292 -- 8O Strains was tested for activity against Western corn rootworm AB294 1OO (WCRW). Three strains, AB433, AB434, and AB435 were AB3OO 8O found to be active on WCRW. Western blot analysis using AB359 1OO VIP2A(a) antisera revealed that strains AB6, AB433, AB434, AB435, AB444, and AB445 produce a protein(s) of Isolates AB289, AB294 and AB359 have been deposited 25 equivalent size to VIP2A(a). in the Agricultural Research Service, Patent Culture Collec Notable among the strains identified was Bacillus thur tion (NRRL), Northern Regional Research Center, 1815 ingiensis strain AB6, (NRRL B-21060) which produced a North University Street, Peoria Ill. 61604, USA and given VIP active against black cutworm (Agrotis ipsilon) as the Accession Numbers NRRL B-21227, NRRL B-21229, described in Example 15. Western blot analysis with poly and NRRL B-21226 respectively. clonal antisera to VIP2A(a) and polyclonal antisera to Bacillus isolates which produce insecticidal proteins dur VIP1A(a) suggests that AB6 produces proteins similar to ing vegetative growth with activity against Diabrotica vir VIP2A(a) and VIP1A(a). Thus, AB6 may contain VIPs gifera virgifera are tabulated below. similar to VIP1A(a) and VIP2A(a), but with a different Spectrum of insecticidal activity. 35 Presence of 8-endotoxin Example 21 Bacillus isolate crystal Percent mortality AB52 50 Cloning of a VIP1A(a)/VIP2A(a) Homolog from AB59 71 Bacillus thuringiensis Var. tenebriones. AB68 -- 60 40 ABF8 1OO Several previously characterized Bacillus Strains were AB122 57 tested for presence of DNA similar to VIP1A(a)/VIP2A(a) AB218 64 by Southern blot analysis. DNA from Bacillus strains AB78, AB256 64 AB188, GC91, HD-1 and ATCC 10876 was analyzed for 45 presence of VIP1A(a)/VIP2A(a) like sequences. DNA from Isolates AB59 and AB256 have been deposited in the Bt strains GC91 and HD-1, and the Bc strain ATCC 10876 Agricultural Research Service, Patent Culture Collection did not hybridize to VIP2A(a)/VIP1A(a) DNA, indicating (NRRL), Northern Regional Research Center, 1815 North they lack DNA sequences similar to VIP1A(a)/VIP2A(a) University Street, Peoria Ill. 61604, USA, and given the genes. Similarly, DNA from the insecticidal strain AB88 Accession Numbers NRRL B-21228 and NRRL B-21230, 50 (Example 16) did not hybridize to VIP1A(a)/VIP2A(a) DNA respectively. region, Suggesting that the VIP activity produced by this strain does not result from VIP1A(a)/VIP2A(a) homologs. Example 20 In contrast, Bacillus thuringiensis war tenebrionis (Bt) contained sequences that hybridized to the VIP1A(a)/VIP2A Identification of Novel VIP1/VIP2 like Genes by 55 (a) region. Further analysis confirmed that Bit contains Hybridization VIP1A(a)/VIP2A(a) like sequences. To identify Strains containing genes related to those found To characterize the Bit homologs of VIP2A(a) and VIP1A in the VIP1A(a)/VIP2A(a) region of AB78, a collection of (a), the genes encoding these proteins were cloned. Southern Bacillus Strains was Screened by hybridization. Independent blot analysis identified a 9.5 kb Eco RI restriction fragment cultures of 463 Bacillus strains were grown in wells of 96 60 likely to contain the coding regions for the homologs. well microtiter dishes (five plates total) until the cultures Genomic DNA was digested with EcoRI, and DNA frag Sporulated. Of the Strains tested, 288 were categorized as ments of approximately 9.5 kb in length were gel-purified. Bacillus thuringiensis, and 175 were categorized as other This DNA was ligated into pBluescript SK(+) digested with Bacillus Species based on the presence or absence of Eco RI, and transformed into E. coli to generate a plasmid Ö-endotoxin crystals. For each microtiter dish, a 96-pin 65 library. Approximately 10,000 colonies were screened by colony Stamper was used to transfer approximately 10 ul of colony hybridization for the presence of VIP2A(a) homolo Spore culture to two 150 mm plates containing L-agar. gous Sequences. Twenty eight positive colonies were iden 5,770,696 35 36 tified. All twenty eight clones are identical, and contain Table 20 discloses the similarity between VIP1A(b) and VIP1A(a)/VIP2A(a) homologs. Clone pCIB7100 has been VIP1A(a) from AB78. This alignment reveals that the amino deposited in the Agricultural Research Service, Patent Cul terminal regions of the two VIP1 proteins share higher ture Collection (NRRL), Northern Regional Research amino acid identity in the amino-terminal region than in the Center, 1815 North University Street, Peoria Ill. 61604, 5 carboxy terminal region. In fact, the amino terminal two USA, and given the Accession Number B-21322. Several thirds (up to aa 618 of the VIP1A(b) sequence shown in subclones were constructed from pCIB7100. A 3.8 kb Xba Table 20) of the two proteins exhibit 91% identity, while the I fragment from pCIB7100 was cloned into pBluescript carboxy-terminal third (from aa 619–833 of VIP1A(b)) SK(+) to yield pCIB7101. A 1.8 kb Hind III fragment and a exhibit only 35% identity. 1.4 kb Hind III fragment from pCIB7100 were cloned into 10 Western blot analysis indicated that Bacillus thuringiensis pBluescript SK(+) to yield pCIB7102 and pCIB7103, var tenebrionis (Bt) produces both VIP1A(a) like and respectively. Subclones pCIB7101, pCIB7102 and VIP2A(a) like proteins. However, these proteins do not pCIB7103 have been deposited in the Agricultural Research appear to have activity against WeStern COrn rootWOrm. Service, Patent Culture Collection (NRRL), Northern Bioassay for activity against Western corn rootworm was Regional Research Center, 1815 North University Street, 15 performed using either a 24 h culture Supernatant from Bit Peoria Ill. 61604, USA, and given the Accession Numbers or E. coli clone pCIB7100 (which contains the entire region B-21323, B-21324 and B-21325 respectively. of the VIP1A(a)/VIP2A(a) homologs). No activity against The DNA sequence of the region of pCIB7100 containing western corn rootworm was detected in either case. the VIP2A(a)/VIP1A(a) homologs was determined by the Given the similarity between the VIP2 proteins from Bitt dideoxy chain termination method (Sanger et al., 1977, Proc. and AB78, the ability of VIP2A(b) from Btt to substitute for Natl. Acad. Sci. USA 74:5463-5467). Reactions were per VIP2A(a) from AB78 was tested. Cells containing formed using PRISM Ready Reaction Dye Deoxy Termina pCIB6206 (which produces AB78 VIP1A(a) but not VIP2A tor Cycle Sequencing Kits and PRISM Sequenase(R) Termi (a) protein) were mixed with Btt culture Supernatant, and nator Double-Stranded DNA Sequencing Kits, and analyzed tested for activity against western corn rootworm. While on an ABI model 373 automated Sequencer. Custom oligo 25 neither Bitt culture Supernatant nor cells containing nucleotides were used as primers to determine the DNA pCIB6206 had activity on WCRW, the mixture of Btt and Sequence in certain regions. The DNA sequence of this pCIB6206 gave high activity against WCRW. Furthermore, region is shown in SEQ ID NO:19. additional bioassay showed that the Bit clone pCIB7100, The 4 kb region shown in SEQ ID NO:19 contains two which contains the Btt VIP1A(b)/VIP2A(b) genes in E. coli, open readings frames (ORFs), which encode proteins with a also confers activity against WCRW when mixed with high degree of similarity to VIP1A(a) and VIP2A(a) proteins pCIB6206. Thus, the VIP2A(b) protein produced by Btt is from strain AB78. The amino acid sequence of the VIP2A(a) functionally equivalent to the VIP2A(a) protein produced by homolog, designated as VIP2A(b) using the Standardized AB78. nomenclature, is found at SEQID NO:20 and the amino acid Thus, the ability to identify new strains with insecticidal sequence of the VIP1A(a) homolog, designated as VIP1A(b) 35 activity by using VIP DNA as hybridization probes has been using the Standardized nomenclature, is disclosed at SEQID demonstrated. Furthermore, Bacillus Strains that contain NO:21. The VIP2A(b) protein exhibits 91% amino acid VIP1A(a)/VIP2A(a) like sequences, produce VIP1A(a)/ identity to VIP2A(a) from AB78. An alignment of the amino VIP2A(a) like protein, yet demonstrate toxicity toward acid sequences of the two VIP2 proteins is provided in Table different insect pests. Similar methods can identify many 19. The VIP1A(b) protein exhibits 77% amino acid identity more members of the VIP1/VIP2 family. Furthermore, use to VIP1A(a) from AB78. An alignment of these two VIP1 of Similar methods can identify homologs of other varieties proteins is provided in Table 20. The alignment shown in of VIPs (for example, the VIPs from AB88).

TABLE 19 Alignment of VIP2 Amino Acid Sequences from Bacillus thuringiensis var. tenebrionis (VIP2A(b) vs. AB78 (VIP2A(a)) Btt MOR ME G. KLF V V S KTL OVVT R T V L L S T V Y S I T L L NNV VI KADOL NI NS O S K 50 SEOID NO:20 Abis Mkkalk hulkki hill kill is he kibb k so stold No. 51 YTNL ONLKI PDNAE D F KEDK GKAKE WG KEK GE E WRP PATE KGEMN NFL DN 100 s, blkirbkvibkhbkikkk Wikki khwkirklik, kill bloo 101 KND I KTNY KEI TFS MAGS CE DEI KD L E E I DKI F D KAN LS S SI I TY KNV EP 150 10 kbix kill MA fiblk blk bk whbkir is SI ITY KNVE P 150 151is ATrh I GF NKkill S L T E GNT I NSDAMAbakh OF khiiKE OF L G KDMKF bik Dby S YLD bill Y H L T AOAbby Q VSS kaoK2OO 2O1 KR VI L. KVTV P S G KGS TT PT KAG WI LNN N E YKML DNG Y V LHW DKW S K V V K 250 20, it k + k + k is kill bivli hk kylk so 251 KGME CLOVE GT L KKS LD F KN DI NAE AHS WG MKI YED WAKNLTAS ORE AL D 300 25 : : | | | | | | | | | | | | | | | | | | | | | | | | | : : . KG VE CLOI E GT L KKS LDF KN DI NAE AHS WG MKNY E E WAKDL TDS ORE AL D 300

5,770,696 39 40

TABLE 21-continued Alignment of VIP1 Amino Acid Sequences from Bacillus thuringiensis var. tenebrionis (VIP1A(b) vs. AB78 (VIP1A(a)) 698 KS AI TS K K V K L N N ONY OR V DI vKs ERN MDK Y R GN GT T N VY GD DWT 747 : . . . : : : . 701 I YP TT KT VNV NKDNY KRLD I AHNI K S N P I S S L H K ND E I T L FWD DI S 750 748 I P E V S AI N P A S L S DE EI QE I F KD S T I EY G NP SF WADAVT FK...... 788 ...... 751. IT VA KP E LTD S E I KOI Y S R Y GI K LED G L I DKKG GI HY GEF I, NEAS 8OO 789 . NI K P L ONY V KEYE I YHK...... S HRYE KKTVF DI MG VHY EY S I ARE O 830 F NI E P L Q NY VT KY KVT Y S S E L G ON V S D TL E SDKI YKD GTI KF DFT KYS KN 850 831 KKA 833 851 E O G 851

Example 22 genes can be designed to be fused at a common restriction Site. Alternatively, the Synthetic fusion gene can be designed Fusion of VIP Proteins to Make a Single Polypeptide to encode a single polypeptide comprised of both VIP1 and VIP proteins may occur in nature as Single polypeptides, VIP2 domains. or as two or more interacting polypeptides. When an active Addition of a peptide linker between the VIP1 and VIP2 VIP is comprised of two or more interacting protein chains, 25 domains of the fusion protein can be accomplished by PCR these protein chains can be produced as a single polypeptide mutagenesis, use of a Synthetic DNA linker encoding the chain from a gene resulting from the fusion of the two (or linker peptide, or other methods known in the art. more) VIP coding regions. The genes encoding the two The fused VIP polypeptides can be comprised of one or chains are fused by merging the coding regions of the genes more binding domains. If more than one binding domain is to produce a single open reading frame encoding both VIP used in the fusion, multiple target pests are controlled using polypeptides. The composite polypeptides can be fused to such a fusion. The other binding domains can be obtained by produce the Smaller polypeptide as the NH2 terminus of the using all or part of other VIPs; Bacillus thuringiensis fusion protein, or they can be fused to produce the larger of endotoxins, or parts thereof, or other proteins capable of the polypeptides as the NH2 terminus of the fusion protein. binding to the target pest or appropriate biding domains A linker region can optionally be used between the two 35 derived from Such binding proteins. polypeptide domains. Such linkers are known in the art. This One example of a fusion construction comprising a maize linker can optionally be designed to contain protease cleav optimized DNA sequence encoding a Single polypeptide age Sites Such that once the Single fused polypeptide is chain fusion having VIP2A(a) at the N-terminal end and ingested by the target insect it is cleaved in the linker region VIP1A(a) at the C-terminal end is provided by pCIB5531. A to liberate the two polypeptide components of the active VIP 40 DNA sequence encoding a linker with the peptide Sequence molecule. PSTPPTPSPSTPPTPS (SEQ ID NO:47) has been inserted VIP1A(a) and VIP2A(a) from B. cereus strain AB78 are between the two coding regions. The Sequence encoding this fused to make a Single polypeptide by fusing their coding linker and relevant cloning sites is 5'-CCC GGG CCTTCT regions. The resulting DNA has the Sequence given in SEQ ACT CCC CCAACT CCCTCTCCTAGC ACG CCT CCG ID NO:22 with the encoded protein given in SEQ ID NO:23. 45 ACACCT AGC GATATCGGATC C-3' (SEQ ID NO:48). In like manner, other fusion proteins may be produced. Oligonucleotides were Synthesized to represent both the The fusion of the genes encoding VIP1A(a) and VIP2A(a) upper and lower Strands and cloned into a puC vector is accomplished using Standard techniques of molecular following hybridization and phosphorylation using Standard biology. The nucleotides deleted between the VIP1A(a) and procedures. The stop codon in VIP2A(a) was removed using VIP2A(a) coding regions are deleted using known mutagen 50 PCR and replaced by the BglI restriction site with a SmaI esis techniqueS or, alternatively, the coding regions are fused Site. A translation fusion was made by ligating the Bam using PCR techniques. HI/PstI fragment of the VIP2A(a) gene from pCIB5522 (see The fused VIP polypeptides can be expressed in other Example 24), a PCR fragment containing the PstI-end organisms using a Synthetic gene, or partially Synthetic gene, fragment of the VIP2A(a) gene (identical to that used to optimized for expression in the alternative host. For 55 construct pCIB5522), a synthetic linker having ends that instance, to express the fused VIP polypeptide from above in would ligate with a blunt site at the 5' end and with BamHI maize, one makes a Synthetic gene using the maize preferred at the 3' end and the modified synthetic VIP1A(a) gene from codons for each amino acid, see for example U.S. Pat. No. pCIB5526 described below (See SEQ ID NO:35). The 5,625,136 herein incorporated by reference. Synthetic DNA fusion was obtained by a four way ligation that resulted in Sequences created according to these methods are disclosed 60 a plasmid containing the VIP2A(a) gene without a transla in SEQ ID NO:17 (maize optimized version of the 100 kDa tion stop codon, with a linker and the VIP1A(a) coding VIP1A(a) coding sequence), SEQ ID NO:18 (maize opti region without the Bacillus secretion signal. The DNA mized version of the 80 kDa VIP1A(a) coding sequence) and sequence for this construction is disclosed in SEQ ID SEQ ID NO:24 (maize optimized version of the VIP2A(a) NO:49, which encodes the fusion protein disclosed in SEQ coding Sequence). 65 ID NO:50. A single polypeptide fusion where VIP1A(a) is at Synthetic VIP1 and VIP2 genes optimized for expression the N-terminal end and VIP2A(a) is at the C-terminal end in maize can be fused using PCR techniques, or the Synthetic can be made in a similar fashion. Furthermore, either one or 5,770,696 41 42 both genes can be linked in a translation fusion with or the mature protein which has the N-terminal Sequence of without a linker at either the 5' or the 3' end to other LKITDKVEDF (amino acid residues 57 to 66 of SEQ ID molecules like toxin encoding genes or reporter genes. NO:2). It is possible to engineer VIP2 to be secreted out of the plant cell or to be targeted to Subcellular organelles Such Example 23 as the endoplasmic reticulum, vacuole, mitochondria or plastids including chloroplasts. Hybrid proteins made by Targeting of VIP2 to Plant Organelles fusion of a Secretion signal peptide to a marker gene have been Successfully targeted into the Secretion pathway. Various mechanisms for targeting gene products are known to exist in plants and the Sequences controlling the (Itirriaga G. et al., The Plant Cell, 1: 381-390 (1989), functioning of these mechanisms have been characterized in Denecke et al., The Plant Cell, 2:51–59 (1990). Amino Some detail. For example, the targeting of gene products to terminal Sequences have been identified that are responsible the chloroplast is controlled by a Signal Sequence found at for targeting to the ER, the apoplast, and extracellular the amino-terminal end of various proteins. This signal is secretion from aleurone cells (Koehler & Ho, Plant Cell 2: cleaved during chloroplast import, yielding the mature pro 769–783 (1990)). 15 The presence of additional Signals are required for the tein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 protein to be retained in the endoplasmic reticulum or the (1988)). These signal Sequences can be fused to heterolo vacuole. The peptide sequence KDEL/HDEL at the carboxy gous gene products Such as VIP2 to effect the import of those terminal of a protein is required for its retention in the products into the chloroplast (van den Broeck et al. Nature endoplasmic reticulum (reviewed by Pelham, Annual 313: 358-363 (1985)). DNA encoding for appropriate signal Review Cell Biol. 5:1-23 (1989). The signals for retention sequences can be isolated from the 5' end of the cDNAS of proteins in the vacuole have also been characterized. encoding the RUBISCO protein, the CAB protein, the EPSP Vacuolar targeting Signals may be present either at the Synthase enzyme, the GS2 protein and many other proteins amino-terminal portion, (Holwerda et al., The Plant Cell, which are known to be chloroplast localized. 4:307-318 (1992), Nakamura et al., Plant Physiol., 101:1-5 Other gene products are localized to other organelles Such 25 (1993)), carboxy- terminal portion, or in the internal as the mitochondrion and the peroxisome (e.g. Unger et al. Sequence of the targeted protein. (Tague et al., The Plant Plant Molec. Biol. 13:411–418 (1989)). The cDNAs encod Cell, 4:307-318 (1992), Saalbach et al., The Plant Cell, ing these products can also be manipulated to effect the 3:695–708 (1991)). Additionally, amino-terminal sequences targeting of heterologous gene products Such as VIP2 to in conjunction with carboxy-terminal Sequences are respon these organelles. Examples of Such Sequences are the Sible for vacuolar targeting of gene products (Shinshi et al. nuclear-encoded ATPases and Specific aspartate amino trans Plant Molec. Biol. 14:357–368 (1990)). Similarly, proteins ferase isoforms for mitochondria. Similarly, targeting to may be targeted to the mitochondria or plastids using cellular protein bodies has been described by Rogers et al. Specific carboxy terminal signal peptide fusions (Heijne et (Proc. Natl. Acad. Sci. USA 82: 6512–6516 (1985)). al., Eur, J. Biochem., 180:535-545 (1989), Archer and By the fusion of the appropriate targeting Sequences 35 Keegstra, Plant Molecular Biology, 23:1105–1115 (1993)). described above to coding Sequences of interest Such as In order to target VIP2, either for secretion or to the VIP2 it is possible to direct the transgene product to any various Subcellular organelles, a maize optimized DNA organelle or cell compartment. For chloroplast targeting, for Sequence encoding a known signal peptide(s) may be example, the chloroplast Signal Sequence from the designed to be at the 5' or the 3' end of the gene as required. RUBISCO gene, the CAB gene, the EPSPsynthase gene, or 40 To secrete VIP2 out of the cell, a DNA sequence encoding the GS2 gene is fused in frame to the amino-terminal ATG the eukaryotic secretion signal peptide MGWSWIFLFLLS of the transgene. The Signal Sequence Selected should GAAGVHCL (SEQID NO:25) from U.S. patent application include the known cleavage Site and the fusion constructed Ser. No. 08/267,641 or any other described in the literature should take into account any amino acids after the cleavage (Itirriaga et al., The Plant Cell, 1:381-390 (1989), Denecke, Site which are required for cleavage. In Some cases this 45 et al., The Plant Cell, 2:51–59 (1990)) may be added to the requirement may be fulfilled by the addition of a small 5' end of either the complete VIP2 gene sequence or to the number of amino acids between the cleavage Site and the Sequence truncated to encode the mature protein or the gene Start codon ATG, or alternatively replacement of Some truncated to nucleotide 286 or encoding a protein to start at amino acids within the coding Sequence. Fusions con amino acid residue 94 (methionine). To target VIP2 to be Structed for chloroplast import can be tested for efficacy of 50 retained in the endoplasmic reticulum, a DNA sequence chloroplast uptake by in Vitro translation of in vitro tran encoding the ER signal peptide KDEL/HDEL, in addition to scribed constructions followed by in vitro chloroplast uptake the Secretion signal, can be added to the 3' end of the gene. using techniques described by (Bartlett et al. In: Edelmann For vacuolar targeting a DNA sequence encoding the Signal et al. (Eds.) Methods in Chloroplast Molecular Biology, peptide SSSSFADSNPIRVTDRAAST (SEQ ID NO:3; Hol Elsevier. pp 1081–1091 (1982); Wasmann et al. Mol. Gen. 55 werda et al., The Plant Cell, 4:307–318 (1992)) can be Genet. 205:446–453 (1986)). These construction techniques designed to be adjacent to the Secretion signal or a sequence are well known in the art and are equally applicable to encoding a carboxyl Signal peptide as described by Dom mitochondria and peroxisomes. browski et al., The Plant Cell, 5:587–596 (1993) or a The above described mechanisms for cellular targeting functional variation may be inserted at the 3' end of the gene. can be utilized not only in conjunction with their cognate 60 Similarly, VIP2 can be designed to be targeted to either the promoters, but also in conjunction with heterologous pro mitochondria or the plastids, including the chloroplasts, by moters So as to effect a specific cell targeting goal under the inserting Sequences in the VIP2 Sequence described that transcriptional regulation of a promoter which has an would encode the required targeting Signals. The bacterial expression pattern different to that of the promoter from Secretion Signal present in VIP2 may be retained or removed which the targeting Signal derives. 65 from the final construction. A DNA sequence encoding a Secretion signal is present in One example of a construction which incorporates a the native Bacillus VIP2 gene. This signal is not present in eukaryotic Secretion signal fused to a coding Sequence for a 5,770,696 43 44 VIP is provided by pCIB5528. Oligonucleotides corre (SEQ ID NO:34) hybridizes on the complementary strand at sponding to both the upper and lower Strand of Sequences nucelotide position 507. A527 bp amplification product was encoding the secretion signal peptide of SEQID NO:25 was obtained containing the restriction sites BamHI at the 5' end synthesized and has the sequence 5'-GGATCCACC ATG and HindIII site at the 3' end. The amplification product was GGCTGGAGCTGG ATC TTC CTG TTC CTG CTGAGC cloned into a T-vector (described in Example 24, below) and sequenced to ensure the correct DNA sequence. The BamHI/ GGC GCC GCG GGC GTG CACTGC CTGCAG-3' (SEQ HindIII fragment was then obtained by restriction digest and ID NO:41). When hybridized, the 5' end of the secretion used to replace the BamHI/HindIII fragment of the maize Signal resembled "Sticky-ends' corresponding to restriction optimized VIP1A(a) gene cloned in the root-preferred pro sites BamHI and Pst. The oligonucleotide was hybridized moter cassette. The construct obtained was designated and phosphorylated and ligated into pCIB5527 (construction pCIB5526. The maize optimized coding region for VIP1A described in Example 23A) which had been digested with (a) with the Bacillus Secretion signal removed is disclosed as BarmHI/Pst using standard procedures. The resulting maize SEQ ID NO:35 and the encoded protein is disclosed as SEQ optimized coding sequence is disclosed in SEQ ID NO:42 ID NO:36. which encodes the protein disclosed in SEQID NO:43. This The gene encoding the processed form of VIP2A(a), i.e., encoded protein comprises the eukaryotic Secretion signal in 15 a coding region with the Secretion Signal removed, was place of the Bacillus Secretion Signal. constructed by a procedure Similar to that described for that One example of a construction which incorporates a used to construct the processed form of VIP1A(a), above. vacuolar targetting Signal fused to a coding Sequence for a The modification was obtained by PCR using the forward VIP is provided by pCIB5533. Oligonucleotides corre primer 5'-GGATCC ACC ATG CTG CAG AAC CTGAAG sponding to both the upper and lower Strand of Sequences ATC AC-3' (SEQ ID NO:37). This primer hybridizes at encoding the vacuolar targetting peptide of SEQ ID NO:3 nucleotide position 150 of the maize optimized VIP2A(a) was synthesized and has the sequence 5'-CCG CGG GCG gene (SEQ ID NO:27). A silent mutation has been inserted TGC ACT GCC TCA GCA GCA GCA GCT TCG CCG at nucleotide position 15 of this primer to obtain a Pst ACA GCA ACC CCA TCC GCG TGA CCG ACC GCG restriction site. The reverse primer has the Sequence 5'-AAG CCG CCA GCACCCTGC AG-3' (SEQ ID NO:44). When 25 CTT CCA CTC CTT CTC-3' (SEQ ID NO:38). A 259 bp hybridized, the 5' end of the vacuolar targetting Signal product was obtained with HindIII restriction site at the 3' resembled "Sticky-ends' corresponding to restriction Sites end. The amplification product was cloned into a T- Vector, SacII and Pst. The oligonucleotide was hybridized and sequenced and ligated to a BamHI/HindIII digested root phosphorylated and ligated into pCIB5528 (construction preferred promoter cassette containing the maize optimized described above) which had been digested with SacII/PstI VIP2A(a). The construct obtained was designated using Standard procedures. The resulting maize optimized pCIB5527. The maize optimized coding region for VIP2A coding sequence is disclosed in SEQ ID NO:45 which (a) with the Bacillus Secretion signal removed is disclosed as encodes the protein disclosed in SEQ ID NO:46. This SEQ ID NO:39 and the encoded protein is disclosed as SEQ encoded protein comprises the vacuolar targetting peptide in ID NO:40. addition to the eukaryotic Secretion Signal. The VIP1 gene can also be designed to be secreted or 35 Example 24 targeted to Subcellular organelles by Similar procedures. Construction and Cloning of the VIP1A(a) and VIP2A(a) Maize Optimized Genes Example 23 Design The maize optimized genes were designed by reverse Removal of Bacillus Secretion Signal from 40 translation of the native VIP1A(a) and VIP2A(a) protein VIP1A(a) and VIP2A(a) Sequences using codons that are used most often in maize VIP1A(a) and VIP2A(a) are secreted during the growth of (Murray et al., Nucleic Acid Research, 17:477-498 (1989)). Strain AB78. The nature of peptide Sequences that act as To facilitate cloning, the DNA sequence was further modi Secretion signals has been described in the literature fied to incorporate unique restriction sites at intervals of (Simonen and Palva, Microbiological reviews, pg. 109-137 45 every 200-360 nucleotides. VIP1A(a) was designed to be (1993)). Following the information in the above publication, cloned in 11 such fragments and VIP2A(a) was cloned in 5 the putative Secretion Signal was identified in both genes. In fragments. Following cloning of the individual fragments, VIP1A(a) this signal is composed of amino acids 1-33 (See adjacent fragments were joined using the restriction Sites SEQ ID NO:5). Processing of the secretion signal probably common to both fragments, to obtain the complete gene. To occurs after the Serine at amino acid 33. The Secretion Signal 50 clone each fragment, oligonucleotides (50-85 nucleotides) in VIP2A(a) was identified as amino acids 1-49 (See SEQ were designed to represent both the upper and the lower ID NO:2). N-terminal peptide analysis of the secreted strand of the DNA. The upper oligo of the first oligo pair was mature VIP2A(a) protein revealed the N-terminal sequence designed to have a 15bp Single Stranded region at the 3' end LKITDKVEDFKEDK. This sequence is found beginning at which was homologous to a similar Single Stranded region of amino acid 57 in SEQ ID NO:2. The genes encoding these the lower Strand of the next oligo pair to direct the orien proteins have been modified by removal of the Bacillus 55 tation and Sequence of the various oligo pairs within a given Secretion signals. fragment. The oligos are also designed Such that when the all A maize optimized VIP1A(a) coding region was con the oligoS representing a fragment are hybridized, the ends Structed which had the Sequences encoding the first 33 have single Stranded regions corresponding to the particular amino acids, i.e., the Secretion Signal, removed from its 5' restriction site to be formed. The Structure of each oligomer end. This modification was obtained by PCR using an 60 was examined for Stable Secondary Structures Such as hairpin forward primer that contained the sequence 5'-GGA TCC loops using the OLIGO program from NBI Inc. Whenever ACC ATG AAG ACC AAC CAG ATC AGC-3' (SEQ ID necceSary, nucleotides were changed to decrease the Stabil NO:33), which hybridizes with the maize optimized gene ity of the Secondary Structure without changing the amino (SEQ ID NO:26) at nucleotide position 100,and added a acid Sequence of the protein. A plant ribosomal binding Site BamHI restriction site and a eukaryotic translation Start Site 65 consensus sequence, TAAACAATG (Joshi et al., Nucleic consensus including a start codon. The reverse primer that Acid Res., 15:6643–6653 (1987)) or eukaryotic ribosomal contained the sequence 5'-AAG CTT CAG CTC CTTG-3' binding site concensus sequence CCACCATG (Kozak, 5,770,696 45 46 Nucleic Acid Research, 12:857-872 (1984)) was inserted at a 2.5% Nusieve (FMC) agarose gel in a TAE buffer system. the translational Start codon of the gene. The correct Size fragment was gel purified and used for Cloning cloning into a PCR cloning vector or T-vector. T-vector Oligos were synthesized by IDT Inc., and were supplied construction was as described by Marchuk et al., Nucleic as lyophilized powders. They were resuspended at a con 5 Acid Research, 19:1154 (1991). pBluescriptsk+ centration of 200 uM. To 30 ul of each oligo formamide was (Stratagene(E), Calif.) was used as the parent vector. Trans added a final concentration of 25-50% and the sample was formation and identification of the correct clone was carried boiled for two minutes before separation on a premade 10% polyacryamide/urea gel obtained from NoveX. After out as described above. electrophoresis, the oligo was detected by UV shadowing by Fragments 1, 3, 4, 5, 6, 8, and 9 of VIP1A(a) and placing the gel on a TLC plate containing a fluorescent fragments 2 and 4 of VIP2A(a) were obtained by cloning of indicator and exposing it to UV light. The region containing PCR amplification products; whereas, fragments 2, 7, 10 and DNA of the correct size was excised and extracted from the 11 of VIP1A(a) and fragments 1, 3, and 5 of VIP2A(a) were polyacryamide by an overnight incubation of the minced gel obtained by hybridization/ligation. fragment in a buffer containing 0.4M LiCl, 0.1 mM EDTA. Once fragments with the desired Sequence were obtained, The DNA was separated from the gel residue by centrifu the complete gene was assembled by cloning together adja gation through a Millipore UFMC filter. The extracted DNA 15 cent fragments. The complete gene was resequenced and was ethanol precipitated by the addition of 2 volumes of tested for activity against WCRW before moving it into plant absolute alcohol. After centrifugation, the precipitate was expression vectors containing the root preferred promoter resuspended in dIO at a concentration of 2.5 uM. Frag (disclosed in U.S. Pat. No. 5,466,785, herein incorporated ments were cloned either by hybridization of the oligos and by reference) and the rice actin promoter. ligation with the appropriate vector or by amplification of One such plant expression vector is pCIB5521. The maize the hybridized fragment using a equimolar mixture of all the optimized VIP1A(a) coding region (SEQ ID NO:26) was oligos for a particular fragment as a template and end cloned in a plant expression vector containing the root specific PCR primers. preferred promoter at the 5' of the gene with the PEP Cloning by hybridization and ligation Carboxylase intron #9 followed by the 35S terminator at the Homologous double Stranded oligo pairs were obtained 25 3' end. The plasmid also contains Sequences for amplicillin by mixing 5ul of the upper and of the lower oligo for each resistance from the plasmid pUC19. Another plant expres oligo pair with buffer containing 1x polynucleotide kinase sion vector is pCIB5522, which contains the maize opti (PNK) buffer (70 mM Tris-HCl (pH 7.6), 10 mM MgCl, 5 mized VIP2A(a) coding region (SEQID NO:27) fused to the mM dithiothreitol (DTT)), 50 mM KC1, and 5% formamide root preferred promoter at the 5' of the gene with the PEP in a final volume of 50 ul. The oligos were boiled for 10 Carboxylase intron #9 followed by the 35S terminator at the minutes and slow cooled to 37 C. or room temperature. 10 3' end. ill was removed for analysis on a 4% agarose in a TAE buffer Example 25 system (Metaphore(R); FMC). Each hybridized oligo pair was kinased by the addition of ATP at a final concentration NAD Affinity Chromatography of 1 mM, BSA at a final concentration of 100 ug per ml and 35 A purification Strategy was used based on the affinity of 200 units of polynucleotide kinase and 1 ul of 10x PNK VIP2 for the substrate NAD. The Supernatant from the pH buffer in a volume of 10 ul. Following hybridization and 3.5 sodium citrate buffer treatment described in Example 4 phosphorylation, the reaction was incubated at 37 C. for 2 was dialyzed in 20 mM TRIS pH 7.5 overnight. The hours to overnight. 10 ul of each of the oligo pairs for a neutralized Supernatant was added to an equal Volume of particular fragment, were mixed in a final Volume of 50 ul. washed NAD agarose and incubated with gentle rocking at The oligo pairs were hybridized by heating at 80° C. for 10 40 4 C. overnight. The resin and protein solution were added minutes and slow cooling to 37 C. 2 ul of oligos was mixed to a 10 ml disposable polypropylene column and the protein with about 100 ng of an appropriate vector and ligated using Solution allowed to flow out. The column was washed with a buffer containing 50 mM Tris-HCl (pH 7.8), 10 mM 5 column volumes of 20 mM TRIS pH 7.5 then washed with MgCl, 10 mM DTT, 1 mM ATP. The reaction was incubated 2–5 column volumes of 20 mMTRIS pH 7.5, 100 mM NaCl, at room temp. for 2 hours to overnight and transformed into 45 followed by 2-5 column volumes of 20 mM TRIS 7.5. The DH5C. Strain of E. coli, plated on L- plates containing VIP proteins were eluted in 20 mM TRIS pH 7.5 supple ampicillin at a concentration of 100 lug/ml using Standard mented with 5 mM NAD. Approximately 3 column volumes procedures. Positive clones were further characterized and of the effluent were collected and concentrated in a Centri confirmed by PCR miniscreen described in detail in U.S. con -10. Yield is typically about 7-15 lug of protein per ml Pat. No. 5,625,136 using the universal primers “Reverse” 50 of resin. and M13 “-20” as primers. Positive clones were identified by When the purified proteins were analyzed by SDS-PAGE digestion of DNA with appropriate enzymes followed by followed by Silver Staining, two polypeptides were visible, Sequencing. Recombinants that had the expected DNA one with Mr of approximately 80,000 and one with Mr of Sequence were then Selected for further work. approximately 45,000. N-terminal Sequencing revealed that PCR Amplification and cloning into T- vector 55 the Mr 80,000 protein corresponded to a proteolytically PCR amplification was carried out by using a mixture of processed form of VIP1A(A) and the Mr 45,000 form all the oligomers that represented the upper and the lower corresponded to a proteolytically processed form of VIP2A Strand of a particular fragment (final concentration 5 mM (a). The co-purification of VIP1A(a) with VIP2A(a) indi each) as template, specific end primers for the particular cates that the two proteins probably form a complex and fragment (final concentration 2 mM) 200 uM of each dATP, have protein-protein interacting regions. VIP1A(a) and dTTP, dCTP and dGTP, 10 mM Tris-HCl (pH 8.3), 50 mM 60 KCl, 1.5 mM MgCl2,0.01% gelatin and 5 units of Taq VIP2A(a) proteins purified in this manner were biologically polymerase in a final reaction Volume of 50 ul. The ampli active against Western corn rootworm. fication reaction was carried out in a Perkin Elmer ther Example 26 mocycler 9600 by incubation at 95 C. for 1 min (1 cycle), followed by 20 cycles of 95°C. for 45 sec., 50° C. for 30 sec. 65 Exprission of Maize Optimized VIP1(a) and VIP2A(a) Finally the reaction was incubated for 5 min at 72 C. before E. coli Strains containing different plasmids comprising analyzing the product. 10 ul of the reaction was analyzed on VIP genes were assayed for expression of VIPs. E. coli 5,770,696 47 48 Strains harboring the individual plasmids were grown over -continued night in L-broth and expressed protein was extracted from the culture as described in Example 3, above. Protein Assay No. 1 Assay No. 2 Protein expression was assayed by Western Blot analysis using Extract Tested %. Mortality Detected antibodies developed using Standard methods known in the Extracts p0IB5522 (p) + 1OO art, Similar to those described in Example 12, above. Also, pCIB6206 (e) combined insecticidal activity of the expressed proteins were tested pCIB6024(e) (native VIP2A(a)) O yes pCIB6206(e) (native VIP1A(a)) 2O yes against Western corn rootworm according to the method in pCIB5521 + pCIB5522 1OO 1OO yes Example 3, above. The results of the E. coli expression (plasmids delivered by assays are described below. cotransformation) (p) = extract of protoplast culture transformed with indicated plasmid Extract of E. coli Strain Assay No. 1 Assay No. 2 Protein (e) = extract of E. coli strain harboring indicated plasmid Harboring Indicated Plasmid %. Mortality Detected The expression data obtained with both E. coli and maize Control O O O 15 protoplasts show that the maize optimized VIP1A(a) and pCIB5521 (maize optimized 47 27 yes VIP1A(a)) VIP2A(a) genes make the same protein as the native VIP1A pCIB5522 (maize optimized 7 7 yes (a) and VIP2A(a) genes, respectively, and that the proteins VIP2A(a)) encoded by the maize optimized genes are functionally pCIB6024 (native VIP2A(a)) 13 13 yes equivalent to the proteins encoded by the native genes. pCIB6206 (native VIP1A(a)) 27 40 yes Extracts p0IB5521 + pCIB5522 87 47 All publications and Pat. applications mentioned in this combined specification are indicative of the level of skill of those Extracts p0IB5521 + pCIB6024 93 1OO skilled in the art to which this invention pertains. All combined publications and patent applications are herein incorporated Extracts p0IB5522 + pCIB6206 1OO 1OO by reference to the same extent as if each individual publi combined cation or patent application was specifically and individually Extracts p0IB6024 + pCIB6206 1OO 1OO 25 indicated to be incorporated by reference. combined The following deposits have been made at Agricultural Research Service, Patent Culture Collection (NRRL), The DNA from these plasmids was used to transiently Northern Regional Research Center, 1815 North University express the VIPs in a maize protoplast expression System. Street, Peoria, Ill. 61604, USA: Protoplasts were isolated from maize 2717 Line 6 suspen Sion cultures by digestion of the cell walls using Cellulase 1. E. coli PL2 Accession No. NRRL B-21221N RS and Macerase R10 in appropriate buffer. Protoplasts 2. E. coli pCIB6022 Accession No. NRRL B-21222 were recovered by Sieving and centrifugation. Protoplasts 3. E. coli pCIB6023 Accession No. NRRL B-21223N were transformed by a Standard direct gene transfer method 35 4. Bacilius thuringiensis HD73- Accession No. NRRL B-21224 using approximately 75 lug plasmid DNA and PEG-40. 78VIP 5. Bacilius thuringiensis AB88 Accession No. NRRL B-21225 Treated protoplasts were incubated overnight in the dark at 6. Bacillus thuringiensis AB359 Accession No. NRRL B-21226 room temperature. Analysis of VIP expression was accom 7. Bacilius thuringiensis AB289 Accession No. NRRL B-21227 plished on protoplast explants by Western blot analysis and 8. Bacillus sp. AB59 Accession No. NRRL B-21228 insecticidal activity against Western corn rootworm as 9. Bacillus sp. AB294 Accession No. NRRL B-21229 described above for the expression in E. coli. The results of 40 10. Bacillus sp. AB256 Accession No. NRRL B-21230 11. E. coli PS-4 Accession No. NRRL B-21059 the maize protoplast expression assays are described below. 12. E. coli P3-12 Accession No. NRRL B-21061 13. Bacilius cereus AB78 Accession No. NRRL B-21058 14. Bacillus thuringiensis AB6 Accession No. NRRL B-21060 Assay No. 1 Assay No. 2 Protein 15. E. coli pCIB6202 Accession No. NRRL B-21321 Extract Tested %. Mortality Detected 45 16. E. coli pCIB7100 Accession No. NRRL B-21322 17. E. coli pCIB7101 Accession No. NRRL B-21323 No DNA control O 1O O 18. E. coli pCIB7102 Accession No. NRRL B-21324 pCIB5521 (p) (maize optimized 20 (O) 3O yes 19. E. coli pCIB7102 Accession No. NRRL B-21325 VIP1A(a)) 20. E. coli pCIB7104 Accession No. NRRL B-21422 pCIB5522 (p) (maize optimizied 20 (O) 2O yes 21. E. coli pCIB7107 Accession No. NRRL B-21423 VIP2A(a)) 50 22. E. coli pCIB7108 Accession No. NRRL B-21438 Extracts p0IB5521 (p) + 87 (82) 90 23. Bacillus thuringiensis AB424 Accession No. NRRL B-21439 pCIB5522 (p) combined Extracts p0IB5521 (p) + 1OO pCIB5522 (e) combined Extracts p0IB5522 (p) + 53 (36) Although the foregoing invention has been described in pCIB5521 (e) combined Some detail by way of illustration and example for purposes Extracts p0IB5521 (p) + 1OO 55 of clarity of understanding, it will be obvious that certain pCIB6024 (e) combined changes and modifications may be practiced within the Scope of the appended claims.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 50 5,770,696 49 SO -continued ( 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.” ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATC GATA CAA T G T T G T TTT A CTTAGA C C G G TA GT C T C T GT AATTT G T TTA A T G CTATATT

CTTT ACTTT G ATA CATTTT A ATA G C CAT TT CAAC CTTATC A GTA T G T TTT T G T G GT CTTC

C T C C T TTTTT T C CAC GAG CT CTA G CT GC GT TTAAT C C T GT TTT G GTAC GT T C G CT AATAA

TAT C T C T TTC TAATT C T G CA ATACT T G C CA T CAT T C GAAA GAA GAA TTT C C C CAT AG CAT

TAGA G G T ATC AA T G T T G T CA T GAAT AGAAA TAA AAT CTAC A C CTA G C T CT TT GAA TTTTT

CA CT TAA CTC AATTAG G T GT TTT G TAGA G C GAGAAATTC G AT CAA GTTT G TAAA CAA CTA

T CT TAT C GCC TTTAC GT AAT A CTTTT A G CA A CT CTTC GAG TT GAG G G C GC T CTTTTTT TA

TT C C T G T TAT TTT C T C C T GA TATA G CCTTT CTA CA C CATA TT G T T G CAAA G CAT CTATTT

G CA. TAT CGA G ATTTT G TT CT T C T G T G CT GA CA C G A G CATA A C CAAAAATC AAA TT GGTTT

CA CTTC CTAT CT AAA. TATAT CTAT TAAAAT A G CAC CAAAA A C C T TAT TAA AT TAA AATAA

G GAACTTT GT TTTT G GATAT G GATTTT G GT A CT CAAT ATG GAT GA GTTTT TAACGC TTTT

GT TAAAAAAC AAA CAA G T G C CATAAA CG GT C GTTTTT G G G A T G A CATAAT AAATAAT CT G

TTT GATT AAC CTAAC CTT GT A T C CTTACA G C C C A GTTTTA TTT G T ACTT C AACT GA CT GA

ATA T GAA AA C AA CAT GAAG G TTT CATAAAA TT TATATATT TT C CATAACG GA T G C T CTAT

CTT TAG GT TA TA GT TAAA TT ATA AGAAAAA AA CAAA CG GA G G GA GT GAAA AAAA G CAT CT

T C T CTATAAT TTT ACA G G CT CTTT AATAAG AAG G G G G GAG AT TA GATAAT AAA. TAT GAAT

AT CTAT CTAT AATT GTTT GC TT CTA CAATA ACT TAT CTA A CTTT CATATA CAA CAA CAAA

A CAG ACT AAA T C C A GATT GT ATA TT CAT TT T CA GT T G TTC CTT TATAAAA TAATTT CATA

A. A T G AAA A GA. A T G G A G G GA AA G T T G T TT A T G G T G T CA. AAA AAA TTA Me t Lys Arg Me t G 1 u G 1 y Lly s Le u Ph e Me t Wa Ser Lys L y s Le u 1. 5 1 O 1 5

CAA GTA GTT ACT AAA ACT G T A TT G CTT A GT ACA GT T TT C T CT ATA T CT G 1 in V a 1 V a 1 T hr Lys Thr V a 1 Le u Le u S e r Thr Wa Ph e S e r I e S e r 2 O 2 5 3 O

TTA TTA AAT AAT GAA G T G ATA AAA GCT GAA CAA TTA AAT ATA AAT T CT Le u Le u As n As n G 1 u V a 1 I le Lys A a G 1 u G in Le u As n I e As n S e r 3 5 4 O 4 5

CAA A GT AAA. TAT ACT AA C T T G CAA AAT CTA AAA A T C A CT GAC AAG G T A G l n Ser Lys Ty r Thr As n Le u G 1 n As n Le u L y s I le Thr As p Lys V a 1 5 O 5 5 6 O

5,770,696 57 58 -continued

S Ty r Thr As n Le u G 1 in As n Le u Lys I le Th r A s p Lys V a 1 G u

6 o G 1 u T r p Lys Le u Th r A la Thr G 1 u Lys G 1 y Lys Me t As in

1 15 1 2 O 1 2 5

1.

1 65 1 7 O 1 7 5

1 9 O

1 9 5 2 O O 2 O 5

Ser G 1 y Ly s G 1 y Ser Thr Thr Pro T hr Lys A 1 a G 1 y V a 1

2 4 5 25 O 25 5

G 1 u G 1 y Th r Le u Lys Lys Ser Le u A s p Ph e Lys As in As p 2 6 O 2 6 2 7 O

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 2 7 5 28 O 28 5

3 2 5 3 3 O 3 3 5

3 5 5 3 6 O 3 65

e 3 7 5 3 8 O

I le Le u A rig Le u G | n V a 1 Pro Lys G 1 y Ser Thr G 1 y A 1 a

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

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

Lys Arg Ty r V a V a 1 A s p A a T h r Le u Le u Thir As in 4 55 4 6 O 5,770,696 59 60 -continued

( 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 Phi e A 1 a As p S e r As in P ro I A rig Wa Th r As p A rig 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) (iii) HYPOTHETICAL: NO ( i v ). ANTI-SENSE: NO ( 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: 1.2652 ( D.) OTHER INFORMATION: product="100 kDa protein VIP1A(a) f note= “This sequence is identical to the portion of SEQ D NO:1 between and including nucleotide 2475 to 5126. ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:4:

A T G AAA AAT A T G AAG AAA AAG TTA G CA A GT GTT GTA A CG T GT A CG TTA 4 8 Me t Lys As n Me t Lys Lys Lys Le u A 1 a S e r Wa Wa Th r Cys Th r Le u 4 6 5 4 7 O 4 75

TTA G C T C C T A T G T TT TT G AAT G GA AAT GT G AAT G CT GTT TAC G CA GAC 9 6 Le u A a Pro Me t P he Le u As in G 1 y As in Wa As in A 1 a Wa Ty r A 1 a As p 4 8 O 48 5 4 9 O

A GC AAA ACA AAT CAA ATT T. CT A CA A CA CAG AAA AAT CAG AAA GAG 1 4 4 S e r Lys Thr As n G 1 n I le S e r Th r Th r G in L y s As in G in L y s G u 4 9 5 5 O O 5 O 5 5 1 O

A T G GA C C GA. AAA G GA TTA. CTT GGG TAT TAT TT C AAA AAA GAT TTT 19 2 Me t A s p A rig L y s G 1 y Le u Le u G 1 y Ty r Ty r P he L y s Ly is As p P he 5 15 5 2 O 5 25

A GT AAT CTT ACT A T G TTT G CA C C G A CA C GT GAT A GT CTT TAT 24 O S e r As in Le u Thr Me t P h e A a P ro Th r A rig As p S e r Le u Ty r 53 O 5 35 5 4 O

GAT CAA CAA ACA G CA. AAT AAA CTA TTA GAT AAA AAA CAA GAA TAT 28 8 As p G 1 in G 1 in Th r A 1 a. As n Lys Le u Le u As p Ly is L y s G in G u Ty r 5 4 5 5 5 O

CAG T CT ATT C GT T G G AT T G GT TT G ATT CA G A GT AAA A CG G GA GAT 33 6 G in S e r I 1 e Arg Tr p I le G 1 y Le u I e G in S e r L y s Th r G 1 y As p

5,770,696 69 70 -continued

Le As in A s p Thir Thr G 1 y P he L y s As p Wa S e r H is Le 5 9 5 6 O O 6 O 5

W a Lys Le u Thr Pro Lys Wa Th r I e Le u S e I 6 1 O

As n S e r I e Th 6 3 5

Gl As in G 1 y G in 6 4.5 6 5 O 65

Le Th r Le u As in Th r A 1 a G G 6 65 6 7

Le T y I e S e r Le u 68 5

I I l e A sp G 1 y G u I e 6 9 O 69 7 O O

Th Th r Lys Thr V a As n W a L y s As p 7 O 5 7 1 O 7 15 2

I I e A a His As n I e As in Pro I e S e r S e r Le 7 25 7 3 O

Th r A s in A s p G 1 u I le Th P h e Tr p I e Th 7 4 O 7 45 7 5

V a 1 A a Se r I 1 e Lys P r As n Le u Th r Gl 7 55 7 6 5

Gl I le Ty r S e r A rig Ty r Gl L y s Le u G u G 1 y I I 7 7 O 7 7

Lys Ly s G 1 y G 1 y I le G 1 y G 1 u P he G 7 8 7 9 O 7 9 5 8 O

Ph As n I I e G u Pro Le u Gl Ty r V a 1 Th r G Th 8 O 5 8 1 O

S e r S e r G 1 u Le u G 1 y P r Wa S e r Th r Gl 8 2 O 8 25 8 3

I le Ty r Lys A s p G 1 y Th Ly s P he P he Th r 8 3 8 45

Ph A s p S e r G 1 y T r Ph 8 5 O 8 5 8 6 O

I e A s in A a I e Thr As p G 1 y Lys G u Me t W a Ph 8 7 5 8 8 Ty r A s in Lys

( 2) INFORMATION FOR SEQ ID NO:6: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 2004 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( i v ). ANTI-SENSE: NO ( 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: 1.2001

5,770,696 78 -continued

I e G 1 in G 1 in I I e Lys A 1 a Th r S e G 29 O s b o

G u A rig V a 1 A a G 1 u Lys Wa A G l 3 O 5 3 1 O 3 2

Gl A s p Lys Thr Pro Se r Le Th r Le Le u S e 3 2 5

Pro A s p G 1 u I le Lys Gl I e Le u 3 4 O 3 4

Pro I le Ty r G 1 u Sle r S e Me Th Th 3 5 5 3 6 O

A Ly s G 1 u V a 1 T her Lys G Le u Th r P he 3 7 O 3 7 o

V a 1 Ser H is Le u T y r Wa Le Th r Me t 3 8 3 9 O 39 5

Th I e Lys Le u Se r I 1 e Le il. A 1 a S e r 4 O 5 4 1 O S e I le G 1 y Lly o T r p Thr Th r I Wa G 1 y

Gl Th r Le 4 4 O

Th r A s p A a G 1 n G 1 u Le u I 4 5 O 4

Le u T y r Me t Lys Ser Th G in G u Th r I 4 75 4 8 G 1 y G 1 u I e Ty r P ro Th r o Th r I e o

I e S e r S e r Le u Hi s Ph

A S e r 53 O

e Th r A s p Ser G 1 u I le G in S 5 5 5 O 55 5

G 1 u A s p G 1 y I le Le u G 1 y I e 5 6 5 o y

Ph e I e A s in G u. A 1 a P he A. I G u Le u O 58 O

Thir Lys Tyr G 1 u V a 1 S e G u W a 59

1. R

G G in Le u 6 3 5

G l y Le u As in Trp As p i. A 1 a Th r

G u Me t As in Wa P he A rig

2 ) INFORMATION FOR SEQ ID NO:8: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single 5,770,696 79 80 -continued (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) ( i i i ) HYPOTHETICAL: NO ( i v ) ANTI-SENSE: NO ( i x ) FEATUR (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 5,770,696 81 82 -continued

( 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

( 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 1 a Le u Ser G 1 u As in Thr G 1 y Lys A s p G 1 y Gil y Ty r I le V a 1 Pro 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 I e A s in G 1 u 5,770,696 83 84 -continued

( 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 E: ( 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 I e A s in G 1 u 1. 5

( 2) INFORMATION FOR SEQ ID NO:16: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 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 ( i x ) FEATURE: ( A ) NAME/KEY: Peptide (B) LOCATION: 1.11 ( D.) OTHER INFORMATION: note= “N-terminal sequence from 60 Da delta- endotoxin

( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:16: Me t As in W a Le u As n Ser G 1 y Arg Th r Thr 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO:17: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 2655 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( i v ) ANTI-SENSE: NO ( i x ) FEATUR (A) NAMEKEY: misc feature (B) LOCATION: 1.2652 ( D.) OTHER INFORMATION: note= “Maize optimized DNA sequence for 100 kd VIP1A(a) protein from AB78 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:17:

5,770,696 99 100 -continued

25 3 O

Le u W a W a I A 1 a A s p G 1 in Le S e G in 4 O

S e r S Th Le Le u L y s I O A G u

Ph Gl S A la Lys G 1 y G

G 1 y G u A 1 a Thr G 1 u Gl G u Me t 8 9 O

P he Le i. I e Lys Thr G u I Th r 1 O 5 1 1 O

P he A G S e G u A s p G 1 u I Le u Gl G u 1 2 O

I e I Ph A 1 a As n Le u S e I e I Th r

T y W a Gl Th r I le G 1 y Ph S e r Le Th r 1 5 1 6 O

G Th e A a Me t A Gl P he Gl G in 1 7 O 1 7

P he Le G 1 y Me P he A s p S e r Le Th r Le u o 18 5 1 9 O

Th r G in W a L y S Ly is Arg W a I W a Th r 1 9 5

Wa S e r Gl G Th r P ro Th G 1 y W a

Gl Me t Le u I e G 1 y W a 2 4 O

W a Wa V a l Lys Gl Me t G u 2 4 25 s

G in W a G u Gl Th S e r Le u Ph 2 6 2 65

G u A Gl Me t Lys I G u T r 2 7 5 28 28 5

Th A A rig G u. A 1 a Le G 1 y A

G in Gl e Ty r Le u G in G 3 O 5

G 1 y G u Le u L y s I S e r A 3 2 3 3 3 3

G 1 y O I I e Thr W a 3 4 5

Me t G u Ph G e S e r A sp P r Le Le 3 5 5

G u Gl Gl Th r I e Gl G 3 7 38

Th r S e A rig L e lu A P he o 39

I e Le Gl Pro Lys Gl Th r A 4 1 4 1

Le u A 1 a e Ph S e r G 1 u I e Le 4 2 4 3 O

L y s S e r I e Ly is A 1 a Th Gl I e I 43 5 4 4 O 4 4 5 5,770,696 101 102 -continued

G 1 y V a 1 Lys Arg Ty W a W a As p A a Th r Le l Le u Th r As i. 4 5 O 4 5 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 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Me t Lys As n Me t Ly S Le A W a Wa Th r Le u 1. 5

Le u A a Pro Me t Ph Gl A 1 a i. 2 O

Ser Lys I e As n S e Th G Gl G u

Gl Gl Ph L y S P he 5

Me P r Th Me t 7

Le Le Gl

G in S e r I e A r Gl Le Th G 1 y 1 1

Ph e Thr Phi e As in A I e Gl I e 1 15 1, 2

G 1 y Lly I e I e G in Wa W a Le u o s 1 4 O

G 1 u Lys G 1 u Lys I G in Th r 1 4 5 1 6 O

Th Ph L y S Le u P he 6 1 7 5

I e A s p Ser G 1 i. G S e Gl Le u i. G u 18 O 18

Ph e As n Ly S L y S G Gl Ph Le u A 1 a Th r 2 O 2 O 5

A r G u Th r

Th r A s p G 1 y As p I G u Th r 2 25 2 3 24 O

I le G 1 in As in Lys W a S e r Le u

G 1 y Ty r T hr Ly W a S e S e r Th Wa G 1 y o 2 7 Gl o Le u S e r

Th Ph Wa A A 1 a S e r Wa 29 O 29 3 O O

W a I S e r G u Le u S e r o 3 1. 3 2 O

S e Th As in T r Th Th r 3 2 3 3 O 3 3 5

G 1 u G 1 y Al a Sle r I e A Gl G G 1 y Le u G 1 y S e r P he 3 4 O 3 4 3 5

5,770,696 115 116 -continued

8 5 9 O 9 5

Ph Le Lys Thr I e Th r 1 O O 1 O 5 1 1

Ph A 1 a S e Ph G l A s p G 1 u I e Le G u 1, 2 O

I Me t Ph Th As n L e u S e r I Th r 1.

Wa Gl Th I le G 1 y P he S e Le u Th r 1 5 5 1 6 O

G Th r e A a Me t A 1 a Gl Ph G u G in 1 7 O 1 7 5

Ph Le Ph A s p S e r Le Th Le u 18 5 1 9 O

Th G in W a G 1 u A rig Wa I Wa Th r 1 9 5

W a S e r Th r P ro Th r S Gl Wa I e

Gl Me Le u I e Me t o 24 O

W a W a V a l Lys Gl W a G C y S Le u 2 4 25

Gl I G u Th S e r Le u Ph i. I e 2 6 2 7

G u Gl Me t Lys G A 1 a 2 7 5 28 28

G I u. A a Le u Gl

Gl Gl e Ty r Le u Gl G G 1 y 3 O

Gl G u I e Lys A 1 a Le u 3 2 3 3 3 3 5

Gl P O I I e Thr Wa G 1 y 3 4 5 5 R

Me G u G S e r A sp P ro Le u 3 5 5

e G u Gl Th r I e l G 1 y 3 7

Th r A rig L e lu A 1 a Ph o 39 5

I e Gl Pro Lys G 1 y Th A 1 a 4 1 4 15

Le A 1 a I e Ph S e r G 1 u I l Le u 4 25 4 3

I Lys V a 1 Th r I e R 4 4

Gl W a A a Thr Le u l Th Me t

Me S e Wa Wa Th r Th Le u A 1 a 4 6 4 7 4 75 4 8 O

P r Me P he W a As in A 1 a Wa 4 9 O 4 9 5

Th G in Th Th G Ly is As in G in Gl G Me t 5 O 5 5 1

5,770,696 119 120 -continued

Th As G I e A s p Thr H is Gl y As I Wa Th 9 45 o 9 5 5 9 6 O

Gl y G W a I G in G in I e Ly S A Th r A 9 7 O 9 7 5

S e I e I e Wa As p Gl G A rig V a 1 A 1 a G Wa 9 8 O 9 8 5

A G u Gl A s p L y s Th r Th r Le 1 O

Le u Le Pro As p G u e G I e G

Gl Le Pro I e T y Gl S e Wa 1 O

Th Le G u Th A Ly s G 1 u Th G 1 O 45 1 O 5 1 O 5

Th Th Gl Ph Wa S e r Le Wa 1 O 1 O 6 5

Le Th W a I e Lys Le u 1 O

A G S e r I le G 1 y Th r 1 O

e W a S e G 1 y G 1 y Ly is Ly s G in l 1 115 Le Thr As p Gl 1 13

I Le u T y r Me t 1 1 4 5

Th G I e Th I G 1 y G 1 u I e Th r s 1. :

Th Wa As n Ty r I e 1 1

A I e I e S e r 1 1

G I e Th r Ph e A s p As p S e 1 2 O 5 1 2 1

A Thr As p G 1 225 3

G 1 y l G 1 u As p I L e 1. 2

G I e Gl Ph e I e Gl P he 1, 2 1, 2 1, 2

I G Le u G in Th r Lys Gl 1, 2

S e G Le G 1 y W a A s p Thr Le u Gl 1 28 5 1, 29

Gl Th r I A s p P he Th r 13 13 O 5

Gl G Le u P he G 1 y Le u As in 13 3 g

A I e Th r Ty r Gl G u Me t As in W a 13 13 13

4 5

5,770,696 133 134 -continued

7 35 7 4 O 7 4 5 7 5 O

GA T G TT T. CT GAA. A T G T T C A CT A CA AAA TTT GAG AAA GAT AAC TTT TAT 23 O 6 A s p V a 1 Ser G 1 u Me t P he Thr Th Ly s P he G u Ly S As p As in P he Ty 7 55 7 6 O 7 6 5

ATA GA G CTT T. CT CAA. GGG AAT AAT TTA TAT C CT ATT GT A CAT 23 5 4 I le G 1 u Le u Ser G 1 in G 1 y As in As i. Le u Ty r Gl y P ro I e Wa Hi S 7 7 O 7 7 5 7 8 O

TTT TAC GAT G T C T C T ATT AAG TAA 23 78 Ph e Ty r A s p V a 1 Se r I 1 e Lys 7 85

( 2) INFORMATION FOR SEQ ID NO:29: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 789 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Me t As n Lys As n As n Th r Lys Le S e r Thr A a Le u Ph 1. 5 1 O

I e A s p Ty r Phe As in G 1 y I 1 e G 1 y P he Th G 1 y I e 2 O 25 I e Me t As in Me t I le P he Ly s o A s p Thr Th r Le

Gl Le u L e u G 1 y 6

Le As in As p I G in G 1 y

Gl I e Le u I G 8 5 9 O

As n L y s A 1 a Th 1 O O 1 O 5 l Me t Le u A rig V a 1 Ty r Le u Pro I e Thr Me Le u W a 1 15 o 1 2 5 Me t Lys G 1 m As n Ty r A 1 a Le u Le u G in Le u S e r 13 O 1 35 1 4

G l n Le u G | n G 1 u I le S e r A sp Le u As p I Wa 1 4 5 1 5 O

Le u I e A s in S e r Th r Le u Thr G I e Thr G in I 1 65 1 7

Gl G u Le u Th Ph A 1 a Th r Th 18 O 18 5 1 9 O

S e r S e r Lys V a l Lys Lys As p Gl S e r P r o A Le u 2 O

Le u Thr G I u. Le u Thr G I u. Le u A Lys S e r W a Th W a 21 O 2 15 2 2

Th r Phe Wa Me t Wa Gl 2 3 2 4

A Le u L y s Th A G u I 2 4 25 25 5

Th r Lys G 1 u As n V a l Lys Thr S e G 1 y Ser G u W a G 1 y Wa 2 6 O 2 65 2 7 O

As in Phi e Le u I e V a Le u Thr A Le u G in A a Gl A 1 a P he Le u Th 2 7 5 28 28 5

5,770,696 147 -continued

Le Th G l Le Th l A Ly S S e Wa Th r Ly W a 2 1 2 1. 22 O

Ph Gl Ph Th Ph H is As p W a Me Gl y o 23 5 2 4 O

Le Ph Gl A Le Th r A 1 a G Le l I 2 4 o 25 5

Th G W a S e G G u Wa G W a 2 6 2 6 2 7

Ph I W a A Le A 1 a A Ph Le l Th 28 28

Le Th Le G Le A 1 a I Th 29 I Me G o G L y S G Ph

I Le P r Th Th Ph e 3 2 3 3 s

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

P r G A Le I Gl Ph G I S e r S e I Th 3 6

W a W a G A Le G in G W a 3 7

Le S e l W a I Gl Le 39 39 5

Gl G Gl I Th r 4 O 4 1 P r G o W a I Th P he Th r o Me

Th Le G W a Th A Ph Th 4 4 Gl e Le s S e r Gl

Th S e i. W a Me t Le Gl 4 6

I S e Th Ph Le Th P he Le 4 8 49 49

G A r Le I Th Th Le 5 O 5 O

Gl Le Le A Th G u Le I 5 1.

W a S e G Ph e I Wa G u Gl I 5 3 5 4 O

Gl G Le Gl l 5 4 5 5 o

W a Th Gl G W a Th A 1 a 5 6 5 7 O

Gl I Gl Ph Gl S 58

Th G W a I G Th G 1 y P I 6 O

Le Gl Th G G u Th o 6 1 6 2 O

G Th I e P he Th Th Gl Th 6 2 63 6 4 5,770,696 149 150 -continued

I Le u L y s S e Gl G 1 y G u 6 4.5 6 5 O 65

A a T r p G 1 y A s p As in Ph e I le I Le u G u. I G 6 6 O 6 65 6 7 O

Le u Le ul S e r Pro G 1 u Le u I e Th r A is in T r Th r S e r Th 6 7 5 68 68 5

Ser Thr As n I le Ser G 1 y As n Th Le u Thr Le G in G 1 y Gl 6 9 O 6 9 5

G 1 y I le Le u L y s G 1 m As n Le u Gl Le u As p Th r 7 O 5 7 1 O 7 1.

V a 1 Ty r P h e Sier V a 1 Ser G 1 y A la As in W a I e 7 25 7 3 O

A rig G 1 u V a 1 Le u Ph e G 1 u Lys Ty r Me t S e Gl 7 4 O 7 4

Ser G 1 u Me t P h e Thr T hr Lys Ph e G 1 u Lys P he G u 7 55 7 6 7 6 5

Le u Ser G 1 n G 1 y As n As n Le u G 1 y G 1 y e Wa 7 7 O 7 7 5

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

( 2) INFORMATION FOR SEQ ID NO:33: ( 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

( 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”

5,770,696 157 -continued

Me Th As i. G in I S e Th Thr G 1 m. Ly S As i. G u 1 O 1.

Me G 1 y Le Le Gl Ty r Ty r P he 25 o

S e Th Me t Ph A O Thr Arg Le 5 i. Th A 1 a Le Le u As p o Gl

I A r T r p e Gl Le I e G 1 in Th

Ph Th Ph Gl G u G in I Gl 9 O

Gl I e Ly s G 1 u Gl W a Le u 1 O 5 1 1

Gl G Le u W a e Lys I le Gl G S e Th r 1 1 1, 2

e P he L y s Gl Le

I S e G in Wa Gl G 1 4 1 5 1 5

G S e r G 1 in G Ph Le P ro 1 7 O y

S e I i. Ph Th G 1 m Me t G e G u 18 5

I e P r o Le 1 9 2 O Gl o Th G in I A a Wa Le u

A Th Ph Wa S e r Gl 2 2 2 3 24 O

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

S e A 1 a Th Ph e A s in Le W a A P he 2 65 2 7

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

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

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

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

Gl G Gl Th r Th Gl As in Th r Gl Ph Th A 1 a 3 4 3 4 5 3 5

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

Gl A I W a P r Th r Th r Ph W a Le 3 7 38

Th I A Th r e Lys S e r Th A Le 38

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

I e Th S e Ph S e r H is P r I Th 4 2 4 25 4 3 5,770,696 159 160 -continued

G W a As p Le Le u As in As Me t Me t Le l G Th 4 3 5 4 4 O 4 4 5

Th Gl y W a T y Th r H is G y I 4 5 4 6 O

Th G 1 y Gl l G in G in I e A S 4 7 4 75

Th r S e I e W a Gl l A rig A 1 a Gl : 49 O s

Wa L y S G u G Th r Le 5 1

Th r Le A Le Le u G u I Gl s 5 2 O 5 25

I e Le u Le T y I e Gl S e 5 3 5 4 O

Me Th Le G u Wa Th i. 5 4 5 5

Le u Th r Th P he S e r Le 5 6

Wa Le Th r P r Me t Le u I 58 O

A 1 a Gl G 1 y Th 59

Th r I Wa Gl Gl G L y S S e 6 1

A Le u Th r Th r A 1 a G G 6 25 6 3 5 6 i. o

Le u Le u Me t

Th G in G I e Th r G 1 y G u I e 6 6 O o Th r Th s Th r W a Wa L y S

I e e L y S I e Le

Gl e Ph I e S e 7 15 o

S e r P ro l Th r Gl 7 3

G in G 1 y G u G 1 y e 7 4 O

G 1 y Gl I P he s 7

P he i. I G u Le G in Th r L y S G W a 7 7 5

S e G u Le P ro W a Gl S e A. 7 9 O R

G Th r e P he Th r 8 O s

G G in Gl y Le P he G 1 y Le u 8 2 O

A 1 a I e Th G u Me t W a Ph 5,770,696 161 162 -continued

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 ) HYPOTHETICAL: 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 ( i i i ) HYPOTHETICAL: NO ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:38:

AAG CTT C C A C T C C T T C T C 18

( 2) INFORMATION FOR SEQ ID NO:39: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1241 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) NAME/KEY: CDS (B) LOCATION: 9.1238 ( D.) OTHER INFORMATION: note= “Maize optimized DNA sequence encoding VIP2A(a) with the Bacillus secretion signal removed as contained in pCIB5527 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:39:

GAT C CA C C A T G CT G CAG AA C C T G AAG AT C A C C GAC AAG G T G GAG GAC TTC 5 O Me t Le u G 1 in As n Le u Lys I le Th r A s p Lys V a 1 G 1 u A s p P he 8 55 8 6 O 8 65

AAG GAG GAC AAG GAG AAG GCC AAG G A G T G G G GC AAG GAG AAG GAG AAG 9 8 Ly s 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 Lys G 1 u Lys 8 7 O 8 75 8 8 O

GA G T G G A A G CTT A C C GCC A C C G A G AAG G GC AAG AT G AAC AAC TT C C T G 1 4 6 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 88 5 8 9 O 8 9 5

GAC AAC AAG AAC GAC AT C AAG ACC AAC TAC AAG GAG AT C A C C T T C A G C 19 4 A s p As n Lys As in A s p I e Lys Thr As in Ty r Lys G 1 u I le Thr Phe S e r 9 O O 9 O 5 9 1 O

ATA G C C G G C A G C T T C G A G GAC GAG AT C AAG GA C C T G AAG GAG AT C GAC 2 4 2

5,770,696 165 166 -continued 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 Ly s G 1 y V a 1 1 2 3 5 1 2 4 O 1 2 4 5 1 2 5 O

AA G C G C T A C G T G G T G GA C GCC A C C C T G C T G A C C AAC TAG 1 2 4 1 Lys Arg Ty r V a l V a 1 A s p A la Thr Le u Le u Thir As n 1 2 5 5 1 2 6 O

( 2) INFORMATION FOR SEQ ID NO:40: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 410 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:40: Me t Le u G | n As n Le u Lys I le Th r A s p Lys V a 1 G 1 u A s p Ph e Ly s G 1 u 1. 5 1 O 1 5 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 G 1 u Lys G 1 u T r p 2 O 25 3

Lys Le u Th r A la Thr G 1 u Lys G 1 y Lys Me t As n As n Ph e Le u A s p As in 3 4 O 4 5

Ph e A s p Lys Thr As n Le u Se r As n Sle r I I e I le Thr Ty r Lys As n 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 G 1 u G 1 1 O O 1 O 5 1 1

I e As n Sle r A s p A 1 a Me t A a G 1 in Ph e Ly 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 Ser Lys V a V a l Lys Lys G 1 y V a 1 G 1 u Cy s Le u G | n I I e G 1 u G 1 y 1 9 2 O 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 21 O 2 15 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 7 5 28 O 28

I e Pro G 1 u As n I e Thr Wa T y A. g T p C y S G Me t Pro G 1 u Ph e

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 Lys G 1 y Ty r Me t S e r Th r S e r 5,770,696 167 168 -continued

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 le I le 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 y 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

Ty r H is I e A s p Lys V a 1 Thr G 1 u V a 1 I I e I le L y s G 1 y V a l Lys Arg 38 5 3 9 O 39 5 4 O O

Ty r V a l V a 1 A s p A a Thr Le u Le u Thir As in 4 O 5 4 1 O

( 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:

G GAT C C A C C A T G G G C T G G A G CT G GAT CTT C C T G T T C C T G C T G A G C G G C G C C GC G G G C G T G 6 O

CA C T G C C T G C A G 7 2

( 2) INFORMATION FOR SEQ ID NO:42: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1241 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) NAME/KEY: CDS (B) LOCATION: 9.1238 ( D.) OTHER INFORMATION: note= “Maize optimized DNA sequence encoding VIP2A(a) with the Bacillus secretion signal removed and the eukaryotic secretion signal inserted as contained in pCIB5528 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:42:

GAT C CA C C A T G CT G CAG AA C C T G AAG AT C A C C GAC AAG G T G GAG GAC TTC 5 O Me t Le u G 1 in As n Le u Lys I le Th r A s p Lys V a 1 G 1 u A s p P he 4 15 4 2 O

AAG GAG GAC AAG GAG AAG GCC AAG G A G T G G G GC AAG GAG AAG GAG AAG 9 8 Ly s 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 Lys G 1 u Lys 4 25 4 3 O 43 5 4 4 O

GA G T G G A A G CTT A C C GCC A C C G A G AAG G GC AAG AT G AAC AAC TT C C T G 1 4 6 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 4 4 5 4 5 O 4 55

GAC AAC AAG AAC GAC AT C AAG ACC AAC TAC AAG GAG AT C A C C T T C A G C 19 4 A s p As n Lys As in A s p I e Lys Thr As in Ty r Lys G 1 u I le Thr Phe S e r 4 6 O 4 6 5 4 7 O

ATA G C C G G C A G C T T C G A G GAC GAG AT C AAG GA C C T G AAG GAG AT C GAC 2 4 2 I e 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 I le. As p

5,770,696 171 172 -continued

7 9 5 8 O O 8 O 5

AA G C G C T A C G T G G T G GA C GCC A C C C T G C T G A C C AAC TAG 1 2 4 1 L y s A r g Ty r Wa W a A s p A 1 a Th Le l Le u Th r As in 8 1 O 8 1 5 8 2 O

( 2) INFORMATION FOR SEQ ID NO:43: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 410 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:43:

G in As in Le l Ly S I e Th Wa G u Ph G u 5

G u L y s A a Ly G l G u G u 2 O o

Le A 1 a Th G Me t Le

I e Th G u P he S e I e

P he G u I Le u G u I e

P he Th r Le S e S e I e I e Th r Wa

G u Th r I G Ph Le u Th r G u Gl Th r 1 O O s 1 1

I e Me A G G u G in P he Le A rig 1, 2 1 2 5

I P he Le Th r Th r A G in G in 13 1 4 O

G I Le Th r Wa P r G 5 1 6

G Th r Th Th G Wa I e Le u 1 6 1 7

G u Le G Me t Wa 18 O 18

Wa Wa W a Gl W a G u Le u G in G u G 1 y 2 O 2 O 5

Th r S e Ph I e G u A 1 a 2 1. 22 O

G 1 y Me G G u A 1 a Le u Th r o 24 O

Gl Le Gl T y G in T y 2 4

G u Le Gl G 1 y S e r G 1 y 2 6

Le u A 1 a I I A 1 a Le u G P ro 2 7 5 28 28

I e O G u I Th G Me t P r G u P he 3 O

Gl G in I e Le S e r Ph G u G u 3 O 3 1. 3 2 O

G in Ph Le u Th I Gl L y s G 1 y Me t S e Th r 3 2 3 3 O 3 3 5

5,770,696 179 18O -continued

2 7 5 28 O 28 5

29 O 2

Gl G 1 y Ser G 1 y As in G 1 u Lys Le u A s p A a G 1 in I I e Lys As n I le S e r 3 O 3 1 O 3 15 3 2 O

3 2 5 3 3 O 3 3 5

T r p Cy s G 1 y Me t Pro G 1 u P he G 1 y Tyr G 1 in I 1 e S e r A s p Pro Le u Pro 3 4 O 3 4 5 3 5 O

Ser Le u Lys A s p P he G 1 u G 1 u G | n Ph e Le u As n Th r I 1 e Ly s G 1 u As p 3 5 5 3 6 O 3 65

Ly s G 1 y Ty r Me t Ser Th r S e r Le u Se r S e r G 1 u A rig Le u A la A 1 a Phe 3 7 O 3 7 3 8 O

38

G 1 y Al a Ty r Le u Se r A la I le G 1 y G 1 y Ph e A a Ser G 1 u Lys G 1 u I le 4 O 5 4 1 O 4 15

Le u Le u A s 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 4 2 O 4 25 4 3 O

I le I le L y ly V a Lys Arg Ty r V a V a 1 A s p A a Thr Le u Le u Thr 43 5 4 4 O 4 4 5

( 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 ( 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

G GAT C C

5,770,696 191 192 -continued

1 4 1 5 1 6 O

Gl Th r e As p A a Me t A a Gl P he Gl l G in 1 7 O 1 7 5

Ph Le P he A s p S e r Le Th Le u 18 5 1 9

Th Gl W a Ly is G 1 u A rig W a I Le u Ly W a Th r 1 9 2 O 5

W a S e Th r Th r P ro Th S Gl W a I e

Gl Me t Le u I e G 1 y Me o 24 O

W a Wa V a l Lys Gl Wa G Le u 2 4 25 s

Gl I Gl Th S e r Le u Ph I e 2 65 2 7

G Gl Me t Lys G u A 1 a 2 7 28 28 5

G I u. A a Le G 1 y

Gl Gl e Ty r Le u G in G 3 O

Gl I e Lys A Le u 3 2 3 3 3 3

Gl P O I I e Thr W a G 1 y 3 4 5 5 R

Me Gl G S e r A sp P r Le 3 5

e G Gl Th r I e l G 3 7 5

Th A rig L e lu A P he o 39

I G in Pro Lys Gl Th r A 4 1 4 1

Le A P he S e r G 1 u I e Le 4 25 4 3

I e Lys V a 1 Th I 4 4 5

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

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

S e G 1 y Th Me t Th r As in Gl S e r Th G in 4 9 O 49

G in Me t A rig L y s G Le u 5 O O 5 O 5

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

Le u I G in G in Th r A Le 5 35 5 4

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

Th r G y P he Th r Ph e As in Le S e G u Gl G in 5 6 5 7 O 5 7