Polypeptide compositions toxic to anthonomus insects, and methods of use

Abstract

A novel gene encoding a Coleopteran inhibitory Bacillus thuringiensis insecticidal crystal protein is disclosed. The protein, tIC851, is insecticidally active and provides plant protection from at least cotton boll weevil, Anthomomus grandis, when applied to plants in an insecticidally effective composition.

What is claimed is:  
     
         1 . An isolated and purified polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 8.  
     
     
         2 . The polypeptide of  claim 1  exhibiting insecticidal activity when provided orally to a susceptible insect larva.  
     
     
         3 . The polypeptide of  claim 2  exhibiting insecticidal activity when provided in an orally administrable diet to a Coleopteran insect larva.  
     
     
         4 . The polypeptide of  claim 3  wherein said insect larva is a cotton boll weevil larva.  
     
     
         5 . The polypeptide of  claim 1  encoded by a nucleic acid sequence comprising at least the open reading frame as set forth in SEQ ID NO: 7 from nucleotide position 28 through nucleotide position 1923.  
     
     
         6 . A composition comprising an insecticidally effective amount of the polypeptide of  claim 1  wherein said composition is a bacterial cell comprising a polynucleotide sequence that encodes said polypeptide, said composition being selected from the group consisting of a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet.  
     
     
         7 . The composition of  claim 6  wherein said bacterial cell is a bacterial species selected from the group consisting of Bacillus, Escherichia, Salmonella, Agrobacterium, and Pseudomonas.  
     
     
         8 . The composition of  claim 7  wherein said bacterial cell is selected from the group consisting of EG4135 and EG4268.  
     
     
         9 . A composition comprising an insecticidally effective amount of the polypeptide of  claim 1  wherein said composition is formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, or solution.  
     
     
         10 . The composition according to  claim 6 , prepared by desiccation lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration.  
     
     
         11 . The composition of  claim 10  wherein said polypeptide is present in a concentration of from about 0.001% to about 99% by weight.  
     
     
         12 . An isolated and purified polynucleotide sequence encoding the polypeptide of SEQ ID NO: 8.  
     
     
         13 . The polynucleotide sequence of  claim 12  wherein said polypeptide exhibits insecticidal activity when provided orally to a susceptible insect larva.  
     
     
         14 . The polynucleotide sequence of  claim 13  wherein said polypeptide exhibits insecticidal activity when provided in an orally administrable diet or composition to a Coleopteran insect larva.  
     
     
         15 . The polynucleotide sequence of  claim 14  wherein said insect larva is a cotton boll weevil larva.  
     
     
         16 . The polynucleotide sequence which is or is complementary to the polynucleotide sequence of  claim 15  and which hybridizes under stringent conditions to a polynucleotide sequence complementary to or encoding the polypeptide as set forth in SEQ ID NO: 8.  
     
     
         17 . A method for protecting a cotton plant from boll weevil infestation comprising providing to a boll weevil in its diet a plant transformed to express a protein toxic to said weevil wherein said protein is expressed in sufficient amounts in said plant's tissues to control boll weevil infestation and wherein said protein is selected from the group consisting of Cry22Aa, ET70, and tIC851.  
     
     
         18 . A method for protecting a cotton plant from boll weevil infestation comprising providing to a boll weevil in its diet a plant or plant tissue transformed to express one or more proteins toxic to said weevil wherein said proteins are expressed in sufficient amounts alone or in combination to control boll weevil infestation and wherein said proteins are selected from the group consisting of Cry22Aa, ET70, and tIC851.  
     
     
         19 . A vector for use in transforming a host cell, wherein said vector comprises a polynucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 8.  
     
     
         20 . The vector of  claim 19 , wherein said vector is plasmid pIC17501.  
     
     
         21 . The vector of  claim 19  wherein said host cell is selected from the group consisting of a plant cell and a bacterial cell.  
     
     
         22 . A plant tissue transformed with a polynucleotide sequence which expresses the polypeptide of  claim 1 , wherein said tissue is selected from the group consisting of a plant cell, an embryonic plant tissue, plant calli, a leaf, a plant stem, a plant root, a plant flower, a fruit, a fruiting body, a boll, and a plant seed.  
     
     
         23 . The plant tissue of  claim 22  wherein said tissue comprises said polypeptide present in a coleopteran insect inhibitory effective amount.  
     
     
         24 . The plant tissue of  claim 23  wherein said coleopteran insect is a cotton boll weevil.  
     
     
         25 . The plant tissue of  claim 22  selected from the group of plants consisting of corn, wheat, cotton, soybean, oat, rice, rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley, turf grass, pasture grass, berry bush, fruit tree, legume, vegetable, ornamental plant, shrub, cactus, succulent, deciduous tree, and evergreen tree.  
     
     
         26 . A method of making a transgenic plant resistant to coleopteran insect infestation comprising incorporating into a genome of a plant cell a polynucleotide comprising at least a plant functional promoter operably linked to a nucleotide sequence encoding the polypeptide of SEQ ID NO: 8, isolating and propagating a plant cell transformed with said polynucleotide, regenerating a plant from said plant cell transformed with said polynucleotide, and propagating said plant from progeny, wherein said plant expresses an insecticidally effective amount of said polypeptide from said polynucleotide.  
     
     
         27 . The method of  claim 26  wherein said plant cell is either a monocot or a dicot plant cell.  
     
     
         28 . The method of  claim 27  wherein said monocot plant cell is selected from the group of plant cells consisting of corn, wheat, rye, barley, rice, banana, sugarcane, oat, flax, turf grass, pasture grass, and sorghum cells.  
     
     
         29 . The method of  claim 27  wherein said dicot plant cell is selected from the group of plant cells consisting of cotton, soybean, canola, potato, tomato, fruit tree, shrub, vegetable, and berry cells.  
     
     
         30 . An isolated and purified antibody which specifically binds to the peptide as set forth in SEQ ID NO: 8 or an epitope therein, said antibody produced from the immune system of a vertebrate in response to the exposure of all or an antigenic part of said peptide to the animal's immune system.  
     
     
         31 . A method for detecting the presence of a peptide as set forth in SEQ ID NO: 8 in a sample comprising obtaining a solution suspected of containing said peptide, probing said solution with the antibody of  claim 30 , and detecting the binding of said antibody to said peptide.  
     
     
         32 . A kit for detecting the presence of the peptide of SEQ ID NO: 8 in a sample comprising, in suitable container means, an antibody that binds to said peptide, reagents necessary for mixing the peptide and antibody in a solution, at least a first immunodetection reagent providing said antibody along with control antibody, control antigen, and the reagents and instructions necessary for detecting said binding.  
     
     
         33 . A plant cell transformed with a polynucleotide sequence that expresses one or more of the polypeptides as set forth in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 10 or insecticidal fragments thereof, wherein said cell produces an amount of said one or more polypeptides effective for controlling a Coleopteran insect pest infestation.  
     
     
         34 . The plant cell of  claim 33  wherein said Coleopteran insect pest is a cotton boll weevil and said plant cell is a cotton plant cell.