Secreted Insecticidal Protein and Gene Compositions From Bacillus Thuringiensis and Uses Therefor

Abstract

The present invention relates to the isolation and characterization of nucleotide sequences encoding novel insecticidal proteins secreted into the extracellular space from Bacillus thuringiensis and related strains. The proteins are isolated from culture supernatants of Bacillus thuringiensis and related strains and display insecticidal activity against lepidopteran insects including European corn borer (ECB), tobacco budworm (TBW) and diamondback moth (DBM). Insecticidal proteins encoded by nucleotide sequences that hybridize under stringent conditions to the isolated and characterized nucleotide sequences are disclosed. Methods are disclosed for making and using transgenic cells and plants comprising the novel nucleotide sequence of the invention.

1 . A recombinant polynucleotide which encodes a  Bacillus thuringiensis  insecticidal toxin protein or insecticidal fragment thereof, active against a lepidopteran insect pest, wherein said insecticidal toxin protein comprises a polypeptide sequence that has at least about 80% sequence identity to SEQ ID NO:6 (TIC402). 
     
     
         2 . The recombinant polynucleotide of  claim 1 , wherein said insecticidal toxin protein comprises SEQ ID NO:6. 
     
     
         3 . The recombinant polynucleotide of  claim 1 , wherein said lepidopteran insect pest is selected from the group consisting of a Noctuidae, a Tortricidae, Epinotia aporema, Anticarsia gemmatalis, Pseudoplusia includens, European Corn Borer (ECB), a Tobacco Budworm (TBW), Black Cutworm (BCW), and a Diamondback Moth (DBM). 
     
     
         4 . The recombinant polynucleotide of  claim 1 , wherein said toxin has a molecular weight between approximately 65 kDa and approximately 70 kDa, and wherein said insecticidal toxin is SEQ ID NO:6 (TIC402). 
     
     
         5 . (canceled) 
     
     
         6 . The recombinant polynucleotide of  claim 1 , wherein said nucleotide sequence has been optimized for expression in plants. 
     
     
         7 . The recombinant polynucleotide of  claim 6 , wherein said nucleotide sequence has been optimized for (a) expression in a monocot plant, said optimization comprising one or more of the steps selected from the group consisting of (i) removing polyadenylation sequences, (ii) adjusting the A and T content of the nucleotide sequence to be from about 40% to about 49% without modifying the amino acid sequence of the protein, and (iii) modifying codons in the coding sequence to be consistent with the steps (i) and (ii), or (b) expression in a dicot plant, said optimization comprising one or more of the steps selected from the group consisting of (i) removing polyadenylation sequences, (ii) adjusting the A and T content of the nucleotide sequence to be from about 40% to about 49% without modifying the amino acid sequence of the protein, and (iii) modifying codons in the coding sequence to be consistent with the steps (i) and (ii). 
     
     
         8 . An insecticidal protein active against Lepidopteran insects, said protein comprising an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:6 (TIC402). 
     
     
         9 . (canceled) 
     
     
         10 . A host cell comprising the recombinant polynucleotide of  claim 1 . 
     
     
         11 . The host cell of  claim 10 , wherein said host cell is a plant cell. 
     
     
         12 . A method for controlling a lepidopteran insect pest, said method comprising contacting said pest with a pesticidal amount of the insecticidal protein of  claim 8  or insecticidal fragment thereof. 
     
     
         13 - 14 . (canceled) 
     
     
         15 . The host cell of  claim 11 , said plant cell selected from a corn plant cell, a wheat plant cell, a rice plant cell, an oat plant cell, an onion plant cell, and a grass plant cell; and wherein said dicot plant cell comprises a cotton plant cell, a canola plant cell, a soybean plant cell, a tobacco plant cell, a fruit tree plant cell, a cruciferous plant cell, a pepper plant cell, an ornamental plant cell, a sunflower plant cell, a cucurbit plant cell, and a melon plant cell. 
     
     
         16 . A method for expressing a lepidopteran-active toxin protein in a plant, comprising the steps of:
 (a) inserting into the genome of a plant cell a nucleic acid sequence comprising in the 5′ to 3′ direction an operably linked recombinant, double-stranded DNA molecule, wherein the recombinant, double-stranded DNA molecule comprises:
 (i) a promoter that functions in the plant cell; 
 (ii) a nucleotide sequence encoding an insecticidal amino acid sequence having at least about 80% sequence identity to SEQ ID NO:6; and 
 (iii) a 3′ non-translated nucleotide sequence that functions in the cells of the plant to cause termination of transcription; 
   (b) obtaining a transformed plant cell containing the nucleic acid sequence of step (a); and   (c) generating from said transformed plant cell a plant that expresses the lepidopteran-active toxin protein in the transformed plant.   
     
     
         17 - 20 . (canceled) 
     
     
         21 . A plasmid vector comprising the recombinant polynucleotide of  claim 1 . 
     
     
         22 . A transformed plant comprising the recombinant polynucleotide of  claim 1 . 
     
     
         23 . A seed from the transformed plant of  claim 22 , wherein said seed comprises said recombinant polynucleotide. 
     
     
         24 . A biological sample derived from the tissues or seed of the plant of  claim 22 , said sample comprising a detectable amount of said recombinant polynucleotide. 
     
     
         25 . A commodity product derived from the plant of  claim 22 , wherein said product comprises a detectable amount of said recombinant polynucleotide. 
     
     
         26 . A composition comprising an insecticidally effective amount of the insecticidal protein of  claim 8 . 
     
     
         27 . The composition of  claim 26 , said composition further comprising an additional insecticidal agent toxic to the same Lepidopteran insect pest but exhibiting a different mode of effectuating its insecticidal activity from said insecticidal protein. 
     
     
         28 . The composition of  claim 27 , wherein said insecticidal agent is selected from the group consisting of a  Bacillus  toxin, a  Xenorhabdus  toxin, a  Photorhabdus  toxin, and a dsRNA specific for suppression of one or more essential genes in said insect pest. 
     
     
         29 . The composition of  claim 28 , wherein said  Bacillus  toxin is selected from the group of proteins consisting of a Cry1, a Cry2, and a Cry9 toxin. 
     
     
         30 . A method of controlling Lepidopteran insect infestation in a crop plant and providing insect resistance management, said method comprising contacting said crop plant with a pesticidal amount of the composition of  claim 26 .