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 . An isolated 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 a polypeptide sequence selected from the group consisting of SEQ ID NO:4 (TIC900), SEQ ID NO:6 (TIC402), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30 (TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16 (TIC963), SEQ ID NO:18 (TIC965) and SEQ ID NO:20 (TIC966), or a lepidopteran-active insecticidal toxin variant or a portion thereof. 
     
     
         2 . The isolated polynucleotide as in  claim 1  wherein said insecticidal toxin protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO:4 (TIC900), SEQ ID NO:6 (TIC402), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30 (TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16 (TIC963), SEQ ID NO:18 (TIC965) and SEQ ID NO:20 (TIC966), or a lepidopteran-active insecticidal toxin variant or a portion thereof. 
     
     
         3 . The isolated polynucleotide of  claim 3  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 isolated 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 selected from the group consisting of SEQ ID NO:4 (TIC900), SEQ ID NO:6 (TIC402), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30 (TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16 (TIC963), SEQ ID NO:18 (TIC965) and SEQ ID NO:20 (TIC966). 
     
     
         5 . The isolated polynucleotide as in  claim 4  wherein said isolated polynucleotide is present within a plasmid vector. 
     
     
         6 . The isolated polynucleotide of  claim 1  wherein said nucleotide sequence has been optimized for expression in plants. 
     
     
         7 . The isolated polynucleotide as in  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 isolated  Bacillus thuringiensis  insecticidal protein selected from the group consisting of TIC900, TIC402, TIC403, TIC404, TIC434, TIC961, TIC962, TIC963, TIC965, and TIC966. 
     
     
         9 . The isolated protein as in  claim 8  wherein said  Bacillus thuringiensis  is selected from the group consisting of EG5438, EG3879, EG4332, EG4971, EG4090, EG4293, EG4611, EG5526, EG5023 and EG4092. 
     
     
         10 . A host cell transformed to contain a polynucleotide encoding an insecticidal protein or insecticidal fragment thereof as set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:30, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, wherein said polynucleotide comprises (1) a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, or (2) a nucleotide sequence that exhibits from about 60% to about 85% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. 
     
     
         11 . The host cell of  claims 10  wherein said host cell is a plant cell. 
     
     
         12 . A method for controlling a lepidopteran insect pest comprising contacting said pest with a pesticidal amount of an approximately about 65 kDa to approximately about 70 kDa  Bacillus thuringiensis  toxin protein or insecticidal fragment thereof, wherein said toxin protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:30, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20. 
     
     
         13 . The method of  claim 12  wherein said toxin protein is encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence comprising at least 18 consecutive nucleotides selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. 
     
     
         14 . A method for detecting a nucleotide sequence in a sample, said method comprising the steps of
 (a) selecting a pair of nucleotide primers for use in producing an amplicon from said nucleotide sequence from an alignment of polynucleotide sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19;   (b) producing an amplicon from said nucleotide sequence in said sample; and   (c) detecting said amplicon.   
     
     
         15 . The host cell of  claim 11  comprising 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 as set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ.ID NO:30, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20; 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   (d) generating from said transformed plant cell a plant that expresses the lepidopteran-active toxin protein in the transformed plant.   
     
     
         17 . A seed from the transformed plant of  claim 16 , wherein said seed comprises said nucleic acid sequence. 
     
     
         18 . Progeny of the seed of  claim 17  wherein said progeny comprise said nucleic acid sequence. 
     
     
         19 . The transformed plant of  claim 16  wherein said plant cell is (a) a monocot plant cell comprising 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, or (b) a dicot plant cell comprising 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. 
     
     
         20 . A biological sample derived from the tissues or seed of the plant of  claim 18  wherein the sample comprises a detectable amount of said nucleotide sequence.