Pesticidal genes from brevibacillus and methods for their use

Abstract

Compositions and methods for conferring insecticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions including a coding sequence for a Brevibacillus -derived delta-endotoxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also include transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated delta-endotoxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for isolated nucleic acid molecules having nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2, 4, 7, or 10, or the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 6, 8, 9, 11, 12, 13, 14, or 15, as well as variants and fragments thereof.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
 a) the nucleotide sequence of any of SEQ ID NO:3, 6, or 9, or a complement thereof;   b) the nucleotide sequence selected from:
 i) nucleotides 61-1953 of SEQ ID NO:3; 
 ii) nucleotides 160-1953 of SEQ ID NO:3; 
 iii) nucleotides 4-1059 of SEQ ID NO:6; 
 iv) nucleotides 13-1059 of SEQ ID NO:6; 
 v) nucleotides 151-1059 of SEQ ID NO:6; 
 vi) nucleotides 19-1893 of SEQ ID NO:9; 
 vii) nucleotides 46-1893 of SEQ ID NO:9; and 
 viii) nucleotides 52-1893 of SEQ ID NO:9; 
   c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:4, 7, or 10;   d) a nucleotide sequence that encodes a polypeptide selected from:
 i) amino acids 54-651 of SEQ ID NO:4; 
 ii) amino acids 2-352 of SEQ ID NO:7; 
 iii) amino acids 5-352 of SEQ ID NO:7; 
 iv) amino acids 51-352 of SEQ ID NO:7; 
 v) amino acids 7-630 of SEQ ID NO:10; 
 vi) amino acids 16-630 of SEQ ID NO:10; and 
 vii) amino acids 18-630 of SEQ ID NO:10; 
   e) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, 7, 10, or any of (d)(1-viii), wherein said amino acid sequence has pesticidal activity against a plant pathogen.   
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant. 
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid sequence is selected from SEQ ID NO:1, 5, 8, 11, 12, 13, 14, or 15. 
     
     
         4 . A vector comprising the nucleic acid molecule of  claim 1 . 
     
     
         5 . The vector of  claim 4 , further comprising a nucleic acid molecule encoding a heterologous polypeptide. 
     
     
         6 . A host cell that contains the vector of  claim 4 . 
     
     
         7 . The host cell of  claim 6  that is a bacterial host cell. 
     
     
         8 . The host cell of  claim 6  that is a plant cell. 
     
     
         9 . A transgenic plant comprising the host cell of  claim 8 . 
     
     
         10 . The transgenic plant of  claim 8 , wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape. 
     
     
         11 . A transgenic seed comprising the nucleic acid molecule of  claim 1 . 
     
     
         12 . An isolated polypeptide with insecticidal activity, selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence of any of SEQ ID NO:4, 7, or 10;   b) a polypeptide that is encoded by the nucleotide sequence of any of SEQ ID NO:3, 6, or 9;   c) a polypeptide selected from:
 i) amino acids 54-651 of SEQ ID NO:4; 
 ii) amino acids 2-352 of SEQ ID NO:7; 
 iii) amino acids 5-352 of SEQ ID NO:7; 
 iv) amino acids 51-352 of SEQ ID NO:7; 
 v) amino acids 7-630 of SEQ ID NO:10; 
 vi) amino acids 16-630 of SEQ ID NO:10; and 
 vii) amino acids 18-630 of SEQ ID NO:10; and 
   d) a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO:4, 7, 10, or any of (c)(i-viii), wherein said polypeptide has pesticidal activity against a plant pathogen.   
     
     
         13 . The polypeptide of  claim 12  further comprising heterologous amino acid sequences. 
     
     
         14 . An antibody that selectively binds to the polypeptide of  claim 12 . 
     
     
         15 . A composition comprising the polypeptide of  claim 12 . 
     
     
         16 . The composition of  claim 15 , wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution. 
     
     
         17 . The composition of  claim 15 , wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of  Bacillus thuringiensis  cells. 
     
     
         18 . The composition of  claim 16 , comprising from about 1% to about 99% by weight of said polypeptide. 
     
     
         19 . A method for controlling a lepidopteran or coleopteran pest population comprising contacting said population with a insecticidally-effective amount of a polypeptide derived from a  Brevibacillus  organism. 
     
     
         20 . The method of  claim 19 , wherein said polypeptide is selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence of any of SEQ ID NO:4, 7, or 10;   b) a polypeptide that is encoded by the nucleotide sequence of any of SEQ ID NO:3, 6, or 9;   c) a polypeptide selected from:
 i) amino acids 21-651 of SEQ ID NO:4; 
 ii) amino acids 54-651 of SEQ ID NO:4; 
 iii) amino acids 2-352 of SEQ ID NO:7; 
 iv) amino acids 5-352 of SEQ ID NO:7; 
 v) amino acids 51-352 of SEQ ID NO:7; 
 vi) amino acids 7-630 of SEQ ID NO:10; 
 vii) amino acids 16-630 of SEQ ID NO:10; and 
 viii) amino acids 18-630 of SEQ ID NO:10; and 
   d) a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO:4, 7, 10, or any of (c)(i-viii), wherein said polypeptide has pesticidal activity against said lepidopteran or coleopteran pest.   
     
     
         21 . A method for killing a lepidopteran or coleopteran pest, comprising contacting said pest with, or feeding to said pest, a insecticidally-effective amount of a polypeptide derived from a  Brevibacillus  organism. 
     
     
         22 . The method of  claim 21 , wherein said polypeptide is selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence of any of SEQ ID NO:4, 7, or 10;   b) a polypeptide that is encoded by the nucleotide sequence of any of SEQ ID NO:3, 6, or 9;   c) a polypeptide selected from:
 i) amino acids 21-651 of SEQ ID NO:4; 
 ii) amino acids 54-651 of SEQ ID NO:4; 
 iii) amino acids 2-352 of SEQ ID NO:7; 
 iv) amino acids 5-352 of SEQ ID NO:7; 
 v) amino acids 51-352 of SEQ ID NO:7; 
 vi) amino acids 7-630 of SEQ ID NO:10; 
 vii) amino acids 16-630 of SEQ ID NO:10; and 
 viii) amino acids 18-630 of SEQ ID NO:10; and 
   d) a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO:4, 7, 10, or any of (c)(i-viii), wherein said polypeptide has pesticidal activity against said lepidopteran or coleopteran pest.   
     
     
         23 . A method for producing a polypeptide with insecticidal activity, comprising culturing the host cell of  claim 6  under conditions in which the nucleic acid molecule encoding the polypeptide is expressed. 
     
     
         24 . A plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having insecticidal activity, wherein said nucleotide sequence is derived from a  Brevibacillus  organism. 
     
     
         25 . The plant of  claim 24 , wherein said nucleotide sequence is selected from the group consisting of:
 a) the nucleotide sequence of any of SEQ ID NO:3, 6, or 9, or a complement thereof;   b) the nucleotide sequence selected from:
 i) nucleotides 61-1953 of SEQ ID NO:3; 
 ii) nucleotides 160-1953 of SEQ ID NO:3; 
 iii) nucleotides 4-1059 of SEQ ID NO:6; 
 iv) nucleotides 13-1059 of SEQ ID NO:6; 
 v) nucleotides 151-1059 of SEQ ID NO:6; 
 vi) nucleotides 19-1893 of SEQ ID NO:9; 
 vii) nucleotides 46-1893 of SEQ ID NO:9; and 
 viii) nucleotides 52-1893 of SEQ ID NO:9; 
   c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:4, 7, or 10;   d) a nucleotide sequence that encodes a polypeptide selected from:
 i) amino acids 21-651 of SEQ ID NO:4; 
 ii) amino acids 54-651 of SEQ ID NO:4; 
 iii) amino acids 2-352 of SEQ ID NO:7; 
 iv) amino acids 5-352 of SEQ ID NO:7; 
 v) amino acids 51-352 of SEQ ID NO:7; 
 vi) amino acids 7-630 of SEQ ID NO:10; 
 vii) amino acids 16-630 of SEQ ID NO:10; and 
 viii) amino acids 18-630 of SEQ ID NO:10; and 
   e) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, 7, 10, or any of (d)(1-viii), wherein said amino acid sequence has pesticidal activity against a plant pathogen;   
       wherein said nucleotide sequence is operably linked to a promoter that drives expression of a coding sequence in a plant cell. 
     
     
         26 . The plant of  claim 24 , wherein said plant is a plant cell. 
     
     
         27 . A method for protecting a plant from an insect pest, comprising introducing into said plant or cell thereof at least one expression vector comprising a nucleotide sequence that encodes a insecticidal polypeptide, wherein said nucleotide sequence is derived from a  Brevibacillus  organism. 
     
     
         28 . The method of  claim 27 , wherein said nucleotide sequence is selected from the group consisting of:
 a) the nucleotide sequence of any of SEQ ID NO:3, 6, or 9, or a complement thereof;   b) the nucleotide sequence selected from:
 i) nucleotides 61-1953 of SEQ ID NO:3; 
 ii) nucleotides 160-1953 of SEQ ID NO:3; 
 iii) nucleotides 4-1059 of SEQ ID NO:6; 
 iv) nucleotides 13-1059 of SEQ ID NO:6; 
 v) nucleotides 151-1059 of SEQ ID NO:6; 
 vi) nucleotides 19-1893 of SEQ ID NO:9; 
 vii) nucleotides 46-1893 of SEQ ID NO:9; and 
 viii) nucleotides 52-1893 of SEQ ID NO:9; 
   c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:4, 7, or 10;   d) a nucleotide sequence that encodes a polypeptide selected from:
 i) amino acids 21-651 of SEQ ID NO:4; 
 ii) amino acids 54-651 of SEQ ID NO:4; 
 iii) amino acids 2-352 of SEQ ID NO:7; 
 iv) amino acids 5-352 of SEQ ID NO:7; 
 v) amino acids 51-352 of SEQ ID NO:7; 
 vi) amino acids 7-630 of SEQ ID NO:10; 
 vii) amino acids 16-630 of SEQ ID NO:10; and 
 viii) amino acids 18-630 of SEQ ID NO:10; and 
   e) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, 7, 10, or any of (d)(1-viii), wherein said amino acid sequence has pesticidal activity against a plant pathogen.   
     
     
         29 . The method of  claim 28 , wherein said plant produces a polypeptide having insecticidal activity against a lepidopteran or coleopteran pest.