Combinations of insecticidal polypeptides having improved activity spectrum and uses thereof

Abstract

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity.

That which is claimed is: 
     
         1 . A DNA construct comprising:
 a) a polynucleotide encoding an IPD103 polypeptide having insecticidal activity; and   b) one or more polynucleotide selected from:
 i) a polynucleotide encoding a PtlP-83 polypeptide having insecticidal activity; 
 ii) a polynucleotide encoding a Cry1B polypeptide having insecticidal activity; 
 iii) a polynucleotide encoding a variant Cry1B polypeptide having insecticidal activity; 
 iv) a polynucleotide encoding a Cry1C polypeptide having insecticidal activity; 
 v) a polynucleotide encoding a Cry1D polypeptide having insecticidal activity and 
 vi) a polynucleotide encoding a Cry1J polypeptide having insecticidal activity. 
   
     
     
         2 . The DNA construct of  claim 1 , wherein the polynucleotides encoding the polypeptides are operably linked to heterologous regulatory elements. 
     
     
         3 . The DNA construct of  claim 1 , wherein:
 a) the IPD103 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2;   b) the PtlP-83 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45;   c) the Cry1B polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 109;   d) the variant Cry1B polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 91;   e) the Cry1C polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of the Cry1Ca polypeptide of SEQ ID NO: 230;   f) the Cry1D polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of the Cry1D polypeptide of SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239 or SEQ ID NO: 241; and   g) the Cry1J polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 213.   
     
     
         4 . The DNA construct of  claim 1 , wherein the DNA construct comprises a molecular stack selected from:
 i) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the variant Cry1B polypeptide of SEQ ID NO: 91; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   ii) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1Ca polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   iii) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1D polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   iv) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1Ca polypeptide; and 
 c) a polynucleotide encoding the Cry1D polypeptide; 
   v) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the variant Cry1B polypeptide of SEQ ID NO: 91; and 
 c) a polynucleotide encoding the Cry1D polypeptide; 
   vi) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1D polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; and 
   vii) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; and 
 b) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45. 
   
     
     
         5 . A transgenic plant comprising a molecular stack comprising:
 a) a polynucleotide encoding an IPD103 polypeptide having insecticidal activity; and   b) one or more polynucleotides selected from:
 i) a polynucleotide encoding a PtlP-83 polypeptide having insecticidal activity; 
 ii) a polynucleotide encoding a Cry1B polypeptide having insecticidal activity; 
 iii) a polynucleotide encoding a variant Cry1B polypeptide having insecticidal activity; 
 iv) a polynucleotide encoding a Cry1C polypeptide having insecticidal activity; 
 v) a polynucleotide encoding a Cry1D polypeptide having insecticidal activity; and 
 vi) a polynucleotide encoding a Cry1J polypeptide having insecticidal activity. 
   
     
     
         6 . The transgenic plant of  claim 5 , wherein the polynucleotide encoding the polypeptides are each operably linked to heterologous regulatory elements. 
     
     
         7 . The transgenic plant of  claim 5 , wherein:
 a) the IPD103 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2;   b) the PtlP-83 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45;   c) the Cry1B polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 109;   d) the variant Cry1B polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 91;   e) the Cry1C polypeptide comprises an amino acid sequence having at least 95% identity to a Cry1Ca polypeptide of SEQ ID NO: 230;   f) the Cry1D polypeptide comprises an amino acid sequence having at least 95% identity to a Cry1D polypeptide of SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239 or SEQ ID NO: 241; and   g) the Cry1J polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 213.   
     
     
         8 . The transgenic plant of  claim 5 , wherein the molecular stack is selected from:
 i) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the variant Cry1B polypeptide of SEQ ID NO: 91; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   ii) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1Ca polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   iii) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1D polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   iv) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1Ca polypeptide; and 
 c) a polynucleotide encoding the Cry1D polypeptide; 
   v) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the variant Cry1B polypeptide of SEQ ID NO: 91; and 
 c) a polynucleotide encoding the Cry1D polypeptide; 
   vi) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1D polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; and 
   vii) a molecular stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; and 
 b) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45. 
   
     
     
         9 . A transgenic plant comprising a breeding stack comprising;
 a) a polynucleotide encoding an IPD103 polypeptide having insecticidal activity; and   b) one or more of polynucleotide selected from:
 i. a polynucleotide encoding a PtlP-83 polypeptide having insecticidal activity; 
 ii. a polynucleotide encoding a Cry1B polypeptide having insecticidal activity; 
 iii. a polynucleotide encoding a variant Cry1B polypeptide having insecticidal activity; 
 iv. a polynucleotide encoding a Cry1C polypeptide having insecticidal activity; 
 v. a polynucleotide encoding a Cry1D polypeptide having insecticidal activity; and 
 vi. a polynucleotide encoding a Cry1J polypeptide having insecticidal activity. 
   
     
     
         10 . The transgenic plant of  claim 9 , wherein the polynucleotides encoding the polypeptides are operably linked to heterologous regulatory elements. 
     
     
         11 . The transgenic plant of  claim 9 , wherein:
 a) the IPD103 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2;   b) the PtlP-83 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45;   c) the Cry1B polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 109;   d) the variant Cry1B polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 91;   e) the Cry1C polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of a Cry1Ca polypeptide;   f) the Cry1D polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of a Cry1Ca polypeptide; and   g) the Cry1J polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 213.   
     
     
         12 . The transgenic plant of  claim 11 , wherein the breeding stack comprises:
 i) a breeding stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the variant Cry1B polypeptide of SEQ ID NO: 91; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   ii) a breeding stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1Ca polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   iii) a breeding stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1D polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; 
   iv) a breeding stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1Ca polypeptide; and 
 c) a polynucleotide encoding the Cry1D polypeptide; 
   v) a breeding stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the variant Cry1B polypeptide of SEQ ID NO: 91; and 
 c) a polynucleotide encoding the Cry1D polypeptide; 
   vi) a breeding stack comprising:
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; 
 b) a polynucleotide encoding the Cry1D polypeptide; and 
 c) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45; and 
   vii) a breeding stack comprising: and
 a) a polynucleotide encoding the IPD103 polypeptide of SEQ ID NO: 2; and 
 b) a polynucleotide encoding the PtlP-83 polypeptide of SEQ ID NO: 45. 
   
     
     
         13 . A transgenic plant or progeny thereof comprising the DNA construct of  claim 1 , wherein said transgenic plant is corn or soy. 
     
     
         14 . A transgenic plant or progeny thereof comprising the molecular stack of  claim 5 , wherein said transgenic plant is corn or soy. 
     
     
         15 . A transgenic plant or progeny thereof comprising the breeding stack of  claim 9 , wherein said transgenic plant is corn or soy. 
     
     
         16 . A method for controlling an insect pest population comprising contacting the insect pest population with the transgenic plant of  claim 5 . 
     
     
         17 . A method for controlling an insect pest population comprising expressing, in the transgenic plant of  claim 5 , said polypeptides. 
     
     
         18 . A method for controlling an insect pest population comprising contacting the insect pest population with the transgenic plant of  claim 9 . 
     
     
         19 . A method for controlling an insect pest population comprising expressing, in the transgenic plant of  claim 9 , said polypeptides. 
     
     
         20 . An insecticidal polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260 or SEQ ID NO: 261. 
     
     
         21 . The insecticidal polypeptide of  claim 20 , wherein the insecticidal polypeptide is selected from amino acid sequence of SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260 or SEQ ID NO: 261. 
     
     
         22 . The insecticidal polypeptide of  claim 20 , wherein the insecticidal polypeptide is operably linked to a heterologous signal peptide or transit peptide. 
     
     
         23 . A polynucleotide encoding the polypeptide of  claim 20 . 
     
     
         24 . The polynucleotide of  claim 23 , wherein the polynucleotide is operably linked to a heterologous regulatory element. 
     
     
         25 . A DNA construct comprising the polynucleotide of  claim 24 . 
     
     
         26 . A transgenic plant comprising the DNA construct of  claim 25 . 
     
     
         27 . A method for controlling an insect pest population comprising contacting the insect pest population with the transgenic plant of  claim 26 . 
     
     
         28 . A method for controlling an insect pest population comprising expressing, in the transgenic plant of  claim 26 , said polypeptide. 
     
     
         29 . The DNA construct of  claim 1 , wherein the IPD103 polypeptide is the insecticidal polypeptide of  claim 20 .