Vip3 Interacting Polypeptides and Methods for Identifying Insecticidal Agents

Abstract

This invention provides polypeptides that were identified as interacting with Vip3 toxin. This invention also provides a method of identifying agents that bind to Vip3 interacting polypeptides, which agents may act as insecticidal agent, cytotoxic agents and/or modulate the activity of Vip3 interacting polypeptides.

What is claimed is: 
     
         1 . A recombinant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and an amino acid sequence that is at least 98% identical to any of the foregoing. 
     
     
         2 . A recombinant polynucleotide comprising a sequence that encodes a polypeptide of  claim 1 . 
     
     
         3 . An expression cassette comprising a polynucleotide sequence of  claim 2 . 
     
     
         4 . A method for identifying a candidate insecticidal agent that interacts with at least one Vip3 interacting polypeptide, said method comprising:
 a. contacting at least one Vip3 interacting polypeptide or fragment thereof to one or more test agents; and   b. detecting binding activity between the at least one Vip3 interacting polypeptide or fragment and the test agent under conditions sufficient for binding,   
       wherein binding between the at least one Vip3 interacting polypeptide or fragment and the test agent indicates that the test agent is a candidate insecticidal agent. 
     
     
         5 . The method of  claim 4 , further comprising assaying the candidate insecticidal agent for insecticidal or cytotoxic activity. 
     
     
         6 . The method of  claim 4  wherein the test agent comprises an extract from an environmental sample. 
     
     
         7 . The method of  claim 4  wherein the test agent comprises an extract from a biological sample. 
     
     
         8 . The method of  claim 4  wherein the test agent comprises members of a chemical library. 
     
     
         9 . The method of  claim 4  wherein the test agent comprises a cellular extract. 
     
     
         10 . The method of  claim 9  wherein the test agent comprises an intracellular protein. 
     
     
         11 . The method of  claim 9  wherein the test agent comprises a secreted protein. 
     
     
         12 . The method of  claim 9  wherein the test agent comprises a protein extract from one or more  Bacillus  ssp. 
     
     
         13 . A method of identifying an agent that modulates the activity of at least one Vip3 interacting polypeptide, the method comprising:
 a. contacting at least one Vip3 interacting polypeptide or fragment thereof with a test agent in an insect cell under conditions sufficient to promote interaction; and   b. detecting cytotoxicity,   
       wherein a change in the level of cytotoxicity as compared with the level of cytotoxicity in the absence of the test agent indicates that the test agent modulates the activity of at least one Vip3 interacting polypeptide. 
     
     
         14 . A method of identifying a cytotoxic agent that interacts with at least one Vip3 interacting polypeptide, said method comprising:
 a. providing at least one Vip3 interacting polypeptide or fragment thereof expressed in an insect cell, wherein the insect cell is not susceptible to Vip3 toxin;   b. contacting the at least one Vip3 interacting polypeptide or fragment with a test agent under conditions sufficient to promote binding, and   c. detecting cytotoxicity in the insect cell,   wherein an increase in the level cytotoxicity as compared with the level of cytotoxicity in the absence of the test agent indicates that the test agent is a cytotoxic agent.   
     
     
         15 . A method for identifying a candidate insecticidal agent that interacts with at least one Vip3 interacting polypeptide or a fragment thereof, said method comprising:
 a. contacting at least one Vip3 interacting polypeptide or fragment with polypeptide from  Bacillus  under conditions sufficient for binding;   b. isolating said at least one Vip3 interacting polypeptide or fragment;   c. identifying a co-purified  Bacillus  polypeptide; and   d. determining insecticidal activity of the co-purified  Bacillus  polypeptide,   
       wherein the presence of insecticidal activity of a co-purified  Bacillus  polypeptide indicates that said polypeptide is a candidate insecticidal agent. 
     
     
         16 . A method of identifying a candidate insecticidal agent that interacts with a Vip3 interacting polypeptide, said method comprising:
 a. providing at least one Vip3 interacting polypeptide or fragment thereof, wherein the Vip3 interacting polypeptide comprises:
 i. ATP synthase α; 
 ii. ATP synthase β, 
 iii. Hsc-70, 
 iv. prohibitin-1; 
 v. prohibitin-2; 
 vi. serpin; or 
 vii. any combination of (i) to (vi) 
   b. contacting said polypeptide with a test agent under conditions sufficient to promote binding, and   c. detecting binding activity between said polypeptide and the test agent,   
       wherein binding between the Vip3 interacting polypeptide or fragment and the test agent indicates that the test agent is a candidate insecticidal agent. 
     
     
         17 . The method of  claim 17 , wherein the Vip3 interacting polypeptide comprises:
 i. the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 1;   ii. the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 2;   iii. the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 3;   iv. the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 4;   v. the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 5;   vi. the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 6;   vii. the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 7;   viii. the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that has at least 80% amino acid sequence similarity with SEQ ID NO: 8; or   ix. any combination of (i) to (viii) above.   
     
     
         18 . The method of  claim 4 , wherein the Vip3 interacting polypeptide is a Vip3A interacting polypeptide.