Receptor for a bacillus thuringiensis toxin

Abstract

The cDNA that encodes a glycoprotein receptor from the tobacco hornworm which binds a Bacillus thuringiensis toxin has been obtained and sequenced. The availability of this cDNA permits the retrieval of DNAs encoding homologous receptors in other insects and organisms as well as the design of assays for the cytotoxicity and binding affinity of potential pesticides and the development of methods to manipulate natural and/or introduced homologous receptors and, thus, to destroy target cells, tissues and/or organisms.

1 . A method to identify agents that bind to a BT-toxin receptor, said method comprising the steps of: 
 (a) contacting an agent with a BT-toxin binding receptor selected from the group consisting of: 
 (i) a cell that has been altered to contain a fragment of a nucleic acid that encodes a BT-toxin binding receptor, wherein: 
 the cell expresses the polypeptide encoded by the fragment;  
 the encoded polypeptide binds to the CryIA(b) toxin;  
 the fragment is about the same length as the protein fragment encoded by a Bam-Sac nucleic acid fragment of SEQ ID NO: 1; and  
 the nucleic acid is selected from the group consisting of: 
 (1) a nucleic acid molecule that encodes a BT-toxin receptor having the amino acid sequence of SEQ ID NO: 2 and expresses said receptor;  
 (2) a nucleic acid molecule encoding a BT-toxin receptor, wherein said nucleic acid molecule hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein said receptor is obtainable from an insect;  
 (3) a nucleic acid molecule that encodes a BT-toxin receptor, wherein said nucleic acid molecule hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein the receptor encoded by the nucleic acid binds to the CryIA(b) toxin;  
 
 
 (ii) an isolated fragment of the BT-toxin binding receptor, wherein: 
 said fragment binds to the CryIA(b) toxin;  
 the fragment is about the same length as the protein fragment encoded by a Bam-Sac nucleic acid fragment of SEQ ID NO: 1; and  
 the BT-toxin binding receptor is selected from the group consisting of: 
 (1) an isolated BT-toxin receptor having an amino acid sequence of SEQ ID NO: 2;  
 (2) an isolated BT-toxin receptor that is encoded by a nucleic acid molecule that hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein said receptor is obtainable from an insect;  
 (3) an isolated BT-toxin receptor encoded by a nucleic acid molecule that hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein the receptor encoded by the nucleic acid binds to the CryIA(b) toxin;  
 
 
   (b) determining whether said agent binds to said BT-toxin receptor; wherein the stringent conditions comprise: 
 50% formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate (pH 6.5), 750 mM NaCl, and 75 mM sodium citrate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS; or  
 50% formamide, 5× SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS; or  
 0.015 M NaCl, 0.0015 M sodium citrate, and 0.1% SDS at 50° C.  
   
     
     
         2 . The method of  claim 1 , wherein said method further comprises the step of determining whether said agent blocks the binding of a BT-toxin to said BT-toxin receptor.  
     
     
         3 . The method of  claim 1 , wherein said cell that has been altered is a eukaryotic cell.  
     
     
         4 . The method of  claim 3 , wherein said eukaryotic cell is an insect cell.  
     
     
         5 . A method to identify agents that block the binding of a BT-toxin to a BT-toxin receptor, said method comprising the steps of: 
 (a) contacting an agent, in the presence and absence of a BT-toxin, to a BT-toxin binding receptor or cell expressing said receptor selected from the group consisting of: 
 (i) a cell that has been altered to contain a fragment of a nucleic acid that encodes a BT-toxin binding receptor, wherein: 
 the cell expresses the polypeptide encoded by the fragment;  
 the encoded polypeptide binds to the CryIA(b) toxin;  
 the fragment is about the same length as the protein fragment encoded by a Bam-Sac nucleic acid fragment of SEQ ID NO: 1; and  
 the nucleic acid is selected from the group consisting of: 
 (1) a nucleic acid molecule that encodes a BT-toxin receptor having the amino acid sequence of SEQ ID NO: 2 and expresses said receptor;  
 (2) a nucleic acid molecule encoding a BT-toxin receptor, wherein said nucleic acid molecule hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein said receptor is obtainable from an insect;  
 ( 3 ) a nucleic acid molecule that encodes a BT-toxin receptor, wherein said nucleic acid molecule hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein the receptor encoded by the nucleic acid binds to the CryIA(b) toxin;  
 
 
 ii) an isolated fragment of the BT-toxin binding receptor, wherein: 
 said fragment binds to the CryIA(b) toxin;  
 the fragment is about the same length as the protein fragment encoded by a Bam-Sac nucleic acid fragment of SEQ ID NO: 1; and  
 the BT-toxin binding receptor is selected from the group consisting of: 
 (1) an isolated BT-toxin receptor having an amino acid sequence of SEQ ID NO: 2;  
 (2) an isolated BT-toxin receptor that is encoded by a nucleic acid molecule that hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein said receptor is obtainable from an insect;  
 (3) an isolated BT-toxin receptor encoded by a nucleic acid molecule that hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein the receptor encoded by the nucleic acid binds to the CryIA(b) toxin;  
 
 
   (b) determining whether said agent binds to said BT-toxin receptor; wherein the stringent conditions comprise: 
 50% formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate (pH 6.5), 750 mM NaCl, and 75 mM sodium citrate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS; or  
 50% formamide, 5× SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS; or  
 0.015 M NaCl, 0.0015 M sodium citrate, and 0.1% SDS at 50° C.  
   
     
     
         6 . The method of  claim 5 , wherein said BT-toxin is a fragment of the BT-cry(1) family of toxins.  
     
     
         7 . The method of  claim 5 , wherein said cell that has been altered is a eukaryotic cell.  
     
     
         8 . The method of  claim 7 , wherein said eukaryotic cell is an insect cell.  
     
     
         13 . A method to produce a BT-toxin receptor protein, or a fragment thereof, said method comprising the steps of: 
 (a) culturing a cell that has been altered to contain a nucleic acid molecule that encodes a BT-toxin receptor protein, or BT-toxin binding fragment thereof, under conditions suitable for expression of said receptor protein or fragment thereof, wherein: 
 said cell has been altered to contain a fragment of a nucleic acid molecule selected from the group consisting of: 
 (i) a nucleic acid molecule that encodes a BT-toxin receptor having the amino acid sequence of SEQ ID NO: 2 and expresses said receptor;  
 (ii) a nucleic acid molecule encoding a BT-toxin receptor, wherein said nucleic acid molecule hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein said receptor is obtainable from an insect;  
 (iii) a nucleic acid molecule that encodes a BT-toxin receptor, wherein said nucleic acid molecule hybridizes to the polynucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein the receptor encoded by the nucleic acid binds to the CryIA(b) toxin;  
 said fragment encodes a polypeptide that binds to the CryIA(b) toxin;  
 said fragment is about the same length as the protein fragment encoded by a Bam-Sac nucleic acid fragment of SEQ ID NO: 1; and  
 
   b) isolating said BT-toxin receptor protein or fragment; wherein the stringent conditions comprise: 
 50% formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate (pH 6.5), 750 mM NaCl, and 75 mM sodium citrate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS; or  
 50% formamide, 5× SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS, or  
 0.015 M NaCl, 0.0015 M sodium citrate, and 0.1% SDS at 50° C.  
   
     
     
         14 . The method of  claim 13 , wherein said cell that has been altered is a eukaryotic cell.  
     
     
         15 . The method of  claim 14 , wherein said eukaryotic cell is an insect cell.