Insecticidal Polypeptides and Methods of Use Thereof

Abstract

A family of insecticidal polypeptides expressed in the venom gland of spiders of the genera Atrax and Hadronyche have been described. Also included are polynucleotides and expression vectors encoding the polypeptides and insect viruses and cells expressing the polypeptides. Transgenic plants and insects expressing the insecticidal polypeptides are also described. The insecticidal polypeptides may be employed in methods and compositions for treating insects, insect larvae, and plants.

1 . A purified polypeptide comprising an amino acid sequence that is greater than or equal to about 70% identical to SEQ ID NO:2, wherein the polypeptide has insecticidal activity. 
     
     
         2 . The purified polypeptide of  claim 1 , comprising any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27. 
     
     
         3 . The purified polypeptide of  claim 2 , further comprising a propeptide sequence, a signal peptide sequence, or a combination thereof. 
     
     
         4 . The purified polypeptide of  claim 1 , comprising SEQ ID NOs: 1, 5, 8, 11, 14, 17, 20, 23 or 26. 
     
     
         5 . The purified polypeptide of  claim 1 , wherein the amino acid sequence is greater than or equal to about 85% identical to SEQ ID NO:2, wherein the polypeptide has insecticidal activity. 
     
     
         6 . An insecticidal composition comprising an insecticidally effective amount of the purified polypeptide of  claim 1 , and an agriculturally acceptable carrier. 
     
     
         7 . An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence have greater than or equal to about 70% identity to SEQ ID NO:2, wherein the polypeptide has insecticidal activity. 
     
     
         8 . The polynucleotide of  claim 7 , encoding a polypeptide comprising any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27. 
     
     
         9 . The isolated polynucleotide of  claim 7 , wherein the polynucleotide further comprises polynucleotide sequences encoding a pro sequence, a signal peptide sequence, or a combination thereof, in frame with the encoded polypeptide. 
     
     
         10 . The isolated polynucleotide of  claim 7 , wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 1, 5, 8, 11, 14, 17, 20, 23 or 26. 
     
     
         11 . The isolated polynucleotide of  claim 7 , wherein the polypeptide comprises an amino acid sequence having greater than or equal to about 80% identity to SEQ ID NO:2, wherein the polypeptide has insecticidal activity. 
     
     
         12 . An expression vector comprising a polynucleotide encoding any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27 operably linked to an expression control sequence. 
     
     
         13 . The expression vector of  claim 12 , wherein the expression vector is suitable for expression in an insect cell or a bacterial cell. 
     
     
         14 . The expression vector of  claim 12 , wherein the expression vector is a baculovirus vector. 
     
     
         15 . A host cell comprising an expression vector comprising a polynucleotide encoding any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27 operably linked to an expression control sequence. 
     
     
         16 . The host cell of  claim 15 , wherein the host cell is a plant cell, a bacterial cell or an insect cell. 
     
     
         17 . An insect virus comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence have greater than or equal to about 70% identity to SEQ ID NO:2, wherein the polypeptide has insecticidal activity. 
     
     
         18 . The insect virus of  claim 17 , wherein the polypeptide comprises any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27. 
     
     
         19 . A method of treating an insect or an insect larva, comprising contacting the insect or insect larva with an insecticidally effective amount of a U-ACTX polypeptide, wherein the U-ACTX polypeptide comprises an amino acid sequence that is greater than or equal to about 70% identical to SEQ ID NO:2, wherein the U-ACTX polypeptide has insecticidal activity. 
     
     
         20 . The method of  claim 19 , wherein the U-ACTX polypeptide comprises any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 24, 24 and 27. 
     
     
         21 . The method of  claim 19 , wherein the U-ACTX polypeptide is in the form of a purified polypeptide, a polynucleotide encoding the U-ACTX polypeptide optionally in an expression vector, an insect virus expressing the U-ACTX polypeptide, a cell expressing the U-ACTX polypeptide, or a transgenic plant expressing the U-ACTX polypeptide. 
     
     
         22 . The method of  claim 19 , wherein the U-ACTX polypeptide is impregnated in a collar. 
     
     
         23 . The method of  claim 19 , wherein contacting comprises external contact with an animal in need of treatment. 
     
     
         24 . A method of treating a plant, comprising contacting the plant with an insecticidally effective amount of a U-ACTX polypeptide, wherein the U-ACTX polypeptide comprises an amino acid sequence that is greater than or equal to about 70% identical to SEQ ID NO:2, wherein the U-ACTX polypeptide has insecticidal activity. 
     
     
         25 . The method of  claim 24 , wherein the U-ACTX polypeptide is in the form of a purified polypeptide, a polynucleotide encoding the U-ACTX polypeptide optionally in an expression vector, an insect virus expressing the U-ACTX polypeptide, or a cell expressing the U-ACTX polypeptide. 
     
     
         26 . A transgenic plant, wherein the transgenic plant expresses a U-ACTX polypeptide, wherein the U-ACTX polypeptide comprises an amino acid sequence that is greater than or equal to about 70% identical to SEQ ID NO:2, wherein the U-ACTX polypeptide has insecticidal activity. 
     
     
         27 . The transgenic plant of  claim 26 , wherein the U-ACTX polypeptide comprises any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27. 
     
     
         28 . A transgenic insect comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence have greater than or equal to about 70% identity to SEQ ID NO:2, wherein the polypeptide has insecticidal activity. 
     
     
         29 . The transgenic insect of  claim 28 , wherein the polypeptide comprises any one of SEQ ID NOs. 2, 6, 9, 12, 15, 18, 21, 24 and 27. 
     
     
         30 . An insecticidal polypeptide that blocks greater than or equal to about 50% of a calcium current in an insect voltage-gated calcium channel and greater than or equal to about 50% of activity of an insect high conductance calcium-activated potassium channel. 
     
     
         31 . The insecticidal polypeptide of  claim 30 , wherein the polypeptide comprises three intrachain disulfide bonds. 
     
     
         32 . The insecticidal polypeptide of  claim 30 , wherein the polypeptide is a component of the venom of a spider of the genera  Atrax  or  Hadronyche.    
     
     
         33 . The insecticidal polypeptide of  claim 30 , wherein the insect voltage-gated calcium channel is one from a DUM neuron of  P. americana.    
     
     
         34 . The insecticidal polypeptide of  claim 30 , wherein the high conductance insect calcium-activated potassium channel comprises a  P. americana  high conductance calcium-activated potassium channel. 
     
     
         35 . A method of selecting a test compound that binds to an insect channel comprising
 providing the insect channel, wherein the insect channel is an insect voltage-gated calcium channel, an insect calcium-activated potassium channel, or a combination comprising one or more of the foregoing insect channels, and   determining if the test compound competes with the binding of a U-ACTX peptide to the insect channel, wherein the U-ACTX peptide is greater than or equal to about 70% identical to SEQ ID NO:2 and has insecticidal activity.   
     
     
         36 . A method of selecting a test compound that binds to an insect channel comprising
 providing the insect channel, wherein the insect channel is an insect voltage-gated calcium channel, an insect calcium-activated potassium channel, or a combination comprising one or more of the foregoing insect channels, and   determining if the test compound releases at least a portion of a U-ACTX peptide pre-bound to the insect channel, wherein the U-ACTX peptide is greater than or equal to about 70% identical to SEQ ID NO:2 and has insecticidal activity.