Abstract:
Disclosed is a novel cadherin peptide that enhances the toxicity of Cry proteins. A novel insecticide composition comprising an effective amount of cadherin peptide having SEQ. ID. NO:2 and an effective amount of  Bacillus thuringiensis  Cry protein wherein the cadherin peptide comprises a Cry3Aa toxin binding region from the full-length  T. molitor  cadherin and has synergistic characteristics of a binary toxin potentiating Cry3 and Cry1 toxins against coleopterans and lepidopteran species, respectively.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Ser. No. 60/988,919, which was filed on Nov. 19, 2007, the disclosure of which is hereby incorporated by reference. 
    
    
     SEQUENCE LISTING SUBMISSION 
     The contents of the following SEQUENCE LISTING submission are incorporated herein by reference in its entirety: a computer readable form of the Sequence Listing submitted via EFS-Web on Mar. 6, 2009, containing the file name: “SequenceListing.txt” as a sequence listing, date recorded: Mar. 6, 2009, size: 29,124 bytes. 
     FIELD OF THE INVENTION 
     The present invention is directed to a novel cadherin fragment peptide derived from  Tenebrio molitor  larvae. Moreover, the peptide comprises a Cry3Aa toxin binding region from the full-length  T. molitor  cadherin and has synergistic characteristics of a binary toxin potentiating Cry3 and Cry1 toxins against coleopterans and lepidopteran species, respectively. Additionally, an identified functional receptor region in the protein can be used to screen insecticidal toxins for activity against various coleopterans and lepidoterans or increased potency. 
     BACKGROUND OF INVENTION 
     Leading biological based pesticide utilizes  Bacillus thuringiensis  (abbreviated herein as Bt) against lepidopterans, coleopterans, and other insect pests. The Gram-positive spore-forming bacterium produces parasporal protein crystals during stationary phase of the growth cycle. Genes encoding the crystals are categorized as cry genes. As a pesticide, the primary mode of action involves protein solubilization, proteolytic activation of the protoxin, binding to epithelial midgut receptor (cadherin), and subsequent pore formation and/or activation of intracellular cell-death signaling pathway. The use of Bt and its effectiveness as an insecticide is largely dependent on receptors in the target insect and the solubility of the Cry protein. 
     Coleopteran pests cause extensive damage to crops in the United States. For example, damage to corn crops occurs when rootworms feed on corn seedling roots. It has been estimated that rootworms cause in excess 1 billion dollars in damage to corn crops in the United States. (Meycalf, R. L., et al., 1986. Drysan, J. L. and T. A. Miller [Eds.], Springer-Verlag, New York, N.Y., pp. vii-xv.) Even with chemical insecticide applications of organophosphate or pyrethroid, rootworm damage still causes an estimated $750 million dollars annual damage to corn crops. One approach to combat rootworm damage while decreasing dependence of chemical pesticides is to express Bt protein in transgenic corn. For instance, Bt strain PS149B1 confers resistance to rootworms in corn plants (Moellenbeck, et al., 2001 . Nature Biotechnology,  19:668-672). Additionally, U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose a  Bacillus thuringiensis  strain  san diego , (NRRL B-15939) that is effective in controlling corn rootworm, among other coleopteran species. Given the widespread damage caused by rootworm, there is continuing need to develop efficient biologically-based insecticides, specifically potentiating the usage of Bt toxin. 
     A lepidopteran pest is the pink bollworm ( Pectinophora gossypiella ). It is estimated that the preventive cost, damage control, and crop lost costs cotton growers over thirty-two million dollars annually (National Cotton Council, 2004). Cotton crop damage occurs when female bollworms lay their eggs in cotton bolls during the summer mating season. Resulting larvae feed on cotton seeds upon chewing and burrowing through cotton lint. Techniques to combat pink bollworm include releasing pheromones to disruption mating, releasing sterile males to disrupt mating patterns, chemical insecticide treatments, and planting of transgenic Bt cotton. 
     While Bt pesticides have been used against a narrow range of lepidopteran pests, the discovery that Bt can have a broader application towards other Orders of insects has prompted its use targeting other pests. (For instance, see U.S. Pat. Nos. 4,797,276 and 4,853,331). Also, it has been reported that the expression of Cry3Aa in transgenic potato cultivars are resistant to  Leptinotarsa decemlineata  while exerting a deleterious effect on the polyphagous moth  Spodoptera littoralis  (Hussein et al., 2006 . Journal of Chemical Ecology,  32:1-13). Novel Bt isolates, new uses of known Bt isolates, and potentiating the toxicity of existing toxins remains an empirical, unpredictable art. 
     One approach to increase potency of Cry proteins against various insect pests is to utilize a Bt toxin receptor to potentiate toxicity to a target pest. The approach uses a peptide fragment derived from an insect cadherin protein combined with Cry protein toxin to increase a synergistic potency that would not be achieved via administration of the partial cadherin fragment or Cry protein individually. 
     BRIEF SUMMARY OF THE INVENTION 
     Disclosed is a novel cadherin peptide that enhances the toxicity of Cry proteins. An embodiment of the invention is a novel insecticide composition comprising an effective amount of cadherin peptide having SEQ. ID. NO:2 and an effective amount of  Bacillus thuringiensis  Cry protein. Furthermore, the effective amount of the composition would comprise a peptide with 90% amino acid sequence identity or greater with SEQ. ID. NO: 2. In one embodiment, the cadherin peptide and  Bacillus thuringiensis  Cry protein is administered at molar ratio range of approximately 1:2.5 to 1:200 respectively. In an embodiment, the Cry protein is a Cry3 protein or a Cry1 protein. In another embodiment, the composition is effective against coleopterans, namely  Tenebrio molitor . It is also contemplated that the composition would be effective against other coleopteran pest such as  Agrilus planipennis, Agrilus marcopoli, Diabrotica  spp., and  Leptinotarsa decemlineata . In another embodiment, the cadherin peptide increases the toxicity of  Bacillus thuringiensis  Cry proteins against lepidopterans, namely  Pectinophora gossypiella  and  Heliothis virescens.    
     It is contemplated that the a composition comprising of cadherin peptide and Cry protein applied to the environment of the coleopteran pests, typically onto the foliage of the plant or crop to be protected by convention methods such as spraying. Other applications include, but are not limited to dusting, sprinkling, aerating, soil soaking, soil injection, seed coating, seedling coating, foliar spraying, misting, fumigating, aerosolizing may be required for application procedure well know to those skilled in the art. 
     The cadherin and Cry protein composition may be formulated for preventive or prophylactic application to an area to prevent infestation of pests. 
     In another embodiment, a method for inhibiting insect pests, the method comprising selecting a  Bacillus thuringiensis  Cry protein, potentiating said protein with an effective amount of cadherin peptide having SEQ. ID. NO: 2, and applying an effective amount of said Cry protein and cadherin peptide to the insect pest, wherein the mortality of said insect increases. It is contemplated that the cadherin peptide and  Bacillus thuringiensis  Cry protein is in a molar ratio range of approximately 1:2.5 to 1:200. For another embodiment, the cadherin peptide potentiates the insecticide activity of Cry proteins. The method is effective against insect pests of the order Coleoptera and Lepidoptera. It is contemplated that the cadherin peptide utilized in conjunction with other Cry proteins would be effective against other coleopteran pest such as  Agrilus planipennis, Agrilus marcopoli, Diabrotica  spp., and  Leptinotarsa decemlineata . More particularly, the cadherin peptide is an isolated polypeptide (amino acid residues 1,322-1,516) comprising the Bt toxin binding site encoded by nucleotides 4,076-4,661 of SEQ ID NO: 3. 
     In another embodiment, a method for screening insecticidal toxins comprising transfecting cells to express base pairs 1322-1626 and 3969-4879 of SEQ. ID. NO: 3 or a fragment thereof sufficient to encode a functional protein, exposing said cells to a Cry toxin; and monitoring said cells for effect attributable to toxin exposure. In an embodiment for screening insecticidal toxins, the cell line is monitored via cytotoxicity assay. In another embodiment, the cells are transfected to express SEQ. ID. NO. 4 or a fragment thereof sufficient to encode a functional protein, exposing said cells to a Cry toxin; and monitoring said cells for effect attributable to toxin exposure. In another embodiment, the cells are transfected to express SEQ. ID. NO.: 28 or a fragment thereof sufficient to encode a functional protein, exposing said cells to a Cry toxin, and monitoring said cells for effect attributable to toxin exposure for screening insecticidal receptor. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
       The present invention together with the above and other objects and advantages may best be understood from the following detailed description of the embodiment of the invention illustrated in the drawings, wherein: 
         FIG. 1A  is a digital image of dot blot assay of 0.1, 0.5, 1, 2.5, 5, and 10 μg of rTmCad1 peptide fragment (rTmCad1p) spotted onto PVDF membrane, blocked with bovine serum albumin, and incubated with Cry3Aa. Binding of rTmCad1p fragment to Cry3Aa toxin was detected by incubations in rabbit anti-Cry3Aa and ECL horseradish peroxidase (HRP)-labeled anti-rabbit antisera (Amersham), followed by detection of HRP activity in ECL substrate solution (Amersham RPN2209). 
         FIG. 1B  is a digital image of an in-gel toxin binding competition assay of 5 μg of rTmCad1p and Cry3Aa recognized by a Cry3Aa-antibody with an infrared labeled dye. 
         FIG. 2  discloses the deduced amino acid sequence of the TmCad1p (SEQ ID NO: 1) as it exists expressed from pET151-D-TOPO expression vector. The TmCad1 peptide fragment corresponds to amino acid residues 1,322-1,516 of the full length protein (translation of nucleotides 4,076-4,661 of SEQ ID NO: 3). Bold letters and underline designates TmCad1 amino acids (195 residues), whereas 37 residues at amino terminus are from pET151-D-TOPO vector, including polyhistidine tag, V5 epitope tag, and TEV protease cleavage site. 
         FIG. 3  is a graph of a percentage change of  Tenebrio molitor  larvae weight when subjected to rTmCad1p premixed with Cry3Aa (1:5 molar ratio of Cry3Aa:rTmCad1p) and fed diet at various mass ratios and toxin concentrations as a function of time (days). rTmCad1p was obtained from  E. coli  BL21 transformation. 
         FIG. 4  is a graph of mortality percentage for  Tenebrio molitor  larvae fed a combination of rTmCad1p peptide premixed with Cry3Aa (Cry3Aa: rTmCad1p molar ratios of 1:2.5 and 1:12.4) and whole grain bread diet at various mass ratios on a microtiter plate over a period of one week. rTmCad1p was obtained from  E. coli  BL21 transformation. 
         FIGS. 5A , B, and C are graphs of mortality percentage for  Tenebrio molitor  neonate larvae fed in combination of rTmCad1p peptide premixed with Cry3AA at various molar ratios of 1:20 and 1:200 (Cry:rTmCad1p) ratios. Specifically,  FIG. 5A  depicts molar ratios of 1:20 Cry3Aa:rTmCad1p, with  FIG. 5B  depicting molar ratios 1:20 and 1:200 Cry3Aa:rTmCad1p, and  FIG. 5C  depicting molar ratios of 1:20 Cry3Aa:rTmCad1p. rTmCad1p utilized in  FIG. 5  were obtained from  E. coli . ArcticExpress (DE3). 
         FIG. 6A  is digital image of immunoblot showing detection of expression of TmCad1(EC12-cyto) in Hi5 insect cells (Invitrogen) transfected with pIZT/TmCad1(EC-12-cyto) (lane 2) in comparison to Hi5 cells transfected with mock plasmid pIZT (lane 1). 
         FIG. 6B  is a graph of mortality of S2 or Hi5 cells transfected with pIZT or pIZT/TmCad1(EC12-cyto) when exposed to approximately 800 nM (S2 cells) or 110 nM (Hi5 cells) of Cry3Aa protoxin. Significant statistical differences between control and experimental cells based on Student&#39;s t-test (P&lt;0.09, n=3) are denoted as different letters. 
         FIG. 7  is a graph of a percentage change of  Pectinophora gossypiella  (APHIS) larvae weight when subjected to combination of a control diet, dialysis buffer, and rTmCad1p premixed with Cry1Ac in a 1:200 molar ratio to toxin:peptide as a function of time (days). Said  Pectinophora gossypiella  (APHIS) larvae are known to be subseptible to Cry1Ac. 
         FIG. 8  is a graph of morality percentages of  Pectinophora gossypiella  (AZP-R) larvae fed a combination of a control diet, dialysis buffer, and rTmCad1p premixed with Cry1Ac in a 1:200 molar ratio to toxin:peptide as a function of time (days). Said  Pectinophora gossypiella  (AZP-R) larvae are known to be resistant to Cry1 Ac. 
         FIG. 9  is a graph of percentages of  Pectinophora gossypiella  (PBW) larval mortality on a Cry1 Ac-resistant strain (AZP-R). Results of single bioassay replicate (n=30 insects per treatment) on Cry1Ac-resistant PBW larvae (from AZP-R strain) with 200-fold (molar ratio) rTmCad1p (expressed from  E. coli  Arctic-Express (DE3)) and Cry1Ac crystalline protoxin (HD-73 preparation). 
         FIG. 10  is a graph of percentages of  Heliothis virescens  larval mortality. Results shown are the means from a bioassay replicated thrice (n=32 larvae per treatment per replicate) with  H. virescens  neonates exposed to HD-73 Cry1Ac protoxin and rTmCad1p (expressed from  E. coli  ArcticExpress (DE3)) using 1:200 (toxin:peptide) molar ratio.  H. virescens  eggs were purchased from Benzon Inc. (Carlisle, Pa.). Each column represents data for the mean±standard errors. 
     
    
    
     BRIEF DESCRIPTION OF THE SEQUENCES 
     SEQ. ID. NO: 1: is the nucleotide sequence that encodes for a cadherin fragment obtained from  Tenebrio molitor  larvae. 
     SEQ. ID. NO: 2: is the deduced amino acid sequence of a cadherin fragment obtained from  Tenebrio molitor  larvae (rTmCad1p). 
     SEQ. ID. NO: 3: is the complementary nucleotide sequence that encodes for a cadherin obtained from  Tenebrio molitor  larvae. 
     SEQ. ID. NO: 4: is the deduced amino acid sequence for a complete cadherin obtained from  Tenebrio molitor  larvae. 
     SEQ. ID. NO: 5: is the nucleotide sequence for PCR primer Tm1. 
     SEQ. ID. NO: 6: is the nucleotide sequence for PCR primer Tm2. 
     SEQ. ID. NO: 7: is the nucleotide sequence for PCR primer Tm3. 
     SEQ. ID. NO: 8: is the nucleotide sequence for PCR primer Tm4. 
     SEQ. ID. NO: 9: is the nucleotide sequence for PCR primer Tm5. 
     SEQ. ID. NO: 10: is the nucleotide sequence for PCR primer Tm6. 
     SEQ. ID. NO: 11: is the nucleotide sequence for PCR primer Tm7. 
     SEQ. ID. NO: 12: is the nucleotide sequence for PCR primer Tm8. 
     SEQ. ID. NO: 13: is the nucleotide sequence for PCR primer Tm9. 
     SEQ. ID. NO: 14: is the nucleotide sequence for PCR primer Tm10. 
     SEQ. ID. NO: 15: is the nucleotide sequence for PCR primer Tm11. 
     SEQ. ID. NO: 16: is the nucleotide sequence for PCR primer Tm12. 
     SEQ. ID. NO: 17: is the nucleotide sequence for PCR primer Tm13. 
     SEQ. ID. NO: 18: is the nucleotide sequence for PCR primer Tm14. 
     SEQ. ID. NO: 19: is the nucleotide sequence for PCR primer Tm15. 
     SEQ. ID. NO: 20: is the nucleotide sequence for PCR primer Tm16. 
     SEQ. ID. NO: 21: is the nucleotide sequence for PCR primer Tm17. 
     SEQ. ID. NO: 22: is the nucleotide sequence for PCR primer Tm18 
     SEQ. ID. NO: 23: is the nucleotide sequence for PCR primer Tm19 
     SEQ. ID. NO: 24: is the nucleotide sequence for PCR primer Tm20. 
     SEQ. ID. NO: 25: is the nucleotide sequence for PCR primer Tm21. 
     SEQ. ID. NO: 26: is the nucleotide sequence for PCR primer Hv1. 
     SEQ. ID. NO: 27: is the nucleotide sequence for PCR primer Hv2. 
     SEQ. ID. NO 28: is the nucleotide sequence listing for TmCad1EC12-cyto. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. 
     “ Bacillus thuringiensis ” or “Bt” refers to Gram-positive bacterium that upon sporulation form proteinaceous delta-endotoxins that are insecticidal towards lepidopterans, dipterans, and coleopterans, depending on the delta-endotoxin. 
     A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant. A compound can comprise multiple moieties, including a mixture of Cry toxin and a potentiating peptide. 
     The term “oligonucleotide” refers to a molecule comprising a plurality of deoxyribonucleotides or ribonucleotides. Oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, polymerase chain reaction (PCR), or a combination thereof. The present invention embodies utilizing the oligonucleotide as a primer for DNA synthesis for cloning purposes or as template for protein synthesis using  Escherichia coli  heterologous expression system. Inasmuch as mononucleotides are synthesized to construct oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. 
     An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. In terms of administering an “effective amount”, such an amount sufficient to reverse, slow, or delay the growth of a coleopteran or lepidopteran pests in an adult stage or a larvae stage would be an effective amount. Table I lists of currently known delta-endotoxins with GenBank accession numbers for sequenced polypeptides and polynucleotides. In a preferred embodiment, the invention discloses a novel peptide, rTmCad1p, is mixed with Cry3Aa to potentiate the Cry3Aa toxin. In another embodiment, rTmCad1p is mixed with Cry1Ac protoxin to potentiate larval mortality. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 List of known  B. thuringiensis  endotoxins 
               
             
          
           
               
                   
                 NAME 
                 GenBank Accession Number 
               
               
                   
               
               
                   
                 Cry1Aa1 
                 M11250 
               
               
                   
                 Cry1Aa2 
                 M10917 
               
               
                   
                 Cry1Aa3 
                 D00348 
               
               
                   
                 Cry1Aa4 
                 X13535 
               
               
                   
                 Cry1Aa5 
                 D17518 
               
               
                   
                 Cry1Aa6 
                 U43605 
               
               
                   
                 Cry1Aa7 
                 AF081790 
               
               
                   
                 Cry1Aa8 
                 I26149 
               
               
                   
                 Cry1Aa9 
                 AB026261 
               
               
                   
                 Cry1Aa10 
                 AF154676 
               
               
                   
                 Cry1Aa11 
                 Y09663 
               
               
                   
                 Cry1Aa12 
                 AF384211 
               
               
                   
                 Cry1Aa13 
                 AF510713 
               
               
                   
                 Cry1Aa14 
                 AY197341 
               
               
                   
                 Cry1Aa15 
                 DQ062690 
               
               
                   
                 Cry1Ab1 
                 M13898 
               
               
                   
                 Cry1Ab2 
                 M12661 
               
               
                   
                 Cry1Ab3 
                 M15271 
               
               
                   
                 Cry1Ab4 
                 D00117 
               
               
                   
                 Cry1Ab5 
                 X04698 
               
               
                   
                 Cry1Ab6 
                 M37263 
               
               
                   
                 Cry1Ab7 
                 X13233 
               
               
                   
                 Cry1Ab8 
                 M16463 
               
               
                   
                 Cry1Ab9 
                 X54939 
               
               
                   
                 Cry1Ab10 
                 A29125 
               
               
                   
                 Cry1Ab11 
                 I12419 
               
               
                   
                 Cry1Ab12 
                 AF059670 
               
               
                   
                 Cry1Ab13 
                 AF254640 
               
               
                   
                 Cry1Ab14 
                 U94191 
               
               
                   
                 Cry1Ab15 
                 AF358861 
               
               
                   
                 Cry1Ab16 
                 AF375608 
               
               
                   
                 Cry1Ab17 
                 AAT46415 
               
               
                   
                 Cry1Ab18 
                 AAQ88259 
               
               
                   
                 Cry1Ab19 
                 AY847289 
               
               
                   
                 Cry1Ab20 
                 DQ241675 
               
               
                   
                 Cry1Ab21 
                 EF683163 
               
               
                   
                 Cry1Ab22 
                 ABW87320 
               
               
                   
                 Cry1Ab-like 
                 AF327924 
               
               
                   
                 Cry1Ab-like 
                 AF327925 
               
               
                   
                 Cry1Ab-like 
                 AF327926 
               
               
                   
                 Cry1Ab-like 
                 DQ781309 
               
               
                   
                 Cry1Ac1 
                 M11068 
               
               
                   
                 Cry1Ac2 
                 M35524 
               
               
                   
                 Cry1Ac3 
                 X54159 
               
               
                   
                 Cry1Ac4 
                 M73249 
               
               
                   
                 Cry1Ac5 
                 M73248 
               
               
                   
                 Cry1Ac6 
                 U43606 
               
               
                   
                 Cry1Ac7 
                 U87793 
               
               
                   
                 Cry1Ac8 
                 U87397 
               
               
                   
                 Cry1Ac9 
                 U89872 
               
               
                   
                 Cry1Ac10 
                 AJ002514 
               
               
                   
                 Cry1Ac11 
                 AJ130970 
               
               
                   
                 Cry1Ac12 
                 I12418 
               
               
                   
                 Cry1Ac13 
                 AF148644 
               
               
                   
                 Cry1Ac14 
                 AF492767 
               
               
                   
                 Cry1Ac15 
                 AY122057 
               
               
                   
                 Cry1Ac16 
                 AY730621 
               
               
                   
                 Cry1Ac17 
                 AY925090 
               
               
                   
                 Cry1Ac18 
                 DQ023296 
               
               
                   
                 Cry1Ac19 
                 DQ195217 
               
               
                   
                 Cry1Ac20 
                 DQ285666 
               
               
                   
                 Cry1Ac21 
                 DQ062689 
               
               
                   
                 Cry1Ac22 
                 EU282379 
               
               
                   
                 Cry1Ac23 
                 AM949588 
               
               
                   
                 Cry1Ac24 
                 ABL01535 
               
               
                   
                 Cry1Ad1 
                 M73250 
               
               
                   
                 Cry1Ad2 
                 A27531 
               
               
                   
                 Cry1Ae1 
                 M65252 
               
               
                   
                 Cry1Af1 
                 U82003 
               
               
                   
                 Cry1Ag1 
                 AF081248 
               
               
                   
                 Cry1Ah1 
                 AF281866 
               
               
                   
                 Cry1Ah2 
                 DQ269474 
               
               
                   
                 Cry1Ai1 
                 AY174873 
               
               
                   
                 Cry1A-like 
                 AF327927 
               
               
                   
                 Cry1Ba1 
                 X06711 
               
               
                   
                 Cry1Ba2 
                 X95704 
               
               
                   
                 Cry1Ba3 
                 AF368257 
               
               
                   
                 Cry1Ba4 
                 AF363025 
               
               
                   
                 Cry1Ba5 
                 ABO20894 
               
               
                   
                 Cry1Ba6 
                 ABL60921 
               
               
                   
                 Cry1Bb1 
                 L32020 
               
               
                   
                 Cry1Bc1 
                 Z46442 
               
               
                   
                 Cry1Bd1 
                 U70726 
               
               
                   
                 Cry1Bd2 
                 AY138457 
               
               
                   
                 Cry1Be1 
                 AF077326 
               
               
                   
                 Cry1Be2 
                 AAQ52387 
               
               
                   
                 Cry1Bf1 
                 AX189649 
               
               
                   
                 Cry1Bf2 
                 AAQ52380 
               
               
                   
                 Cry1Bg1 
                 AY176063 
               
               
                   
                 Cry1Ca1 
                 X07518 
               
               
                   
                 Cry1Ca2 
                 X13620 
               
               
                   
                 Cry1Ca3 
                 M73251 
               
               
                   
                 Cry1Ca4 
                 A27642 
               
               
                   
                 Cry1Ca5 
                 X96682 
               
               
                   
                 Cry1Ca6 [1] 
                 AF215647 
               
               
                   
                 Cry1Ca7 
                 AY015492 
               
               
                   
                 Cry1Ca8 
                 AF362020 
               
               
                   
                 Cry1Ca9 
                 AY078160 
               
               
                   
                 Cry1Ca10 
                 AF540014 
               
               
                   
                 Cry1Ca11 
                 AY955268 
               
               
                   
                 Cry1Cb1 
                 M97880 
               
               
                   
                 Cry1Cb2 
                 AY007686 
               
               
                   
                 Cry1Cb3 
                 EU679502 
               
               
                   
                 Cry1Cb-like 
                 AAX63901 
               
               
                   
                 Cry1Da1 
                 X54160 
               
               
                   
                 Cry1Da2 
                 I76415 
               
               
                   
                 Cry1Db1 
                 Z22511 
               
               
                   
                 Cry1Db2 
                 AF358862 
               
               
                   
                 Cry1Dc1 
                 EF059913 
               
               
                   
                 Cry1Ea1 
                 X53985 
               
               
                   
                 Cry1Ea2 
                 X56144 
               
               
                   
                 Cry1Ea3 
                 M73252 
               
               
                   
                 Cry1Ea4 
                 U94323 
               
               
                   
                 Cry1Ea5 
                 A15535 
               
               
                   
                 Cry1Ea6 
                 AF202531 
               
               
                   
                 Cry1Ea7 
                 AAW72936 
               
               
                   
                 Cry1Ea8 
                 ABX11258 
               
               
                   
                 Cry1Eb1 
                 M73253 
               
               
                   
                 Cry1Fa1 
                 M63897 
               
               
                   
                 Cry1Fa2 
                 M73254 
               
               
                   
                 Cry1Fb1 
                 Z22512 
               
               
                   
                 Cry1Fb2 
                 AB012288 
               
               
                   
                 Cry1Fb3 
                 AF062350 
               
               
                   
                 Cry1Fb4 
                 I73895 
               
               
                   
                 Cry1Fb5 
                 AF336114 
               
               
                   
                 Cry1Fb6 
                 EU679500 
               
               
                   
                 Cry1Fb7 
                 EU679501 
               
               
                   
                 Cry1Ga1 
                 Z22510 
               
               
                   
                 Cry1Ga2 
                 Y09326 
               
               
                   
                 Cry1Gb1 
                 U70725 
               
               
                   
                 Cry1Gb2 
                 AF288683 
               
               
                   
                 Cry1Gc 
                 AAQ52381 
               
               
                   
                 Cry1Ha1 
                 Z22513 
               
               
                   
                 Cry1Hb1 
                 U35780 
               
               
                   
                 Cry1H-like 
                 AF182196 
               
               
                   
                 Cry1Ia1 
                 X62821 
               
               
                   
                 Cry1Ia2 
                 M98544 
               
               
                   
                 Cry1Ia3 
                 L36338 
               
               
                   
                 Cry1Ia4 
                 L49391 
               
               
                   
                 Cry1Ia5 
                 Y08920 
               
               
                   
                 Cry1Ia6 
                 AF076953 
               
               
                   
                 Cry1Ia7 
                 AF278797 
               
               
                   
                 Cry1Ia8 
                 AF373207 
               
               
                   
                 Cry1Ia9 
                 AF521013 
               
               
                   
                 Cry1Ia10 
                 AY262167 
               
               
                   
                 Cry1Ia11 
                 AJ315121 
               
               
                   
                 Cry1Ia12 
                 AAV53390 
               
               
                   
                 Cry1Ia13 
                 ABF83202 
               
               
                   
                 Cry1Ia14 
                 EU887515 
               
               
                   
                 Cry1Ib1 
                 U07642 
               
               
                   
                 Cry1Ib2 
                 ABW88019 
               
               
                   
                 Cry1Ib3 
                 EU677422 
               
               
                   
                 Cry1Ic1 
                 AF056933 
               
               
                   
                 Cry1Ic2 
                 AAE71691 
               
               
                   
                 Cry1Id1 
                 AF047579 
               
               
                   
                 Cry1Ie1 
                 AF211190 
               
               
                   
                 Cry1If1 
                 AAQ52382 
               
               
                   
                 Cry1I-like 
                 I90732 
               
               
                   
                 Cry1I-like 
                 DQ781310 
               
               
                   
                 Cry1Ja1 
                 L32019 
               
               
                   
                 Cry1Jb1 
                 U31527 
               
               
                   
                 Cry1Jc1 
                 I90730 
               
               
                   
                 Cry1Jc2 
                 AAQ52372 
               
               
                   
                 Cry1Jd1 
                 AX189651 
               
               
                   
                 Cry1Ka1 
                 U28801 
               
               
                   
                 Cry1La1 
                 AAS60191 
               
               
                   
                 Cry1-like 
                 I90729 
               
               
                   
                 Cry2Aa1 
                 M31738 
               
               
                   
                 Cry2Aa2 
                 M23723 
               
               
                   
                 Cry2Aa3 
                 D86064 
               
               
                   
                 Cry2Aa4 
                 AF047038 
               
               
                   
                 Cry2Aa5 
                 AJ132464 
               
               
                   
                 Cry2Aa6 
                 AJ132465 
               
               
                   
                 Cry2Aa7 
                 AJ132463 
               
               
                   
                 Cry2Aa8 
                 AF252262 
               
               
                   
                 Cry2Aa9 
                 AF273218 
               
               
                   
                 Cry2Aa10 
                 AF433645 
               
               
                   
                 Cry2Aa11 
                 AAQ52384 
               
               
                   
                 Cry2Aa12 
                 DQ977646 
               
               
                   
                 Cry2Aa13 
                 ABL01536 
               
               
                   
                 Cry2Aa14 
                 ACF04939 
               
               
                   
                 Cry2Ab1 
                 M23724 
               
               
                   
                 Cry2Ab2 
                 X55416 
               
               
                   
                 Cry2Ab3 
                 AF164666 
               
               
                   
                 Cry2Ab4 
                 AF336115 
               
               
                   
                 Cry2Ab5 
                 AF441855 
               
               
                   
                 Cry2Ab6 
                 AY297091 
               
               
                   
                 Cry2Ab7 
                 DQ119823 
               
               
                   
                 Cry2Ab8 
                 DQ361266 
               
               
                   
                 Cry2Ab9 
                 DQ341378 
               
               
                   
                 Cry2Ab10 
                 EF157306 
               
               
                   
                 Cry2Ab11 
                 AM691748 
               
               
                   
                 Cry2Ab12 
                 ABM21764 
               
               
                   
                 Cry2Ab13 
                 EU909454 
               
               
                   
                 Cry2Ab14 
                 EU909455 
               
               
                   
                 Cry2Ac1 
                 X57252 
               
               
                   
                 Cry2Ac2 
                 AY007687 
               
               
                   
                 Cry2Ac3 
                 AAQ52385 
               
               
                   
                 Cry2Ac4 
                 DQ361267 
               
               
                   
                 Cry2Ac5 
                 DQ341379 
               
               
                   
                 Cry2Ac6 
                 DQ359137 
               
               
                   
                 Cry2Ac7 
                 AM292031 
               
               
                   
                 Cry2Ac8 
                 AM421903 
               
               
                   
                 Cry2Ac9 
                 AM421904 
               
               
                   
                 Cry2Ac10 
                 BI 877475 
               
               
                   
                 Cry2Ac11 
                 AM689531 
               
               
                   
                 Cry2Ac12 
                 AM689532 
               
               
                   
                 Cry2Ad1 
                 AF200816 
               
               
                   
                 Cry2Ad2 
                 DQ358053 
               
               
                   
                 Cry2Ad3 
                 AM268418 
               
               
                   
                 Cry2Ad4 
                 AM490199 
               
               
                   
                 Cry2Ad5 
                 AM765844 
               
               
                   
                 Cry2Ae1 
                 AAQ52362 
               
               
                   
                 Cry2Af1 
                 EF439818 
               
               
                   
                 Cry2Ag 
                 ACH91610 
               
               
                   
                 Cry2Ah 
                 EU939453 
               
               
                   
                 Cry3Aa1 
                 M22472 
               
               
                   
                 Cry3Aa2 
                 J02978 
               
               
                   
                 Cry3Aa3 
                 Y00420 
               
               
                   
                 Cry3Aa4 
                 M30503 
               
               
                   
                 Cry3Aa5 
                 M37207 
               
               
                   
                 Cry3Aa6 
                 U10985 
               
               
                   
                 Cry3Aa7 
                 AJ237900 
               
               
                   
                 Cry3Aa8 
                 AAS79487 
               
               
                   
                 Cry3Aa9 
                 AAW05659 
               
               
                   
                 Cry3Aa10 
                 AAU29411 
               
               
                   
                 Cry3Aa11 
                 AY882576 
               
               
                   
                 Cry3Aa12 
                 ABY49136 
               
               
                   
                 Cry3Ba1 
                 X17123 
               
               
                   
                 Cry3Ba2 
                 A07234 
               
               
                   
                 Cry3Bb1 
                 M89794 
               
               
                   
                 Cry3Bb2 
                 U31633 
               
               
                   
                 Cry3Bb3 
                 I15475 
               
               
                   
                 Cry3Ca1 
                 X59797 
               
               
                   
                 Cry4Aa1 
                 Y00423 
               
               
                   
                 Cry4Aa2 
                 D00248 
               
               
                   
                 Cry4Aa3 
                 AL731825 
               
               
                   
                 Cry4A-like 
                 DQ078744 
               
               
                   
                 Cry4Ba1 
                 X07423 
               
               
                   
                 Cry4Ba2 
                 X07082 
               
               
                   
                 Cry4Ba3 
                 M20242 
               
               
                   
                 Cry4Ba4 
                 D00247 
               
               
                   
                 Cry4Ba5 
                 AL731825 
               
               
                   
                 Cry4Ba-like 
                 ABC47686 
               
               
                   
                 Cry4Ca1 
                 EU646202 
               
               
                   
                 Cry5Aa1 
                 L07025 
               
               
                   
                 Cry5Ab1 
                 L07026 
               
               
                   
                 Cry5Ac1 
                 I34543 
               
               
                   
                 Cry5Ad1 
                 EF219060 
               
               
                   
                 Cry5Ba1 
                 U19725 
               
               
                   
                 Cry5Ba2 
                 EU121522 
               
               
                   
                 Cry6Aa1 
                 L07022 
               
               
                   
                 Cry6Aa2 
                 AF499736 
               
               
                   
                 Cry6Aa3 
                 DQ835612 
               
               
                   
                 Cry6Ba1 
                 L07024 
               
               
                   
                 Cry7Aa1 
                 M64478 
               
               
                   
                 Cry7Ab1 
                 U04367 
               
               
                   
                 Cry7Ab2 
                 U04368 
               
               
                   
                 Cry7Ab3 
                 BI 1015188 
               
               
                   
                 Cry7Ab4 
                 EU380678 
               
               
                   
                 Cry7Ab5 
                 ABX79555 
               
               
                   
                 Cry7Ab6 
                 FJ194973 
               
               
                   
                 Cry7Ba1 
                 ABB70817 
               
               
                   
                 Cry7Ca1 
                 EF486523 
               
               
                   
                 Cry8Aa1 
                 U04364 
               
               
                   
                 Cry8Ab1 
                 EU044830 
               
               
                   
                 Cry8Ba1 
                 U04365 
               
               
                   
                 Cry8Bb1 
                 AX543924 
               
               
                   
                 Cry8Bc1 
                 AX543926 
               
               
                   
                 Cry8Ca1 
                 U04366 
               
               
                   
                 Cry8Ca2 
                 AAR98783 
               
               
                   
                 Cry8Ca3 
                 EU625349 
               
               
                   
                 Cry8Da1 
                 AB089299 
               
               
                   
                 Cry8Da2 
                 BD133574 
               
               
                   
                 Cry8Da3 
                 BD133575 
               
               
                   
                 Cry8Db1 
                 AB303980 
               
               
                   
                 Cry8Ea1 
                 AY329081 
               
               
                   
                 Cry8Ea2 
                 EU047597 
               
               
                   
                 Cry8Fa1 
                 AY551093 
               
               
                   
                 Cry8Ga1 
                 AY590188 
               
               
                   
                 Cry8Ga2 
                 DQ318860 
               
               
                   
                 Cry8Ga3 
                 FJ198072 
               
               
                   
                 Cry8Ha1 
                 EF465532 
               
               
                   
                 Cry8Ia1 
                 EU381044 
               
               
                   
                 Cry8Ja1 
                 EU625348 
               
               
                   
                 Cry8 like 
                 ABS53003 
               
               
                   
                 Cry9Aa1 
                 X58120 
               
               
                   
                 Cry9Aa2 
                 X58534 
               
               
                   
                 Cry9Aa like 
                 AAQ52376 
               
               
                   
                 Cry9Ba1 
                 X75019 
               
               
                   
                 Cry9Bb1 
                 AY758316 
               
               
                   
                 Cry9Ca1 
                 Z37527 
               
               
                   
                 Cry9Ca2 
                 AAQ52375 
               
               
                   
                 Cry9Da1 
                 D85560 
               
               
                   
                 Cry9Da2 
                 AF042733 
               
               
                   
                 Cry9Db1 
                 AY971349 
               
               
                   
                 Cry9Ea1 
                 AB011496 
               
               
                   
                 Cry9Ea2 
                 AF358863 
               
               
                   
                 Cry9Ea3 
                 EF157307 
               
               
                   
                 Cry9Ea4 
                 EU760456 
               
               
                   
                 Cry9Ea5 
                 EU789519 
               
               
                   
                 Cry9Ea6 
                 EU887516 
               
               
                   
                 Cry9Eb1 
                 AX189653 
               
               
                   
                 Cry9Ec1 
                 AF093107 
               
               
                   
                 Cry9Ed1 
                 AY973867 
               
               
                   
                 Cry9 like 
                 AF093107 
               
               
                   
                 Cry10Aa1 
                 M12662 
               
               
                   
                 Cry10Aa2 
                 E00614 
               
               
                   
                 Cry10Aa3 
                 AL731825 
               
               
                   
                 Cry10A like 
                 DQ167578 
               
               
                   
                 Cry11Aa1 
                 M31737 
               
               
                   
                 Cry11Aa2 
                 M22860 
               
               
                   
                 Cry11Aa3 
                 AL731825 
               
               
                   
                 Cry11Aa-like 
                 DQ166531 
               
               
                   
                 Cry11Ba1 
                 X86902 
               
               
                   
                 Cry11Bb1 
                 AF017416 
               
               
                   
                 Cry12Aa1 
                 L07027 
               
               
                   
                 Cry13Aa1 
                 L07023 
               
               
                   
                 Cry14Aa1 
                 U13955 
               
               
                   
                 Cry15Aa1 
                 M76442 
               
               
                   
                 Cry16Aa1 
                 X94146 
               
               
                   
                 Cry17Aa1 
                 X99478 
               
               
                   
                 Cry18Aa1 
                 X99049 
               
               
                   
                 Cry18Ba1 
                 AF169250 
               
               
                   
                 Cry18Ca1 
                 AF169251 
               
               
                   
                 Cry19Aa1 
                 Y07603 
               
               
                   
                 Cry19Ba1 
                 D88381 
               
               
                   
                 Cry20Aa1 
                 U82518 
               
               
                   
                 Cry21Aa1 
                 I32932 
               
               
                   
                 Cry21Aa2 
                 I66477 
               
               
                   
                 Cry21Ba1 
                 AB088406 
               
               
                   
                 Cry22Aa1 
                 I34547 
               
               
                   
                 Cry22Aa2 
                 AX472772 
               
               
                   
                 Cry22Aa3 
                 EU715020 
               
               
                   
                 Cry22Ab1 
                 AAK50456 
               
               
                   
                 Cry22Ab2 
                 AX472764 
               
               
                   
                 Cry22Ba1 
                 AX472770 
               
               
                   
                 Cry23Aa1 
                 AAF76375 
               
               
                   
                 Cry24Aa1 
                 U88188 
               
               
                   
                 Cry24Ba1 
                 BAD32657 
               
               
                   
                 Cry24Ca1 
                 AM158318 
               
               
                   
                 Cry25Aa1 
                 U88189 
               
               
                   
                 Cry26Aa1 
                 AF122897 
               
               
                   
                 Cry27Aa1 
                 AB023293 
               
               
                   
                 Cry28Aa1 
                 AF132928 
               
               
                   
                 Cry28Aa2 
                 AF285775 
               
               
                   
                 Cry29Aa1 
                 AJ251977 
               
               
                   
                 Cry30Aa1 
                 AJ251978 
               
               
                   
                 Cry30Ba1 
                 BAD00052 
               
               
                   
                 Cry30Ca1 
                 BAD67157 
               
               
                   
                 Cry30Da1 
                 EF095955 
               
               
                   
                 Cry30Db1 
                 BAE80088 
               
               
                   
                 Cry30Ea1 
                 EU503140 
               
               
                   
                 Cry30Fa1 
                 EU751609 
               
               
                   
                 Cry30Ga1 
                 EU882064 
               
               
                   
                 Cry31Aa1 
                 AB031065 
               
               
                   
                 Cry31Aa2 
                 AY081052 
               
               
                   
                 Cry31Aa3 
                 AB250922 
               
               
                   
                 Cry31Aa4 
                 AB274826 
               
               
                   
                 Cry31Aa5 
                 AB274827 
               
               
                   
                 Cry31Ab1 
                 AB250923 
               
               
                   
                 Cry31Ab2 
                 AB274825 
               
               
                   
                 Cry31Ac1 
                 AB276125 
               
               
                   
                 Cry32Aa1 
                 AY008143 
               
               
                   
                 Cry32Ba1 
                 BAB78601 
               
               
                   
                 Cry32Ca1 
                 BAB78602 
               
               
                   
                 Cry32Da1 
                 BAB78603 
               
               
                   
                 Cry33Aa1 
                 AAL26871 
               
               
                   
                 Cry34Aa1 
                 AAG50341 
               
               
                   
                 Cry34Aa2 
                 AAK64560 
               
               
                   
                 Cry34Aa3 
                 AY536899 
               
               
                   
                 Cry34Aa4 
                 AY536897 
               
               
                   
                 Cry34Ab1 
                 AAG41671 
               
               
                   
                 Cry34Ac1 
                 AAG50118 
               
               
                   
                 Cry34Ac2 
                 AAK64562 
               
               
                   
                 Cry34Ac3 
                 AY536896 
               
               
                   
                 Cry34Ba1 
                 AAK64565 
               
               
                   
                 Cry34Ba2 
                 AY536900 
               
               
                   
                 Cry34Ba3 
                 AY536898 
               
               
                   
                 Cry35Aa1 
                 AAG50342 
               
               
                   
                 Cry35Aa2 
                 AAK64561 
               
               
                   
                 Cry35Aa3 
                 AY536895 
               
               
                   
                 Cry35Aa4 
                 AY536892 
               
               
                   
                 Cry35Ab1 
                 AAG41672 
               
               
                   
                 Cry35Ab2 
                 AAK64563 
               
               
                   
                 Cry35Ab3 
                 AY536891 
               
               
                   
                 Cry35Ac1 
                 AAG50117 
               
               
                   
                 Cry35Ba1 
                 AAK64566 
               
               
                   
                 Cry35Ba2 
                 AY536894 
               
               
                   
                 Cry35Ba3 
                 AY536893 
               
               
                   
                 Cry36Aa1 
                 AAK64558 
               
               
                   
                 Cry37Aa1 
                 AAF76376 
               
               
                   
                 Cry38Aa1 
                 AAK64559 
               
               
                   
                 Cry39Aa1 
                 BAB72016 
               
               
                   
                 Cry40Aa1 
                 BAB72018 
               
               
                   
                 Cry40Ba1 
                 BAC77648 
               
               
                   
                 Cry40Ca1 
                 EU381045 
               
               
                   
                 Cry40Da1 
                 EU596478 
               
               
                   
                 Cry41Aa1 
                 AB116649 
               
               
                   
                 Cry41Ab1 
                 AB116651 
               
               
                   
                 Cry42Aa1 
                 AB116652 
               
               
                   
                 Cry43Aa1 
                 AB115422 
               
               
                   
                 Cry43Aa2 
                 AB176668 
               
               
                   
                 Cry43Ba1 
                 AB115422 
               
               
                   
                 Cry43-like 
                 AB115422 
               
               
                   
                 Cry44Aa 
                 BAD08532 
               
               
                   
                 Cry45Aa 
                 BAD22577 
               
               
                   
                 Cry46Aa 
                 BAC79010 
               
               
                   
                 Cry46Aa2 
                 BAG68906 
               
               
                   
                 Cry46Ab 
                 BAD35170 
               
               
                   
                 Cry47Aa 
                 AY950229 
               
               
                   
                 Cry48Aa 
                 AJ841948 
               
               
                   
                 Cry48Aa2 
                 AM237205 
               
               
                   
                 Cry48Aa3 
                 AM237206 
               
               
                   
                 Cry48Ab 
                 AM237207 
               
               
                   
                 Cry48Ab2 
                 AM237208 
               
               
                   
                 Cry49Aa 
                 AJ841948 
               
               
                   
                 Cry49Aa2 
                 AM237201 
               
               
                   
                 Cry49Aa3 
                 AM237203 
               
               
                   
                 Cry49Aa4 
                 AM237204 
               
               
                   
                 Cry49Ab1 
                 AM237202 
               
               
                   
                 Cry50Aa1 
                 AB253419 
               
               
                   
                 Cry51Aa1 
                 DQ836184 
               
               
                   
                 Cry52Aa1 
                 EF613489 
               
               
                   
                 Cry53Aa1 
                 EF633476 
               
               
                   
                 Cry54Aa1 
                 EU339367 
               
               
                   
                 Cry55Aa1 
                 EU121521 
               
               
                   
                 Cry55Aa2 
                 AAE33526 
               
               
                   
               
             
          
         
       
     
     It should similarly be noted that one skilled in the art, having the benefit of the subject disclosure, will recognize that the subject peptides potentially have a variety of functions, uses, and activities. As stated herein, the subject peptides can be administered together with a Cry protein. When used in this manner, peptides of the subject invention can effect a faster kill of the targeted insects, and/or they can enable less Cry protein to be required for killing the insects. Complete lethality, however, is not required. The ultimate preferred goal is to prevent insects from damaging plants of interest. Thus, prevention of feeding is sufficient. Thus “inhibiting” the insects is all that is required. This can be accomplished by making the insects “sick” or by otherwise inhibiting (including killing) them so that damage to the plants being protected is reduced. This includes inhibiting larval growth of target pests or causing larval mortality. Thus, the inhibitory function of the subject peptides can be achieved by any mechanism of action, directly or indirectly related to the Cry protein, or completely independent of the Cry protein. 
     It is contemplated TmCad1 would potentiate toxicity of Cry delta-endotoxins such as but not limited to Cry1A, Cry1B, Cry1I, Cry1J, Cry2A, Cry3A, Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, Cry8C, Cry8D, Cry15A, Cry18A, Cry34A, Cry34B, Cry35A, Cry 35B. Additionally, polynucleotide of Bt toxins yet to be discovered or active fragments thereof would potentiate toxicity with the novel peptide. Accordingly, the skilled artisan would potentiate Bt toxins with the teachings disclosed herein. 
     Those trained in the art will recognize that nucleotide sequences including that encoding for TmCad1 will encode for amino acids with equivalent biological activity. Allelic variation may occur in the DNA sequences but will likely not change toxin-binding or potentiation activity of rTmCad1. DNA sequences having at least 90% identity to the included sequences are considered equivalent sequences and are included in the subject invention. 
     Chymotrypsin was from Worthington (Lakewood, N.J.). All other chemicals were from Sigma Chemical Co. (St. Louis, Mo.). 
     This invention is directed to a novel polypeptide that potentiates Bt toxin. The cDNA that encodes this polypeptide was derived from a  Tenebrio molitor  larval midgut library, which is of the Order Coleoptera. Gene-specific primers were designed and the complete coding sequence (tmcad1) was obtained from larval midgut cDNA by 5′- and 3′-RACE using the GeneRacer kit from Invitrogen (Carlsbad, Calif.) and SuperTaq Plus DNA polymerase (Ambion, Austin, Tex.). Tm1 and Tm2 nucleotide primers (Table 2) were designed from a partial tmcad1 cDNA, originally obtained from randomly selected clones from a  T. molitor  larval midgut library. Tm1 and Tm2 are in the sense orientation and were used with the GeneRacer 3′-primer to amplify the 3′ end. Similarly, PCR primers were designed in the antisense orientation (Tm3, Tm4, Tm5, Tm6, Tm7, Tm8, Tm9, Tm10; Table 2) and were used with the GeneRacer 5′-primer and GeneRacer 5′-nested primer to amplify the missing 5′ cDNA fragments. PCR products were gel-purified and inserted into pCR2.1-TOPO or pCR4-TOPO cloning vectors. Oligonucleotide primers (Tm11, Tm12, Tm13, Tm14, Tm15) were designed from known tmcad1 and used to sequence missing internal regions of subcloned cDNA. A cDNA containing the entire  T. molitor  cadherin (tmcad1) coding sequence was obtained by RT-PCR and confirmed that our results from RACE are consistent with a single, continuous cadherin cDNA. DNA sequencing was performed using the GenomeLab DTCS Quick Start Kit on a CEQ8000 DNA sequencer (Beckman-Coulter, Fullerton, Calif.). The complete tmcad1 cDNA sequence was deposited in the NCBI database (accession DQ988044). 
     
       
         
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Primer 
                 Orientation 
                 Position 
                 Primer DNA Sequence 
               
               
                   
               
             
             
               
                 Tm1-SEQ. ID. 
                 Sense 
                 4538-4563 
                 5′-TGAAAGCGTGGTTGATCGGTGTTTCG-3′ 
               
               
                 NO: 5 
                   
                   
                   
               
               
                   
               
               
                 Tm2-SEQ. ID. 
                 Sense 
                 4648-4676 
                 5′-TCCAGTACCAAATTCGGGTCGCAAGAG-3′ 
               
               
                 NO: 6 
                   
                   
                   
               
               
                   
               
               
                 Tm3-SEQ. ID. 
                 Antisense 
                 4152-4179 
                 5′-GGCATCAGCTTTGTGATTTTCCGGCTCT-3′ 
               
               
                 NO: 7 
                   
                   
                   
               
               
                   
               
               
                 Tm4-SEQ. ID. 
                 Antisense 
                 4018-4042 
                 5′-TGTCCAGGTCGAGGTTAGATGGAGT-3′ 
               
               
                 NO: 8 
                   
                   
                   
               
               
                   
               
               
                 Tm5-SEQ. ID. 
                 Antisense 
                 4055-4079 
                 5′-TCTCCGGATTGCGTATTCATGGTAA-3′ 
               
               
                 NO: 9 
                   
                   
                   
               
               
                   
               
               
                 Tm6-SEQ. ID. 
                 Antisense 
                 3864-3893 
                 5′-TCAAACACTGGAGATTCGTCGTTCTGGTCT-3′ 
               
               
                 NO: 10 
                   
                   
                   
               
               
                   
               
               
                 Tm7-SEQ. ID. 
                 Antisense 
                 3788-3811 
                 5′-GCTTGTCAGCGTTAGATGACTGAA-3′ 
               
               
                 NO: 11 
                   
                   
                   
               
               
                   
               
               
                 Tm8-SEQ. ID. 
                 Antisense 
                 3734-3753 
                 5′-GAGCGGTTGTTTAAGGGTGA-3′ 
               
               
                 NO: 12 
                   
                   
                   
               
               
                   
               
               
                 Tm9-SEQ. ID. 
                 Antisense 
                 2905-2928 
                 5′-TGTCACCTTCATCGTCATCTTTCC-3′ 
               
               
                 NO: 13 
                   
                   
                   
               
               
                   
               
               
                 Tm10-SEQ. ID. 
                 Antisense 
                 1388-1412 
                 5′-TCATCGTTGCATATCATTTAGGTTGA-3′ 
               
               
                 NO: 14 
                   
                   
                   
               
               
                   
               
               
                 Tm11-SEQ. ID. 
                 Sense 
                 1830-1853 
                 5′-CGACGCAGATTTGGAGTTCTCGAT-3′ 
               
               
                 NO: 15 
                   
                   
                   
               
               
                   
               
               
                 Tm12-SEQ. ID. 
                 Antisense 
                 2267-2290 
                 5′-CAACCCAGTCGGGAGTGTTCTCAT-3′ 
               
               
                 NO: 16 
                   
                   
                   
               
               
                   
               
               
                 Tm13-SEQ. ID. 
                 Sense 
                 377-404 
                 5′-TCAAGAACTTGGACGACGAACATCCGAC-3′ 
               
               
                 NO: 17 
                   
                   
                   
               
               
                   
               
               
                 Tm14-SEQ. ID. 
                 Antisense 
                 883-909 
                 5′-GGCATCCACCGTAGCGAAGTTGTTCTC-3′ 
               
               
                 NO: 18 
                   
                   
                   
               
               
                   
               
               
                 Tm15-SEQ. ID. 
                 Antisense 
                 1023-1044 
                 5′-AATGTCTTCAAGGATCAGCAGT-3′ 
               
               
                 NO: 19 
                   
                   
                   
               
               
                   
               
               
                 Tm16-SEQ. ID. 
                 Sense 
                 Adapter 
                 5′-CACCGAGCACGAGGACACTGACAT-3′ 
               
               
                 NO: 20 
                   
                   
                   
               
               
                   
               
               
                 Tm17-SEQ. ID. 
                 Antisense 
                 4526-4548 
                 5′-CTACCACGCTTTCAAAATTGCTTCCA-3′ 
               
               
                 NO: 21 
                   
                   
                   
               
               
                   
               
               
                 Tm18-SEQ. ID. 
                 Sense 
                 3964-3990 
                 5′-ACTGACAAGGATACAACTAGTAAGGAC-3′ 
               
               
                 NO: 22 
                   
                   
                   
               
               
                   
               
               
                 Tm19-SEQ. ID. 
                 Antisense 
                 4852-4878 
                 5′-TTCAAACTGATCATCTTTAGTTGGGTA-3′ 
               
               
                 NO: 23 
                   
                   
                   
               
               
                   
               
               
                 Tm20-SEQ. ID. 
                 Sense 
                 3961-4005 
                 5′-CGAATTCGCCATGGCCACTGACAAGGATACA 
               
               
                 NO: 24 
                   
                   
                 ACTAGTAAGGACAAGTTGCAATACAAC-3′ 
               
               
                   
               
               
                 Tm21-SEQ. ID. 
                 Antisense 
                 4861-4878 
                 5′-GCGGCGGCGCGGCCGCCTTCAAACTGATCAT 
               
               
                 NO: 25 
                   
                   
                 CTTT-3′ 
               
               
                   
               
               
                 Hv1-SEQ. ID. 
                 Sense 
                  1-31 
                 5′-GGGGTACCAACTATGAGATGGCAGTCGACGT 
               
               
                 NO: 26 
                   
                   
                 GAGAATAC-3′ 
               
               
                   
               
               
                 Hv2-SEQ. ID. 
                 Antisense 
                 59-81 
                 5′-GGAATTCATCTTGCGCGACCGTTAAATGA-3′ 
               
               
                 NO: 27 
               
               
                   
               
             
          
         
       
     
     The full-length cDNA, tmcad1, is 5,095 bp and contains an open reading frame of 4,881 bp that encode for 1,626 amino acid residues. TmCad1 has a predicted pI of 4.13 and expected molecular mass of 179,341 kDa. TmCad1 was predicted to have extracellular, transmembrane, and intracellular domains using TMHMM Server v 2. (http://www.cbs.dtu.dk/services/TMHMM-2.0/; an available web-based server for the prediction of transmembrane helices in proteins) and 12 cadherin repeat domains using Motif Scan of PROSITE database (http://myhits.isb-sib.ch/cgi-bin/motif_scan; an available web-based server for scanning sequences for all known protein motifs). 
     PCR and KOD high-fidelity DNA polymerase (EMD Biosciences, San Diego, Calif.) was used to amplify 585 bp product from cDNA encoding for the partial TmCad1 (nucleotides 4,076-4,661 from SEQ. ID. NO: 3 that correspond to amino acid residues 1,322-1,516). PCR product generated using the primers Tm16 and Tm17 was gel-purified and inserted into the  Escherichia coli  expression vector pET151-D-TOPO (Invitrogen). Insertion of the correct sequence into the expression vector was confirmed by sequencing DNA in both directions with T7 and T7rev vector primers. 
     Peptide Expression Via  E. Coli  BL21 Transformation 
     For expression of the rTmCad1 peptide fragment, BL21 Star (DE3)  E. coli  was transformed and cultures were grown as previously outlined (Fabrick and Tabashnik, 2007 , Insect Biochem. Mol. Biol.  37(2):97-106). Because the pET151-D-TOPO vector produces recombinant protein containing an amino-terminal six-histidine tag (see  FIG. 2 ), Ni 2+ -affinity chromatography was used to purify 6His-rTmCad1 peptide (6His-rTmCad1p). Protein was extracted from  E. coli  inclusion bodies and purification was performed under hybrid denaturing/native conditions as previously described in Fabrick and Tabashnik, 2007 , Insect Biochem. Mol. Biol.  37(2):97-106 and incorporated herein by reference. Elution fractions containing 6His-rTmCad1p were pooled and dialyzed against 0.01 M Tris-HCl, pH 8.0, 0.01% Triton X100. 
     rAcTEV protease (Invitrogen) can be used to remove 27 amino acid residues at the amino terminus of 6His-rTmCad1p, which included the six histidine tag and a V5 epitope (See  FIG. 2 ). Bioassays as detailed infra, utilized unhydrolyzed rTmCAD1p. 
     Purification of rAcTEV protease-treated rTmCad1p was conducted per manufacturer recommendation. Purified rTmCad1p was analyzed by SDS-PAGE, and the protein concentration was determined with Coomassie Plus Protein Assay Reagent (Pierce, Rockford, Ill.). Concentration and buffer exchange of rTmCad1p was performed using Centricon centrifugal filters (Millipore, Bedford, Mass.). 
     Peptide Expression Via  E. Coli  ArcticExpress™ (DE3) Transformation 
     Additional rTmcad1p protein was expressed. in  E. coli  ArcticExpress™ (DE3). Tmcad1p/pET151-D-TOPO plasmid was transformed into  E. coli  ArcticExpress (DE3) host strain and transformants were selected by Ampicillin resistance. Single colony of  E. coli  host was inoculated in Luria broth containing 0.05 mg/mL of Ampicillin. The culture tube was shaken at 37° C. at rpm. The overnight culture was added into fresh LB media in a ratio of 1:100. Once cell density reached 0.6 to 0.8 O.D. at 600 nm, IPTG of 1 mM was added for induction at 25° C. One liter of  E. coli  was cultured and pelleted by centrifugation. Cell pelleted centrifugation was washed with 20 mM sodium phosphate and 500 mM NaCl at pH of 7.8. Cells were also lysed by sonication in lysis buffer (6 M guanidine hydrochloride, 20 mM sodium phosphate and 500 mM NaCl at pH of 7.8). The supernatant was collected by centrifugation. Two mL of Ni-NTA resin was equilibrated in buffer containing 8M urea and chromatography was conducted by batch elution under hybrid conditions and following the Ni-NTA purifications protocols. A elution fraction containing the peptide was pooled and dialyzed against 10 mM Tris-HCl, 0.01% Triton X-100 and pH of 8.0, with two separate buffer changes. Concentration of rTmcad1p was determined by comparing with standard BSA (1 mg/mL), yielding a concentration of about 1 mg/mL with a volume of 5 mL. 
     Cry3Aa protoxin used was purified from sporulated cultures of  Bacillus thuringiensis  var.  tenebrionis . Bacterial cultures were grown for three days at 28° C. Spore-crystal mixtures were collected by centrifugation and washed with 1 M NaCl 0.1% Triton-X-100 and then water. Cry3Aa protoxin was solubilized in 50 mM Na 2 CO 3  0.1 M NaCl 0.1% β-mercaptoethanol pH 9.8 and further purified using anion exchange chromatography (AKTA FPLC, GE Healthcare, Uppsala, Sweden). Purified Cry3Aa protoxin was quantified using the Coomassie Protein Assay kit (Pierce) using BSA as standard. 
     Dot-Blot Assay 
     Example 1 
     In dot blot assays, 0.1, 0.5, 1, 2.5, 5, and 10 μg of rTmCad1p obtained from  E. coli  BL21 was spotted and dried on Immobilon-P PVDF membrane (Millipore, Billerica, Mass.). The membrane was blocked with 3% bovine serum albumin in PBS (0.08 M Na 2 HPO 4 , 0.02 M NaH 2 PO 4 , 0.1 M NaCl, pH 7.4) for at least one hour. Incubations with target ligands were done for 2 h in PBS, pH 7.4, 0.1% BSA, 0.1% Tween-20. Blots were washed between each step three times for 5 min in wash buffer (PBS buffer, pH 7.4, 0.2% BSA, 0.1% Tween-20). Blots were incubated with 100 nM Cry3Aa (spore/crystalline toxin preparation from  Bacillus thuringiensis  var.  tenebrionis ) in PBS buffer, pH 7.4, 0.1% BSA, 0.3% Tween-20 followed by 1:5000-diluted rabbit anti-Cry3Aa sera (in wash buffer) and 1:5000-diluted ECL peroxidase-labeled anti-rabbit sera (in wash buffer). ECL Western blotting detection reagent (Amersham RPN2209) was used to visualize peroxidase activity on a Fluor Chem imager (Innotech). All steps were carried out at room temperature on an orbital shaker. rTmCad1p bound Cry3Aa but not BSA, indicating specificity for binding to this peptide ( FIG. 1A ). Binding was detected with 10 μg of rTmCad1p, but toxin binding to 1 μg peptide was barely detectable. 
     Toxin Binding in-Gel Assay 
     Example 2 
     Toxin binding in-gel assays were according to the manufacturer recommendation (LI-COR Biosciences, Lincoln, Nebr.). Briefly, rTmCad1p obtained from  E. coli  BL21, was separated by SDS-PAGE on a 10-20% Tricine gel with Tricine sample and running buffers (Invitrogen, Carlsbad, Calif.). After electrophoresis, gels were fixed in 45% methanol/10% acetic acid for 15 min. Separate gels were either stained with Coomassie blue (Imperial Protein Stain, Pierce Chemical Co., Rockford, Ill.), or were incubated with 2.35 μg IR-labeled toxin in 10 mL 1% BSA in 1× wash buffer (0.002 M imidazole-buffered saline with 0.02% Tween 20, KPL, Gaithersburg, Md.), with or without 100-fold excess rTmCad1p, overnight at room temperature with gentle shaking. Gels were washed thrice in wash buffer and scanned at 800 nm on an Odyssey Imager using v. 1.2.15 Odyssey software (LI-COR). 
     Chymotrypsin-activated Cry3Aa was labeled with a fluorescent dye using the IRDye 800CW Protein Labeling Kit (LI-COR Biosciences, Lincoln, Nebr.). The dye forms a stable ester conjugate with the toxin and has an emission maximum of 789 nm in 1×PBS. IR-labeled toxin was used in toxin-binding assays. 
     The IR-labeled Cry3Aa bound to peptide ( FIG. 1B , lane 2), and this binding was completely inhibited by addition of rTmCad1p peptide to the labeled toxin ( FIG. 1B , lane 3), suggesting specificity in the peptide/toxin interaction. These results are evidence that Cry3Aa binds specifically to this region of TmCad1. 
       Tenebrio Molitor  Larvae Inhibition Bioassays 
     Example 3 
     rTmCad1p obtained from  E. coli  BL21, was premixed with Cry3Aa and added to the diet (10 mg total comprising of 50% glucose/30% yeast/20% wheat germ) with a  T. molitor  larva, as indicated in  FIG. 3 . rTmCad1p was mixed with 10 ppm (0.154 μM) or 100 ppm (1.54 μM) Cry3Aa to maintain a molar ratio of 1:5 toxin:rTmCad1p.  T. molitor  larvae aged approximately 1 month and weighed 1.4-4.3 mg when placed on diets. Larvae were weighed at regular intervals, and the percent change in the mass±S.D. were determined. Although the difference between treatments and control were not statistically significant (one-way ANOVAs with Holm-Sidak comparisons) because of the large variation in larval weights, larvae fed rTmCad1p were smaller than those without peptide or control. 
     Bioassay results of  T. molitor  using whole grain bread discs cut using a 2 mm cork borer and placed into a microtiter plate well are shown in  FIG. 4 . Doses of Cry3Aa toxin, rTmCad1p peptide, or toxin and peptide at Cry3Aa:rTmCad1p molar ratios of 1:2.5 and 1:12.4 were added to each bread disc in 5 μl total volume as indicated, and the plate was equilibrated at 25° C., 60% RH., for 24 h. Molar ratios were calculated using molecular weight of Cry3Aa as 65,000 Da and that of rTmCad1p as 26,200 Da. Newly hatched  T. molitor  larvae were added with gentle forceps to each well, and wells were covered with an air-permeable membrane (Breathe-easier, DIVBIO). Mortality was evaluated after 7 days. 
     rTmCad1p obtained from  E. coli  ArcticExpress (DE3), was premixed with Cry3Aa and added to the diet (10 mg total comprising of 50% glucose/30% yeast/20% wheat germ) with  T. molitor  larvae as indicated supra. Molar ratios of 1:20 and 1:200 of CryAa:rTmCad1p were evaluated as indicated in  FIG. 5A-C . In trial 1, at a molar ratio of 1:20 CryAa:rTmCad1p, had a increase in mortality percentage against solely Cry3Aa. Specifically, as indicated in  FIG. 5A , potentiating occurred most following 2 days post treatment with three-fold increase in mortality with respect to 200 ppm of Cry3Aa against 200 ppm Cry3Aa mixed with 800 ppm of rTmCad1p. 
     Inhibition of Subseptible  Pectinophora Gossypiella  with rTmCad1p and Cry1Ac 
     Example 4 
     Larval mortality and weight bioassays were conducted with 40 ppm of rTmCad1p (1.48 μM) was derived from  E. coli  BL21 and was premixed with 1 ppm (7.41 nM) Cry1Ac and added to the diet of  Pectinophora gossypiella  first-instar larva. As indicated in  FIG. 7  and  FIG. 8  different diet treatments were tested, including control diet with dialysis buffer (0.01 M Tris-HCl, pH 8.0, 0.01% Triton X-100), control diet with TmCad1p, Cry1Ac protoxin-treated diet with dialysis buffer, and Cry1Ac protoxin-treated diet with rTmCad1p. Bioassays show that the combination of Cry1Ac and TmCad1p results in a decrease in weight gained by developing pink bollworm larvae ( FIG. 7 ) compared to the Cry1Ac toxin alone over time. Cry1Ac-susceptible  Pectinophora gossypiella  larvae (APHIS-S or APHIS) were used in bioassays as indicated in  FIG. 7  and  FIG. 8 . Susceptible  Pectinophora gossypiella  is known to be Bt cotton and Cry1Ac (LC50 of approximately 0.25 ug Cry1Ac per mL of artificial diet). The source of Cry1Ac protoxin was produced as a recombinant protoxin in  E. coli . The molar concentration of Cry1Ac protoxin was determined using 135,000 Da as its molecular weight.  Pectinophora gossypiella  larvae were reared on wheat germ artificial diet as described previously (Bartlett and Wolf, 1985. In R. F. Moore and P. Singh [eds.], Handbook of Insect Rearing, Vol. 2: 415-430. Elsevier Science, Amsterdam). 
     Inhibition of Resistant  Pectinophora gossypiella  with rTmCad1p and Cry1Ac 
     rTmCad1p obtained from  E. coli  ArcticExpress (DE3), was premixed with Cry1Ac and added to the diet with  Pectinophora gossypiella  larvae as indicated supra, however  Pectinophora gossypiella  larvae used in  FIG. 9  is a Cry1Ac resistant strain (AZP-R). The AZP-R resistant stain was provided by the University of Arizona and is published characterizing the resistance (Tabashnik et al., 2004 J. Econ. Ento. 97(3), 721-726.) A bioassay with 30 Cry1Ac-resistant larvae were treated with 1 ppm Cry1Ac inconjuction with 40 ppm rTmCad1p for a molar ratio of 1:200 of Cry1Ac:rTmCad1p as indicated in  FIG. 9 . After 21 day post initiation, the combination of rTmCad1p mixed with Cry1Ac increased mortality percentage against solely Cry1Ac with and increase of approximately 1.5-fold (19%) in mortality with respect to 1 ppm of Cry1Ac against 1 ppm Cry1Ac mixed with 40 ppm of rTmCad1p. 
     Inhibition of  Heliothis Virescens  with rTmCad1p and Cry1Ac 
     Larvae of insects of the genus constitute agricultural pests for major agricultural crops such as tobacco, cotton, soybean. A bioassay with  Heliothis virescens  neonates having no known  Bacillus thuringiensis  resistance, were obtained from Benzon Inc. (Carlisle, Pa.). A plurality of treatments (n=3) were conducted against 32 larvae per treatment to determine whether HD-73 Cry1Ac would be potentiate with a mixture of rTmCad1p. Specifically, rTmCad1p obtained from  E. coli  ArcticExpress (DE3) vector was mixed at a 1:200 Cry1Ac to rTmCad1p molar ratio. As detailed in  FIG. 10 , the mean percentage of mortality of neonates increased with a toxin/peptide combination rather than solely peptide or Cry1Ac toxin. Artificial diet (tobacco budworm diet, Bio-Serv, Frenchtown, N.J.) was prepared following manufacturer&#39;s instructions. Approximately 1 mL of diet was poured per well of a bioassay tray (BAW-128, C-D International, Pitman, N.J.) and cooled down at room temperature until diet solidified. Toxin or toxin plus rTmCad1p solutions were diluted in buffer (50 mM Na 2 CO 3  pH 9.8, 0.3 M NaCl) and 50 μl homogeneously overlayed per well containing solidified artificial diet. Controls included buffer or rTmCad1p alone. After the solutions dried on the diet surface, a single neonate larva of  H. virescens  was placed in each well with a fine brush. Wells were sealed with adhesive plastic lids with small holes to allow gas exchange. Larvae were held at 28° C. with a 16L:8D photoperiod. Larvae were scored for mortality after seven days. 
     Transient Expression of TmCad1(EC12-Cyto) in Cultured Insect Cells and Cytotoxicity Assays 
     Example 5 
     Heterologous Expression of TmCad1(EC12-Cyto) in Insect Cell Cultures 
     To test the receptor function of SEQ. ID. NO: 3, and based on previous identification of Bt toxin functional receptor sites in cadherins (Hua et al., 2004 . Insect Biochem. Mol. Biol.,  34(3):193-202), nucleotides 3,964-4,879 of SEQ. ID. NO: 3 corresponding to the homologous region to Cry1 functional receptor region in lepidopteran cadherin were cloned. The partial TmCad1 sequence including amino acids 1322-1626 of SEQ. ID. NO: 3 is referred as TmCad1(EC12-cyto) (SEQ. ID. NO.: 28). 
     For expression of the partial rTmCad1 corresponding to extracellular domain 12 through the end of the cytoplasmic domain (rTmCad1(EC12-cyto)) in insect cell culture, Tm18 and Tm19 were used to PCR amplify cDNA corresponding to nucleotides 3,964-4,878 and subcloned into pCR2.1-TOPO. Using TmCad1(EC12-cyto) cloned in pCR2.1 as template and the PCR supermix (Invitrogen), TmCad1(EC12-cyto) was amplified using PCR primers (Table 2) containing EcoRI (Tm20) or NotI (Tm21) restriction sites at the 5′ position. The 938 bp TmCad1(EC12-cyto) PCR amplicon was gel-purified using the S.N.A.P. gel purification kit (Invitrogen) and digested with EcoRI and NotI (Invitrogen) overnight at 37° C. Products were separated by 1% agarose gel electrophoresis, and DNA purified as for PCR amplicons. TmCad1(EC12-cyto) was cloned into pIZT/V5/H is vector predigested with EcoRI and NotI using T4 DNA ligase (Invitrogen) following manufacturer&#39;s instructions. Ligation reactions were used to transform chemically competent One Shot cells (Invitrogen) following manufacturer&#39;s suggested protocol to obtain pIZT/TmCad1(EC12-cyto). Transformants were selected on LB plates containing 50 μg/mL zeocin. The presence of TmCad1(EC12-cyto) insert was tested with restriction digestion assays and by DNA sequencing in both directions (UT sequencing facility, Knoxyille, Tenn.). 
     To target expression of TmCad1 (EC12-cyto) to the cell membrane of insect cells, a fragment corresponding to the signal peptide of  Heliothis virescens  cadherin (HevCaLP) was inserted using engineered KpnI and EcoRI sites. The signal peptide of HevCadLP was cloned using PCR with specific primers (Table 2) containing KpnI (Hv1) or EcoRI (Hv2) restriction sites at the 5′ ends. PCR amplicons were purified using the Qiaquick Nucleotide removal kit (Qiagen). After digestion with EcoRI and KpnI, fragments were ligated using T4 ligase into pIZT/TmCad1(EC12-cyto) previously digested with KpnI and EcoRI to obtain the pIZT/Hvseq/TmCad1(EC12-cyto) construct. Ligation reactions were used to transform competent DH5a  E. coli  cells. Clones containing pIZT/Hvseq/TmCad1(EC12-cyto) were selected with zeocin (50 μg/mL) on LB plates. Selected clones were checked with restriction enzymes for correct insert orientation and used for midipreps. Plasmid DNA was purified from midipreps using Qiagen HiSpeed plasmid purification kit following manufacturer&#39;s instructions. Purified plasmid was sequenced in both directions at the UT sequencing facility (Knoxyille, Tenn.) to confirm insertion and for correct reading frame. 
     For transient expression of TmCad1(EC12-cyto) in insect cell cultures,  Trichoplusia ni  Hi5 (Invitrogen) was used. Insect cell cultures were grown in serum-free insect cell media (Hyclone). For lipofection, approximately 1.5×10 6  cells from a confluent culture were resuspended in 5 mL fresh media and allowed to adhere overnight to 60×15 mm polystyrene dishes (Falcon). Plasmid transfection mixtures were prepared by mixing either 2.5 μg of pIZT/V5/His or 5 μg of pIZT/Hvseq/TmCad1(EC12-cyto) plasmid with 1 mL of serum-free insect medium (Hyclone) and 20 μl of Cellfectin reagent (Invitrogen). Cells were incubated for four hours with the transfection mixture and then changed to fresh media and incubated at 26° C. for 2 days. 
     Immunoblotting with rTmCad1p antisera was used to test for TmCad1(EC12-cyto) expression ( FIG. 6A ). Approximately 1×10 6  cells were pelleted by centrifugation at 14,500×g for 2 min. and then washed twice with 1 mL of PBS buffer (135 mM NaCl, 2 mM KCl, 10 mM Na 2 HPO 4 , 1.7 mM KH 2 PO 4 , pH 7.5). Final pellets were solubilized in 50 μl of SDS-PAGE buffer and separated in 8% SDS-PAGE electrophoresis. Proteins were transferred to PVDF filters and filters blocked with PBS plus 1% Tween-20 (PBST) plus 3% BSA. Blots were probed sequentially with 1:5,000 dilution of rTmCad1p antisera and 1:20,000 dilution of goat anti-rabbit-HRP conjugate. Cross-reacting proteins were detected using enhanced chemiluminescence substrates (Western pico, Pierce). pIZT/Hvseq/TmCad1 (EC12-cyto)-transfected  T. ni  Hi5 cells expressed on their membrane a protein of about 44 kDa, the predicted size for TmCad1(EC12-cyto) plus tags, that cross-reacted with rTmcad1p antisera ( FIG. 6A ). 
     To test the functional receptor function of TmCad1(EC12-cyto), cytotoxicity assays were performed via fluorescent microscopy and flow cytometry assays. Briefly, insect cells were transfected and incubated for a period of 2 days, followed by a media change and transfer of cells to a 12-well tissue culture plate (Falcon). Upon incubation at 26° C. overnight, 501β/mL for S2 cells or 10 μg/mL for Hi5 cells of Cry3Aa protoxin (approximately 800 nM and 160 nM protoxin concentration, respectively) was added and the cells incubated at 26° C. for four hours. Cells were stained with 1 μg/mL of propidium iodide (PI) for 5 min, then immediately observed for GPF fluorescence (green fluorescent protein) and PI (propidium iodide) staining using a flow cytometer (LSR benchtop flow cytometer, Beckton Dickinson, USA). To calculate the percentage of GFP positive cells in the pIZT/Hv/TmCad1(EC12-cyto) cell population killed by Cry3A toxins ( FIG. 6B ), the formula in Table 3 was utilized. The formula accounts for the dead cells (PI-positive) in an untreated population, GFP positive dead cells that lost GFP due to cell leakage, and the observed transfection efficiency. 
                     TABLE 3                           =       [               (         GFP   +     ⁢     PI   +     ⁢     Cells   Toxin       +       GFP   -     ⁢     PI   +     ⁢     Cells   Toxin         )     -               (         GFP   +     ⁢     PI   +     ⁢     Cells   control       +       GFP   -     ⁢     PI   +     ⁢     Cells   control         )               GFP   +     ⁢     Cells   Toxin         ]     ×   100                              
As shown in  FIG. 6B , Cry3Aa treatment induced 40% and almost 25% cytotoxicity in S2 and Hi5 cells expressing TmCad1(EC12-cyto), respectively and provides evidence for the functional role of this peptide as Cry3Aa receptor.
 
     While the invention has been described with reference to details of the illustrated embodiment, these details are not intended to limit the scope of the invention as defined in the appended claims. The embodiment of the invention in which exclusive property or privilege is claimed is defined as follows: