Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解CrylAc毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigern（Hǔbner）与甜菜夜蛾Spodoptera exigm（Hǔbner）的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 °C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.     

Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices 1 and 2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the 1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Activation pattern and toxicity of the Cry11Bb1 toxin of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis protoxins undergo proteolytic processing in the midgut of susceptible insects to become active. The ability to process the Cry11Bb1 protoxin by trypsin and Culex quinquefasciatus larval gut extracts was tested. The protease activity indicated by the appearance of proteolytic products increased with an increment in pH, with the highest activity being observed at pH 10.6. A time course study showed the proteolysis of the 94-kDa Cry11Bb protein ending with the production of fragments of relative molecular mass of 30 and 35 kDa within 5 min. In vitro, gut proteases extract cleaved the solubilized toxin between Ser59 and Ile60 and between Ala395 and Asn396, generating a 30-kDa N-terminal and a 35-kDa C-terminal fragment, respectively. Similarly, mosquito larvae processed in vivo the parasporal inclusions, generating the same fragments as those observed in vitro. The Cry11Bb1 protoxin activated with trypsin or gut proteases showed larvicidal activity against C. quinquefasciatus first instar larvae. The data suggest that gut proteases participate in the activation of CryllBbl protoxin, generating at least two different fragments on which the activity could reside.     

Processing of Cry1Ab δ-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices α1 and α2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the α1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.     

Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: Activation process and toxicity against Prays oleae

Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC₅₀ of 189 and 116 ng/cm², respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na₂CO₃ pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut.     

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.     

Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb,Cry3Ca,and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu⁴⁷ for the 70-kDa form or Ile⁸⁸ for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.     

Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ~97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ~45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.     

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.     

Proteolytic processing of native Cry1Ab toxin by midgut extracts and purified trypsins from the Mediterranean corn borer Sesamia nonagrioides

The proteolytic processing of native Cry1Ab toxin by midgut extracts from the Mediterranean corn borer, Sesamia nonagrioides, takes place in successive steps. Several cuts occur until a 74 kDa protein is obtained; this is further digested to give rise to an active form of 69 kDa, which can be again processed to fragments of 67, 66 and 43 kDa. We have shown that three different trypsins (TI, TIIA and TIII) purified from the S. nonagrioides midgut were able to digest Cry1Ab protoxin to obtain the active form of 69 kDa. Interestingly, TI and TIII further hydrolyzed the 69 kDa protein to a fragment of slightly lower molecular mass (67 kDa), while TIIA was able to continue digestion to give fragments of 46 and 43 kDa. These results contrast with those obtained using bovine trypsin, in which the main product of Cry1Ab digestion is a 69 kDa protein. The digestion of the toxin with a "non-trypsin" fraction from S. nonagrioides midgut lumen, mostly containing chymotrypsins and elastases and free of trypsin-like activity, resulted in a different processing pattern, yielding fragments of 79, 77, 71, 69 and 51 kDa. Our results indicate that trypsins and other proteases are involved in the first steps of protoxin processing, but trypsins play the most important role in obtaining the 74 and 69 kDa proteins. All the digestion products, including the proteins of 46 and 43 kDa obtained from the digestion of Cry1Ab by TIIA, were toxic to neonate larvae, indicating that none of the tested proteases contribute to toxin degradation in a significant manner.     

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.     

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.     

Partial Purification and Characterization of Bacillus thuringiensis Cry1A Toxin Receptor A from Heliothis virescens and Cloning of the Corresponding cDNA

Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.     

Differences in the midgut proteolytic activity of two Heliothis virescens strains,one susceptible and one resistant to Bacillus thuringiensis toxins

The development of resistance to Bacillus thuringiensis toxic proteins is a growing concern because it could threaten both conventional and gene transfer use of this environmentally safe biological insecticide. The most common mechanism of resistance involves changes in binding affinity of toxin receptors in the insect midgut membrane. This has not been the case in Heliothis virescens. We have investigated changes in midgut proteolytic activity as a possibility to explain the resistance observed in this insect species. We have developed an improvement of known methods to demonstrate proteolytic activity in crude extracts. Using this method we have found differences in the proteolytic activity profile of midgut extracts of a susceptible and a resistant H. virescens strain. We also have studied the in vitro processing of CrylA(b) toxin and protoxin by midgut contents of both strains. SDS-PAGE of the in vitro degradation products showed differences between the strains. The resistant strain degrades protoxin more slowly and processes the active toxin more quickly than the susceptible strain. © 1996 Wiley-Liss, Inc.     

Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb Toxicities to Coleopteran Larvae by a Toxin-Binding Fragment of an Insect Cadherin

The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis are used in biopesticides and transgenic crops to control larvae of leaf-feeding beetles and rootworms. Cadherins localized in the midgut epithelium are identified as receptors for Cry toxins in lepidopteran and dipteran larvae. Previously, we discovered that a peptide of a toxin-binding cadherin expressed in Escherichia coli functions as a synergist for Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae. Here we report that the fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm binds toxin and enhances Cry3 toxicity to larvae of naturally susceptible species. The cadherin fragment (CR8 to CR10 [CR8-10]) of western corn rootworm Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. By an enzyme-linked immunosorbent microplate assay, we demonstrated that the CR8-10 peptide binds α-chymotrypsin-treated Cry3Aa and Cry3Bb toxins at high affinity (11.8 nM and 1.4 nM, respectively). Coleopteran larvae ingesting CR8-10 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxin. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle Leptinotarsa decemlineata, southern corn rootworm Diabrotica undecimpunctata howardi, and western corn rootworm. The extent of Cry3 toxin enhancement, which ranged from 3- to 13-fold, may have practical applications for insect control. Cry3-containing biopesticides that include a cadherin fragment could be more efficacious. And Bt corn (i.e., corn treated with B. thuringiensis to make it resistant to pests) coexpressing Cry3Bb and CR8-10 could increase the functional dose level of the insect toxic activity, reducing the overall resistance risk.The Cry3 class of Bacillus thuringiensis Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. Cry3Aa and Cry3Bb proteins are highly toxic to Colorado potato beetle (CPB) Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), and both were used for the development of Bt crops (crops treated with B. thuringiensis to make them resistant to pests) and Bt biopesticides. Due to the limited efficacy of Cry3-based biopesticides/plants and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (12). Cry3Bb is toxic to corn rootworms (8, 17), and a modified version is expressed in commercialized MON863 corn hybrids (26).Cry3 toxins have a mode of action that is similar to, yet distinct from, the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73 kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. The Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (5, 18). Activated Cry3A toxin binds to brush border membrane vesicles with a K_d (dissociation constant) of ∼37 nM (19) and recognizes a 144-kDa binding protein in brush border membrane vesicles prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (2). Recently, Ochoa-Campuzano et al. (20) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.Structural differences between Cry3Bb and Cry3Aa toxins must underlie the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (11), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (27). Those authors introduced a chymotrypsin/cathepsin G cleavage site into domain 1 of Cry3Aa that allowed the processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.Cadherins function as receptors for Cry toxins in lepidopteran and dipteran larvae. A critical Cry1 toxin binding site is localized within the final cadherin repeat (CR), CR12, of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) (14, 28). Unexpectedly, a fragment of B. thuringiensis R₁ cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (6). If the binding residues within CR12 were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. Recently, we identified a cadherin from mosquito Anopheles gambiae (Diptera: Culicidae) that binds Cry4Ba toxin and probably functions as a receptor. We discovered a similar effect where a fragment of a cadherin from A. gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (15). Sayed et al. (22) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in the midgut tissue of larval stages. The encoded protein is conserved in structure relative to that of other insect midgut cadherins.In this study, we hypothesized that a fragment from a beetle cadherin that contains a putative Bt toxin binding region might enhance the insecticidal toxicities of Cry3Aa and Cry3Bb toxins. The region spanning CR8 to CR10 (CR8-10) of the WCRW cadherin (22) was cloned and expressed in E. coli. This cadherin fragment significantly enhanced the toxicities of Cry3Aa and Cry3Bb toxins to CPB and rootworms.