Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.     

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa,Cry1Ab,Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests,Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with ¹²⁵I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.     

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.     

Binding of Bacillus thuringiensis Cry1 Toxins to the Midgut Brush Border Membrane Vesicles of Chilo suppressalis (Lepidoptera: Pyralidae): Evidence of Shared Binding Sites

Binding and competition among Cry1Aa, Cry1Ac, and Cry1Ba toxins were analyzed quantitatively in vitro by using (sup125)I-labeled activated toxins and brush border membrane vesicles isolated from Chilo suppressalis larval midguts. The three toxins bound specifically to the midgut brush border membrane vesicles. Direct binding experiments showed that Cry1Aa and Cry1Ba recognized a single class of binding sites with different affinities, whereas Cry1Aa recognized two classes of binding sites, one with a high affinity and a low concentration and the other with a lower affinity but higher concentration. Competition experiments showed that toxins Cry1Ac and Cry1Ba shared a binding site in the C. suppressalis midgut membranes and that this site was also the low-affinity binding site for Cry1Aa.     

Binding of Bacillus thuringiensis Cry1 Toxins to the Midgut Brush Border Membrane Vesicles of Chilo suppressalis (Lepidoptera: Pyralidae): Evidence of Shared Binding Sites

Volume 62, no. 5, p. 1544, Abstract, line 4: "Cry1Aa" should read "Cry1Ac." [This corrects the article on p. 1544 in vol. 62.].     

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.     

Role of helix 3 in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1Aa

Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.     

Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris)

Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60 kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37 kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.     

Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.

Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.     

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.     

Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Background

Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F₂ screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.

Methodology/Principal Findings

Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with ¹²⁵I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in ¹²⁵I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.

Conclusion/Significance

This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.     

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Binding of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis serovar israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera) and subsequent pore formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with ¹²⁵I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with ¹²⁵I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K_d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R_t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.     

Toxicity and Mode of Action of Bacillus thuringiensis Cry Proteins in the Mediterranean Corn Borer, Sesamia nonagrioides (Lefebvre)

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with ¹²⁵I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC₃(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.     

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.     

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130 kDa Cry7Ab3 protoxin was degraded into the ～75 kDa proteinase-resistant fragments. In vivo analysis, 130 kDa Cry7Ab3 protoxin was also processed into ～75 kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220 kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.