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.     

Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella)

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.     

Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells

A 120-kDa protein was purified from brush border membrane vesicles of the tortricid moth Epiphyas postvittana (Walker) based both on its activity as an aminopeptidase and the ability to bind the Bacillus thuringiensis delta-endotoxin Cry1Ac. The purified enzyme had a pI of 5.6 and was a leucine aminopeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptidase activity. Further characterisation showed that the protein was also able to bind Cry1Ba. During purification, the molecular weight of the protein decreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl anchor. The protein was N-terminally sequenced and, using this information and conserved regions within other insect aminopeptidase-N (APN) sequences, redundant primers were designed to amplify the aminopeptidase coding sequence from E. postvittana midgut cDNA. The predicted protein sequence from the full-length cDNA was most closely related to the APN protein sequence from Heliothis virescens (61% identity) and shared other features of insect APNs including a Zn(2+) binding site motif and four conserved cysteines. The E. postvittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence. Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays. However, despite the protein being expressed on the external surface of the Sf9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo.     

Partial purification of Cry 1A toxin-binding proteins from Helicoverpa armigera larval midgut

Toxicity of insecticidal endotoxins produced by Bacillus thuringiensis correlates with the presence of specific proteins in the midgut of susceptible larvae. This study was aimed at identifying and purifying Cry 1A binding proteins from Helicoverpa armigera, an important crop pest of India. B. thuringiensis strain HD 73 which produces Cry 1Ac toxin, specific for H. armigera was used in this study. Toxin-binding proteins from insect larvae were detected by employing a toxin overlay assay using both radiolabelled as well as unlabelled toxin. Detergent-solubilized fractions of larval brush border membranes were subjected to soybean agglutinin (SBA) chromatography, from which N-acetylgalactosamine (NAG)-containing proteins were eluted. Analysis of the SBA-purified proteins indicated that four proteins of approximately 97, 120, 170 and 200 kDa could bind to Cry 1Ac toxin, and three proteins of 97, 170 and 200 kDa proteins could bind to Cry 1Ab. Furthermore, in the presence of excess Cry 1Ab toxin, the labelled Cry 1Ac toxin could bind only to 170 and 200 kDa proteins, implying that Cry 1Ab can also bind the 120 kDa protein. This study therefore demonstrates that in H. armigera, midgut proteins of 97, 120, 170 and 200 kDa have the ability to bind both Cry 1Ab and Cry 1Ac. Furthermore, while the 170 and 200 kDa proteins have higher affinity for Cry 1Ac, the 97 kDa has higher affinity for Cry1 Ab. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis

Proteins in the brush border membrane (BBM) of the midgut binding to the insecticidal Cry1Ac toxin from Bacillus thuringiensis were investigated to examine the lower sensitivity of Bombyx mori to Cry1Ac, and new aminopeptidase N that bound to Cry1Ac was discovered. DEAE chromatography of Triton X-100-soluble BBM proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4-kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to approximately 190-kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of the amino acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. K(D) of the APN96-Cry1Ac interaction was determined to be 1.83 +/- 0.95 microM. The lectin binding assay suggested that APN96 had an N-linked bi-antennal oligosaccharide or an O-linked mucin type one. The role of APN96 was discussed in relation to the insensitivity of B. mori to Cry1Ac.     

Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under nondenaturing conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with (125)I-labeled Cry1Ac, Cry1Ac mutant (509)QNR-AAA(511) (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both (125)I-Cry1Ac and (125)I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots (125)I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, (125)I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. (125)I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured (125)I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) (125)I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.     

Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with ¹²⁵I-labeled Cry1Ac, Cry1Ac mutant ⁵⁰⁹QNR-AAA⁵¹¹ (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both ¹²⁵I-Cry1Ac and ¹²⁵I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots ¹²⁵I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, ¹²⁵I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. ¹²⁵I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured ¹²⁵I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) ¹²⁵I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.     

Bacillus thuringiensis Cry toxins bound specifically to various proteins via domain III, which had a galactose-binding domain-like fold

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.     

Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K(com)] = 1.1 nM) for (125)I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM. Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.     

Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.     

Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.     

Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin.     

Purification and Characterization of Cry1Ac Toxin Binding Proteins from the Brush Border Membrane of Helicoverpa armigera Midgut

Several Cry1Ac binding proteins from midgut of Helicoverpa armigera were purified using toxin-affinity chromatography. Enzyme assays showed that the purified proteins had strong aminopeptidase activity. The N-terminal sequences confidently identified a 124-kDa binding protein as an aminopeptidase N (APN), and some similarity suggests that a 162-kDa binding protein may also be an APN. Two minor binding proteins were not characterized.     

Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.     

Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K⁺ ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K⁺ ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.     

Disruption of Ha_BtR alters binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to midgut BBMVs of Helicoverpa armigera

Disruption of the Ha_BtR (a cadherin gene) is genetically linked to resistance to Cry1Ac delta-endotoxin of Bacillus thuringiensis in the GYBT strain of Helicoverpa armigera. Brush border membrane vesicles (BBMVs) prepared from midguts of both the Cry1Ac-resistant GYBT strain (homozygous for a deletion knockout of Ha_BtR) and the susceptible GY strain (homozygous for the wild type of Ha_BtR) possessed saturable and specific binding ability to (125)I-Cry1Ac. The binding constant (K(d)) of the GY strain was significantly lower than that of the resistant GYBT strain, whereas their binding site concentrations (B(max)) were similar. When midgut BBMVs were reacted directly with streptavidin conjugated to horseradish peroxidase, the GY strain had very clear 120- and 85-kDa protein bands, which indicated that the 120- and 85-kDa bands are endogenous biotin-containing proteins. However, the GYBT strain almost completely lost these two biotin-containing proteins. Ligand blotting with biotinylated Cry1Ac toxin showed midgut BBMVs of the GY strain contain five protein bands of 210-, 190-, 150-, 120-, and 85-kDa, respectively, while BBMVs of the GYBT strain contain only two protein bands of 150- and 120-kDa. 120-kDa bands may consist of two proteins with coincidentally the same molecular weight (putatively, an APN and a biotin-containing protein). Our results showed that the binding pattern of Cry1Ac to midgut BBMVs of H. armigera was altered quantitatively and qualitatively by knockout of Ha_BtR. There are multiple Cry1Ac-binding proteins in the midgut of susceptible H. armigera, but only the Ha_BtR can be considered as a putative functional receptor of Cry1Ac. Possible involvement of other receptor proteins in the intoxication process in vivo could not be excluded.     

Cloning of a Heliothis virescens 110 kDa aminopeptidase N and expression in Drosophila S2 cells

We previously identified a novel Heliothis virescens 110 kDa aminopeptidase N (APN) that binds Bacillus thuringiensis (Bt) Cry1Ac and Cry1Fa delta-endotoxins, and cloned an internal region of the 110 kDa APN gene (Banks et al., 2001). Here we describe the RACE-PCR cloning and sequence of a cDNA encoding 110 kDa APN. The 110 kDa APN gene was transiently co-expressed with green fluorescent protein (GFP) in Drosophila S2 cells using the pIZT expression vector. Enrichment of total membranes purified from S2 cells transfected with the 110 kDa APN gene had 3.3 fold increased APN enzymatic activity relative to enriched total membranes purified from S2 cells transfected with vector alone. Whereas the majority of S2 cells transfected with the 110 kDa APN gene bound rhodamine-labeled Cry1Ac toxin, no S2 cells transfected with vector alone bound rhodamine-labeled Cry1Ac toxin. This indicates that toxin binding to whole cells is APN mediated. However, flow cytometry and microscopy indicated that 110 kDa APN transfected S2 cells exposed to Cry1Ac or Cry1Fa toxin did not experience an increase in membrane permeability, indicating that APN transfected cells were resistant to toxin. This suggests while the H. virescens 110 kDa APN functions as a Bt toxin binding protein, it does not mediate cytotoxicity when expressed in S2 cells.     

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.     

Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with ¹²⁵I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K_com] = 1.1 nM) for ¹²⁵I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for ¹²⁵I-Cry1Ab binding sites, though the K_com values ranged from 179 to 304 nM. Cry1Ab competed for ¹²⁵I-Cry1Ac binding sites (K_com = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the ¹²⁵I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.