Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae

Cry toxins produced by Bacillus thuringiensis bacteria are insecticidal proteins used worldwide in the control of different insect pests. Alterations in toxin-receptor interaction represent the most common mechanism to induce resistance to Cry toxins in lepidopteran insects. Cry toxins bind with high affinity to the cadherin protein present in the midgut cells and this interaction facilitates the proteolytic removal of helix ??-1 and pre-pore oligomer formation. Resistance to Cry toxins has been linked with mutations in the cadherin gene. One strategy effective to overcome larval resistance to Cry1A toxins is the production of Cry1AMod toxins that lack helix ??-1. Cry1AMod are able to form oligomeric structures without binding to cadherin receptor and were shown to be toxic to cadherin-silenced Manduca sexta larvae and Pectinophora gossypiella strain with resistance linked to mutations in a cadherin gene.We developed Cry1AbMod tobacco transgenic plants to analyze if Cry1AMod toxins can be expressed in transgenic crops, do not affect plant development and are able to control insect pests. Our results show that production of the Cry1AbMod toxin in transgenic plants does not affect plant development, since these plants exhibited healthy growth, produced abundant seeds, and were virtually undistinguishable from control plants. Most importantly, Cry1AbMod protein produced in tobacco plants retains its functional toxic activity against susceptible and tolerant M. sexta larvae due to the silencing of cadherin receptor by RNAi. These results suggest that CryMod toxins could potentially be expressed in other transgenic crops to protect them against both toxin-susceptible and resistant lepidopteran larvae affected in cadherin gene.     

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.     

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R₁, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gómez, I., Sánchez, J., Miranda, R., Bravo A., Soberón, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R₁ in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R₁. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R₁ is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R₁ was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.     

Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R(1) receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R(1) receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae.     

Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda

Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The LC–MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda.     

Enhancement of insecticidal activity of Bacillus thuringiensis Cry1A toxins by fragments of a toxin-binding cadherin correlates with oligomer formation

Cry1A toxins produced by Bacillus thuringiensis bind a cadherin receptor that mediates toxicity in different lepidopteran insect larvae. Insect cadherin receptors are modular proteins composed of three domains, the ectodomain formed by 9-12 cadherin repeats (CR), the transmembrane domain and the intracellular domain. Cry1A toxins interact with three regions of the Manduca sexta cadherin receptor that are located in CR7, CR11 and CR12 cadherin repeats. Binding of Cry1A toxin to cadherin induces oligomerization of the toxin, which is essential for membrane insertion. Also, it has been reported that cadherin fragments containing the CR12 region enhanced the insecticidal activity of Cry1Ab toxin to M. sexta and other lepidopteran larvae. Here we report that cadherin fragments corresponding to CR7 and CR11 regions also enhanced the activity of Cry1Ac and Cry1Ab toxin to M. sexta larvae, although not as efficient as the CR12 fragment. A single point mutation in the CR12 region (I1422R) affected Cry1Ac and Cry1Ab binding to the cadherin fragments and did not enhance the activity of Cry1Ab or Cry1Ac toxin in bioassays. Analysis of Cry1Ab in vitro oligomer formation in the presence of wild type and mutated cadherin fragments showed a correlation between enhancement of Cry1A toxin activity in bioassays and in vitro Cry1Ab-oligomer formation. Our data shows that formation of Cry1A toxin oligomer is in part responsible for the enhancement of Cry1A toxicity by cadherin fragments that is observed in vivo.     

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxin-receptor interactions in cytotoxicity, we established a cell-based system utilizing High Five insect cells stably expressing BT-R1, the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R1, which apparently activates a Mg2+-dependent cellular signaling pathway.     

Efficient Production of Bacillus thuringiensis Cry1AMod Toxins under Regulation of cry3Aa Promoter and Single Cysteine Mutations in the Protoxin Region

Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity to P. xylostella. These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis.     

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.     

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.     

Aggregation of Bacillus thuringiensis Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles

During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.     

Structural changes of the Cry1Ac oligomeric pre-pore from bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.     

Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.     

A trimeric building block model for Cry toxins in vitro ion channel formation

The crystal (Cry) insecticidal toxins, or δ-endotoxins, are lethal to a wide variety of insect larvae, and are therefore very important in insect control. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and, more recently, by the ability of the monomeric toxin to subvert cellular signaling pathways. The structure, topology, and precise role of the putative pore in toxicity are not known. However, in vitro biophysical studies suggest that helices α4 and α5 in domain I insert into the lipid bilayer as an α-helical hairpin. Mutagenesis studies have assigned an important role to α5 in maintaining oligomerization, and to α4 in channel formation. To detect the possible homo-oligomerizing tendencies of these two helices, we have used the evolutionary conservation data contained in sixteen Cry homologs in order to filter non-native interactions found during a global conformational search. No conserved homo-oligomer was found for α4, but a right handed trimeric α5 model was present in the simulations of all Cry sequences. We propose a model for Cry toxin oligomerization based on sequence analysis and available mutagenesis data.     

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.     

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.     

Expression of Cry1Ac cadherin receptors in insect midgut and cell lines

Cadherin-like proteins have been identified as putative receptors for the Bacillus thuringiensis Cry1A proteins in Heliothis virescens and Manduca sexta. Immunohistochemistry showed the cadherin-like proteins are present in the insect midgut apical membrane, which is the target site of Cry toxins. This subcellular localization is distinct from that of classical cadherins, which are usually present in cell-cell junctions. Immunoreactivity of the cadherin-like protein in the insect midgut was enhanced by Cry1Ac ingestion. We also generated a stable cell line Flp-InT-REX-293/Full-CAD (CAD/293) that expressed the H. virescens cadherin. As expected, the cadherin-like protein was mainly localized in the cell membrane. Interestingly, toxin treatment of CAD/293 cells caused this protein to relocalize to cell membrane subdomains. In addition, expression of H. virescens cadherin-like protein affects cell-cell contact and cell membrane integrity when the cells are exposed to activated Cry1Ab/Cry1Ac.     

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion. For formation of these oligomers coiled-coil structures are important, and helix alpha-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix alpha-3 are affected in pore formation and toxicity. Mutants affected in toxicity bind Bt-R(1) receptor with a similar K(D) as the wild type toxin but do not form oligomers nor induce pore formation in planar lipid bilayers, indicating that the pre-pore oligomer is an obligate intermediate in the intoxication of Cry1Ab toxin and that interaction of monomeric Cry1Ab with Bt-R(1) is not enough to kill susceptible larvae.