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.     

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC–MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.     

Loop residues of the receptor binding domain of Bacillus thuringiensis Cry11Ba toxin are important for mosquitocidal activity

Using a Cry11Ba toxin model, predicted loops in domain II were analyzed for their role in receptor binding and toxicity. Peptides corresponding to loops α8, 1 and 3, but not loop 2, competed with toxin binding to Aedes midgut membranes. Mutagenesis data reveal loops α8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae than to Anopheles. Cry11Ba binds the apical membrane of larval caecae and posterior midgut, and binding can be competed by loop 1 but not by loop 2 peptides. Cry11Ba binds the same regions to which anti-cadherin antibody binds, and this antibody competes with Cry11Ba binding suggesting a possible role of cadherin in toxication.     

Three toxins,two receptors,one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens

Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a ��Ping Pong�� Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R₁) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R₁ promotes cleavage of the amino-terminal end, including helix α-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7–12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R₁ fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a “ping pong” binding mechanism with both receptors is involved in toxin action.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.     

Expression of recombinant and mosaic Cry1Ac receptors from Helicoverpa armigera and their influences on the cytotoxicity of activated Cry1Ac to Spodoptera litura Sl-HP cells

Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells.     

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.     

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism.     

棉铃虫中肠钙粘蛋白、氨肽酶N及碱性磷酸酯酶的抗血清对Cry1Ac和Cry2Aa毒力的影响

【目的】Cry1A和Cry2A类Bt蛋白通过特异性地与昆虫中肠上的受体蛋白结合而发挥杀虫作用,现已广泛应用于转基因抗虫作物。本研究旨在进一步明确Cry2A类蛋白的作用机制和Cry1A受体蛋白在Cry2A发挥毒力中的作用。【方法】本研究首先提取了棉铃虫Helicoverpa armigera的BBMV,制备了钙粘蛋白(CAD)、氨肽酶N(APN)和碱性磷酸酯酶(ALP)3种受体蛋白的抗体和抗血清;然后,利用Western blot检测BBMV上这3种受体蛋白后,利用抗体封闭技术比较了敏感棉铃虫和Cry1Ac抗性棉铃虫(BtR)中3种受体蛋白的抗血清对Cry1Ac和Cry2Aa毒力的影响。【结果】对敏感品系棉铃虫,这3种已知的Cry1Ac受体蛋白抗血清显著地降低了Cry1Ac和Cry2Aa的毒力。其中APN抗血清对Cry1Ac毒力的影响最大,棉铃虫幼虫的死亡率降低了84.44%;ALP抗血清对Cry2Aa的毒力影响最大,棉铃虫幼虫死亡率比对照降低了71.04%。Cry1Ac对Cry1Ac抗性棉铃虫(BtR)的毒力显著降低,Cry2Aa的毒性也减弱。在Cry1Ac抗性棉铃虫(BtR)中,3种受体抗血清对Cry1Ac的影响比在敏感棉铃虫中的影响小,尤其是CAD和APN抗血清对Cry1Ac毒力的抑制率显著低于在敏感棉铃虫中的抑制作用;CAD和ALP抗血清对Cry2Aa毒力的影响与在敏感棉铃虫中的影响差异不显著,但APN抗血清可以显著降低Cry2Aa对Cry1Ac抗性棉铃虫(BtR)的毒力。【结论】棉铃虫CAD,APN和ALP不仅参与了Cry1Ac的杀虫过程,也对Cry2Aa毒力有一定的影响,而且这3种蛋白可能与棉铃虫对Cry1Ac和Cry2Aa产生抗性及交互抗性相关。     

Cover Caption

Previous studies have confirmed HaCad (cadherin), HaABCC2 and HaABCC3 are functional receptors of Bt toxin Cry1Ac in cotton bollworm, Helicoverpa armigera. Aminopeptidase N1 (APN1) has been suggested as a putative receptor in several lepidopteran insects including H. armigera through evidence from RNAi‐based gene silencing approaches. In the current study, we tested the role of APNs in the mode of action of Bt toxins using CRISPR/Cas9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible SCD strain of H. armigera to establish three homozygous knockout strains. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera (see pages 440–448). Photo by Yi‐Dong Wu.     

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.     

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.     

Role of Bacillus thuringiensis Cry1 δ Endotoxin Binding in Determining Potency during Lepidopteran Larval Development

Five economically important crop pests, Manduca sexta, Pieris brassicae, Mamestra brassicae, Spodoptera exigua, and Agrotis ipsilon, were tested at two stages of larval development for susceptibility to Bacillus thuringiensis toxins Cry1Ac, Cry1Ca, Cry1J, and Cry1Ba. Bioassay results for M. sexta showed that resistance to all four Cry toxins increased from the neonate stage to the third-instar stage; the increase in resistance was most dramatic for Cry1Ac, the potency of which decreased 37-fold. More subtle increases in resistance during larval development were seen in M. brassicae for Cry1Ca and in P. brassicae for Cry1Ac and Cry1J. By contrast, the sensitivity of S. exigua did not change during development. At both larval stages, A. ipsilon was resistant to all four toxins. Because aminopeptidase N (APN) is a putative Cry1 toxin binding protein, APN activity was measured in neonate and third-instar brush border membrane vesicles (BBMV). With the exception of S. exigua, APN activity was found to be significantly lower in neonates than in third-instar larvae and thus inversely correlated with increased resistance during larval development. The binding characteristics of iodinated Cry1 toxins were determined for neonate and third-instar BBMV. In M. sexta, the increased resistance to Cry1Ac and Cry1Ba during larval development was positively correlated with fewer binding sites in third-instar BBMV than in neonate BBMV. The other species-instar-toxin combinations did not reveal positive correlations between potency and binding characteristics. The correlation between binding and potency was inconsistent for the species-instar-toxin combinations used in this study, reaffirming the complex mode of action of Cry1 toxins.     

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.     

Helicoverpa armigera cadherin fragment enhances Cry1Ac insecticidal activity by facilitating toxin-oligomer formation

The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity. It has been reported that Manduca sexta cadherin fragment CR12-MPED and Anopheles gambiae cadherin fragment CR11-MPED enhance the insecticidal activity of Cry1Ab and Cry4Ba to certain lepidopteran and dipteran larvae species, respectively. This study reports that a Helicoverpa armigera cadherin fragment (HaCad1) containing its toxin binding region, expressed in Escherichia coli, enhanced Cry1Ac activity against H. armigera larvae. A binding assay showed that HaCad1 was able to bind to Cry1Ac in vitro and that this event did not block toxin binding to the brush border membrane microvilli prepared from H. armigera. When the residues ¹⁴²³GVLSLNFQ¹⁴³⁰ were deleted from the fragment, the subsequent mutation peptide lost its ability to bind Cry1Ac and the toxicity enhancement was also significantly reduced. Oligomerization tests showed that HaCad1 facilitates the formation of a 250-kDa oligomer of Cry1Ac-activated toxin in the midgut fluid environment. Oligomer formation was dependent upon the toxin binding to HaCad1, which was also necessary for the HaCad1-mediated enhancement effect. Our discovery reveals a novel strategy to enhance insecticidal activity or to overcome the resistance of insects to B. thuringiensis toxin-based biopesticides and transgenic crops.     

Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured ¹²⁵I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for ¹²⁵I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit ¹²⁵I-Cry1Ab binding to BBMV. Cry1F inhibited ¹²⁵I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.