Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.     

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC₅₀, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC₉₀, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC₉₀ level than at the LC₅₀ level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.     

Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation

Helicoverpa armigera is one of the most harmful pests in China. Although it had been successfully controlled by Cry1A toxins, some H. armigera populations are building up resistance to Cry1A toxins in the laboratory. Vip3A, secreted by Bacillus thuringiensis, is another potential toxin against H. armigera. Previous reports showed that activated Vip3A performs its function by inserting into the midgut brush border membrane vesicles (BBMV) of susceptible insects. To further investigate the binding of Vip3A to BBMV of H. armigera, the full-length Vip3Aa10 toxin expressed in Escherichia coli was digested by trypsin or midgut juice extract, respectively. Among the fragments of digested Vip3Aa10, only a 62 kDa fragment (Vip3Aa10-T) exhibited binding to BBMV of H. armigera and has insecticidal activity. Moreover, this interaction was specific and was not affected by the presence of Cry1Ab toxin. Binding of Vip3Aa10-T to BBMV resulted in the formation of an ion channel. Unlike Cry1A toxins, Vip3Aa10-T was just slightly associated with lipid rafts of BBMV. These data suggest that although activated Vip3Aa10 specifically interacts with BBMV of H. armigera and forms an ion channel, the mode of action of it may be different from that of Cry1A toxins.     

Toxicity, Binding, and Permeability Analyses of Four Bacillus thuringiensis Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with ¹²⁵I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. ¹²⁵I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.     

Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

Background

Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.

Methodology/Principal Findings

Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that ¹²⁵I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances ¹²⁵I-Cry35Ab1 specific binding, and that ¹²⁵I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with ¹²⁵I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with ¹²⁵I-Cry3Aa, or ¹²⁵I-Cry8Ba.

Conclusions/Significance

Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.     

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.     

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

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

The Mode of Action of the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Ab δ-Endotoxin

The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.     

Integrative Model for Binding of Bacillus thuringiensis Toxins in Susceptible and Resistant Larvae of the Diamondback Moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

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.     

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.     

Toxicity and Receptor Binding Properties of Bacillus thuringiensisδ-Endotoxins to the Midgut Brush Border Membrane Vesicles of the Rice Leaf Folders, Cnaphalocrocis medinalis and Marasmia patnalis

Pesticidal activity and receptor-binding properties of Bacillus thuringiensis toxins to rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis, were investigated. Saturation and competition binding experiments were done with iodine (¹²⁵1)-labeled Bt proteins and brush border membrane vesicles prepared from the midgut of C. medinalis and M. patnalis. The results show saturable, specific, and high-affinity binding of all toxins except Cry2A toxin. Cry1Aa and Cry2A toxins were bound with low affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab, and Cry1Ac recognized or shared the same binding site that is different from the binding site for Cry2A toxin. Iodine (¹²⁵I)-labeled Cry1Ac and Cry1Ab toxins were used in ligand blot experiments to detect specific binding proteins in brush border membrane vesicles of C. medinalis and M. patnalis. Cry1Ab toxin protein binds to 205-kDa and 200-kDa proteins respectively in case of C. medinalis and M. patnalis. The apparent molecular mass of the protein bound to labeled Cry1Ac toxins was identified as a 120-kDa protein in both C. medinalis and M. patnalis. Received: 10 April 2000 / Accepted: 23 May 2000     

Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins

Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.     

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.     

Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm

Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa–QD probes compared to CrylAc–QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa–QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins.     

Interaction of Bacillus thuringiensis δ-endotoxins with Midgut Brush Border Membrane Vesicles of Helicoverpa armigera

Pesticidal activity of different Bacillus thuringiensis (Bt) δ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac and Cry2A, were investigated against Helicoverpa armigera infesting cotton crop worldwide. Cry1Ac toxin was found to be the most potent toxin towards H. armigera. All selected Bt toxins were found stable in vitro processing by midgut juice of H. armigera. Saturation and competition binding experiments were performed with iodine-125 labeled proteins and brush border membrane vesicles prepared from the midgut of H. armigera. The results show saturable, specific and high affinity of all toxins except for Cry2A. Both the toxins were bound with low binding affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab and Cry1Ac recognized or share the same binding site which is different from that of Cry2A. The data suggest that development of multiple toxin system in transgenic plants with toxin pyramiding, which recognize different binding sites, may be useful in the deployment strategies to decrease the rate of pest adaptation to Bt toxins in transgenic plants.     

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.     

Altered Glycosylation of 63- and 68-Kilodalton Microvillar Proteins in Heliothis virescens Correlates with Reduced Cry1 Toxin Binding, Decreased Pore Formation, and Increased Resistance to Bacillus thuringiensis Cry1 Toxins

The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of Heliothis virescens. ¹²⁵I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and ¹²⁵I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the N-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of H. virescens and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.