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.     

Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

Evidence of Field-Evolved Resistance of Spodoptera frugiperda to Bt Corn Expressing Cry1F in Brazil That Is Still Sensitive to Modified Bt Toxins

Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.     

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.     

The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens

Retrotransposon-mediated disruption of the BtR-4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to high levels of resistance in the YHD2 strain to Cry1Ac toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a Cry1Ac toxin receptor on the surface of midgut cells in susceptible larvae and that the BtR-4 gene disruption eliminates this protein in resistant larvae. However, Cry1Ac toxin binding to HevCaLP is yet to be reported. We used the polymerase chain reaction and immunoblotting as tools to discriminate between individual H. virescens larval midguts from susceptible (YDK) and resistant (CXC, KCBhyb, and YHD2-B) strains according to their BtR-4 gene disruption genotype and phenotype. This approach allowed us to test the correlation between BtR-4 gene disruption, lack of HevCaLP, and altered Cry1A toxin binding. Toxin-binding assays using brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and Cry1Aa toxin binding, while most of Cry1Ab and Cry1Ac binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to midguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of Cry1A binding in YHD2 and supports our earlier suggestion that a separate genetic change occurred in YHD2 after appearance of the cadherin disruption, conferring even higher resistance in the resulting YHD2-B strain as well as a large reduction in Cry1Ab and Cry1Ac binding.     

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.     

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.     

Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae.

We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [125I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.     

Binding Site Concentration Explains the Differential Susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-Producing Rice

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.     

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

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

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.     

Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

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.     

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.     

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.     

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.     

Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae)

Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) Heliothis virescens strains were compared. The overall pH of gut extracts from YDK and CXC were statistically similar (9.56 and 9.62, respectively), while the pH of extracts from KCBhyb and YHD2-B were significantly more alkaline (9.81 and 10.0, respectively). Gut extracts from YHD2-B and CXC larvae processed Cry1Ac and Cry2Aa protoxin slower than extracts from YDK larvae, suggesting that differences in proteolysis contribute to resistance in these strains. Casein zymogram analysis of gut extracts revealed both qualitative and quantitative differences in caseinolytic activities among all strains, but the overall caseinolytic activity of YHD2-B gut extract was lower. Kinetic microplate assays with a trypsin substrate (l-BApNA) demonstrated that proteinases in YDK gut extract had increased alkaline pH optima compared to resistant strains YHD2-B, CXC and KCBhyb. Gut extracts from YHD2-B had reduced trypsin-like activity, and activity blots indicated that YHD2-B had lost a trypsin-like proteinase activity. In assays with a chymotrypsin substrate (SAAPFpNA), enzymes from all Bt-resistant strains had increased pH optima, especially those from KCBhyb. Activity blots indicated that CXC had lost a chymotrypsin-like proteinase activity. Because serine proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.     

Binding analyses of Cry1Ab and Cry1Ac with membrane vesicles from Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis

The binding properties of Bacillus thuringiensis toxins to brush border membrane vesicles of Dipel-resistant and -susceptible Ostrinia nubilalis larvae were compared using ligand-toxin immunoblot analysis, surface plasmon resonance (SPR), and radiolabeled toxin binding assays. In ligand-toxin immunoblot analysis, the number of Cry1Ab or Cry1Ac toxin binding proteins and the relative toxin binding intensity were similar in vesicles from resistant and susceptible larvae. Surface plasmon resonance with immobilized activated Cry1Ab toxin indicated that there were no significant differences in binding with fluid-phase vesicles from resistant and susceptible larvae. Homologous competition assays with radiolabeled Cry1Ab and Cry1Ac toxin and vesicles from resistant and susceptible larvae resulted in similar toxin dissociation constants and binding site concentrations. Heterologous competition binding assays indicated that Cry1Ab and Cry1Ac completely competed for binding, thus they share binding sites in the epithelium of the larval midguts of O. nubilalis. Overall, the binding analyses indicate that resistance to Cry1Ab and Cry1Ac in this Bt-resistant strain of O. nubilalis is not associated with a loss of toxin binding.     

Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens

Membrane-bound alkaline phosphatases (mALPs, EC 3.1.3.1) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac-binding protein that is down-regulated in Cry1Ac-resistant insects. To further characterize HvALP, we localized mALP protein to foregut and midgut tissues using anti-mALP serum and then cloned five mALPs from H. virescens larval midgut. All five clones displayed high levels of sequence identity (above 90%), suggesting that they may represent allelic variants, and grouped with other lepidopteran mALPs in sequence alignments. All these cloned ALPs were predicted to contain a glycosylphosphatidylinositol (GPI) anchor and were named HvmALP1–5. We expressed two of the most diverse HvmALPs in a heterologous system to test binding of Cry1Ac and recognition by HvALP cross-reacting antiserum. Our data highlight the importance of glycosylation for Cry1Ac binding to HvALP and suggest that, depending on glycosylation, all the identified HvmALPs may be synonymous with HvALP, the Cry1Ac-binding phosphatase identified in H. virescens midgut epithelium.