Analyses of Cry1Ab binding in resistant and susceptible strains of the European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae)

Cry1Ab toxin binding analysis was performed to determine whether resistance in laboratory-selected Ostrinia nubilalis strains is associated with target site alteration. Brush border membrane vesicles were prepared using dissected midguts from late instars of susceptible and resistant strains (Europe-R and RSTT) of O. nubilalis. Immunoblot analysis indicated that three different proteins bound to Cry1Ab toxin and were recognized by an anticadherin serum. In a comparison of resistant and susceptible strains, reduced Cry1Ab binding was apparent for all three bands corresponding to cadherin-like proteins in the Europe-R strain, while reduced binding was apparent in only one band for the RSTT strain. Real-time analysis of Cry1Ab binding to gut receptors using surface plasmon resonance suggested slight differences in affinity in both resistant strains. Additional binding analysis was conducted using 125I-labeled Cry1Ab, Cry1Ac, and Cry1Aa. Slight differences were again observed between the resistant and susceptible strains for Cry1Ab binding. However, when binding of 125I-labeled Cry1Aa was tested, a 10-fold reduction in the concentration of binding sites was observed in the Europe-R strain. Expression of the O. nubilalis cadherin gene was similar in both the resistant and susceptible strains and did not account for differences in binding. In combination, the results of the present work suggest that differences in susceptibility to Cry1A toxins in the Europe-R strain of O. nubilalis are associated with altered receptor binding, although the precise nature of this mechanism is still uncertain.     

Binding analyses of Bacillus thuringiensis Cry delta-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 (125)I-Cry1Ab binding to BBMV. Cry1F inhibited (125)I-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.     

Sequence variation in the cadherin gene of Ostrinia nubilalis: a tool for field monitoring

Toxin-binding proteins of insect midgut epithelial cells are associated with insect resistance to Bacillus thuringiensis (Bt) Cry toxins. A 5378 nt cDNA encoding a 1717 amino acid putative midgut cadherin-like glycoprotein and candidate Cry1Ab toxin-binding protein was characterized from Ostrinia nubilalis. Intraspecific alignment of partial O. nubilalis cadherin gene sequences identified variance within proposed Cry1A toxin binding region 2 (TBR2), 1328IPLQTSILVVT[I/V] N1340, and flanking Cry1A toxin binding region 1 (TBR1), 861DIEIEIIDTNN871. DNA sequence and PCR-RFLP detected single nucleotide polymorphism between cadherin alleles, and pedigree analysis demonstrated Mendelian inheritance. A population sample from Mead, Nebraska showed allelic polymorphism. These assays may be useful for linkage mapping and field surveillance of wild populations and of O. nubilalis.     

Growth, development, and survival of Nosema pyrausta-infected European corn borers (Lepidoptera: Crambidae) reared on meridic diet and Cry1Ab

Transgenic corn, Zea mays L., hybrids expressing crystal protein endotoxin genes from Bacillus thuringiensis Berliner are an increasingly popular tactic for managing the European corn borer, Ostrinia nubilalis (Hübner), in North America. O. nubilalis populations also are often vulnerable to the ubiquitous entomopathogenic microsporidium Nosema pyrausta (Paillot). We examined the effect of feeding meridic diet incorporated with purified Cry1Ab on growth, development, and survival of Nosema-infected and uninfected neonate O. nubilalis. Infected larvae developed more slowly than uninfected larvae. Increasing the concentration of Cry1Ab in diet reduced larval development, and this effect was amplified by microsporidiosis. Infected larvae weighed significantly less than uninfected larvae. The relationship among Nosema infection, Cry1Ab concentration, and larval weight was fitted to an exponential function. The LC50 of infected larvae was one-third that of uninfected larvae, indicating that infected larvae are more vulnerable to toxin. This work has implications for resistance management of O. nubilalis and demonstrates that it is important to determine whether N. pyrausta is present when testing susceptibility of larvae to transgenic corn hybrids.     

Analyses of Cry1Ab Binding in Resistant and Susceptible Strains of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae)

Cry1Ab toxin binding analysis was performed to determine whether resistance in laboratory-selected Ostrinia nubilalis strains is associated with target site alteration. Brush border membrane vesicles were prepared using dissected midguts from late instars of susceptible and resistant strains (Europe-R and RSTT) of O. nubilalis. Immunoblot analysis indicated that three different proteins bound to Cry1Ab toxin and were recognized by an anticadherin serum. In a comparison of resistant and susceptible strains, reduced Cry1Ab binding was apparent for all three bands corresponding to cadherin-like proteins in the Europe-R strain, while reduced binding was apparent in only one band for the RSTT strain. Real-time analysis of Cry1Ab binding to gut receptors using surface plasmon resonance suggested slight differences in affinity in both resistant strains. Additional binding analysis was conducted using ¹²⁵I-labeled Cry1Ab, Cry1Ac, and Cry1Aa. Slight differences were again observed between the resistant and susceptible strains for Cry1Ab binding. However, when binding of ¹²⁵I-labeled Cry1Aa was tested, a 10-fold reduction in the concentration of binding sites was observed in the Europe-R strain. Expression of the O. nubilalis cadherin gene was similar in both the resistant and susceptible strains and did not account for differences in binding. In combination, the results of the present work suggest that differences in susceptibility to Cry1A toxins in the Europe-R strain of O. nubilalis are associated with altered receptor binding, although the precise nature of this mechanism is still uncertain.     

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.     

Susceptibility of Spanish populations of the corn borers Sesamia nonagrioides (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Lepidoptera: Crambidae) to a Bacillus thuringiensis endotoxin

Baseline susceptibility to the Cry1Ab delta-endotoxin from Bacillus thuringiensis (Berliner) was determined for four populations of Sesamia nonagrioides (Lefebvre) and two populations of Ostrinia nubilalis (Hübner) from Spain. This study shows that S. nonagrioides is at least as susceptible as O. nubilalis to B. thuringiensis Cry1Ab protein. We found small differences in susceptibility among the Spanish populations of S. nonagrioides that can be attributed to natural variation, because there are no records of B. thuringiensis products being used on corn crops in Spain. There were no differences in susceptibility to Cry1Ab toxin between the two populations of O. nubilalis.     

Comparative susceptibility of European corn borer, southwestern corn borer, and sugarcane borer (Lepidoptera: Crambidae) to Cry1Ab protein in a commercial Bacillus thuringiensis corn hybrid

One field strain each of the European corn borer, Ostrinia nubilalis (Hübner); southwestern corn borer, Diatraea grandiosella Dyar; and sugarcane borer, Diatraea saccharalis (F.); were collected from cornfields in northeastern Louisiana. Susceptibilities of the field strain and a corresponding laboratory strain of the three borer species to Cry1Ab protein in DK69-70 Bacillus thuringiensis (Bt) corn hybrid were determined by exposing neonates to intact leaf tissues from whorl stage plants or by feeding neonates or third instars on a meridic diet treated with different concentrations of Cry1lAb protein extracted from Bt corn leaves. Mortality and growth of larvae were evaluated after 2 and 4 d posttreatment in the bioassays by using intact leaf tissues or after 7 d in the bioassays by using diet incorporating Cry1Ab protein. D. saccharalis was the least susceptible species to Cry1Ab protein among the three species, followed by D. grandiosella, whereas O. nubilalis was most susceptible. The 2-d mortality of D. saccharalis neonates on intact Bt leaf tissues was lower than that of O. nubilalis and D. grandiosella. All neonates of O. nubilalis were killed on the diet treated with Cry1Ab protein at 0.5 and 1 mg/kg. The mortality of D. grandiosella was > 75% at 1 mg/kg, but it was < 6% for D. saccharalis at 1 mg/kg. The LC50 values of D. saccharalis were 3- and 11-fold higher than those of D. grandiosella and O. nubilalis, respectively. The LC90 values of D. saccharalis were 8- and 32-fold higher than those of D. grandiosella and O. nubilalis, respectively. Larval growth of the three species on Cry1Ab-treated diet was inhibited, but the inhibition was greater for O. nubilalis and D. grandiosella than for D. saccharalis. The lower susceptibility of D. saccharalis to Cry1Ab protein suggests that it is necessary to verify if a high-dose Bt corn for O. nubilalis and D. grandiosella is also a high dose for D. saccharalis.     

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance.     

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.     

DiPel-selected Ostrinia nubilalis larvae are not resistant to transgenic corn expressing Bacillus thuringiensis Cry1Ab

The survival of KS-SC DiPel-resistant and -susceptible European corn borer, Ostrinia nubilalis (Hübner), was evaluated on different tissues from corn, Zea mays L., hybrids, including a nontransgenic and two transgenic corn plants (events MON810 and Bt11) expressing high doses of Bacillus thuringiensis (Bt) Cry1Ab. The survival of Bt-resistant and -susceptible third instars was similar after a 5-d exposure to transgenic plant tissues. Survivors eventually died when returned to Bt corn tissues, but many were able to continue development when transferred to non-Bt corn tissues. Survival of resistant and susceptible larvae also was evaluated in bioassays with dilutions of leaf extracts from the three corn hybrids incorporated in an artificial diet. In these assays, survival was significantly higher for resistant O. nubilalis neonates at three of the five dilutions compared with the susceptible strain, but the resistance ratio was only 2.2- and 2.4-fold for MON810 and Bt11, respectively. The data demonstrate that Bt-resistant and unselected control O. nubilalis larvae were similar in susceptibility to MON810 and Bt11 event corn hybrids. Although we were unable to evaluate the Cry1Ab protein that larvae were exposed to in the transgenic tissue because of company restrictions, Cry1Ab protoxin produced in Escherichia coli was incubated with extracts from non-Bt corn leaves to simulate the in planta effect on the transgenic protein. Cry1Ab protoxin was hydrolyzed rapidly by enzymes in the corn extract into peptide fragments with molecular masses ranging from 132 to 74 kDa, and eventually 58 kDa. Overall, these data suggest that plant enzymes hydrolyze transgenic toxin to one that is functionally activated. Therefore, resistant insect populations with reduced proteinase activity do not seem to pose a threat to the efficacy of commercial MON810 and Bt11 corn hybrids.     

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130 kDa Cry7Ab3 protoxin was degraded into the ～75 kDa proteinase-resistant fragments. In vivo analysis, 130 kDa Cry7Ab3 protoxin was also processed into ～75 kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220 kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.     

Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display

In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K(d)) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20-51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R(1)), Bombyx mori (Bt-R(175)), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R(1) or Bt-R(175) are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R(1) or Bt-R(175) receptors. Finally, we showed that synthetic peptides homologous to Bt-R(1) and scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R(1) and Bt-R(175) involved in Cry1A toxin interaction.     

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.     

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.     

Cross-pollination of nontransgenic corn ears with transgenic Bt corn: efficacy against lepidopteran pests and implications for resistance management

The efficacy of nontransgenic sweet corn, Zea mays L., hybrids cross-pollinated by Bacillus thuringiensis (Bt) sweet corn hybrids expressing Cry1Ab toxin was evaluated in both field and laboratory studies in Minnesota in 2000. Non-Bt and Bt hybrids (maternal plants) were cross-pollinated with pollen from both non-Bt and Bt hybrids (paternal plants) to create four crosses. Subsequent crosses were evaluated for efficacy in the field against European corn borer, Ostrinia nubilalis (Hübner), and corn earworm, Helicoverpa zea (Boddie), and in laboratory bioassays against O. nubilalis. Field studies indicated that crosses with maternal Bt plants led to low levels of survival for both O. nubilalis and H. zea compared with the non-Bt x non-Bt cross. However, the cross between non-Bt ears and Bt pollen led to survival rates of 43 and 63% for O. nubilalis and H. zea larvae, respectively. This intermediate level of survival also was reflected in the number of kernels damaged. Laboratory bioassays for O. nubilalis, further confirmed field results with larval survival on kernels from the cross between non-Bt ears and Bt pollen reaching 60% compared with non-Bt crossed with non-Bt. These results suggest that non-Bt refuge plants, when planted in proximity to Bt plants, and cross-pollinated, can result in sublethal exposure of O. nubilalis and H. zea larvae to Bt and may undermine the high-dose/refuge resistance management strategy for corn hybrids expressing Cry1Ab.     

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 (125)I- 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 DiSC(3)(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.     

Hydropathic complementarity determines interaction of epitope (869)HITDTNNK(876) in Manduca sexta Bt-R(1) receptor with loop 2 of domain II of Bacillus thuringiensis Cry1A toxins

In susceptible insects, Cry toxin specificity correlates with receptor recognition. In previous work, we characterized an scFv antibody (scFv73) that inhibits binding of Cry1A toxins to cadherin-like receptor. The CDR3 region of scFv73 shared homology with an 8-amino acid epitope ((869)HITDTNNK(876)) of the Manduca sexta cadherin-like receptor Bt-R(1) (Gomez, I., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2001) J. Biol. Chem. 276, 28906-28912). In this work, we show that the previous sequence of scFv73 CDR3 region was obtained from the noncoding DNA strand. However, most importantly, both scFv73 CDR3 amino acid sequences of the coding and noncoding DNA strands have similar binding capabilities to Cry1Ab toxin as Bt-R(1) (869)HITDTNNK(876) epitope, as demonstrated by the competition of scFv73 with binding to Cry1Ab with synthetic peptides with amino acid sequences corresponding to these regions. Using synthetic peptides corresponding to three exposed loop regions of domain II of Cry1Aa and Cry1Ab toxins, we found that loop 2 synthetic peptide competed with binding of scFv73 to Cry1A toxins in Western blot experiments. Also, loop 2 mutations that affect toxicity of Cry1Ab toxin are affected in scFv73 binding. Toxin overlay assays of Cry1A toxins to M. sexta brush border membrane proteins showed that loop 2 synthetic peptides competed with binding of Cry1A toxins to cadherin-like Bt-R(1) receptor. These experiments identified loop 2 in domain II of as the cognate binding partner of Bt-R(1) (869)HITDTNNK(876). Finally, 10 amino acids from beta-6-loop 2 region of Cry1Ab toxin ((363)SSTLYRRPFNI(373)) showed hydropathic pattern complementarity to a 10-amino acid region of Bt-R(1) ((865)NITIHITDTNN(875)), suggesting that binding of Cry1A toxins to Bt-R(1) is determined by hydropathic complementarity and that the binding epitope of Bt-R(1) may be larger than the one identified by amino acid sequence similarity to scFv73.     

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.