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 CrylAc toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a CrylAc 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, CrylAc 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 CrylA toxin binding. Toxin-binding assays using, brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and CrylAa toxin binding, while most of CrylAb and CrylAc binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to niidguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of CryIA 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 YHD2-13 strain as well as a large reduction in CrylAb and CrylAc binding.