Intra- and extracellular domains of the Helicoverpa armigera cadherin mediate Cry1Ac cytotoxicity

Abstract

Diverse midgut cadherin mutations confer resistance to Cry1A toxins in at least three lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Most of these cadherin mutations are inherited as recessive alleles and result in changes within the cadherin repeat (CR) regions of the extracellular protein domain. However, the H. armigera r15 cadherin mutation results in a deletion of 55 amino acid residues within the cytoplasmic domain, and Cry1A resistance is inherited as a non-recessive trait. Here, eight recombinant H. armigera cadherin (HaCad) proteins, including seven variants containing different combinations of CRs and the cytoplasmic domain, were expressed in cultured insect cells using a baculovirus expression system and were analyzed for Cry1Ac binding and toxicity. Cells expressing either the wild-type HaCad or a mutant lacking only the region corresponding to the first nine CRs bound Cry1Ac and were equally susceptible to Cry1Ac. Cells expressing mutant HaCad proteins without the Cry1A toxin binding region (TBR) located in the CR nearest the plasma membrane did not bind Cry1Ac and were not killed by the toxin. Among the mutant proteins, loss of toxicity was observed in all cells producing HaCad variants lacking the amino acids 1422–1440, indicating that this TBR motif is important for both toxin binding and to confer susceptibility to Cry1Ac. Cells expressing the HaCad variant lacking the entire cytoplasmic domain retained Cry1Ac binding, but were significantly less susceptible to Cry1Ac than the cells producing either wild-type HaCad or HaCad lacking the first nine CRs. These results suggest that both the extracellular and the cytoplasmic domains of HaCad participate in Cry1Ac intoxication.