Functional analysis of cadherin as a receptor to Cry1Ac toxin in polyphagous insect pest, Helicoverpa armigera

Insecticidal crystal toxins (Cry) from B. thuringiensis (Bt) are widely used as sprays or expressed in transgenic crops to control diverse insect pests worldwide. The cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) is a serious polyphagous pest, which is reported to cause huge economic losses (~1 billion US$). Cry1Ac is the most effective Bt toxin in controlling H. armigera. Aminopeptidase N (APN) and cadherin located at the insect midgut epithelium are implicated as receptors to several Cry toxins. We have earlier reported that HaAPN1 functions as a receptor to Cry1Ac. In an effort to further study the mode of action of Cry1Ac, we have identified H. armigera cadherin (HaCAD) as putative Cry1Ac-binding protein by ligand-blot analysis. The RNAi knockdown of HaCad resulted in decreased susceptibility to Cry1Ac demonstrating the functional role of cadherin as receptor mediating Cry1Ac toxicity. To investigate the specific toxin-binding region, we have isolated cadherin fragments, expressed in E. coli and evaluated the interaction with Cry1Ac by in-vitro pull down assays and gel-filtration chromatography. Our results demonstrated that fragment containing cadherin repeats CR9-CR11 and membrane proximal region (MPR), acts as a synergist for Cry1Ac toxicity.  The specific amino acids related to binding with Cry1Ac were identified by Phage display, oligomerization and fluorescence assays. In addition, in silico evaluation of interaction was elaborated to predict the specific interactions between Cry1Ac and the cadherin. Overall, our results showed that amino acids at CR10 and MPR of HaCAD are core-binding regions, which act as scaffold for oligomerization of Cry1Ac.