Two parallel pathways mediate Cry1Ac toxicity in the Asian corn borer: one dependent on ABCC2 and one requiring synergistic action of cadherin and ABCC3.

Transgenic crops expressing Bacillus thuringiensis (Bt) toxins are central to global pest management, but their long-term efficacy is threatened by the evolution of insect resistance. Elucidating the genetic basis of resistance is essential for early detection and for designing proactive resistance management strategies. In lepidopterans, cadherin (Cad) and the ATP-binding cassette transporters ABCC2 and ABCC3 function as key midgut receptors for Cry1 toxins, although their contributions vary among toxins and species. Following recent studies on Cry1Ab and Cry1Fa receptor pathways in the Asian corn borer (Ostrinia furnacalis), we employed an established platform of six single- and double-knockout strains targeting Cad, ABCC2, and ABCC3 to define the receptor network mediating Cry1Ac toxicity. Bioassays showed that the double-knockout strains for Cad and ABCC2 (Cad + C2-KO) and for ABCC2 and ABCC3 (C2 + C3-KO) exhibited completely recessive resistance to Cry1Ac, with resistance ratios of 4984- and > 7732-fold, respectively. In contrast, all single-gene knockouts and the double-knockout strain for Cad and ABCC3 (Cad + C3-KO) remained susceptible or showed only marginal resistance. Resistance phenotypes, together with genetic linkage analyses, support a model in which Cry1Ac kills larvae either through a pathway requiring ABCC2 alone, or through an alternative route that depends on the concerted action of Cad and ABCC3. This model was further corroborated by cytotoxicity assays in Sf9 cells and electrophysiological pore-formation analyses in Xenopus oocytes expressing these receptors singly or in combination. Collectively, our results define the functional contributions of OfCad, OfABCC2, and OfABCC3 to Cry1Ac toxicity. When considered alongside previous findings on Cry1Ab pathways, this study extends our understanding of the insecticidal mechanisms of Bt toxins and offers practical insights for the sustainable deployment of Bt corn.