Two pathways mediate toxicity of Cry1Ac in Mythimna separata: one is ABCC2-dependent and the other involves ABCC3-CAD interaction.

Mythimna separata is an important pest. The ATP-binding cassette (ABC) transporter proteins have been implicated in mediating toxicity of Bacillus thuringiensis Cry1 toxins in lepidopteran insects. Here we investigated the role of MsABCC3 in mediating toxicity of multiple Bt toxins by MsABCC3-expression in Hi5 insect cells and by gene-editing knockout in M. separata larvae. We assessed Cry1Ac toxicity in Hi5 cells expressing different putative M. separata receptors, including MsABCC3, MsABCC2 and cadherin (MsCAD). The cytotoxicity of activated Cry1Ac mediated by MsABCC3 was lower than that mediated by MsABCC2, but significantly higher than that mediated by MsCAD. In addition, co-expression of MsABCC3 and MsCAD resulted in Cry1Ac susceptibility comparable to that of MsABCC2, indicating a synergistic or cooperation interaction of MsCAD with the MsABCC3 transporter, but not with MsABCC2. Interestingly, co-expression of both MsABCC2 and MsABCC3 in Hi5 cells did not show a synergistic interaction. Bioassays revealed that MsABCC3 knockout in M. separata larvae conferred low resistance to Cry1Ac. Our results suggest that MsABCC3 along with MsCAD cooperatively participates as receptors of Cry1Ac and also that ABCC2 alone is involved in Cry1Ac toxicity. These findings provide new insight on the mechanism of resistance against Bt toxins in M. separata.