Differential roles of ABCC2, ABCC3, and cadherin in mediating Cry1Ac toxicity in Spodoptera exigua

Abstract

Bacillus thuringiensis Cry toxins have been widely used to control major lepidopteran pests. Interaction of Cry toxins with larval midgut receptors is a critical step in exerting toxicity. Receptor alternations are the major mechanisms of Bt resistance in the target pests. Identification of the critical receptors is essential for early warning of Bt resistance and for developing adaptive resistance management strategies. Two ATP-binding cassette transporters, ABCC2 and ABCC3, as well as cadherin, are important receptors for Cry1A toxins in lepidopterans, but they play different roles across various species. In the present study, the differential roles of SeABCC2, SeABCC3, and SeCad1 in mediating Cry1Ac toxicity in Spodoptera exigua, one of the most serious crop pests worldwide, were investigated through CRISPR-mediated gene knockouts and in vitro testing of Sf9 cell toxicity. Six knockouts were established by knocking out SeABCC2, SeABCC3, or SeCad1 individually and in pairs. Bioassay results demonstrated that knockout of SeABCC2 alone or in combination with either SeABCC3 or SeCad1 resulted in significant resistance to Cry1Ac by 1180-, 990-, and 7540-fold in the SeC2-KO, SeC2/Cad1-KO, and SeC2/C3-KO strains, respectively. In contrast, the other three knockouts (SeC3-KO, SeCad1-KO, and SeC3/Cad1-KO) exhibited less than two-fold change in Cry1Ac toxicity compared to the wild-type strain. Cell toxicity results with Sf9 cells infected to produce recombinant SeABCC2, SeABCC3, and SeCad1, individually or in pairs, generally aligned with the gene knockout findings. Our results demonstrate that SeABCC2 is the most critical receptor for Cry1Ac in S. exigua, and that Cry1Ac exerts its toxicity primarily through one major toxic pathway mediated by SeABCC2. SeABCC3 and SeCad1 played minor roles in mediating Cry1Ac toxicity. Therefore, resistance surveillance and management efforts should focus on SeABCC2 mutations in field populations of S. exigua.