ABCC3 mediates insecticide metabolism and functions as a receptor for Bt toxins in Plutella xylostella

Abstract Plutella xylostella is a major global pest with increasing resistance to insecticides. ATP‐binding cassette (ABC) transporters are implicated in the metabolism of insecticides, but their molecular mechanisms remain poorly understood. Here, we identified and characterized the role of P. xylostella ABCC3 ( Px ABCC3) in mediating the metabolism of chemical insecticides and its function as a receptor for Bacillus thuringiensis (Bt) toxins. Px ABCC3 and its mutant variants were cloned, expressed, and purified, followed by biochemical and structural analyses. Our results showed that Px ABCC3 exhibits typical ABC transporter structural features and its ATPase activity is significantly modulated by chlorpyrifos and pyriproxyfen, and other insecticides. Surface plasmon resonance confirmed direct insecticide binding. Overexpression of Px ABCC3 in insect cells enhanced survival under chlorpyrifos and pyriproxyfen exposure, suggesting an efflux‐mediated detoxification mechanism. The analysis of predicted complex structures of Px ABCC3 with chlorpyrifos or pyriproxyfen identified overlapping substrate‐binding pockets within the transmembrane channel. Furthermore, Px ABCC3 functions as a receptor for Bt toxins Cry1Ac and Cry1Ab, as evidenced by toxin‐induced ATPase activation, binding affinity, and cellular swelling in transfected cells. We further predicted the complex structure of Px ABCC3–Cry1Ab, revealing that Cry1Ab binds to the extracellular side of Px ABCC3 via domain II through electrostatic complementarity and hydrogen‐bond networks. These findings provide novel insights into mechanistic understanding at the protein level, especially with respect to structural characteristics and substrate‐binding specificity, and highlight Px ABCC3 as a potential target for pest control.