Functional characterization of ABC transporters in chlorpyrifos resistance in Nilaparvata lugens.

ATP-binding cassette (ABC) transporters play important roles in insecticide resistance, but their functional contributions and structural basis of substrate recognition remain poorly understood. Here, we demonstrate that ABCG9 is an important determinant of chlorpyrifos resistance in Nilaparvata lugens, a destructive rice pest throughout Asia. Verapamil synergism suggested the involvement of active efflux, and expression profiling identified ABCB7, ABCC5, and ABCG9 as constitutively upregulated in the resistant strain and inducible by chlorpyrifos. However, only RNAi-mediated knockdown of ABCG9 significantly increased chlorpyrifos susceptibility, suggesting that this transporter plays a critical role in chlorpyrifos resistance in N. lugens. Structural analysis revealed that ABCG9 adopts a canonical ABCG half-transporter architecture with a conserved N-terminal nucleotide-binding domain (NBD) and a C-terminal transmembrane domain (TMD) containing six helices. Molecular docking and dynamics simulations showed that chlorpyrifos binds within a hydrophobic pocket at the TMD cavity entrance, stabilized primarily by van der Waals interactions (ΔGvdW = -39.04 kcal/mol), with a total binding free energy of -33.46 kcal/mol. Notably, the predicted hydrogen bond was transient, whereas hydrophobic contacts persisted throughout the 100 ns simulation, highlighting the importance of dynamic approaches over static docking alone. These findings identify ABCG9 as a phase III efflux transporter completing the chlorpyrifos detoxification cascade and provide structural insights into substrate recognition by insect ABCG transporters.