Functional and structural characterization of OBP7 reveals a sequestration-based chlorpyrifos resistance mechanism in...

The evolution of insecticide resistance threatens sustainable pest control, urging the discovery of mechanisms beyond classical metabolic detoxification and target-site mutations. In this study, we report a sequestration-based resistance mechanism mediated by odorant-binding protein 7 (OBP7) in the brown planthopper, Nilaparvata lugens. Through a combination of in vivo functional genetics and in vitro biochemical assays, we demonstrate that OBP7 plays a critical role by acting as a high-affinity sequestrant of chlorpyrifos and its more toxic metabolite, chlorpyrifos-oxon, to confer insecticide resistance. RNA interference knockdown of OBP7 significantly increased susceptibility to chlorpyrifos in both susceptible and resistant strains, while heterologous expression of OBP7 in Escherichia coli conferred enhanced tolerance to this insecticide. Fluorescence competitive binding assays revealed that OBP7 binds directly to both chlorpyrifos and chlorpyrifos-oxon, exhibiting a significantly stronger binding affinity for the latter. Furthermore, molecular docking identified Gln53 as a key residue interacting with both ligands. Site-directed mutagenesis uncovered a compensatory binding mechanism: mutation of Gln53 prompted the adjacent Gln54 to form alternative interactions, while dual mutation of Gln53 and Gln54 eliminated hydrogen bonding with chlorpyrifos but preserved binding to chlorpyrifos-oxon through interactions with other residues, notably His181 and Ser270. These results illustrate a redundant, ligand-selective, and remarkably robust binding mechanism that prioritizes the sequestration of the activated toxin. Our findings establish OBP7 as a crucial component in insecticide resistance and provide a molecular basis for developing novel strategies that disrupt olfactory protein-insecticide interactions.