CSP15 attenuates chlorpyrifos toxicity through sequestration and bioactivation suppression in Nilaparvata lugens

The escalating problem of insecticide resistance in agricultural pests highlights the need to identify non-canonical resistance mechanisms. In this study, comparative transcriptional analysis identified five chemosensory protein (CSP) genes significantly overexpressed in a chlorpyrifos (CPF)-resistant strain of Nilaparvata lugens. RNA interference and heterologous expression confirmed their contribution to resistance, with CSP15, exhibiting 42.98-fold overexpression, identified as a key mediator. CSP15 knockdown increased CPF susceptibility 1.76-fold, while its bacterial expression raised tolerance 5.47-fold. Competitive fluorescence binding assays showed that CSP15 binds CPF (K_i = 3.32 μM) with higher affinity than its toxic metabolite chlorpyrifos-oxon (CPO; K_i = 6.00 μM). Structural modeling revealed a CPF-binding pocket mediated by Arg84 and Lys118. CPO binding utilized four hydrogen bonds with Lys28; the K28A mutation induced structural rearrangement through neo-formed interactions with Arg84 and Lys118, reducing the binding free energy by -40.95 kcal mol^-1. Mutagenesis revealed that R84A/K118A double mutants lost 62.85-64.37 % of CPF-binding capacity, whereas the K28A variant increased CPO affinity by 22.65 %. In vivo metabolic profiling indicated that CSP15 knockdown promoted CPF degradation and CPO accumulation, supporting its role in suppressing bioactivation. Yeast two-hybrid assays confirmed a direct interaction between CSP15 and CYP6BD12, the P450 enzyme responsible for CPF bioactivation. Additionally, CSP15 expression was regulated by the transcription factor Lim1β through two conserved promoter cis-elements. Together, these findings reveal a novel dual resistance mechanism wherein CSP15 acts synergistically by sequestering CPF and binding CYP6BD12, thereby effectively suppressing bioactivation and identifying promising targets for pest management.