Molecular dynamics and mutagenesis reveal OBP2-mediated imidacloprid resistance in Nilaparvata lugens

The evolution of insecticide resistance in insect pests severely threatens global food security. Recently, sequestration by odorant-binding proteins (OBPs) has emerged as a functionally important yet mechanistically unresolved resistance pathway. In this study, we demonstrated that specific OBPs contribute to imidacloprid resistance in the brown planthopper, Nilaparvata lugens, a major rice pest. Transcriptional analysis revealed consistent overexpression of five OBP genes in resistant strains, with OBP2 expression increased by 2.5-fold and 3.8-fold in laboratory-selected and field-collected resistant strains, respectively. Tissue-specific profiling further indicated predominant expression of OBP2 in the leg, head, and antennae, the primary insecticide contacting tissues. Functional studies showed that RNAi-mediated knockdown of OBP2, OBP5, or OBP7 significantly enhanced imidacloprid susceptibility, with mortality increases exceeding 60% in resistant insects. Heterologous expression of these OBPs in Escherichia coli conferred protection, increasing bacterial survival more than two-fold under imidacloprid stress. Competitive binding assays confirmed high-affinity OBP-imidacloprid interactions, with OBP2 exhibiting the strongest binding (K_i = 2.84 ± 0.12 μM). Molecular dynamics simulations and site-directed mutagenesis identified two hydrophobic residues, Met71 and Leu139, as critical for complex stabilization. Their mutation to alanine substantially impaired binding affinity (K_i = 3.80 μM and 4.05 μM, respectively) without altering the overall protein structure. Collectively, these results provide mechanistic insights into OBP-mediated insecticide sequestration and lay the groundwork for novel resistance management strategies.