New insights into chlorantraniliprole metabolic resistance mechanisms mediated by the striped rice borer cytochrome P450 monooxygenases: A case study of metabolic differences

Abstract

The anthranilic diamide insecticide chlorantraniliprole has been extensively applied to control Lepidoptera pests. However, its overuse leads to the development of resistance and accumulation of residue in the environment. Four P450s (CYP6CV5, CYP9A68, CYP321F3, and CYP324A12) were first found to be constitutively overexpressed in an SSB CAP-resistant strain. It is imperative to further elucidate the molecular mechanisms underlying P450s-mediated CAP resistance for mitigating its environmental contamination. Here, we heterologously expressed these four P450s in insect cells and evaluated their abilities to metabolize CAP. Western blotting and reduced CO difference spectrum tests showed that these four P450 proteins had been successfully expressed in Sf9 cells, which are indicative of active functional enzymes. The recombinant proteins CYP6CV5, CYP9A68, CYP321F3, and CYP324A12 exhibited a preference for metabolizing the fluorescent P450 model probe substrates EC, BFC, EFC, and EC with enzyme activities of 0.54, 0.67, 0.57, and 0.46 pmol/min/pmol P450, respectively. In vitro metabolism revealed distinct CAP metabolic rates (0.97, 0.86, 0.75, and 0.55 pmol/min/pmol P450) and efficiencies (0.45, 0.37, 0.30, and 0.17) of the four recombinant P450 enzymes, thereby elucidating different protein catalytic activities. Furthermore, molecular model docking confirmed metabolic differences and efficiencies of these P450s and unveiled the hydroxylation reaction in generating N-demethylation and methylphenyl hydroxylation during CAP metabolism. Our findings not only first provide new insights into the mechanisms of P450s-mediated metabolic resistance to CAP at the protein level in SSB but also demonstrate significant differences in the capacities of multiple P450s for insecticide degradation and facilitate the evaluation and mitigation of toxic risks associated with CAP application in the environment.