Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua

Insect cytochrome P450 s (P450 s) are associated with the metabolic detoxification of toxic xenobiotics and their constitutive upregulation is often associated with resistance to natural and synthetic toxicants. The P450 s CYP321A16 and CYP332A1 are constitutively overexpressed in an insecticide-resistant strain of beet armyworm, Spodoptera exigua. However, the function and upstream regulation of these two P450 s remain unknown. Here, we investigated the function of CYP321A16 and CYP332A1 in resistance to the organophosphate insecticide, chlorpyrifos and their regulation by the transcription factors CncC and Maf. Transgenic strains of Drosophila melanogaster expressing CYP321A16 or CYP332A1 showed higher levels of tolerance to chlorpyrifos than the control flies with the same genetic background. Furthermore, recombinant CYP321A16 and CYP332A1 proteins metabolized chlorpyrifos. Analysis of the putative promoter sequences of the genes coding for CYP321A16 and CYP332A1 revealed conserved CncC/Maf binding sites. Transfection of luciferase reporter plasmids containing the promoter of CYP450 gene together with CncC and Maf expression plasmids significantly enhanced the activity of the reporter. Promoter truncation identified a site in the promoter of CYP321A16 that is critical for the CncC/Maf binding. These data demonstrate that resistance to chlorpyrifos in S. exigua is conferred by the combined action of CYP321A16 and CYP332A1 and uncovered their regulation by the transcription factors CncC and Maf.