Two independent regulatory mechanisms synergistically contribute to P450-mediated insecticide resistance in a lepidopteran pest, Spodoptera exigua.

Background: Cytochrome P450 enzymes play a pivotal role in the detoxification of plant allelochemicals and insecticides. Overexpression of P450 genes has been proven to be involved in insecticide resistance in insects. However, the molecular mechanisms underlying the regulation of P450 genes in insects are poorly understood.

Results: Here, we determine that upregulation of CYP321B1 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in the resistant Spodoptera exigua strain. Enhanced expression of transcription factors CncC/Maf contributes to the increase in the expression of CYP321B1 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses confirm that a specific binding site is crucial for binding CncC/Maf to activate the expression of CYP321B1. In addition, creation of a new binding site resulting from the cis-mutations in the promoter region of CYP321B1 in the resistant strain facilitates the binding of the POU/homeodomain transcription factor Nubbin, and further enhances the expression of this P450 gene. Furthermore, we authenticate that changes in both trans- and cis-regulatory elements in the promoter region of CYP321B1 act in combination to modulate the promoter activity in a synergistic manner.

Conclusions: Collectively, these results demonstrate that two distinct but synergistic mechanisms coordinately result in the overexpression of CYP321B1 involved in insecticide resistance in an agriculturally important insect pest, S. exigua. The information on mechanisms of metabolic resistance could help to understand the development of resistance to insecticides by other pests and contribute to designing effective integrated pest management strategies for the pest control.