MAPK-directed activation of the whitefly transcription factor <i>CREB</i> leads to P450-mediated imidacloprid resistance

The evolution of insect resistance to pesticides poses a continuing threat to agriculture and human health. While much is known about the proximate molecular and biochemical mechanisms that confer resistance, far less is known about the regulation of the specific genes/gene families involved, particularly by <i>trans</i>-acting factors such as signal-regulated transcription factors. Here we resolve in fine detail the <i>trans</i>-regulation of <i>CYP6CM1</i>, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly <i>Bemisia tabaci,</i> by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor <i>cAMP-response element binding protein</i> (<i>CREB</i>). Reporter gene assays were used to identify the putative promoter of <i>CYP6CM1</i>, but no consistent polymorphisms were observed in the promoter of a resistant strain of <i>B. tabaci</i> (imidacloprid-resistant, IMR), which overexpresses this gene, compared to a susceptible strain (imidacloprid-susceptible, IMS). Investigation of potential <i>trans</i>-acting factors using in vitro and in vivo assays demonstrated that the bZIP transcription factor <i>CREB</i> directly regulates <i>CYP6CM1</i> expression by binding to a cAMP-response element (CRE)-like site in the promoter of this gene. <i>CREB</i> is overexpressed in the IMR strain, and inhibitor, luciferase, and RNA interference assays revealed that a signaling pathway of MAPKs mediates the activation of <i>CREB</i>, and thus the increased expression of <i>CYP6CM1</i>, by phosphorylation-mediated signal transduction. Collectively, these results provide mechanistic insights into the regulation of xenobiotic responses in insects and implicate both the MAPK-signaling pathway and a transcription factor in the development of pesticide resistance.