In this work, we discovered that overexpression of a P450 that confers resistance to neonicotinoid insecticides in whitefly is trans-regulated by the transcription factor CREB. Further studies demonstrated that exposure to neonicotinoid insecticides activates a key pathway involved in the cellular response to extracellular signals, the MAPK signaling pathway, that activates CREB by phosphorylation. CREB then binds to a specific site on the promoter of CYP6CM1 resulting in its increased expression. These findings reveal mechanisms underlying the regulation of P450-mediated pesticide resistance and also provide a potential target for pest control.
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 trans-acting factors such as signal-regulated transcription factors. Here we resolve in fine detail the trans-regulation of CYP6CM1, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly Bemisia tabaci, by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor cAMP-response element binding protein (CREB). Reporter gene assays were used to identify the putative promoter of CYP6CM1, but no consistent polymorphisms were observed in the promoter of a resistant strain of B. tabaci (imidacloprid-resistant, IMR), which overexpresses this gene, compared to a susceptible strain (imidacloprid-susceptible, IMS). Investigation of potential trans-acting factors using in vitro and in vivo assays demonstrated that the bZIP transcription factor CREB directly regulates CYP6CM1 expression by binding to a cAMP-response element (CRE)-like site in the promoter of this gene. CREB is overexpressed in the IMR strain, and inhibitor, luciferase, and RNA interference assays revealed that a signaling pathway of MAPKs mediates the activation of CREB, and thus the increased expression of CYP6CM1, 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.
Figure 1: CYP6CM1 contributes to imidacloprid (IM) resistance in the B. tabaci strain IMR. (A) Relative expression of CYP6CM1 mRNA (A) and protein (B) in the imidacloprid-resistant strain IMR and the susceptible strain IMS as determined by qPCR and Western blot, respectively (qPCR: n = 3, mean ± SE; ***P < 0.001, two-tailed Student’s t test). Actin was used as a loading control in Western blot. (C) Life span of adults of the IMR and IMS strains exposed to 2.0 mM imidacloprid (2) or no imidacloprid (0) after RNAi knockdown of CYP6CM1. Adults fed on dsEGFP were used as a negative control.