Multi-site phosphorylation of a transcription factor orchestrates imidacloprid resistance in whitefly

Protein phosphorylation plays a key role in regulating cellular function. However, the role of this post-translational modification in the evolution of adaptive traits such as insecticide resistance remains poorly resolved. Here, we show that two protein kinases, belonging to components of different signaling pathways, act antagonistically via phosphorylation to regulate a single transcription factor, which in turn trans-regulates a resistance gene in the global pest Bemisia tabaci. We reveal that the transcription factor Eagle is constitutively overexpressed and trans-activates the 20E-related gene CYP306A1, enhancing imidacloprid detoxification and metabolic resistance. Eagle activity is antagonistically regulated by the protein kinase mitogen-activated protein kinase p38 and the cyclic adenosine monophosphate-dependent protein kinase A PKA_C1 via phosphorylation at two distinct phosphoacceptor sites, with p38 upregulation and PKA_C1 downregulation sustaining resistance. These findings reveal the role of antagonistic phosphorylation in the evolution of adaptive phenotypes and uncover a complex trans-regulatory network underpinning insecticide resistance.