HaGATAe regulates chitin synthase 2 and insect intestinal mucin expression with ecdysone responsive transcription...

The peritrophic matrix (PM) serves as a crucial intestinal barrier in insects, with Chitin Synthase 2 (CHS2) and Insect Intestinal Mucin (IIM) being its essential structural components. However, the regulatory mechanisms controlling their expression remain poorly understood. Here, we identified HaIIM80 as the most highly expressed chitin-binding protein gene in the midgut of Helicoverpa armigera larvae. Through dual-luciferase assays, DNA pull-down, RNA interference, histological assays, and functional studies, we demonstrated that the transcription factor HaGATAe binds to promoters of both HaCHS2 and HaIIM80 via multiple GATA sites, upregulating their expression and promoting PM integrity. Knockdown of HaGATAe reduced midgut chitin abundance and PM thickness, increased susceptibility to HaNPV, but decreased sensitivity to Vip3Aa toxin. Furthermore, the ecdysone-responsive transcription factors HaEcRb enhanced the HaGATAe-mediated activation of HaCHS2 and HaIIM80, and HaHR3 suppressed them, which is consistent with their expression patterns during pupation period. This regulatory mode was further supported by RNAi of HR3, starvation, and treatment with the ecdysone agonist methoxyfenozide, collectively indicating that ecdysone signaling regulates the expression of both HaCHS2 and HaIIM80. Besides, HaE75C (an HR3 repressor) and its ligand heme upregulated the expression of both HaCHS2 and HaIIM80. These findings reveal a transcriptional regulatory network affecting PM formation and provide potential targets for pest management strategies aiming at disrupting gut barrier function.