MAP4K4 controlled transcription factor POUM1 regulates PxABCG1 expression influencing Cry1Ac resistance in Plutella xylostella (L.)

Deciphering the molecular mechanisms of insect resistance to Bacillus thuringiensis (Bt) based biotechnology products including Bt sprays and Bt crops is critical for the long-term application of Bt technology. Previously, we established that down-regulation of the ABC transporter gene PxABCG1, trans-regulated by the MAPK signaling pathway, contributed to high-level resistance to Bt Cry1Ac toxin in diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulatory mechanism was unknown. Herein, we identified putative binding sites (PBSs) of the transcription factor (TF) POUM1 in the PxABCG1 promoter and used a dual-luciferase reporter assay (DLRA) and yeast one-hybrid (Y1H) assay to reveal that POUM1 activates PxABCG1 via interaction with one of these sites. The expression of POUM1 was significantly decreased in the midgut tissue of Cry1Ac-resistant P. xylostella strains compared to a Cry1Ac-susceptible P. xylostella strain. Silencing of POUM1 expression resulted in reduced expression of the PxABCG1 gene and an increase in larval tolerance to Bt Cry1Ac toxin in the Cry1Ac-susceptible P. xylostella strain. Furthermore, silencing of PxMAP4K4 expression increased the expression of both POUM1 and PxABCG1 genes in the Cry1Ac-resistant P. xylostella strain. These results indicate that the POUM1 induces PxABCG1 expression, while the activated MAPK cascade represses PxABCG1 expression thus reducing Cry1Ac susceptibility in P. xylostella. This result deepens our understanding of the transcriptional regulatory mechanism of midgut Cry receptor genes and the molecular basis of the evolution of Bt resistance in insects.