MAPK-mediated transcription factor GATAd contributes to Cry1Ac resistance in diamondback moth by reducing PxmALP expression

Author summaryGene expression and regulation are associated with adaptive evolution in living organisms. The rapid evolution of insect resistance to Bt insecticidal Cry toxins is frequently associated with reduced expression of diverse midgut genes that code for Cry-toxin receptors. Nonetheless, our current knowledge about the regulation of gene expression of these pivotal receptor genes in insects is limited. Membrane-bound alkaline phosphatase (mALP) is a known receptor for Cry1Ac toxin in diverse insects and here, we report the transcriptional regulatory mechanism of the PxmALP gene related to Cry1Ac resistance in P. xylostella. We identified a MAPK signaling pathway that negatively regulates the PxGATAd transcriptional factor which is involved in the differential expression of PxmALP via interacting with the PxmALP promoter. Furthermore, a cis-acting element mutation repressing the regulatory activity of PxGATAd for PxmALP expression in the Cry1Ac resistant strain was identified. Our study provides an insight into the precise transcriptional regulatory mechanism that regulates PxmALP expression and is involved in the evolution of Bt Cry1Ac resistance in P. xylostella, which provides a paradigm for decoding the regulation landscape of midgut Cry-toxin receptor genes in insects. The benefits of biopesticides and transgenic crops based on the insecticidal Cry-toxins from Bacillus thuringiensis (Bt) are considerably threatened by insect resistance evolution, thus, deciphering the molecular mechanisms underlying insect resistance to Bt products is of great significance to their sustainable utilization. Previously, we have demonstrated that the down-regulation of PxmALP in a strain of Plutella xylostella (L.) highly resistant to the Bt Cry1Ac toxin was due to a hormone-activated MAPK signaling pathway and contributed to the resistance phenotype. However, the underlying transcriptional regulatory mechanism remains enigmatic. Here, we report that the PxGATAd transcription factor (TF) is responsible for the differential expression of PxmALP observed between the Cry1Ac susceptible and resistant strains. We identified that PxGATAd directly activates PxmALP expression via interacting with a non-canonical but specific GATA-like cis-response element (CRE) located in the PxmALP promoter region. A six-nucleotide insertion mutation in this cis-acting element of the PxmALP promoter from the resistant strain resulted in repression of transcriptional activity, affecting the regulatory performance of PxGATAd. Furthermore, silencing of PxGATAd in susceptible larvae reduced the expression of PxmALP and susceptibility to Cry1Ac toxin. Suppressing PxMAP4K4 expression in the resistant larvae transiently recovered both the expression of PxGATAd and PxmALP, indicating that the PxGATAd is a positive responsive factor involved in the activation of PxmALP promoter and negatively regulated by the MAPK signaling pathway. Overall, this study deciphers an intricate regulatory mechanism of PxmALP gene expression and highlights the concurrent involvement of both trans-regulatory factors and cis-acting elements in Cry1Ac resistance development in lepidopteran insects.