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.

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Published in PLOS Genetics, v. 18, issue 2, 1010037.

© 2022 Guo et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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This work was supported by the Laboratory of Lingnan Modern Agriculture Project (NT2021003) to YZ, the National Natural Science Foundation of China (32022074; 32172458) to ZG, the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS) to YZ.

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The final cloned full-length cDNA sequence of P. xylostella GATAd gene was deposited in the GenBank database (accession no. MZ712004). The raw data of the figures and statistical analyses in this study are provided in S1 Data.

pgen.1010037.s001.pdf (109 kB)
S1 Fig. Sequence alignment of the 5′-flanking regions of PxmALP cloned from Cry1Ac susceptible DBM1Ac-S and resistant NIL-R strains. https://doi.org/10.1371/journal.pgen.1010037.s001

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S2 Fig. Multiple amino acid sequence alignment of different GATAd proteins. https://doi.org/10.1371/journal.pgen.1010037.s002

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S3 Fig. Phylogenetic relationship of GATAd proteins in insects and mammals. https://doi.org/10.1371/journal.pgen.1010037.s003

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S4 Fig. Role of PxGATAd on the transcriptional regulation of other Cry1Ac-receptors. https://doi.org/10.1371/journal.pgen.1010037.s004

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S1 Table. Sequence of the primers used for cloning the promoter region of receptor genes and construction of pGL4.10 recombinant plasmids. https://doi.org/10.1371/journal.pgen.1010037.s005

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S2 Table. Sequence of the primers used for site-directed mutagenesis. https://doi.org/10.1371/journal.pgen.1010037.s006

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S3 Table. Sequence of the primers used for cloning TFs from P. xylostella. https://doi.org/10.1371/journal.pgen.1010037.s007

pgen.1010037.s008.pdf (144 kB)
S4 Table. Primers used for constructing the recombinant plasmids of TFs. https://doi.org/10.1371/journal.pgen.1010037.s008

pgen.1010037.s009.pdf (134 kB)
S5 Table. Sequence of the primers used for real time qPCR analyzes and dsRNA synthesis. https://doi.org/10.1371/journal.pgen.1010037.s009

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S1 Data. Raw data used in the figures and statistical analysis. https://doi.org/10.1371/journal.pgen.1010037.s010