The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.
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The work was funded by the National Natural Science Foundation of China (No.32000333) and China Postdoctoral Science Foundation (2020M671264) to BH. The work was supported by the National Natural Science Foundation of China (No.32072452) and Provincial Key Research and Development Program of Jiangsu (BE2019396) to JS. The project was funded by the European Research Council under the European Union's Horizon 2020 research and innovation program (n° 646625) to CB. The project was supported by National Institute of Food and Agriculture of US Department of Agriculture, HATCH Project 2351177000 and Agriculture and Food Research Initiative Competitive Grant no. 2019-67013-29351 to SRP.
All gene accession numbers are available from the Gene Bank (accession number(s) MK327547 and MK327548).
Hu, Bo; Huang, He; Hu, Songzhu; Ren, Miaomiao; Wei, Qi; Tian, Xiangrui; Esmail Abdalla Elzaki, Mohammed; Bass, Chris; Su, Jianya; and Palli, Subba Reddy, "Changes in Both Trans- and Cis-Regulatory Elements Mediate Insecticide Resistance in a Lepidopteron Pest, Spodoptera exigua" (2021). Entomology Faculty Publications. 213.
S1 Table. Resistance and synergism of PBO in insecticide resistant strain of S. exigua. https://doi.org/10.1371/journal.pgen.1009403.s001
journal.pgen.1009403.s002.docx (18 kB)
S2 Table. Primers used in quantitative real-time PCR of Cytochrome P450 genes. https://doi.org/10.1371/journal.pgen.1009403.s002
journal.pgen.1009403.s003.docx (14 kB)
S3 Table. Primers used in quantitative real-time PCR of transcription factors. https://doi.org/10.1371/journal.pgen.1009403.s003
journal.pgen.1009403.s004.docx (14 kB)
S4 Table. Primers used for gene amplification of transcription factors. https://doi.org/10.1371/journal.pgen.1009403.s004
journal.pgen.1009403.s005.docx (14 kB)
S5 Table. Primers used for Constuction of transgenic Drosophila and eukaryotic expression. https://doi.org/10.1371/journal.pgen.1009403.s005
journal.pgen.1009403.s006.docx (14 kB)
S6 Table. Primers used for cloning 5’-flanking regions. https://doi.org/10.1371/journal.pgen.1009403.s006
journal.pgen.1009403.s007.docx (15 kB)
S7 Table. Primers used for reporter and promoter constructs. https://doi.org/10.1371/journal.pgen.1009403.s007
journal.pgen.1009403.s008.tif (772 kB)
S1 Fig. Relative expression of the CYP321A8 transgene in the transgenic D. melanogaster Act5C-CYP321A8 strain and the control sample with no transgene expression. https://doi.org/10.1371/journal.pgen.1009403.s008
journal.pgen.1009403.s009.tif (1436 kB)
S2 Fig. CYP321A8 expression and functional analysis. https://doi.org/10.1371/journal.pgen.1009403.s009
journal.pgen.1009403.s010.tif (10191 kB)
S3 Fig. The alignment of upstream sequences of CYP321A8 gene from susceptible and resistant strains of S. exigua. https://doi.org/10.1371/journal.pgen.1009403.s010