Fig 1.
Overexpression of CYP321A8 confers resistance to multiple insecticides in S. exigua.
(A) P450 activity in the larvae of the susceptible and resistant strains of S. exigua. P450 monooxygenase activity was evaluated by measuring ethoxycoumarin-O-deethylase (ECOD) activity. A significant difference in enzymatic activities is indicated using an asterisk (Student’s t-test, p < 0.05). Error bars indicate SD. (B) Relative expression of P450 genes in the susceptible and resistant strains of S. exigua as determined by RT-qPCR. Error bars display SD. A significant difference in expression between the susceptible and resistant strains is indicated using an asterisk (ANOVA with post-hoc Tukey’s HSD, p < 0.05). The sensitivity of transgenic D. melanogaster to chlorpyrifos (C), cypermethrin (D) and deltamethrin (E). Error bars display SD. Significant differences in mortality between lines expressing CYP321A8 and control flies without the transgene are indicated using an asterisk (Student’s t-test, p < 0.05). (F) Metabolic activity of recombinant CYP321A8 enzyme against a model fluorescent substrate. The rate of O-dealkylation of 7-ethoxy coumarin (ECOD) is shown. Error bars display SD. Metabolism of chlorpyrifos (G), cypermethrin (H) and deltamethrin (I) metabolism by recombinant CYP321A8. Error bars display SD values. Significant differences (p < 0.05) in metabolism are denoted using letters above bars (ANOVA with post-hoc Tukey’s HSD).
Fig 2.
Overexpression of CncC and Maf increases the promoter activity of CYP321A8.
(A) Prediction of transcription factor binding sites in a ~2 kb region of the promoter of CYP321A8 gene. Nucleotides are numbered relative to the translation start site (ATG) indicated by +1. The predicted binding sites for transcription factors sites are underlined. The predicted transcription initiation site (Inr) is underlined. (B) Relative expression of transcription factors in the susceptible and resistant strains of S. exigua as determined by RT-qPCR. Error bars display SD. A significant difference (p < 0.05) in expression between the Sus and Res strains is indicated using an asterisk (ANOVA with post-hoc Tukey’s HSD). (C) The Luciferase activity in cells transfected with the reporter construct (the luciferase gene is under the control of CYP321A8 promoter) and CncC or Maf and both CncC and Maf expression constructs. Different letters above the bars indicate significant differences based on ANOVA followed by post-hoc Tukey’s HSD (p < 0.05).
Fig 3.
CncC and Maf regulate the expression of CYP321A8 by binding to proximal and/or distal sites.
(A) Schematic representation of the CYP321A8 promoter deletions. (B) Analysis of the activity of CYP321A8 promoter deletions in reporter gene assays in the presence and absence of CncC/Maf. The luciferase activity was normalized with the Renilla luciferase activity. Error bars display SD. The full-length promoter and each truncated version were compared using Student’s t-test. Significant differences (p < 0.05) in the luciferase activity are indicated using an asterisk.
Fig 4.
Mutations in CncC/Maf binding sites abolishes the ability of these transcription factors to induce the expression of CYP321A8.
(A) Schematic representation of mutated CYP321A8 promoter constructs. (B) The activity of these constructs in reporter assays. Error bars indicate SD. The activity of each mutated construct was compared with the corresponding wild-type control by Student’s t-test and significant differences (p < 0.05) are indicated with an asterisk.
Fig 5.
A cis-acting mutation in the promoter of CYP321A8 enhances the expression of this P450 gene in the resistant strain of S. exigua.
(A) Phylogenetic relationship of CYP321A8 promoter sequences obtained from the susceptible and resistant strains. The phylogeny was inferred by the maximum likelihood and percentage bootstrap values from 1000 replicates are displayed. (B) Analysis of the activity of the CYP321A8 resistant promoter (DNA5’-R) and susceptible promoter (DNA5’-S). Error bars display SD. Letters above bars denote a significant difference at p < 0.05 (ANOVA with post-hoc Tukey HSD). (C) The activity of progressive 5’ deletion constructs of the CYP321A8 promoter. Error bars display SD. Letters to the right of bars denote significant difference at p < 0.05 (ANOVA with post-hoc Tukey HSD). (D) The activity of progressive 5’-deletion constructs from -522 to -142 of the CYP321A8 promoter from the susceptible and resistant strains. Error bars display SD. Letters to the right of bars denote significant difference at p < 0.05 (ANOVA with post-hoc Tukey HSD).
Fig 6.
A cis-acting mutation in the CYP321A8 promoter of the resistant strain facilitate binding of nuclear receptor, Knirps and contributes to upregulation of CYP321A8.
(A) Alignment of a region of the CYP321A8 promoter of the resistant and susceptible strains. Mutations at five positions are indicated above the alignment. (B) Activity of mutant constructs of the CYP321A8 promoter from the resistant strain, where mutations in the region from -385 to -142 were reverted to wild-type. Error bars display SD. Letters to the right of bars denote significant difference at p < 0.05 (ANOVA with post-hoc Tukey HSD). (C) Activity of the promoter constructs P(-385/-1)S (susceptible strain) and P(-385/-1)R (resistant strain) in reporter gene assays in the presence and absence of Knirps. PIB represents the empty vector pIB/V5-His. Error bars display SD. Letters above bars denote significant difference at p < 0.05 (ANOVA with post-hoc Tukey HSD). (D) Relative expression of Knirps in the susceptible and resistant strains of S. exigua as determined by RT-qPCR. Error bars display SD. (E) The activity of the promoter constructs (DNA5’-R and DNA5’-S) in the presence and absence of CncC/Maf constructs. Error bars display SD. Letters above bars denote a significant difference at p < 0.05 (ANOVA with post-hoc Tukey HSD).
Fig 7.
Schematic of the regulation of CYP321A8 by cis- and trans-acting factors.
In the resistant strain, constitutive overexpression of the trans-acting factors CncC and Maf act in combination with a cis-acting mutation in the promoter of CYP321A8, which facilitates binding of nuclear receptor, Knirps, enhancing the expression of CYP321A8 leading to insecticide resistance. In the susceptible strain, CncC and Maf are expressed at lower levels and there is no binding site for Knirps therefore, CYP321A8 expression is low making them susceptible to insecticides.