MAPK-Activated Transcription Factor PxJun Suppresses PxABCB1 Expression and Confers Resistance to Bacillus thuringiensis Cry1Ac Toxin in Plutella xylostella (L.)

Abstract

Deciphering the molecular mechanisms underlying insect resistance to Cry toxins produced by Bacillus thuringiensis (Bt) is pivotal for the sustainable utilization of Bt biopesticides and transgenic Bt crops. Previously, we identified that mitogen-activated protein kinase (MAPK)-mediated reduced expression of the PxABCB1 gene is associated with Bt Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulation mechanism remains enigmatic. Here, the PxABCB1 promoter in Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella strains was cloned and analyzed and found to contain a putative Jun binding site (JBS). A dual-luciferase reporter assay and yeast one-hybrid assay demonstrated that the transcription factor PxJun repressed PxABCB1 expression by interacting with this JBS. The expression levels of PxJun were increased in the midguts of all resistant strains compared to the susceptible strain. Silencing of PxJun expression significantly elevated PxABCB1 expression and Cry1Ac susceptibility in the resistant NIL-R strain, and silencing of PxMAP4K4 expression decreased PxJun expression and also increased PxABCB1 expression. These results indicate that MAPK-activated PxJun suppresses PxABCB1 expression to confer Cry1Ac resistance in P. xylostella, deepening our understanding of the transcriptional regulation of midgut Cry receptor genes and the molecular basis of insect resistance to Bt Cry toxins. IMPORTANCE The transcriptional regulation mechanisms underlying reduced expression of Bt toxin receptor genes in Bt-resistant insects remain elusive. This study unveils that a transcription factor PxJun activated by the MAPK signaling pathway represses PxABCB1 expression and confers Cry1Ac resistance in P. xylostella. Our results provide new insights into the transcriptional regulation mechanisms of midgut Cry receptor genes and deepen our understanding of the molecular basis of insect resistance to Bt Cry toxins. To our knowledge, this study identified the first transcription factor that can be involved in the transcriptional regulation mechanisms of midgut Cry receptor genes in Bt-resistant insects.

Keywords: ABCB1; Bacillus thuringiensis; Cry1Ac resistance; Jun; Plutella xylostella; transcription factor.

FIG 1

Transcriptional activity of the PxABCB1 promoter in susceptible and resistant P. xylostella. (A) Diagram of sequence alignment of the 5′-flanking regions of PxABCB1 in susceptible DBM1Ac-S and resistant NIL-R strains. The right-angled arrow denotes the TSS. The numbers with arrows specify the 5′ and 3′ positions of the corresponding nucleotide. The green/white rectangles indicate DNA fragment insertion/deletion (Ins/Del). (B) Detection of PxABCB1 promoter activities in susceptible and resistant P. xylostella. All the fragment constructs are named with “P” as the starting letter, followed by a pair of parentheses that contain two numerals, separated by a slash (/), to specify the 5′ and 3′ positions of the corresponding promoter fragment. Relative luciferase (Luc) activities were detected at 48 h posttransfection in S2 cells. The relative luciferase activity (fold) of different promoter recombinants was calculated based on the value of the pGL4.10 control vector. The values shown are means and corresponding standard errors (SEM) for three independent experiments. The significance of differences was determined by one-way ANOVA with Duncan’s test (P < 0.05). Different letters used to mark bars denote significant differences.

FIG 2

Relative luciferase activity analysis of the fragments between positions −765 and +125. Progressive 5′ deletion constructs from positions −765 to +125 were transfected into S2 cells, and luciferase activity was detected. The relative luciferase activity (fold) of different constructs was calculated based on the value of the pGL4.10 vector. The values shown are means and the corresponding SEM. One-way ANOVA followed by Duncan’s test was used for comparison (P < 0.05, n = 3). Different letters used to mark bars denote significant differences.

FIG 3

Effects of potential TFs on the activity of the PxABCB1 promoter. (A) TFBS in critical positive and negative regulatory regions (see Fig. S1 for detailed DNA motifs). Different colored ellipses represent different TFBSs. (B) Effects of different predicted TFs on PxABCB1 promoter activity. Every cloned TF was subcloned into the pAc5.1 expression vector to generate a recombinant vector, which was then cotransfected with P(–1122/+125) to determine the luciferase activity. The empty pAc5.1 without PxJun was used as a control. The relative luciferase activity (fold) was calculated based on the value of the empty pAc5.1 vector. The values shown are means and the corresponding SEM. One-way ANOVA followed by Duncan’s test was used for statistical analysis (P < 0.05, n = 3). Different letters used to mark bars denote significant differences.

FIG 4

PxJun represses PxABCB1 promoter activity through the Jun binding site (JBS). (A) Effect of PxJun on PxABCB1 promoter activity with either a deleted or a mutated form of the JBS via the dual-luciferase reporter assay. The JBS motif (AAGAAATGAGAGAT, −107 to −94) in P(–1122/+125) was deleted or mutated to GGAGGGCAGAGAGC. PxJun was cotransfected with P(–1122/+125) containing normal JBS (red ellipse), deleted JBS (black ellipse), or mutated JBS (green ellipse). The empty pAc5.1 without PxJun was used as a control. Three biological replicates were performed for all experiments. The values shown are means and the corresponding SEM. One-way ANOVA followed by Duncan’s test was used for statistical analysis (P < 0.05). Different letters used to mark bars denote significant differences. (B) Verification of direct binding of PxJun to the JBS by the yeast one-hybrid (Y1H) assay. Three tandem repeats containing wild-type or mutated JBS sequences were fused to the pABAi vector, which was subsequently integrated into the Y1HGold yeast strain to generate the “bait strain.” A critical aureobasidin A (AbA) concentration of 500 ng/ml was detected to completely repress the growth of the bait strains on SD/–Ura media. PxJun was fused to the pGATD7 vector and then transferred into the “bait strain.” which was grown on SD/–Leu selective media with or without AbA. EV, empty vector; positive control, using pGADT7-p53 + pABAi-p53.

FIG 5

Effects of PxJun and/or PxFos on the activity of the PxABCB1 promoter. PxJun and/or PxFos were cotransfected with P(–1122/+125), and the luciferase activity was measured. The empty pAc5.1 without TF was used as a control. The relative luciferase activity (fold) was calculated based on the value of the empty pAc5.1 vector. The values shown are the means and the corresponding SEM for three independent experiments. One-way ANOVA followed by Duncan’s test was used for statistical analysis (P < 0.05, n = 3). Different letters used to mark bars denote significant differences.

FIG 6

Relative expression levels of the PxJun gene in the midgut tissues of fourth-instar larvae in five P. xylostella strains as detected by qPCR. The RPL32 gene was used as an internal control. The relative expression level (fold) is presented as the ratio to the value of the lowest expression level, observed in the DBM1Ac-S strain. The average relative expression levels and SEM of three independent replicates are presented. Different letters used to mark bars denote significant differences (P < 0.05, Duncan’s test, n = 3).

FIG 7

Effect of PxJun gene silencing on PxABCB1 expression and Cry1Ac resistance in resistant NIL-R larvae. (A) Relative expression of PxJun and PxABCB1 at 48 h postinjection with buffer, dsEGFP or dsPxJun. The expression levels of PxJun or PxABCB1 in the control larvae injected with buffer were set as 1. (B) Silencing of PxJun expression decreased the resistance of NIL-R larvae to Cry1Ac protoxin. At 48 h after microinjection with buffer, dsEGFP or dsPxJun, the larvae were fed diets with or without Cry1Ac protoxin (1,000 mg/liter). The percentage of larval mortality was then counted at 72 h posttreatment. The data are presented as mean values ± the SEM for three biologically independent experiments. Different letters in each group indicate statistically significant differences between treatments (P < 0.05; Duncan’s test; n = 3).

FIG 8

Effect of PxMAP4K4 gene silencing on the expression levels of PxJun and PxABCB1 in the midgut tissues at different periods. The data are presented as mean values ± the SEM for three biologically independent experiments. Asterisks (*) indicate significant differences among periods for each gene (P < 0.05; Duncan’s test; n = 3).

FIG 9

Proposed model for the transcriptional regulation of reduced PxABCB1 expression by the MAPK-activated TF PxJun. The activated MAPK signaling pathway increases the expression of PxJun, which in turn represses the transcript level of Bt Cry1Ac receptor gene PxABCB1 and enhances larval resistance to Bt Cry1Ac toxin in P. xylostella.