BpWRKY6 regulates insect resistance by affecting jasmonic acid and terpenoid synthesis in Betula platyphylla

Abstract

Forest pests and diseases pose serious threats to the sustainable development of forestry. Plants have developed effective resistance mechanisms through long-term evolution. Jasmonic acid and terpenoids play important roles in the defence response of plants against insects. Here, we discovered a transcription factor of the WRKY IIa subgroup, BpWRKY6, which is located in the nucleus, and the overexpression of BpWRKY6 in birch (Betula platyphylla) can increase resistance to gypsy moths (Lymantria dispar). The selective feeding results indicated that the gypsy moth tends to feed more on wild-type (WT) and mutant birch. The overexpression of BpWRKY6 decreased feeding, delayed development, inhibited CarE activity, and increased the activities of GST and CYP450 in gypsy moth larvae, whereas gypsy moth larvae that fed on the mutant birch presented the opposite trend. Further analysis revealed that BpWRKY6 directly binds to the promoters of jasmonic acid (JA) synthesis genes, including BpLOX15, BpAOC4, and BpAOS1, and the terpenoid synthesis gene BpCYP82G1, promoting their expression and increasing the contents of JA, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and total terpenoids, thus affecting birch resistance to insects. In addition, BpWRKY6 was phosphorylated as a substrate for BpMAPK6, suggesting that BpWRKY6 functions through the MAPK signalling pathway. In conclusion, this study further improves the understanding of the insect defence response mechanism of plants to achieve green pest control and provide insect-resistant germplasm resources.

Keywords: Betula platyphylla; Lymantria dispar; WRKY; biotic stress.

Figure 1

Analysis of BpWRKY6 gene expression pattern. (a) Schematic diagram of the leaf area fed by the larvae of the gypsy moth. (b) BpWRKY6 expression pattern after feeding on leaves by the gypsy moth. (c) BpWRKY6 expression pattern after treatment with MeJA. * represents P‐value < 0.05, ** represents P‐value < 0.01.

Figure 2

BpWRKY6 sequence characteristics analysis. (a) Phylogenetic analysis of BpWRKY6 and WRKYs in Arabidopsis. (b) Multiple sequence alignment analysis of WRKYs between BpWRKY6 and other species. (c) Cis‐elements in the promoter sequence of BpWRKY6. (d) Subcellular localization analysis of BpWRKY6. (e) Transcription activation activity analysis of BpWRKY6.

Figure 3

Monitor and comparison of the insect resistance of BpWRKY6 transgenic birch and control birch. (a) Selective‐feeding experiment between leaves of BpWRKY6 transgenic birch and control. (b) Selective antifeedant rate of gypsy moth larvae feeding on control and BpWRKY6 transgenic birch leaves. (c) Development of larvae feeding on control and transgenic leaves for 7 days. (d) No‐selective antifeedant rate of gypsy moth larvae feeding on control and BpWRKY6 transgenic birch leaves. (e) JA content analysis in BpWRKY6 transgenic birch and control. (f–h): Analysis of GST activity (f), CarE activity (g), and CYP450 activity (h) in gypsy moth larvae feeding BpWRKY6 transgenic birch and control leaves. * Represents P‐value < 0.05, ** represents P‐value < 0.01.

Figure 4

Transcriptome analysis of overexpression‐BpWRKY6 in birch. (a) Volcano plot of DEGs between overexpress line and control. (b) The Venn diagram between TFs and DEGs in birch. (c) GO enrichment analysis of DEGs in birch. (d) KEGG enrichment analysis of DEGs in birch.

Figure 5

Analysis of target genes of BpWRKY6. (a) Expression level of target genes screened from overexpressing BpWRKY6 birch. (b) LUC activity detection of BpWRKY6 and target gene promoter co‐transgenic tobacco, and standardization was carried out uniformly through the control. (c) The distribution of W‐box in the promoter of the target gene. (d) ChIP‐qPCR analysis of binding between BpWRKY6 and target genes. (e, f) Y1H analysis (e) and EMSA experiment (f) proves the combination of BpWRKY6 and W‐box. * Represents P‐value < 0.05, ** represents P‐value < 0.01.

Figure 6

Analysis of the interaction between BpWRKY6 and BpMAPK6. (a) Y2H analysis of the interaction between BpWRKY6 and BpMAPK6. (b) Pull‐down analysis of interaction between BpWRKY6 and BpMAPK6 in vitro. (c) Phosphorylation in vitro. Phos‐tag represents phosphorylation specific antibody detection. CBB represents Coomassie brilliant blue staining. (d) BpWRKY6^S253A/S288A cannot be phosphorylated by BpMAPK6 in vitro. (e) The impact of the interaction between BpMAPK6 and BpWRKY6 on their target genes, and the LUC/REN of BpMAPK6 cotransfected with BpWRKY6 and BpWRKY6 target genes were standardized using only BpWRKY6 and BpWRKY6 target genes as controls. * Represents P‐value < 0.05, ** represents P‐value < 0.01.

Figure 7

Schematic diagram of BpWRKY6 regulating the gypsy moth resistance mechanism of birch. The green arrow and dashed line propose the hypothesis of the signal transduction process, while the black arrow indicates that it has been validated.