A WRKY transcription factor, PgWRKY4X, positively regulates ginsenoside biosynthesis by activating squalene epoxidase transcription in Panax ginseng

Ginsenosides are important metabolites synthesized by the traditional Chinese medicinal plant, Panax ginseng. The market demand for ginsenosides is increasing. Fungal elicitor is effective for improving ginsenosides biosynthesis. Here, we isolated an effective fungal elicitor, Chaetomium globosum from Panax notoginseng. Total saponins content in adventitious roots of P. ginseng was 3.94 times higher than that in control under C. globosum treatment. Correspondingly, the antioxidant activity of C. globosum-stressed roots is significantly increased than control adventitious roots and cultivated ginseng roots. In this study, PgWRKY4X, a novel pathogen-related gene encoding WRKY transcription factor from P. ginseng was isolated and functionally characterized. PgWRKY4X was responsive to the treatment of C. globosum. Subcellular localization assay indicated PgWRKY4X located in the nucleus. Electrophoretic mobility shift assay showed PgWRKY4X binds to the W-box of squalene epoxidase (PgSE) promoter. The interaction between PgWRKY4X and PgSE was discovered by glutathione S-transferase pull-down assay. Homology modeling and molecular docking was also performed to reveal the putative spatial interaction between PgWRKY4X and PgSE. Overexpression of PgWRKY4X in P. ginseng transgenic cells could significantly enhance ginsenosides accumulation by comprehensively upregulating ginsenosides biosynthetic genes, especially PgSE. Our study suggested that PgWRKY4X may be a potential target for metabolic engineering of ginsenosides biosynthesis in P. ginseng.