A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon-VIGS) approach to study specialized metabolism in medicinal plants

作者：Yongliang Liu, Ruiqing Lyu, Joshua J. Singleton, Barunava Patra, Sitakanta Pattanaik, Ling Yuan · 发表于：Plant Methods · 年份：2024 · DOI：10.1186/s13007-024-01154-x · 被引用次数：14 · 研究领域：Plant tissue culture and regeneration、Plant Virus Research Studies、Toxin Mechanisms and Immunotoxins

BACKGROUND: Virus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites. RESULTS: We evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGB...