Characterization of two glycosyltransferases that modulate withanolide biosynthesis and defense in Withania somnifera.

Background: The medicinal properties of Ashwagandha (Withania somnifera) are attributed to the presence of triterpenoid withanolides and their glycosylated forms, withanosides. Withanosides are proposed to be formed from withanolides by the action of glycosyltransferases (GTs).

Methods And Results: Two genes encoding GTs (WsGT4 and WsGT6) from W. somnifera were identified and characterized using Escherichia coli expressed recombinant proteins and in-planta studies. Biochemical assays with recombinant proteins showed that WsGT4 catalyzed product formation using withanolide A, withanolide B, withanone, and 12-deoxywithastramonolide as substrates and UDP-glucose serving as the glucose donor. While, WsGT6 catalyzed the product formation only with withaferin A as a substrate employing either UDP-glucose or UDP-galactose as sugar donors. Quantitative real-time analysis showed that transcripts of WsGT4 and WsGT6 were induced in response to methyl jasmonate treatment and prominent in leaves as compared to other tissues. Modulating the expression of WsGT4 and WsGT6 by virus-induced gene silencing (VIGS) and transient overexpression significantly affected the levels of withanolides and withanosides in the leaves. Furthermore, silencing either WsGT4 or WsGT6 in W. somnifera reduced the tolerance to Pseudomonas syringae DC3000 growth, while their overexpression enhanced the tolerance to the bacterium.

Conclusions: Our results indicate the role of WsGT4 and WsGT6 in withanoside biosynthesis and in defense against a bacterial pathogen in W. somnifera. These GTs could be utilized for targeted modulation of withanolides in cell cultures or at the whole-plant level and for enhancing tolerance against bacterial pathogens and improving the yield of withanosides.