Fig. 8: Virus-induced gene silencing in W. somnifera confirms the role of the biosynthetic gene cluster for withanolide biosynthesis.

a Construct design to co-silence all homologues of target genes and the visual marker gene phytoene desaturase (PDS). b Representative pictures of W. somnifera plants infiltrated with silencing constructs four weeks after infiltration. c Quantitative reverse transcription polymerase chain reaction analysis of target gene expression in silenced leaves. The bar plot shows mean ± SEM and data points of three biological replicates. d Relative quantification of withaferin A levels in silenced plants. The bar plot shows mean ± SEM and individual data points; the sample size of replicates showing successful silencing as judged by a photobleaching phenotype is shown above the bars. e, f Representative GC-MS chromatograms showing accumulation of intermediates in silenced plants (target gene plus PDS) in comparison to PDS-GFP-silenced plants. Chromatograms were normalised by leaf sample dry weight. Comparable data was obtained in two independent experiments. Compounds: 24-methylenecholesterol (1), 24-methyldesmosterol (2), ergosta-5,25-diene-3β,24ξ-diol (physalindicanol A) (7), ergosta-5,24(28)-diene-3β,25-diol (physalindicanol B) (8), (22R)-ergosta-5,24-diene-3β,22-diol (10), (22R)-ergosta-5,24(28)-diene-3β,22,25-triol (phyministerol A) (12), (22R)-ergosta-5,24-diene-1α,3β,22-triol (14), (22R)-ergosta-5,24(28)-diene-1α,3β,22,25-tetrol (15). Source data are provided as a Source Data file.