Analysis of the mechanisms regulating the expression of isoprenoid biosynthesis genes in hydroponically-grown Nicotiana benthamiana plants using virus-induced gene silencing

Secondary metabolites in plants play important roles in defence against biotic and abiotic stresses. Although the biosynthesis pathways of secondary metabolites have been extensively studied, the regulatory mechanism of gene expression involved in these pathways remains poorly understood. In this study, we develop a virus-induced gene silencing (VIGS) system that enables a rapid analysis of the regulatory mechanism of genes involved in the biosynthesis of isoprenoids, one of the largest groups in secondary metabolites, using hydroponically-grown Nicotiana benthamiana. Using VIGS, we successfully reduced the transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMGR1), cycloartenol synthase 1 (CAS1), sterol side chain reductase 2 (SSR2) and S-adenosyl-L-Met-dependent C-24 sterol methyltransferase 1 (SMT1) in leaf, stem and root tissues in approximately 2 weeks. We identified novel feedback and feed-forward regulation of isoprenoid biosynthesis genes when CAS1, which encodes a key enzyme involved in the biosynthesis of sterols and steroidal glycoalkaloids, was down-regulated. Furthermore, the regulation of these genes differed among different tissues. These results demonstrate that our system can rapidly analyse the regulatory mechanisms involved in the biosynthesis of secondary metabolites.