Transient gene expression of recombinant terpenoid indole alkaloid enzymes inCatharanthus roseus leaves

We developed a transient expression assay for Madagascar periwinkle (Catharanthus roseus [L.] G. Don.) that is based on vacuum infiltration of intact leaves with recombinantAgrobacterium tumefaciens. This simple and rapid technique was used to overexpresstryptophan decarboxylase (tdc) andstrictosidine synthase (str1) genes, which encode 2 key enzymes of the terpenoid indole alkaloid (TIA) biosynthesis pathway. Immunoblot analysis of crude leaf extracts demonstrated that recombinant TDC and STR1 accumulated to detectable levels when targeted to their native subcellular compartments (i.e., the cytosol and vacuole, respectively) or to the chloroplast. In this article, we discuss possible applications of the transient assay in studies on the overexpression of enzymes of the TIA pathway in intactC. roseus leaves.