Gene discovery in Catharanthus roseus using virus induced gene silencing
This thesis presents the use of Virus Induced Gene Silencing (VIGS) for the discovery of enzymes and transporters involved in monoterpene indole alkaloid (MIA) metabolism in the medicinal plant Catharanthus roseus. C. roseus is the source of a number of MIAs that are used as chemotherapeutic agents in the treatment of a variety of cancers, however the complete biosynthetic pathway for these metabolites remains to be elucidated. Additionally, this metabolic pathway is subcellulary compartmented with the key branch point enzyme, strictosidine synthase, localised to the plant vacuole. There is therefore a need for the import of the substrates for strictosidine biosynthesis; secologanin and tryptamine, across the vacuolar membrane, and export of the product, strictosidine, for synthesis of the downstream alkaloids. This thesis presents the identification of two proteins that act as trans-tonoplastic transporters in MIA metabolism. The multidrug and toxic compound extrusion (MATE) protein, CrMATE1952, was localised to the vacuolar membrane and silencing its expression in planta resulted in the accumulation of a secologanin derivative. This implicates CrMATE1952 in the transport of secologanin into the vacuole and highlights the importance of the spatial organisation of the pathway in preventing secologanin derivatisation. Secondly silencing the expression of a tonoplast localised nitrate/peptide (NPF) transporter, CrNPF2.9, resulted in the 20-fold accumulation of strictosidine, suggesting this transporter is the exporter of strictosidine from the vacuole. Furthermore, VIGS also allowed the identification of a reticuline oxidase like protein, CrRO, which resulted in the accumulation of two new MIAs in leaf tissue upon silencing. This thesis highlights a reverse genetics strategy for gene identification in metabolic pathways and is the first time the MATE and NPF transporters, and the reticuline oxidase like enzymes, have been shown to be involved in MIA metabolism in C. roseus.
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