Gene Discovery and in Vivo Characterization of the Iridoid Biosynthesis Pathway in Nepeta

This thesis presents the characterization of the Nepeta iridoid biosynthetic pathway via gene discovery and the development of a functional genomics tool for in planta genetic characterization. The Lamiaceae plant family, colloquially known as the mint family, is well known for its chemical diversity and economical importance, especially amongst members of the Nepetoideae sub-family. Most members of this sub-family are well known for their diverse terpene-based natural products; however, one genus, Nepeta, is unique amongst the Nepetoideae for its ability to produce nepetalactone, an iridoid-scaffold compound known for its psychoactive effect on cats and potential use as a bio-based pest control in agriculture due to its influence on various insect species. Chemical profiling on Nepeta spp. and within varieties of a single species have revealed the production of different nepetalactone stereoisomers varies widely across plants. Previous work has identified Nepeta spp. biosynthetic enzymes that can synthesize different stereoisomers of nepetalactones in vitro. Work in this thesis also presents the gene discovery and biochemical characterization of the early steps of this pathway (Chapter 2). The role these biosynthetic genes play in planta leading to the synthesis of different stereochemical ratios of nepetalactone has not been addressed. This thesis presents the development of a virus-induced gene silencing (VIGS) tool for N. cataria (Chapter 3) to explore the in vivo function of the putative biosynthetic genes (Chapter 4). Simultaneously targeting a visual marker gene, magnesium chelatase subunit H (ChlH), and the genes involved in the production of the various nepetalactone stereoisomers, allows the precise selection of the tissue under the knockdown phenotype of VIGS and characterisation of this pathway in vivo. Furthermore, VIGS provides the possibility to untangle the mechanisms behind isomer regulation and gene expression in nepetalactone production, as well as to understand the effect of this pathway on other physiological processes.