Development of an In Vivo Method in Nepeta Cataria (catnip) to Characterize the Biosynthetic Pathway behind Nepetalactone Stereoisomer Production

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 sub-family the Nepetoideae. Most members of this sub-family are well known for their diverse terpene-based natural products; however, one genus, the Nepeta, stands out amongst the Nepetoideae for its unique 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. The role these biosynthetic genes play in planta to synthesize different stereochemical ratios of nepetalactone has not been addressed. I developed a virus-induced gene silencing (VIGS) tool for N. cataria to explore the in vivo function of the putative biosynthetic genes. By simultaneously targeting a visual marker gene, magnesium chelatase subunit H (CHLH), and each member of the genes involved in the production of the various nepetalactone stereoisomers, I am able to precisely select the tissue under the knockdown phenotype of VIGS and characterize this pathway in vivo. Furthermore, using this tool, along with expression analysis tools such as qPCR and RNAseq, I aim 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.