Sweet potato is among the top 10 most cultivated crops globally and is valuable for not just avariety of food products but also for hunger elimination efforts worldwide. Despite its yieldbeing affected by numerous pests and pathogens, its defense metabolites are poorly understood.Here we focus on a hyperdiverse class of its defense compounds called resin glycosides (RGs).Despite its vast structural diversity and known pesticidal/herbicidal activities, the core RGbiosynthetic enzymes are completely unknown. We recently developed metabolomic,computational and functional genomic tools to probe RG diversity and characterize itsbiosynthesis, which identified multiple candidate genes. In proposed research, we will (Objective1)apply these tools to survey 55 diverse accessions from the USDA sweet potato germplasmcollection and associate RG levels with their previously scored insect/nematode susceptibilities.(Objective2) To prioritize genes for functional validation, we will perform RNA-seq from high- andlow-RG cultivars as well as proteomics from sweet potato roots. (Objective3) Role of predictedcandidates in RG biosynthesis will be validated through in vitro and in vivo assays, including viapathway reconstruction in Nicotiana benthamiana. These experiments will identify high-RGlines for sweet potato breeding, generate hypotheses about RG relationship with pestsusceptibility, and enable reconstitution of this pathwayin other crops. Foundational knowledgeof this metabolite class with herbicidal/pesticidal activities will contribute to discoveries tosafeguard sweet potato and the long-term demand for sweet potato-based products.