Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata
17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) are abundant and potent anti-herbivore defense metabolites in Nicotiana attenuata whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 members of family-1 UGTs. Tissue-specific time-course transcriptional profiling revealed that the transcripts of three UGTs were highly correlated with two HGL-DTG key biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLSs role in HGL-DTG biosynthesis was confirmed by virus-induced gene-silencing. Silencing the UDP-rhamnosyltransferase, UGT91T1, indicated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. UGT74P3 and UGT74P5 stably silenced plants were severely developmentally deformed, suggesting a phytotoxic effect of 17-HGL. Applications of synthetic 17-HGL and silencing of these UGTs in HGL-DTG-free plants confirmed the phytotoxic effect of 17-HGL. Feeding assays with Manduca sexta larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation is a critical step that contributes to the metabolites defensive function and solves the autotoxicity problem of this potent chemical defense.