Terpene Synthase Gene Family in Chinese Chestnut ( BL.) Harbors Two Sesquiterpene Synthase Genes Implicated in Defense against Gall Wasp .

Chinese chestnut ( BL.) is a well-known fruit tree that has been cultivated in East Asia for millennia. Leaves and buds of the plant can become seriously infested by the gall wasp (GWDK), which results in gall formation and associated significant losses in fruit production. Herbivore-induced terpenes have been reported to play an important role in plant-herbivory interactions, and in this study, we show that upon herbivory by GWDK, four terpene-related compounds were significantly induced, while the concentrations of these four compounds in intact buds were relatively low. Among these compounds, ()-nerolidol and (, )-α-farnesene have frequently been reported to be involved in plant herbivory defenses, which suggests direct and/or indirect functions in chestnut GWDK defenses. Candidate terpene synthase (TPS) genes that may account for ()-nerolidol and (, )-α-farnesene terpene biosynthesis were characterized by transcriptomics and phylogenetic approaches, which revealed altered transcript levels for two TPSs: , a TPS-g subfamily member, and CmNES/AFS, a TPS-b clade member. Both genes were dramatically upregulated in gene expression upon GWDK infestation. Furthermore, -mediated transient overexpression in showed that catalyzed the formation of (, )-α-farnesene, while / showed dual ()-nerolidol and (, )-α-farnesene synthase activity. Biochemical assays of the recombinant and / proteins confirmed their catalytic activity in vitro, and the enzymatic products were consistent with two of the major volatile compounds released upon GWDK-infested chestnut buds. Subcellular localization demonstrated that and / were both localized in the cytoplasm, the primary compartment for sesquiterpene synthesis. In summary, we show that two novel sesquiterpene synthase genes and are inducible by herbivory and can account for the elevated accumulation of (, )-α-farnesene and ()-nerolidol upon GWDK infestation and may be implicated in chestnut defense against GWDK herbivores.