Characterization of defensive cadinenes and a novel sesquiterpene synthase responsible for their biosynthesis from the invasive Eupatorium adenophorum

• Eupatorium adenophorum is a malignant invasive plant possessing extraordinary defense potency, but its chemical weaponry and formation mechanism have not yet been extensively investigated. • We identified six cadinene sesquiterpenes, including two volatiles (amorpha-4,7(11)-diene and (–)-amorph-4-en-7-ol) and four nonvolatiles (9-oxo-10,11-dehydroageraphorone, muurol-4-en-3,8-dione, 9-oxo-ageraphorone and 9β-hydroxy-ageraphorone), as the major constitutive and inducible chemicals of E. adenophorum. All cadinenes showed potent antifeedant activity against a generalist insect Spodoptera exigua, indicating that they have significant defensive roles. • We cloned and functionally characterized a sesquiterpene synthase from E. adenophorum (EaTPS1), catalyzing the conversion of farnesyl diphosphate to amorpha-4,7(11)-diene and (–)-amorph-4-en-7-ol, which were purified from engineered Escherichia coli and identified by extensive nuclear magnetic resonance (NMR) spectroscopy. EaTPS1 was highly expressed in the aboveground organs, which was congruent with the dominant distribution of cadinenes, suggesting that EaTPS1 is likely involved in cadinene biosynthesis. Mechanical wounding and methyl jasmonate negatively regulated EaTPS1 expression but caused the release of amorpha-4,7(11)-diene and (–)-amorph-4-en-7-ol. Nicotiana benthamiana transiently expressing EaTPS1 also produced amorpha-4,7(11)-diene and (–)-amorph-4-en-7-ol, and showed enhanced defense function. • The findings presented here uncover the role and formation of the chemical defense mechanism of E. adenophorum – which probably contributes to the invasive success of this plant – and provide a tool for manipulating the biosynthesis of biologically active cadinene natural products.