The Sesquiterpene Synthase PtTPS5 Produces (1S,5S,7R,10R)-Guaia-4(15)-en-11-ol and (1S,7R,10R)-Guaia-4-en-11-ol in Oomycete-Infected Poplar Roots

Abstract

Pathogen infection often leads to the enhanced formation of specialized plant metabolites that act as defensive barriers against microbial attackers. In this study, we investigated the formation of potential defense compounds in roots of the Western balsam poplar (Populus trichocarpa) upon infection with the generalist root pathogen Phytophthora cactorum (Oomycetes). P. cactorum infection led to an induced accumulation of terpenes, aromatic compounds, and fatty acids in poplar roots. Transcriptome analysis of uninfected and P. cactorum-infected roots revealed a terpene synthase gene PtTPS5 that was significantly induced upon pathogen infection. PtTPS5 had been previously reported as a sesquiterpene synthase producing two unidentified sesquiterpene alcohols as major products and hedycaryol as a minor product. Using heterologous expression in Escherichia coli, enzyme assays with deuterium-labeled substrates, and NMR analysis of reaction products, we could identify the major PtTPS5 products as (1S,5S,7R,10R)-guaia-4(15)-en-11-ol and (1S,7R,10R)-guaia-4-en-11-ol, with the former being a novel compound. The transcript accumulation of PtTPS5 in uninfected and P. cactorum-infected poplar roots matched the accumulation of (1S,5S,7R,10R)-guaia-4(15)-en-11-ol, (1S,7R,10R)-guaia-4-en-11-ol, and hedycaryol in this tissue, suggesting that PtTPS5 likely contributes to the pathogen-induced formation of these compounds in planta.

Isotopic Labeling Experiments Solve the Hedycaryol Problem

“…Together with the results from the NMR analyses discussed as follows, the previously tentatively identified product 7 of HcS 23 must be reassigned as 1. On the basis of the known absolute configurations of 8 and 9 24 5 this first isolation paper shows the wrong absolute configurations for 1 and 2, despite the fact that the absolute configuration of 2 had been clarified 4 years before 38 ). These data confirmed the structural reassignment for the HcS product because the previously assigned 7 is known to form cis-isoelemol through Cope rearrangement, 14 a stereoisomer of 2, but not 2.…”

“…Plant sesquiterpene synthases (STPSs) converting farnesyl diphosphate (FPP) into 1 as the main product are known from Populus trichocarpa (PtTPS7), Camellia brevistyla (CbTPS1), and Liquidambar formosana (LfTPS01), whereas the 2 Z ,6 E stereoisomer 7 was tentatively assigned by GC/MS as the product of a bacterial STPS from Kitasatospora setae (HcS), in which case the crystal structure has also been obtained . We have recently identified another STPS from P. trichocarpa (PtTPS5) that produces a mixture of (1 S ,5 S ,7 R ,10 R )-guaia-4­(15)-en-11-ol ( 8 ) and (1 S ,7 R ,10 R )-guaia-4-en-11-ol ( 9 ) and studied the cyclization mechanism that proceeds through ( R )- 1 . Now we report on site-directed mutagenesis experiments on PtTPS5 yielding inter alia an enzyme variant (PtTPS5-C403A) that selectively produces ( R )- 1 .…”

“…Together with the results from the NMR analyses discussed as follows, the previously tentatively identified product 7 of HcS must be reassigned as 1 . On the basis of the known absolute configurations of 8 and 9 the product of PtTPS5-C403A can be assigned as ( R )- 1 . The optical rotation of the HcS product, [α] D 25 = −21.3 ( c 0.29, C 6 H 6 ), in comparison with that for ( R )- 1 from H. angustifolia , [α] D 25 = +30.8 (CHCl 3 ), pointed to the absolute configuration of ( S )- 1 for the bacterial compound.…”

The establishment ofPopulusxLaccaria bicolorectomycorrhiza requires the inactivation of MYC2 coordinated defense response with a key role for root terpene synthases

“…By contrast, little is known about the role(s) of terpenes produced by roots on the belowground microbial communities (54). The products of PtTPS16 an PtTPS21, in addition to the product of the sesquiterpene synthase PtTPS5, have previously shown inhibitory activity against oomycete ingress in poplar roots (37, 55). Other previously characterized poplar TPSs play a role in leaf herbivore defense (56, 57), although our results suggest that they could also be involved in root defense against fungi.…”

Taxonomic Insights and Its Type Cyclization Correlation of Volatile Sesquiterpenes in Vitex Species and Potential Source Insecticidal Compounds: A Review

“…Studies showed that the gene expression in Aquilaria species was up-regulated, encoding δ-guiene synthase in response to mechanical injury and MeJa treatment and inducing δ-guaiene production [126,187,188]. Recently, transcriptome analysis of western aspenbalsam infected roots (Populus trichocarpa) by Phytophthora cactorum (Oomycetes) revealed the induction of the PtTPS5 gene, forming the compounds (1S, 5S, 7R, 10R)-guaia-4(15)-en-11-ol and (1S, 7R, 10R)-guaia-4-en-11-ol [189].…”