A barley gene cluster for the biosynthesis of diterpenoid phytoalexins

Phytoalexins are specialized metabolites that are induced upon pathogen infection and contribute to the defense arsenal of plants. Maize and rice produce multiple diterpenoid phytoalexins and there is evidence from genomic sequences that other monocots may also produce diterpenoid phytoalexins. Here we report on the identification and characterization of a gene cluster in barley ( Hordeum vulgare cv. Golden Promise) that is involved in the production of a set of labdane-related diterpenoids upon infection of roots by the fungal pathogen Bipolaris sorokiniana . The cluster is localized on chromosome 2, covers over 600 kb and comprises genes coding for a (+)-copalyl diphosphate synthase (HvCPS2), a kaurene synthase like (HvKSL4) and several cytochrome P450 oxygenases (CYPs). Expression of HvCPS2 and HvKSL4 in yeast and Nicotiana benthamiana resulted in the production of a single major product, whose structure was determined to be of the cleistanthane type and was named hordediene. Co-expression of HvCPS2, HvKSL4 and one of the CYPs from the cluster (CYP89E31) afforded two additional products, hordetriene and 11-hydroxy-hordetriene. Both of these compounds could be detected in extracts of barley roots infected by B. sorokiniana , validating the function of these genes in planta . Furthermore, diterpenoids with multiple oxidations and with molecular masses of 316, 318 and 332 were induced in infected barley roots and secreted in the medium, indicating that additional oxidases, possibly from the same genomic cluster are involved in the production of these phytoalexins. Our results provide the basis for further investigation of the role of this gene cluster in the defense of barley against pathogens and more generally in the interaction with the microbiome. ### Competing Interest Statement The authors have declared no competing interest.