Transcriptomics-Driven Discovery of New Meroterpenoid Rhynchospenes Involved in the Virulence of the Barley Pathogen Rhynchosporium commune.

Rhynchosporium commune, the causal agent of barley scald disease, poses a major threat to global barley production. Despite its significant impact, the molecular mechanisms underlying R. commune's infection process remain largely unexplored. To address this, we analyzed the differential gene expression data of R. commune WAI453 cultivated under both in planta and in vitro conditions, aiming to identify secondary metabolite biosynthetic gene clusters that are potentially involved in the pathogenicity of R. commune. Our analysis revealed increased expression of a polyketide-terpene gene cluster (the rhy cluster), containing a specific myeloblastosis (MYB)-type transcription factor gene rhyM, during in planta growth. Overexpression of rhyM in an axenic culture activated the expression of the rhy cluster, resulting in the production of a series of new meroterpenoid metabolites, which we named rhynchospenes A-E. Their structures were elucidated through a combination of spectroscopic methods and single crystal X-ray diffraction analysis. Infiltration of rhynchospenes into barley leaves resulted in strong necrosis, with rhynchospene B demonstrating the highest phytotoxicity and causing necrosis at a minimum concentration of 50 ppm. Silencing rhyM in R. commune WAI453 confirmed the role of rhynchospenes as virulence factors in barley disease. The resulting mutant showed significantly reduced expression of the rhy cluster in planta compared to the wild-type strain and decreased virulence in seedling pathogenicity assays on barley. The characterization of the rhy cluster and rhynchospenes provided insights into the role of secondary metabolites in R. commune virulence and barley scald disease development. The study also highlights the potential use of MYB-type transcription factor overexpression in uncovering cryptic SMs involved in pathogenicity and host adaptations.