Transcriptomics-DrivenDiscovery of New MeroterpenoidRhynchospenes Involved in the Virulence of the Barley Pathogen <i>Rhynchosporium commune</i>

<i>Rhynchosporium commune</i>, the causal agent of barley scald disease, poses a major threat to global barley production. Despite its significant impact, the molecular mechanisms underlying <i>R. commune</i>’s infection process remain largely unexplored. To address this, we analyzed the differential gene expression data of <i>R. commune</i> WAI453 cultivated under both <i>in planta</i> and <i>in vitro</i> conditions, aiming to identify secondary metabolite biosynthetic gene clusters that are potentially involved in the pathogenicity of <i>R. commune</i>. Our analysis revealed increased expression of a polyketide-terpene gene cluster (the <i>rhy</i> cluster), containing a specific myeloblastosis (MYB)-type transcription factor gene <i>rhyM</i>, during <i>in planta</i> growth. Overexpression of <i>rhyM</i> in an axenic culture activated the expression of the <i>rhy</i> 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 <i>rhyM</i> in <i>R. commune</i> WAI453 confirmed the role of rhynchospenes as virulence factors in barley disease. The resulting mutant showed significantly reduced expression of the <i>rhy</i> cluster <i>in planta</i> compared to the wild-type strain and decreased virulence in seedling pathogenicity assays on barley. The characterization of the <i>rhy</i> cluster and rhynchospenes provided insights into the role of secondary metabolites in <i>R. commune</i> 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.