IN-DEPTH FUNCTIONAL CHARACTERIZATION OF GMC OXIDOREDUCTASES IN CLONOSTACHYS ROSEA

Enzymes from the glucose-methanol-choline structural family of flavoproteins (GMC oxidoreductases) are often linked to enzymatic mechanisms of lignin and lignocellulose degradation involving oxidative enzymes. Such mechanisms are well known and described in basidiomycetes, mainly for white rot, but also for brown rot fungi, but are largely absent in Ascomycota, which are generally non-ligninolytic. Clonostachys rosea is a filamentous ascomycete that colonizes living plants, digests material in soil and is known as a parasite of other fungi and of nematodesa member of Hypocreales, one of the largest orders of filamentous ascomycetes with a wide range of ecological specializations from plant pathogens, saprophytes, animal pathogens to mycoparasites. In the genome of C. rosea, the gene family encoding of GMC oxidoreductases was found to be significantly expanded, with a particularly large number of genes encoding aryl alcohol oxidases. Ecologically similar fungi like the saprotrophs Neurospora crassa and Trichoderma reesei and the mycoparasitices Trichoderma atroviride and Trichoderma virens showedexhibit a reduced content of such genes, suggesting that the respective enzymes are not essential for these fungal life-styles. This poses the question why C. rosea contains this many orthologs in its genome, and what is their function, as this fungus is neither a prominent lignocellulose decomposer nor a plant pathogen, that would demand such an arsenal of genes for (ligno)cellulose degradation. TheUsing transcriptomic, molecular and biochemical techniques the aim of this studyour project is to elucidate the functionality and role of C. rosea GMC oxidoreductases, and identify the nutritional strategies they are involved in. Our research plan includes a study on the expression of GMC oxidoreductase genes, to identify strongly expressed and regulated ones, an investigation of the functional significance of GMC oxidoreductases in C. rosea (including inactivation of single and multiple genes using state-of-the-art CRISPR/Cas9 engineering tools) and biochemical characterization of selected GMC oxidoreductase genes that encode functional enzymes. Our large-scale approach will also provide important insights in the regulation of gene families supporting different life-styles in C. rosea and highlight mechanisms of mycoparasitism and fungus-plant interactions.