Research

1) Wider research context / theoretical framework The cohesin complex mediates sister chromatid cohesion to ensure proper segregation of chromosomes. Sororin protein stabilizes cohesin on chromatin, thus enabling cohesive cohesin complexes to maintain sister chromatid cohesion from S phase until the onset of anaphase. Although sororin is a key regulator of cohesin in animal cells, sororin was thought to be absent in lower eukaryotes. 2) Hypothesis / research questions / objectives Our recent data suggest that sororin is also present in the fission yeast Schizosaccharomyces pombe. We identified previously uncharacterized S. pombe protein, namely Sor1, which shares similarities with known sororin proteins. Our recent results are consistent with the notion that Sor1 regulates cohesin in fission yeast but the function of Sor1 is not clear. The aim of this project is to test the hypothesis that this protein is a functional homolog of sororin in the fission yeast S. pombe. 3) Approach / methods Phenotypic analysis will aim to reveal whether the absence of the S. pombe Sor1 results in phenotype consistent with defects in regulation of cohesin’s function. Biochemical experiments will analyze physical interactions between the S. pombe Sor1 and cohesin as well as their localization on chromatin. 4) Level of originality / innovation Until recently, the consensus in the field was that sororin is present only in metazoans and that lower eukaryotes rely on other mechanisms that substitute for sororin’s function. Therefore, identification of yeast sororin can be seen as a breakthrough that will have an important impact on future studies of cohesin and chromosome biology. 5) Primary researchers involved As the principal investigator of this grant proposal, Dr. Juraj Gregan will be responsible for the project management and he will also perform the experimental work.

Self-resistance to trichothecenes in fungi Trichothecene mycotoxins are inhibitors of eukaryotic protein biosynthesis – they are therefore toxic to humans and animals, to plants but also to fungi. Some species, e.g. Trichothecium roseum (grape dry rot pathogen) or Trichoderma species (e.g. T. brevicompactum) produce large amounts of highly fungitoxic compounds such as trichothecin and trichodermin, respectively. The goal of the project is to get insights into the molecular mechanisms allowing the toxin producing fungi to resist to their own toxin. Several hypotheses should be tested. One is that the ribosomes of the producers show target insensitivity due to amino-acid changes in the ribosomal protein L3 as described for mutants of Saccharomyces cerevisiae with increased toxin resistance. This will be tested by functional replacement of the yeast gene with the orthologs from resistant (and susceptible) filamentous fungi. Another hypothesis is that adaptive modifications dependent on S-adenosylmethionine occur by methylation of either ribosomal RNA or certain ribosomal proteins. The effect of two candidate methyltransferases should be tested. A further hypothesis is that the fungi can cope with toxin by being able chemically modify the toxin structure. An epoxide opening GST from F. graminearum that is able to protect transformed yeast against toxicity of trichothecin and trichodermin will be characterized.

Wider research context: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave glycosidic bonds in polysaccharides, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. The process involves molecular oxygen/hydrogen peroxide and an electron donor, such as enzymes of GMC oxidoreductase family (e.g. cellobiose dehydrogenase), small-molecule reductants or photoactive pigments. Clonostachys rosea (Hypocreales, Ascomycota) is a filamentous fungus that colonizes living plants as an endophyte, and parasitizes on and kills other fungi (necrotrophic mycoparasite). Upon sequencing of the C. rosea genomes, the gene family encoding LPMOs (AA9), as well as the GMC oxidoreductase family (AA3) were found to be significantly expanded. In contrast, the genomes of the saprotrophic and mycoparasitic Trichoderma species (ecologically very similar to C. rosea) have significantly low number of AA9 and AA3 genes. Research objectives: We aim to unravel the catalytic activities and substrate specificities of C. rosea novel LPMOs, identify new structure-function aspects in combination with biochemical characterizations, and study their synergies with other native C. rosea enzymes such as GMC oxidoreductases.