Research

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.

The funds of the third BiMM funding period will be used to complete the ongoing work in the field of new bioactive substances and to establish an insect screening procedure and for “exploratory research”, which will then serve as the basis for further research applications to other funds. The research plan contains an “open part” for the topics ‘insect screening’ and “exploratory work” as well as a series of work packages for the more detailed characterization of the 5 candidates for new bioactive substances. One of them is BiMM20 (bimmycin), which comes from a new fungal species isolated in a BiMM project and is already well characterized. It has a fungicidal effect. One of the four other molecules has an antibacterial effect against Gram+ organisms; further research is being carried out into its mode of action. Another molecule can break the resistance of bacteria to chloramphenicol and thus restore sensitivity to this important antibiotic. Another molecule from a new, previously undescribed species of fungus from BiMM's own collection has a strong phytotoxic effect. The identity of this molecule and its properties are also being determined in this BiMM III project. The last of the five new molecules has already been tested in collaboration with the FH IMC Krems and found to have anti-inflammatory properties. Funding from the BiMM III project is also required to finance further characterization with a view to medical application. In addition to the specific studies on the new metabolites and their activities, the funds will also be used for exploratory research aimed at establishing new screening systems within BiMM. This includes, for example, the possibility of testing extracts and molecules for their insecticidal effect with a higher throughput.