Dissecting functional roles of different chitin synthases in Trichoderma atroviride

The ascomycete Trichoderma is long known for its mycoparasitic behavior towards plant pathogenic fungi and thus biocontrol. It has recently become evident that cell wall turn over might also contribute to the mycoparasitic ability of Trichoderma spp. as these fungi substantially (re)model their cell periphery during the interaction with host fungi. Since information on the mechanisms of cell wall synthesis in filamentous fungi such as in Trichoderma spp. is still scarce, it shall be the primary focus of the proposed project. Chitin and particularly the deacetylated form chitosan are critical components of the fungal cell wall. Since they play a major role in the defense mechanism of various fungi, they might critically account for specific aspects of mycoparasitism. Hence, we will analyze the synergistic behavior of chitin and chitosan remodeling enzymes, during vegetative growth and mycoparasitism. This shall be accomplished by functional characterization (generation of knock-out lines) of genes involved in chitin biosynthesis such as chitin synthases, which determine the degree of polymerization. The required in-depth characterization of fungal chitin and chitosan formation will largely enhance our knowledge on fungal cell wall synthesis, and as such yet remains to be investigated in Trichoderma species. This is of further importance as chitin synthases are attractive therapeutic targets to battle fungal infections. Taken together, the proposal is designed to mine truly novel insights into the principles of enzymatic cell walls synthesis during vegetative growth and host attack in mycoparasitic Trichoderma species and thereby contributes, at least, to improve biological control on field.

The use of biological pesticides is an environmentally friendly and sustainable alternative to petrochemical pesticides in agriculture. The filamentous fungi Trichoderma spp. are effective, promising alternatives to chemical fungicides by parasitic destruction of plant pathogens. The ability to invade host organisms during parasitism requires adaptive remodeling of the cell wall to prevent recognition by the host and its defensive responses. The most important polysaccharides therefore are chitin, chitosan and glucan. Strategies by which the cell wall evades the host's immune system are common to all plant and human pathogens, but have not yet been investigated in mycoparasites. In this project we have shown that an abundance of chitin- and chitosan-modulating enzymes play a major role in the Trichoderma atroviride mycoparasitism. Our studies suggest that the conversion of chitin into chitosan is important in mycoparasites, in particular as a masking / defense strategy, and thus as a radical scavenger to compensate for the oxidative stress caused by the host fungus. Mycoparasitism depends on the ability to invasively feed on the host organisms and requires adaptive cell wall remodeling. In the project we describe the entire spectrum of chitin / chitosan-modifying enzymes in the mycoparasite Trichoderma atroviride with a central role in cell wall remodeling. Rapid adaptation to a variety of growth conditions, environmental stresses and host defense mechanisms such as oxidative stress depend on the interaction of these enzymes and are ultimately necessary for the success of the mycoparasitic attack. In the context of Trichoderma research, these findings about chitin and the associated polymer synthesis play a fundamentally important role in mycoparasitism. Eight chitin synthases, six chitin deacetylases, other chitinolytic enzymes, including six chitosanases, transglycosylases and accessory proteins, are involved in this complex, regulated process. In the project, systematic and biochemical classification, phenotypic characterization and in vitro assays to understand the mycoparasitic interaction were performed and the importance of chitin and chitosan in vegetative development and in biocontrol in T. atroviride was confirmed. Our results generally contribute to the understanding of the molecular mechanism of chitin synthesis in filamentous fungi and in particular in mycoparasites, with the overarching goal to further improve bio-control strategies.