Hydrophobins: the vital invention of fungi

Higher filamentous fungi decompose organic matter, interact with other organisms and produce metabolites, many of which are used by humankind in industry and medicine. They rely on absorptive nutrition and require a large surface area. Therefore, their body is shaped as a network of thin and long filaments, or hyphae. The tubular lifestyle, however, brings such biological problems as overexposure to the environment, the loss of water/nutrients and vulnerability to pathogens and abiotic stresses. Thus, the physicochemical properties of the hyphal surface are essential for the adaptation of a fungus to its ecological niche. In this project, we will focus on hydrophobins (HFB) that are small cysteine-rich proteins secreted exclusively by filamentous microbes. The amphiphilic nature of these proteins and their superior surface activity allow them to self-assemble in monolayers at liquid/air and liquid/solid interfaces to change the interface surface energy (hydrophobicity) and chemistry. This project hypothesizes that HFBs are crucially important for fungal fitness because they can modulate the physiochemical properties of the hyphal surface and thus play a role in the establishment of a fungus in its ecological niche. We propose using the genus Trichoderma because these fungi are ecologically versatile and have a high diversity of HFBs. The study will consist of (i) an in-depth molecular evolutionary analysis of HFBs in Trichoderma and related fungi. It will reveal possible mechanisms that resulted in the enrichment of HFB- encoding genes in Trichoderma; (ii) the homology modeling and heterologous production of the selected extant and resurrected Trichoderma HFBs. We will investigate the properties of HFBS and engineer of tailor-made HFBs; (iii) the physiochemical characterization of the produced HFBs by the standard proteomic techniques and with the use of a comprehensive toolkit of physiochemical and surface chemical methods; (iv) the analysis of HFB-gen regulation in different conditions of growth in the presence of plants, fungi, bacteria and on a diversity of natural polymers, at various developmental stages and under stress conditions. (v) The functional molecular biological analysis of the selected HFB-encoding genes in at least two Trichoderma spp. by gene deletion, gene overexpression, and expression of fluorescently tagged HFBs for microscopic detection. The applicability of the CRISPR/Cas9 system for hfb silencing will be tested. Thus, the novelty of our approach is the establishment of a systems biological view on HFBs in a given fungus using a combination of tools from fungal genomics, molecular biology, and physical chemistry. We hope to resolve the involvement of HFBs in the ecophysiological adaptations of fungi, i.e. in fungal fitness. This project will also lead to the detection of HFBs with unknown properties that may then be used for a diversity of applications in industry and medicine.