HFB7 - A novel orphan hydrophobin of the Harzianum and Virens clades of Trichoderma, is involved in response to biotic and abiotic stresses.

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity.

COMPARATIVE PHYSIOCHEMICAL ANALYSIS OF HYDROPHOBINS PRODUCED IN ESCHERICHIA COLI AND PICHIA PASTORIS.

Hydrophobins (HFBs) are small surface-active proteins secreted by filamentous fungi. Being amphiphilic, they spontaneously form layers that convert surfaces from hydrophilic to hydrophobic and vice versa. We have compared properties of the class II HFB4 and HFB7 from Trichoderma virens as produced in Escherichia coli and Pichia pastoris. Since the production in E. coli required denaturation/renaturation steps because of inclusion bodies, this treatment was also applied to HFBs produced and secreted in yeast. The protein yields for both systems were similar. Both HFBs produced by E. coli proved less active on PET compared to HFBs produced in P. pastoris. HFBs produced in E. coli decreased the hydrophilicity of glass the most, which correlated with the adsorption of a more dense protein layer on glass compared to HFBs produced in P. pastoris. The hydrophobins produced in P. pastoris formed highly structured monolayers. Layers of hydrophobins produced in E. coli were less prone to self-organization. Our data suggests that irrespective of the production host, the HFBs could be used in various applications that are based on their surface activity. However, the production host and the subsequent purification procedure will influence the stability of HFB layers. In the area of high-value biomedical devices and nanomaterials, where the formation of highly ordered protein monolayers is essential, our results point to P. pastoris as the preferred production host. Furthermore, the choice of an appropriate hydrophobin for a given application appears to be equally important.