Functional analysis of the RHOIII and 14-3-3 proteins of Trichoderma reesei: Dissertation

The filamentous fungus Trichoderma reesei is a biotechnologically important organism having an excellent capability to secrete hydrolytic enzymes e.g. cellulases.Allthough the protein production technology is well established, the protein secretion apparatus of T. reesei is still insufficiently known. To isolate and characterise genes involved in secretion in T. reesei, a complementation screening in a sec15-1 Saccharomyces cerevisiae strain with a T. reesei expression cDNA library was performed.Sec15p is a component of the exocyst complex that is essential for the orientation of the secretion pathway and thus for polarised growth and secretion.Two signal transduction genes encoding the RHOIII and a truncated 14-3-3 protein (FTT1DC) were obtained as suppressors of the yeast sec15-1 mutation.Two full-length FTT genes (FTTI and FTTII) were also cloned from T. reesei but they did not suppress this mutation. Several other genetic interactions between late-acting secretory genes of S. cerevisiae and T. reesei rho3 or ftt1DC were also detected and this further implies the involvement of these genes in regulation of protein secretion.The expression of rho3 gene was shown to rescue the growth of mutations in genes encoding other exocyst components and Sec1p that is a regulator of secretory vesicle docking to the plasma membrane.Ftt1DC appeared to interact with genes involved in vesicle targeting along actin cables in yeast. These T. reesei genes were also able to suppress the secretion defects of sec15-1 or sec2-41 strains of S. cerevisiae.The full-length ftt1 enhanced the secretion of invertase in a wild type yeast although it could not suppress the growth of the yeast secretion mutations.The ftt2 did not have any clear effect on secretion in yeast.Moreover, expression of ftt1 slightly retarded the growth of wild type yeast cells while expression of ftt2 enhanced it. The rho3 gene was disrupted in the T. reesei genome.The disruptants were able to grow as well as the parental strain in media with either glucose or cellobiose as the carbon source.However, in a medium with cellulose as the carbon source both growth and secretion were reduced in the disruptants.The morphology or actin organisation was not affected by the disruption.Therefore, the rho3 gene would appear to be more involved in the regulation of secretion than morphogenesis in T. reesei.