Die Auswirkung der posttranslationellen Aktivierung des Transkriptionsfaktors Prf1 auf die Pheromonantwort in Ustilago maydis

Prerequisite for successful infection of the phytopathogenic fungus Ustilago maydis are recognition and fusion of two compatible sporidia on the plant surface. In this process the reciprocal pheromone stimulation constitutes a critical signal which is transmitted within the cell via at least two signal transduction pathways, a MAP kinase cascade (MAPK) and a cAMP-dependent protein kinase A (PKA). Target protein of both pheromone-activated kinases is the transcription factor Prf1 which regulates the expression of many factors crucial for the subsequent developmental steps. Using mutation analyses it could be shown that the respective MAPK and PKA phosphorylation sites in Prf1 are essential for its activation. Interestingly, both kinases influenced the function of this transcription factor by different means. In particular, the phosphorylation pattern on Prf1 seemed to allow to discriminate between the promoters of the genes of both mating type loci. In this thesis, the tetracycline-regulated expression system could be established as an important extension of the promoter repertoire available for U. maydis. Tetracycline-mediated regulation of the prf1 gene confirmed utilisability and efficiency of this heterologous system. First indications concerning the mechanism underlying the pheromone-mediated activation of Prf1 could be gained from localisation studies as well as by uncoupling its functional modules. These experiments indicated that DNA binding properties as well as transactivation potential could be potential targets of this regulation. Characterisation of constitutively activated Prf1 variants verified the functional relevance of PKA phosphorylation. Subsequent microarray analyses allowed a transcriptome-wide investigation of the impact of PKA and MAPK modification on Prf1. Thereby, it was possible to define destinct regulatory classes within the pheromone-regulated genes whose expression is directed via the phosphorylation status of Prf1. While transcriptional induction of the prf1 gene is sufficient to induce e. g. expression of factors implicated in pheromone maturation and secretion, MAPK or PKA phosphorylation determine the regulation of destinct subclasses. These results emphasise that the transcription factor Prf1 is capable to alter its target promoter specificity on a posttranslational level. This represents a novel concept of crosslinking different signal transduction pathways during the fungal pheromone response.