Identifizierung von Effektoren der Pheromon-MAPK-Kaskade in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis, the causative agent of corn smut disease, sexual development is initiated by the fusion of two compatible haploid sporidia. The resulting filamentous dikaryon is then able to infect the host plant maize. These processes are genetically controlled by the two mating type loci a and b. The biallelic a-locus encodes a pheromone/ pheromone receptor system, which controls cell-cell recognition and fusion of compatible sporidia. Subsequent filamentous growth and pathogenic development are regulated by the multiallelic b-locus. This locus codes for two homeodomain proteins, bE and bW, which heterodimerize to generate an active transcription factor when derived from different alleles. During cell-cell recognition the pheromone signal triggers the formation of conjugation hyphae and the enhanced expression of the a- and b- mating type genes. Basal as well as pheromone-induced expression of these genes require the HMG domain transcription factor Prf1. Consequently, prf1 deletion mutants are sterile and non-pathogenic. To perform its function Prf1 needs to be activated by a conserved cAMP-cascade and a conserved MAP kinase module. Both pathways activate Prf1 on the post-transcriptional level. In addition, activation of the MAP kinase cascade results in the elevated transcription of prf1 and triggers the formation of conjugation hyphae. Interestingly, previous studies have shown that prf1 is dispensable for this morphological transition. These results pointed towards the existence of additional effectors of the pheromone MAP kinase cascade in U. maydis. In this thesis, rop1 and hmg3 encoding two additional HMG domain transcription factors were identified. Like Prf1, they belong to the sequence-specific class of this protein family. While hmg3 deletion strains are slightly impaired in mating and pathogenicity and show reduced expression of the pheromone gene, these strains developed conjugation hyphae in response to pheromone. In contrast to that the deletion of rop1 results in a complete loss of pheromone-induced gene expression and conjugation hyphae formation. Analyses of strains that express a constitutively active variant of the pheromone MAPK kinase Fuz7 showed that rop1 is still required for the transcription of prf1 and prf1-induced genes when the need for the perception of a pheromone signal is bypassed. This approach also indicated that rop1 affects conjugation tube formation only indirectly. Since constitutive expression of prf1 complements the mating and conjugation defects of rop1 deletion strains, rop1 appeared to be an essential regulator of prf1 gene expression. Indeed, electrophoretic mobility shift assays demonstrated that Rop1 directly binds to the prf1 promoter in vitro and led to the identification of a specific DNA binding motif, which is present in a number of additional putative promoter regions of the U. maydis genome. Furthermore, Northern analyses revealed that rop1 gene expression is positively regulated by the pheromone MAP kinase cascade, while active cAMP signalling represses rop1 transcription. Surprisingly, on the plant surface rop1 deletion strains do form conjugation hyphae and express sufficient prf1 to cause full pathogenicity. This indicates the involvement of addititional components in the regulation of prf1 gene expression during pathogenic growth.