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The phytopathogenic fungus Ustilago maydis, the causal agent of corn smut disease, displays a complex life cycle, which is linked to distinct morphological changes. In its haploid form U. maydis divides by budding and is non-pathogenic. After fusion of two compatible haploid cells the pathogenic dikaryon is formed which grows filamentously. Compatibility is genetically regulated by two mating type loci. The biallelic a locus controls recognition and fusion while the multiallelic b locus regulates filamentous growth and pathogenic development. To exert their regulatory function the bE and bW homeodomain proteins encoded by the b locus have to dimerize. A prerequisite for this is that they are derived from different alleles. The a locus encodes pheromone precursor and receptor genes that allow recognition and fusion with non-self partners. In response to the pheromone signal, conjugation tubes are formed and pheromone-responsive gene expression is elevated. Among the induced genes are the pheromone, the pheromone receptor as well as the b genes. Transcriptional activation and basal expression of these genes require the HMG-protein Prf1. Interestingly, Prf1 activity is controlled by two signalling pathways, a cAMP and a MAP-kinase cascade. In previous studies it could be shown that the MAP-kinase Kpp2 and the MAPK-kinase Fuz7 are involved in regulating the mating process. However, these components could not be placed in one cascade.
In this thesis kpp4 encoding a MAPKK-kinase was identified and shown to be necessary for the pheromone response of U. maydis. Use of epistasis and biochemical analyses made it possible to demonstrate that Kpp4, Fuz7 and Kpp2 act in one cascade which transmits the pheromone signal. Disrupting signalling in the Kpp4/Fuz7/Kpp2-cascade results in loss of conjugation tube formation and pheromone-responsive b gene expression. In contrast, switching on the cascade by expression of dominant-active alleles of fuz7 or kpp4 stimulates conjugation tube formation and pheromone-responsive gene expression. Moreover, Fuz7 interacts with and activates Kpp2 which phophorylates Prf1 in vitro. Finally, pheromone stimulation increases Kpp2 kinase activity. Interestingly, Prf1 is dispensable for conjugation tube formation while it is essential for pheromone-responsive gene expression. Hence, the pheromone-signalling route bifurcates downstream of Kpp2.
In addition to the Kpp4/Fuz7/Kpp2 module cAMP-signalling is involved in transmission of the pheromone signal. The cAMP-signalling cascade controls the pheromone-responsive a gene expression as well as the process of cell-cell fusion. These data provide evidence that in U. maydis two independent signalling cascades transmit the pheromone signal, whereas in related fungi only one module is involved.
In this examination it could also be demonstrated that the Kpp4/Fuz7/Kpp2 MAPK module plays a crucial role during pathogenic development of U. maydis even in situations in which cell-cell fusion is not required. Detailed studies suggest that signalling via this MAPK module is responsible for development of the appressoria, which are formed by the infectious dikaryotic hyphae to penetrate the leaf surface.