Regulation des Kpp2/Kpp6-MAPK-Moduls in Ustilago maydis durch Phosphatasen

ation of the Kpp2/Kpp6 MAPK Module in U. maydis by Phosphatases In the phytopathogenic fungus Ustilago maydis a conserved Mitogen-activated-protein-kinase (MAPK) module is required for sexual and pathogenic development. The central MAPK of this module, Kpp2, regulates the transcriptional and morphological responses to pheromone. Kpp2 and the related MAP kinase Kpp6 (involved in plant penetration) are activated via phosphorylation by the MAPK kinase Fuz7 upon perception of the pheromone signal. However, it is still unknown how these kinases are deactivated and return to their inactive state. In the studies underlying this thesis the role of Rok1 (Regulator of Kpp2) annotated as a dual specificity phosphatase (DSP) in U. maydis has been analysed. It could be shown that rok1 mRNA level are increased upon pheromone stimulation. rok1 deletion strains show increased filamentation and induce dramatically increased pathogenicity symptoms compared to wild type infections. In line with these results, overexpression of rok1 reduces conjugation hyphae formation and abolishes mating. Furthermore, rok1 overexpression strains were severely reduced in pathogenicity, similar to kpp2 deletion strains. These results demonstrate a role for rok1 in pheromone signaling, most likely by controlling Kpp2 and Kpp6 phosphorylation status and hence activity. Western analysis revealed that rok1 indeed controls the phophorylation of Kpp2 and Kpp6. In line with this result, the deletion of rok1 partially suppressed the pathogenicity defect of kpp2 and kpp6 mutant strains. In experiments using an appressorial-marker strain (SG200AM1) repression of appresoria-formation by rok1 was demonstrated in hyphae developing on the leaf surface as well as by using an in vitro system. Localization assays using Rok1 fused to the red fluorescent mCherry protein showed a membrane-associated localization of Rok1 in budding cells. After stimulating cells with compatible pheromone a transition of the DSP towards structures that could represent endoplasmatic reticulum was observed. Furthermore a second DSP, Mkp2 (MAP kinase phosphatase 2) was identified. Mkp2 is also involved in filamentation and pathogenic development of U. maydis. In contrast to the situation in Drok1 strains, the deletion of mkp2 caused a filamentation defect in the solopathogenic SG200 strain and lead to reduced virulence. This suggests that Mkp2 and Rok1 have distinct substrates.