Appressorienbildung von Ustilago maydis auf hydrophoben Oberflächen: Regulation durch Membranproteine

Ustilago maydis is the causative agent of corn smut disease. Pathogenic development is initiated by fusion of compatible cells and the formation of a filamentous dikaryon. On the plant surface U. maydis differentiates appressoria enabling the fungus to penetrate the plant epidermis. Appressorium formation is stimulated by the hydrophobicity of the leaf surface and cutin monomers. Pathogenic development of U. maydis is regulated by a conserved MAP-kinase cascade homologues to the FG (filamentous growth)-pathway in Saccharomyces cerevisae. In yeast, two transmembrane proteins, Sho1p and Msb2p, act at the head of this MAP-kinase cascade. In this study Sho1 and Msb2-related proteins in U. maydis were characterized. It could be shown that Sho1 and Msb2 are essential for virulence of U. maydis. Genetic data provided evidence that Sho1 and Msb2 act upstream of the pathogenicity-relevant MAP-kinase cascade. Furthermore, Sho1 was shown to destabilize the MAP-kinase Kpp6 through direct interaction with the unique N-terminal domain in Kpp6, indicating a role of Sho1 in fine-tuning Kpp6 activity. It could be demonstrated that Sho1 and Msb2 specifically regulate appressorium formation and are neither required for cell fusion nor for filamentous growth. Moreover, sho1 and msb2 mutants were affected in their morphological response to hydrophobic surfaces, but responded normally to cutin monomers. This suggests that Sho1 and Msb2, which localize in the plasma membrane, are involved in surface perception. The Msb2 protein, which belongs to the family of transmembrane mucins, is processed into a cellular and an extracellular fragment. Both domains are essential for function of Msb2. Since the highly glycosylated extracellular domain is shed to the environment, additional roles of Msb2 in the extracellular matrix are assumed, i.e. mediation of adhesion between filaments and surfaces. Transcriptional profiling under appressorium inducing in vitro conditions revealed that Sho1 and Msb2 are necessary for the expression of potential secreted cell wall degrading enzymes. Furthermore, secreted effectors, known to be essential for the biotrophic interaction between U. maydis and its host plant, were found to be Sho1 and Msb2 dependently expressed. This indicates that Sho1 and Msb2 prime U.maydis for biotrophic development when hyphae are growing on the plant surface. Since Sho1 and Msb2 are conserved proteins in phytopathogenic fungi, they could represent general virulence factors.