Das AUACCC-bindende Protein Khd4 kontrolliert Morphogenese und Pathogenität in Ustilago maydis

The phytopathogenic basidiomycete Ustilago maydis is the causative agent of corn smut disease. To penetrate the plant surface of its host plant Zea mays formation of a dikaryotic filament is obligatory. Only at this stage tumors are formed. U. maydis is, exclusive of pathogen/host-interaction, also ideal for, e.g. investigations in DNA repair or RNA biology. Initial characterizations regarding the influence on RNA-binding proteins on pathogenicity indicated that post-transcriptional regulation plays a key role in the infectious phase. So far, only a few RNA-binding proteins in filamentous fungi could be investigated concerning regulatory functions. In this thesis, the role of the RNA-binding protein Khd4 in U. maydis was analyzed in detail. Deletion of khd4 leads to a pleiotropic phenotype resulting in a cytokinesis defect of haploid sporidia, in disturbed filamentous growth and in severely reduced tumor formation. To investigate the influence of Khd4 on cell morphology and pathogenicity characterizations of RNA/protein interaction and identification of putative target mRNAs were determined. In this dissertation, it was shown that the tandem-KH-domains 3 and 4 are essential for function of Khd4 and for RNA binding in vivo. Point mutations of essential amino acids within these domains caused khd4-deletion phenotype and inhibited binding to AUACCC-containing RNAs using the yeast three- hybrid system. The presence of the cis-acting motif AUACCC is thereby necessary and sufficient. In order to investigate whether or not the motif AUACCC is a regulatory RNA element in U. maydis two independent approaches were performed: (1) microarray analysis revealed that AUACCC is enriched in differentially expressed mRNAs (29 out of 79 differentially expressed mRNAs); (2) bioinformatic analysis uncovered that AUACCC is significantly enriched in first 150 nucleotides (nt) of 3untranslated region (UTR) of mRNAs. Additionally, the experimentally determined 3UTR lengths confirmed the enrichment of AUACCC. Furthermore, the majority of differentially expressed mRNAs exhibited increased mRNA-amounts in khd4-deletion strain leading to the assumption that Khd4 might play a role in destabilizing mRNAs. The partial co-localization of Khd4 and the processing body (P-body) marker protein Dhh1 supported this hypothesis. P-bodies are known to be sites for mRNA degradation. The 29 AUACCC-containing mRNAs represent target mRNAs of Khd4, which possibly can be connected to the khd4-deletion phenotype. Hence, RNA-binding of Khd4 seems to be essential to post-transcriptionally regulate AUACCC-containing target mRNAs, e.g. via degradation in P-bodies. Thus, Khd4-dependent regulation is an important process for accurate development of cell morphology and pathogenicity in U. maydis.