Funktionelle Charakterisierung des Rum1-Proteins aus dem phytopathogenen Pilz Ustilago maydis

Ustilago maydis is a dimorphic fungus with a yeast-like non-pathogenic form and a filamentous (hyphal) pathogenic form that induces tumor formation in maize. The transition from yeast to hyphal form entails several steps: formation of conjugation tubes by haploid sporidia of opposite mating type, fusion of the cells to form a dikaryon, and finally filamentous (hyphal) growth of the dikaryon. This transition is regulated by two mating type loci, a and b, and requires that mating partners carry different alleles at both of these loci. The biallelic a-locus encodes a pheromone/pheromon-receptor system that is responsible for the recognition of sporidia of different mating type, the formation of konjugation tubes and the fusion of the sporidia. The multiallelic b-locus encodes a pair of unrelated homeodomain proteins, bE and bW, that interact to form a heterodimer. The bE-bW-heterodimer is a transcriptional regulator that is essential for the biotrophic stage of the fungus. In a screen for components of the b-dependent regulatory cascade, the genes rum1 and hda1 could be identified, that both play a role in the repression of b-regulated genes in the absence of a bE-bW-heterodimer. The hda1 gene encodes a histone deacetylase (HDAC), and for Rum1 the domain structure suggests, similar to Hda1, a role in chromatin mediated transcriptional regulation. The deletion of either gene leads to a discrete block during teliospore development. Database searches revealed similarities between Rum1 and the RBP2 protein (retinoblastoma binding protein 2) in mouse and humans. The Rum1 protein, as well as RBP2, includes a highly conserved DNA-binding motif, termed ARID-domain, three PHD-domains, that are responsible for protein-protein interactions, a C5HC2 zincfinger motif and a JmjC- and a JmjN-domain. Based on these data, it was hypothesized that Rum1 and Hda1, in the saprophytic stage of U. maydis, are part of a common complex that could be a direct antagonist of the b heterodimer. Three questions arouse from this hypothesis, that should be investigated: 1. Which of the conserved domains are essential for the function of Rum1? 2. Are Rum1 and Hda1 components of a common protein-complex during the saprophytic stage of U. maydis? 3. Is the Rum1-Hda1-complex a direct antagonist of the b-heterodimer? Functional analyses of domain deletion mutants revealed, that the JmjC-domain is essential for the function of Rum1. Further on, a Coimmunoprecipitation showed, that during the saporphytic stage of U. maydis, Rum1 and Hda1 are part of a common complex. By the use of DNA-Microarray analyses, 286 deregulated genes in the rum1 deletion mutant and 486 genes in the hda1 deletion mutant could be identified. More than 50% of these genes were coregulated in both strains. Comparison of the genes regulated by Rum1, Hda1 and the b-heterodimer revealed that only few b-regulated genes are regulated by Rum1 and/or Hda1. Therefore it is unlikely that the Rum1-Hda1-complex is a direct, global antagonist of the b-heterodimer.