Die Rolle von RNA-bindenden Proteinen bei der pathogenen Entwicklung von Ustilago maydis

Ustilago maydis is the causative agent of corn smut disease. Successful infection of corn plants by this phytopathogenic basidiomycete is dependent on various morphological transitions including pheromone-induced conjugation hyphae formation, formation of a dicaryotic filament after fusion of two compatible haploid sporidia and the branched mycelial growth after plant penetration. In previous analysis 24 open reading frames coding for putative RNA-binding proteins could be identified using a bioinformatic candidate gene approach. On this basis the effect of 18 genes coding for proteins that harbouring PUM-, KHD- and RRM domains, has been studied using a revers-genetic approach. Gene deletion mutants were generated and subsequently characterised by extensive phenotypic analysis. Three genes could be identified having an influence on development of U. maydis designated khd1, khd4 and rrm4. Deletion of khd1 leads to cold sensitivity, whereas deletion of khd4 influences morphology of the haploid sporidia, growth rate, pheromone response as well as pathogenicity. Deletion of rrm4 leads to a loss of fast polar growth and reduced virulence. Rrm4 harbours three modules of RRM-type RNA-binding domains. The RRM domain architecture shows similarity to ELAV-like (embryonic lethal abnormal vision) proteins. In addition to the RNA-binding domains, Rrm4 contains a C-terminal PABC domain (poly[A]-binding protein C-terminal domain), that is known as a protein interaction interface. Rrm4 accumulates PABC-dependent in cytoplasmic particles that move bidirectionally along the microtubule cytoskeleton. Investigating the mechanism of Rrm4 particle movement, an indirect influence of conventional kinesin Kin2 of U. maydis could be shown. Therefore, a participation of another plus-end directed kinesin motorprotein as well as the minus-end directed dynein motorprotein is expected. By analysis of strains expressing site-mutagenised Rrm4 proteins RNA binding is expected to be carried out by the first and second RRM domains. The importance of Rrm4 for pathogenic development and its movement along the microtubule cytoskeleton leads to the assumption that this protein plays a crucial role in RNA transport during pathogenic development of U. maydis.