Charakterisierung der Tryptophan-abhängigen Pigmentsynthese am Modellorganismus Ustilago maydis und Identifizierung von Wirtsspezifitätsfaktoren in den phytopathogenen Brandpilzen Ustilago maydis und Sporisorium reilianum
Im ersten Teil der vorliegenden Arbeit wurde am Modellorganismus Ustilago maydis der Tryptophan-abhängige Biosyntheseweg untersucht, der zur Generierung von Indolpigmenten mit biologischen Effekten führt, die erstmals für den Pityriasis versicolor-assoziierten Hautpilz Malassezia furfur nachgewiesen...
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Table of Contents: In the first part of the thesis the model organism Ustilaog maydis was used to study the tryptophan dependent biosynthesis that leads to the generation of indole pigments with biological effects. These indole pigments were originally found in the fungus Malassezia furfur associated with the human skin disease pityriasis versicolor. Using a reverse genetic approach it could be shown that the tryptophan aminotransferase Tam1 represents the only enzyme responsible for pigment biosynthesis. Heterologously expressed and purified Tam1 from U. maydis catalyzed the conversion from tryptophan and phenyl pyruvate into indole pyruvate and phenyl alanine, respectively. Using a forward genetic approach mutants were identified that were incapable of producing pigments in the presence of tryptophan. These mutants were affected in the sulphite reductase gene sir1. In vitro experiments with purified Tam1 showed that also 2-oxo-methylthiobutanoate, the oxoacid of methionine can serve as a substrate for Tam1, thereby coupling tryptophan deamination to sulfur metabolism. The experiments show that indole pyruvate in an aqueous environment spontaneously reacts (either alone or in combination with tryptophan) to form numerous colourful compounds. At least four compounds could be detected that had been described for M. furfur and seem to be associated with biological effects. Experiments with human keratinocytes showed that indole pyruvate dependent biological effects such as impaired proliferation could be directed via the human aryl hydrocarbon receptor. In the second part of the thesis candidate genes for host specificity from Sporisorium reilianum were identified using genome comparison. S. reilianum exists in two varieties (SRM and SRS) of which SRM produces spores in the flower of maize and SRS produces spores in the flower of sorghum. To identify factors in S. reilianum that support virulence on maize and at the same time reduce virulence on other host plants, genes present in the genomes of the maize pathogens SRM and U. maydis were tested for absence in the genomes of related pathogens that have different hosts. Thereby one gene from S. reilianum was identified, sr13864, that presumably codes for a secreted protein. Using RT-PCR it was shown that sr13864 is expressed during the biotrophic phase. U. maydis strains in which the homolog of sr13864, um00823, was deleted, showed reduced virulence on maize. While expression of um00823 in the um00823 deletion mutant could fully complement the virulence defect, heterologous eexpression of sr13864 in the um00823 deletion mutant could only partially restore the virulence. Expression of sr13864 in SRS led to an enhanced defence reaction on sorghum along the vacular bundles as could be deduced from the production of red spots. These red spots most likely represent 3-Deoxyanthocyanidine phytoalexins. Thus, sr13864 seems to be a host specificity factor with a dual function as virulence factor for maize and avirulence factor for sorgum.