Virulence in smut fungi: Insights from evolutionary comparative genomics

Fungi and plants show a long history of co-evolution since about 400 million years. This lead to the development of diverse types of interactions which include for example parasitism, in which fungi reduce the fitness of their host. Parasitic fungi can establish biotrophic interactions, which require living plant tissues for successful colonization. To establish biotrophy, fungi secret effectors, which are proteins that prevent or mediate plant immune responses. They can also contribute to virulence by changing the host physiology towards the needs of the pathogen. Effectors and their plant targets evolve in a molecular arms race, where both pathogen and plants evolve new effectors and plant interactors, respectively. In this process, single nucleotide polymorphisms and species-specific orphan genes can play an important role. Smut fungi (order: Ustilaginales) are biotrophic pathogens, which parasitize mostly sweet grasses, including wheat, oat, barley, maize, sugar cane and Sorghum grass. The genomes of five related species with different host plants or colonization strategies (Ustilago hordei, Ustilago maydis, Sporisorium scitamineum, Sporisorium reilianum f. sp. zeae and S. reilianum f. sp. sorghi) were sequenced. Furthermore, methods allowing geneitc manipulations were developed, which makes this group of smut fungi an interesting model system for studying virulence and/or host specificity. The aim of the present work was to investigated to which extent positively selected or species-specific effectors contribute to virulence of the respective species. To detect positive selection, families of homologous proteins were built. Positive selection was then inferred by applying a non-homogenous branch model of sequence evolution. Most genes under positive selection were found in both formae speciales of S. reilianum. A role in virulence could be shown for sr10529 in S. reilianum f. sp. zeae. This gene is orthologous to pit2 of U. maydis, where it encodes an inhibitor of cysteine proteases. To get insights in differences in the inhibition of maize cysteine proteases by Pit2-orthologues, a yeast-2-hybrid assay was conducted In contrast to the expectaion that Pit2-orthologues of maize pathogens can better interact with maize cysteine proteases compared to Pit2 of the Sorghum pathogen, no host/pathogen-specific interaction could be observed. Besides this, a contribution to virulence could be demonstrated for three gene clusters containing positively selected genes inS. reilianum f. sp. zeae. Besides positively selected genes, species-specific orphan genes were bioinformatically identified. Most candidates could be detected in Pseudocyma flocculosa. Deletion of the orphan gene um02193 in U. maydis did not reveal a contribution to virulence for this protein