Charakterisierung eines Ustilago maydis Genclusters, das für drei neuartige sekretierte Effektoren kodiert

For the biotrophic interaction with its host plant maize the corn smut fungus U. maydis depends on a variety of novel secreted effector proteins. A significant number of the corresponding genes are arranged in clusters and their expression is induced specifically during biotrophic growth (Kämper et al., 2006). This study focuses on the characterization of the gene cluster 5B. Deletion of this cluster results in a complete loss of virulence symptoms in maize infections (Kämper et al., 2006). Cluster 5B encodes three novel proteins that are predicted to be secreted via canonical N-terminal signal peptides. Deletion analyses revealed that only one of the genes, stp1, is responsible for the phenotype. Since stp1 is also present in other closely related smut fungi this effector may represent a general pathogenicity factor. Microscopic analyses revealed that stp1 deletion mutants arrest directly after penetration in the maize epidermis. Furthermore, a strong plant defense response is elicited by stp1 deletion mutants, that is characterized by the formation of papillae, H2O2, induction of pr genes and finally by death of infected plant cells. This suggests that Stp1 is able to suppress plant defense reactions. Using promoter:gfp fusions stp1 expression was found to be induced during penetration and persists during growth in the maize epidermis. Since there were no bioinformatic hints towards a function of Stp1, domain analyses were performed. It could be shown that the central glycine-rich region of Stp1 is not needed for protein function while the N- and C-terminal parts are essential during biotrophic growth. Investigation of culture supernatants revealed that Stp1 is secreted in a glycosylated form by fungal hyphae. However, during expression in haploid sporidia the protein is processed by the Kex2 protease. These results demonstrate that Stp1 secretion is controlled at multiple levels including transcriptional as well as post-transcriptional mechanisms. Using confocal microscopy Stp1 fusion proteins could be localised to the apoplastic interaction zone of infected plant cells. Five intracellular plant proteins were identified to interact with Stp1 in a yeast-two hybrid screen. After transient expression in Nicotiana benthamina Stp1 lacking the signal peptide specifically localized to sub-compartments of the nucleus. Moreover, one of the interactors co-localized with Stp1 lacking the signal peptide after expression in N. benthamiana. These results might indicate that Stp1 is transferred to the plant cell where it is involved in regulation of defense responses.