Dokument

Summary:
Ustilago maydis is a biotrophic fungal pathogen that causes smut disease in its host plant maize. During colonization, U. maydis secretes effector proteins to suppress plant defense responses and manipulate the host physiology for its own benefit. The majority of these proteins lack functional annotations and their role in virulence remains to be determined. Transcriptional profiling defined a set of effectors whose expression is linked to the developmental stage in which biotrophy is established. Systematic deletion of these effectors identified three mutants that were no longer able to cause disease. Mutants of these three effectors, named stp2, stp3 and stp4 (stop after penetration), were still able to form appressoria and penetrate the plant, but arrested in the epidermal cell layer. The arrest was accompanied by plant defense responses, including a disruption of the plant plasma membrane surrounding the fungal hyphae. A similar phenotype was observed for the previously described effectors stp1 and pep1. All five effectors are highly conserved among related smut fungi infecting different hosts, suggesting a essential function. Using live cell confocal microscopy, in vitro and in vivo assays, it could be demonstrated that Stp2, Stp3, and Stp4 are secreted by the fungal hyphae, but are not translocated into the plant cell. Confocal microscopy of mCherry fusion strains revealed that all five essential effectors form a speckled pattern on the surface of the biotrophic hyphae. Co-immunoprecipitation/mass spectrometry experiments using each of these essential effectors revealed that Stp1, Stp3, Stp4 and Pep1 form an effector complex. The four complex members did not interact with Stp2 or plant proteins. Recent experiments suggest that Stp2 interacts with at least two other U. maydis effectors, which have a virulence phenotype comparable to stp2 deletion strains. Attempts to visualize the Stp1, Stp3, Stp4 and Pep1 effector complex through bimolecular fluorescence complementation (BiFC) interfered with the complex formation and caused a complete loss of virulence. The subsequent overexpression of two BiFC-fragment tagged complex members in the wild type allele resulted in a dominant negative phenotype. This provides evidence that not only the presence of the individual complex members, but the formation of the complex itself is necessary for a successful colonization. However, using full-length versions of fluorescent proteins enabled the co-localization of complex members and located them in the speckles. Finally, interaction studies in Saccharomyces cerevisiae confirmed the formation of the complex and demonstrated pairwise interactions and subcomplex formations among the complex members. Based on these results, it is proposed that the effector complex forms a structure, whose function is essential for the virulence of U. maydis and could be used as a potential drug target in the future.

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