Funktionelle Analyse des Ustilago maydis Effektorproteins Tin3 im Gencluster 19A

Ustilago maydis is a fungal plant pathogen, which causes smut disease in maize. The pathogenic development of all smut fungi is coupled to sexual development, which is initiated by the fusion of two compatible haploid cells with different mating types (Bölker et al., 1992). This leads to the formation of a dicaryotic filament that is able to penetrate plant cells with a special infection structure called appressorium (Feldbrügge et al., 2004). During this process the host plasma membrane invaginates and encases the fungal hyphae. A special interacting zone between plant and fungus is created. Biotrophic pathogens like U. maydis are dependent on the survival of their host plants. Therefore, it is essential for the pathogen to avoid or suppress recognition by the plant immune system in order to establish a compatible interaction (Doehlemann et al., 2008b). To accomplish this interaction with its host plant U. maydis depends on a variety of novel secreted proteins. In this study the U. maydis effector Tin3 was characterised that contributes significantly to tumor formation and interacts with a plant cysteine protease and the plant autophagy protein Beclin1 in yeast two-hybrid-assays. Expression studies showed that tin3 is highly and specificly upregulated during the biotrophic phase. Functional studies revealed a dual function of Tin3 via interaction with two maize proteins. After secretion of Tin3 into the apoplastic interaction zone it inhibits defense related secreted maize cysteine proteases. In vitro protease activity assays combined with in vivo studies showed that Tin3 is able to inhibit cysteine protease Mir3 and closely related proteases via binding to the active site. The biological relevance of this function was shown in experiments that were able to partially complement the virulence phenotype of the tin3 mutant by protease inhibitors. Furthermore, a second function of Tin3 as autophagy repressor could be postulated, which was revealed by a yeast assay and in planta stainings of autophagosomes. Expression of four plant autophagy genes was induced after deletion of tin3, a finding that supports inhibition of autophagy by Tin3. H2O2 accumulations in infections with the tin3 deletion mutant indicated that the Tin3 effector actively participates in suppression of plant defense responses via its dual function and is therefore needed during the establishment of biotrophy.