Plants are resistant to the majority of potential pathogenic microbes. Adapted pathogens can however overcome plant defense and induce susceptibility. The molecular processes underlying this adaptation are only partially understood. Obligate biotrophic pathogens, which require a living host for growth and reproduction, establish especially intimate relationships with their plant hosts. A crucial aspect of this lifestyle is the formation of a specialized infection structure termed the haustorium. Haustoria are believed to represent pivotal sites of nutrient uptake and deliver effectors, proteins that manipulate the host cell during infection to promote susceptibility. While the effector arsenal of pathogenic bacteria has been investigated intensively, the repertoires and host targets of fungal effectors are currently underexplored. The work presented here thus aims at characterizing virulence mechanisms employed by the obligate biotrophic Ascomycete Golovinomyces orontii, the causal agent of the powdery mildew disease in Arabidopsis thaliana (hereafter Arabidopsis). To this end, the haustorial transcriptome of G. orontii was obtained by pyrosequencing of a cDNA library generated from isolated haustorial complexes. Transcripts coding for gene products with roles in protein turnover, detoxification of reactive oxygen species and fungal pathogenesis were abundant, while surprisingly transcripts encoding presumptive nutrient transporters were not highly represented in the haustorial cDNA library.
A substantial proportion (~38%) of transcripts encoding predicted secreted proteins comprised effector candidates. These candidates were cloned and found to frequently suppress induced plant cell death. A subset of effectors enhanced bacterial virulence and could suppress callose deposition, indicating a role in defense suppression. Transcript profiling of these effectors suggested their sequential delivery during pathogenesis. Furthermore, subcellular localization revealed diverse target compartments in the host. In a complementing approach, a large-scale yeast 2-hybrid (Y2H) assay was performed on the 84 cloned effector candidates and revealed convergence onto 61 potential host targets. These targets were enriched in transcription factors and components involved in development and cellular trafficking. Bimolecular fluorescence complementation assays confirmed the interaction of selected effectors with their host interactors. Finally, the Y2H targets of effectors were used to construct an integrated protein-protein interaction network of Arabidopsis and the three adapted pathogens Pseudomonas syringae (Psy), Hyaloperonospora arabidopsidis (Hpa) and G. orontii. This network revealed pathogen-specific as well as nine common host targets. These common targets are highly connected in the Arabidopsis cellular network. After the development of suitable quantitative methods, the important role of these common targets in the Arabidopsis immune response was validated by screening respective T-DNA insertion lines. In sum, my work supports the hypothesis that pytopathogenic microbes target hubs in the host cellular network to promote susceptibility. The effector targets identified will therefore form the basis of subsequent effector research in G. orontii
