The molecular basis of symptom formation in Sporisorium reilianum
Zhao, Yulei; Schirawski, Jan (Thesis advisor); Panstruga, Ralph (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2015)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2015
The fungal pathogen Sporisorium reilianum parasites maize at the seedling stage, then replaces the cob with spores, which causes a huge loss in corn production. Former studies showed that deletion of the gene cluster 19A in S. reilianum dramatically reduces virulence and leads to wilting of inoculated leaves. This study focussed on the identification of effectors in cluster 19A that modulate symptom formation of S. reilianum. Several effector-encoding genes were identified by subdeletion analysis of cluster 19A that affected virulence of the fungus on maize. However, only slight changes in virulence were observed in most single gene deletion mutants, possibly suggesting functional redundancy of effectors in S. reilianum. One effector (vag2, virulence associated gene 2) could be identified whose individual deletion led to a significant reduction in spore formation and an increase in leaf tip death of inoculated maize. Bioinformatic analysis predicted that Vag2 carries a signal pepetide for secretion at the N-terminus. A Vag2:GFP fusion did not complement the vag2 deletion phenotype. However, GFP fluorescence was observed around the fungal cells, indicating that Vag2:GFP may be secreted by the fungus. The vag2 deletion mutants colonized leaves and ears of its host plant, but also induced H2O2 production during penetration, and hyperproliferated at the penetration site. Yeast two-hybrid analysis identified different maize proteins as potential Vag2 interaction partners, including defense response proteins and metabolic enzymes. Interaction of Vag2 with the maize chorismate mutase (ZmCm2) in planta was confirmed by bimolecular fluorescence complementation. ZmCm2 is predicted to function in the production of prephenate from chorismate that is also a precursor for salicylic acid (SA). Interaction of Vag2 with ZmCm2 may alter the SA level in the host. Although no significant differences in SA level of wild-type and vag2 deletion mutant-colonized leaves and ears could be observed, expression of the SA indicator genes PR1 and PR5 was induced in tissues colonized with vag2 deletion strains. Collectively, these data suggest that the S. reilianum effector Vag2 is secreted from fungal hyphae, may enter host cells, and may interfere with the host hormone balance, which suppresses host defense and supports full virulence of S. reilianum on maize.