Das Effektorprotein Pep1 und seine Rolle in der Biotrophie von Brandpilzen

For establishment of the biotrophic interaction with its host plant maize, the corn smut fungus Ustilago maydis depends on a variety of secreted effector proteins. A deletion of um01987, the gene encoding for the secreted effector Pep1 (protein essential during penetration 1), leads to a complete loss of pathogenicity. Penetration attempts of the deletion mutant elicit strong plant defense responses such as the accumulation of reactive oxygen species (ROS), papilla formation and necrotic lesions. Pep1 localizes in the biotrophic interface and is conserved within different smut fungi. This study focuses on the functional characterization of Pep1 with biochemical and molecular biological methods. It was demonstrated that heterologously expressed Pep1 protein from Escherichia coli inhibits the oxidative burst in maize. Treatment of maize plants with the ROS scavenger ascorbate led to reduced defense responses in reaction to Δpep1 infections. In addition, Pep1 was shown to inhibit activity of a class III peroxidase from horseradish in vitro. Class III peroxidases play a pivotal role in ROS production during the oxidative burst. In maize plants, inoculated with the pep1 deletion mutant, the secreted peroxidase 12 (POX12) is transcriptionally induced. Bimolecular fluorescence complementation and yeast-2-hybrid experiments revealed a direct interaction of Pep1 with POX12. Virus induced gene silencing of pox12 in maize partially restored the Δpep1 phenotype. Silencing of pox12 led to reduced plant defense responses, such as papilla formation at penetration sites. As a consequence, hyphae of the pep1 mutant penetrated epidermal cells of pox12 silencing plants. Furthermore, orthologs of pep1 in additional smut species were identified. In vitro studies showed peroxidase inhibition by Pep1 orthologs from Ustilago avenae, Ustilago nuda and Melanopsichium pennsylvanicum. Additionally, inhibition of apoplastic peroxidases from different host plants by Pep1 was demonstrated. Maize infection studies with U. maydis confirmed the biological function of the identified pep1 orthologs. In summary, these results suggest a role of Pep1 as inhibitor of peroxidases, a core component of the basal plant defense response.