Funktionelle Charakterisierung von Pep1, einem sekretierten Effektorprotein von Ustilago maydis

Ustilago maydis, the causative agent of corn smut disease, depends on a variety of secreted effector proteins, to establish a biotrophic interaction with its host plant maize. The deletion of the gene um01987, which encodes the effector protein Pep1, leads to a complete loss of pathogenicity. Moreover the plant reacts to penetration attempts of the Δpep1-mutant strain with the induction of different defense reactions. This results in e.g. the formation of necrotic lesion as well as in the production of reactive oxygen species at the penetration sites. Pep1 is conserved among different species of smut fungi and contains a N-terminal signal peptide, a conserved core region as well as a low conserved glycine- and serine-rich C-terminus. In the present study, the effector protein Pep1 was heterologously expressed and purified. Via biochemical and molecular biological methods, two plant interaction partners of Pep1 were identified. By Activity based protein profiling it could be demonstrated, that Pep1 specifically inhibits the activity of papaine like cysteine proteases. Particularly, the activity of papaine like cysteine proteases, induced by the salicylic acid signaling pathway, was inhibited by Pep1 very efficiently. Obviously, these proteases are critical for the induction of maize plant defense. Additionally, Pep1 binds and inhibits plant class-III-heme-peroxidases. In cooperation with Christoph Hemetsberger, the in vitro inhibition of the horseradish peroxidase (HRP) could be shown. Moreover the physical interaction of Pep1 with the HRP was demonstrated. During the Δpep1-infection, a single peroxidase, the maize peroxidase-12, is transcriptionally upregulated. Virus induced gene silencing of the peroxidase-12 lead to a partial complementation of the Δpep1-mutant phenotype on POX12si plants. Thus, silencing of peroxidase-12 leads to the suppression of plant defense reactions, such as the formation of papillae, which enables the Δpep1-mutant strain to successfully penetrate the plant tissue. Taken together these results show, that Pep1 inhibits enzymes of the basal plant defense and therefore suppressing the innate immunity of maize against U. maydis.