Functional characterisation of the Ustilago maydis effector Pit2

André Nicolai Müller
Ustilago maydis, the causative agent of corn smut disease, is a biotrophic plant pathogen that relies on living plant tissue to fulfill its life cycle. This lifestyle requires an efficient suppression of the plant's immune system which is accomplished by the secretion of so-called effector proteins. One of these effectors is Pit2 (Protein important for tumors 2, Um01375). Deletion mutants for pit2 are still able to penetrate and colonize host plants but fail to induce tumor formation in
more » ... ormation in infected plants. Instead, chlorosis can be observed at infection sites, indicating that an increased activity of plant defense reactions is elicited. Aim of this thesis was the functional characterization of Pit2. Yeast-two-hybrid analyses showed that Pit2 interacts with a set of defense-associated apoplastic maize cysteine proteases (CP1A/B, CP2 and XCP2) and inhibits their activity. Sequence comparison with Pit2 orthologs from related smuts identified a conserved 14 amino acid motif (PID14), which is required for protease inhibition and interaction. This domain is essential for virulence of U. maydis. Moreover, synthetic UmPID14-peptides are able to inhibit the activity of the tested proteases and expression of PID14 in U. maydis SG200Δpit2 partially restores fungal virulence. These results indicate that Pit2 is essential for U. maydis to establish and maintain a biotrophic interaction with maize and that this is determined by the inhibition of apoplastic cysteine proteases. Furthermore it was demonstrated that Pit2 orthologs from the related smuts Ustilago hordei, Sporisorium reilianum or Melanopsichium pennsylvanicum are not functional in the U. maydis-maize pathosystem. These findings were further confirmed by showing that the orthologous PID14-regions are not able to replace the function of the UmPID14-region inside UmPit2 in planta. In [...]
doi:10.17192/z2014.0071 fatcat:zkjwyasorfcybnhoxatsz27g54