Functional characterization of the Ustilago maydis effector protein Ten1

Ustilago maydis, the causal agent of corn smut disease, is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the host plant is largely governed by a plethora of secreted effector proteins, many of which are encoded in gene clusters. The deletion of cluster 10A consisting of 10 effector-encoding genes results in strongly reduced virulence after maize seedling infection. In the present study, the gene UMAG_03744 (termed ten1) could be identified as a major virulence factor of gene cluster 10A. Via quantitative reverse transcription PCR an induction of ten1 during the biotrophic development of the fungus was detected. ten1 deletion strains showed a virulence phenotype mainly reflected by a reduced tumor size on seedling leaves. Moreover, by complementing the cluster 10A deletion strain for ten1, the strong virulence defect of the cluster mutant was partially rescued. After overexpression in U. maydis hyphae, secreted Ten1 protein could be detected in axenic culture supernatant. Furthermore, using immunoelectron microscopy, the translocation of secreted Ten1 to plant cells could be shown after maize seedling infection, manifested by a significant accumulation of the protein in the plant cytoplasm and especially in plant nuclei. Through a yeast two-hybrid screen ZmPP26, a type 2C maize protein phosphatase (PP2C) could be identified as interaction partner of Ten1. This interaction was supported by coimmunoprecipitation experiments after transient co-expression of Ten1 and ZmPP26 in Nicotiana benthamiana. Moreover, ZmPP26 could be detected by mass spectrometry after immunoprecipitation of Ten1 from U. maydis-infected leaf tissue. Via yeast two-hybrid assays the ZmPP26-interacting domain of Ten1 was mapped. The engineered protein Ten1m, harboring amino acid substitutions in the interacting domain, showed no interaction with ZmPP26 in yeast two-hybrid assays. By complementing the cluster 10A deletion strain with Ten1m, the virulence defect of the cluster mutant could not be rescued, suggesting that the interaction of Ten1 and ZmPP26 may be biologically relevant