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Abstract

The plant pathogen Ustilago maydis is the causative agent of maize smut disease and serves as a model system to study plant-fungal interactions. In this pathogen, a mitogen-activated protein kinase (MAPK) cascade controls mating, invasive growth and virulence on maize plants. The key players for infection-related processes and pathogenicity are the conserved mitogen-activated protein kinases (MAPKs) Kpp2 and Kpp6. Specifically, the MAP kinase Kpp2 is involved in appressorium development while Kpp6 is required for penetration of plant epidermal cells. Neither for Kpp2 nor for Kpp6 have the immediate downstream phosphorylation targets been identified. The aim of this work was to identify crucial virulence factors which act downstream of the MAP kinases Kpp2 and Kpp6. To artificially induce MAP kinase signaling we used the strain FB1fuz7DD in which a constitutive active allele of the MAP kinase-kinase Fuz7 (Fuz7DD) is expressed under the control of an arabinose inducible promoter. Using a phospho-proteomic approach we detected phosphorylated proteins upon induction of the MAP kinase cascade in the presence and absence of kpp2 and kpp6. Enrichment of phosphorylated proteins involved a two-step chromatographic procedure, using Al(OH)3-based metal oxide affinity chromatography (MOAC), tryptic digestion of enriched phospho-proteins, and TiO2-based MOAC for phospho-peptide enrichment. LC-MS/MS analysis of the phospho-peptide fraction yielded 111 potential MAP kinase substrates that were differentially phosphorylated in strains FB1fuz7DD and FB1Δkpp6Δkpp2fuz7DD. Fifteen of these differentially phosphorylated proteins, that could possibly be targets of Kpp2 and Kpp6, were selected for further studies based on extensive bioinformatic analysis. To assess a possible contribution of the selected genes to mating and virulence, the respective genes were deleted in a solopathogenic strain and for some of the genes also in compatible haploid strains. Analysis of the respective deletions strains showed that, um12335 was required for virulence. Subsequent studies suggest that Um12235 is a microtubule-associated protein that is a direct substrate of the MAP kinase Kpp2 and/or Kpp6.