title: Identification of O-mannosylated virulence factors in Ustilago maydis.
abstract: The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.
authors.affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Consejo Superior de Investigaciones Científicas, Sevilla, Spain.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Fernández-Álvarez
authors.forename: Miriam
authors.initials: M
authors.lastname: Marín-Menguiano
authors.forename: Daniel
authors.initials: D
authors.lastname: Lanver
authors.forename: Alberto
authors.initials: A
authors.lastname: Jiménez-Martín
authors.forename: Alberto
authors.initials: A
authors.lastname: Elías-Villalobos
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pérez-Pulido
authors.forename: Regine
authors.initials: R
authors.lastname: Kahmann
authors.forename: José I
authors.initials: JI
authors.lastname: Ibeas
mesh_terms: D019295:Computational Biology / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015394:Molecular Structure; D009183:Mycotoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology*; D010940:Plant Proteins / Q000378:metabolism; D040901:Proteomics; D013329:Structure-Activity Relationship; D050817:Transcription Factor Pit-1 / Q000378:metabolism; D014588:Ustilago / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003313:Zea mays / Q000382:microbiology / Q000648:ultrastructure
chemical_list: D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009183:Mycotoxins; D010940:Plant Proteins; D050817:Transcription Factor Pit-1; D037521:Virulence Factors; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase