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Identification of O-mannosylated virulence factors in Ustilago maydis.

Fernández-Álvarez A, Marín-Menguiano M, Lanver D, Jiménez-Martín A, Elías-Villalobos A, Pérez-Pulido AJ, Kahmann R, Ibeas JI - PLoS Pathog. (2012)

Bottom Line: We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4.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.Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Consejo Superior de Investigaciones Científicas, Sevilla, Spain.

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.

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Msb2 is a Pmt4 substrate in U. maydis.A. Domain architecture of the Msb2-HA-GFP fusion protein. The HA-epitope is integrated at amino acid 709 and the C-terminus is fused to GFP. The Ser/Thr rich region as well as the predicted proteolytic cleavage site is indicated. B. Western Blot analysis of Msb2-HA-GFP isolated from SG200Δmsb2/msb2-HA-GFP (WT) and two independent clones of SG200Δmsb2Δpmt4/msb2-HA-GFP (#1 and #2, respectively). SG200 was used as a control. The first gel (above) contains 6% of polyacrylamide and α-HA antibody was used to detect the N-terminal part of Msb2. The other gel (below) contains 10% of polyacrylamide and was treated with α-GFP antibody to detect the C-terminus of Msb2. Equal amounts of proteins of total cell extracts were loaded in each lane.
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ppat-1002563-g002: Msb2 is a Pmt4 substrate in U. maydis.A. Domain architecture of the Msb2-HA-GFP fusion protein. The HA-epitope is integrated at amino acid 709 and the C-terminus is fused to GFP. The Ser/Thr rich region as well as the predicted proteolytic cleavage site is indicated. B. Western Blot analysis of Msb2-HA-GFP isolated from SG200Δmsb2/msb2-HA-GFP (WT) and two independent clones of SG200Δmsb2Δpmt4/msb2-HA-GFP (#1 and #2, respectively). SG200 was used as a control. The first gel (above) contains 6% of polyacrylamide and α-HA antibody was used to detect the N-terminal part of Msb2. The other gel (below) contains 10% of polyacrylamide and was treated with α-GFP antibody to detect the C-terminus of Msb2. Equal amounts of proteins of total cell extracts were loaded in each lane.

Mentions: In S. cerevisiae, Msb2p is cleaved upstream of the transmembrane domain releasing an extracellular N-terminal part and a cellular C-terminal fragment [45]. To analyse the Msb2 protein in U. maydis we used a differentially tagged protein, which was C-terminal fused to GFP and carried an internal HA-epitope between amino acids 709 and 710 in the extracellular region (Figure 2A). The msb2-HA-GFP construct was placed under the control of the otef promoter and integrated into the ip locus of SG200Δmsb2. Western blot analysis revealed that the Msb2 protein is processed into two distinct fragments. The extracellular domain, detected by the anti-HA-antibody, migrated at a molecular weight of >250 kDa, while the anti-GFP-antibody detected a product of approximately 65 kDa. The size of the latter fragment allowed us to predict a cleavage site, situated between the Ser/Thr rich region and the transmembrane domain (Figure 2). In S. cerevisae, cleavage of Msb2 results in the secretion of the N-terminal extracellular domain [45]. Similar to the situation in S. cerevisiae we could detect the extracellular N-terminal domain of U. maydis Msb2 in the culture supernatant, while the C-terminal fragment was exclusively detected in the cellular fraction (Figure S5).


Identification of O-mannosylated virulence factors in Ustilago maydis.

Fernández-Álvarez A, Marín-Menguiano M, Lanver D, Jiménez-Martín A, Elías-Villalobos A, Pérez-Pulido AJ, Kahmann R, Ibeas JI - PLoS Pathog. (2012)

Msb2 is a Pmt4 substrate in U. maydis.A. Domain architecture of the Msb2-HA-GFP fusion protein. The HA-epitope is integrated at amino acid 709 and the C-terminus is fused to GFP. The Ser/Thr rich region as well as the predicted proteolytic cleavage site is indicated. B. Western Blot analysis of Msb2-HA-GFP isolated from SG200Δmsb2/msb2-HA-GFP (WT) and two independent clones of SG200Δmsb2Δpmt4/msb2-HA-GFP (#1 and #2, respectively). SG200 was used as a control. The first gel (above) contains 6% of polyacrylamide and α-HA antibody was used to detect the N-terminal part of Msb2. The other gel (below) contains 10% of polyacrylamide and was treated with α-GFP antibody to detect the C-terminus of Msb2. Equal amounts of proteins of total cell extracts were loaded in each lane.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295589&req=5

ppat-1002563-g002: Msb2 is a Pmt4 substrate in U. maydis.A. Domain architecture of the Msb2-HA-GFP fusion protein. The HA-epitope is integrated at amino acid 709 and the C-terminus is fused to GFP. The Ser/Thr rich region as well as the predicted proteolytic cleavage site is indicated. B. Western Blot analysis of Msb2-HA-GFP isolated from SG200Δmsb2/msb2-HA-GFP (WT) and two independent clones of SG200Δmsb2Δpmt4/msb2-HA-GFP (#1 and #2, respectively). SG200 was used as a control. The first gel (above) contains 6% of polyacrylamide and α-HA antibody was used to detect the N-terminal part of Msb2. The other gel (below) contains 10% of polyacrylamide and was treated with α-GFP antibody to detect the C-terminus of Msb2. Equal amounts of proteins of total cell extracts were loaded in each lane.
Mentions: In S. cerevisiae, Msb2p is cleaved upstream of the transmembrane domain releasing an extracellular N-terminal part and a cellular C-terminal fragment [45]. To analyse the Msb2 protein in U. maydis we used a differentially tagged protein, which was C-terminal fused to GFP and carried an internal HA-epitope between amino acids 709 and 710 in the extracellular region (Figure 2A). The msb2-HA-GFP construct was placed under the control of the otef promoter and integrated into the ip locus of SG200Δmsb2. Western blot analysis revealed that the Msb2 protein is processed into two distinct fragments. The extracellular domain, detected by the anti-HA-antibody, migrated at a molecular weight of >250 kDa, while the anti-GFP-antibody detected a product of approximately 65 kDa. The size of the latter fragment allowed us to predict a cleavage site, situated between the Ser/Thr rich region and the transmembrane domain (Figure 2). In S. cerevisae, cleavage of Msb2 results in the secretion of the N-terminal extracellular domain [45]. Similar to the situation in S. cerevisiae we could detect the extracellular N-terminal domain of U. maydis Msb2 in the culture supernatant, while the C-terminal fragment was exclusively detected in the cellular fraction (Figure S5).

Bottom Line: We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4.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.Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Consejo Superior de Investigaciones Científicas, Sevilla, Spain.

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.

Show MeSH
Related in: MedlinePlus