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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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The pmt4 mutant appressorium shows outgrowing as a consequence of its incapability to penetrate the plant cuticle.Crosses of the sexually compatible strains FB1 and FB2, and FB1Δpmt4 and FB2Δpmt4 were inoculated into maize seedlings. Leaves were fixed and analysed by Scanning Electron Microscopy (SEM) one day post infection. In these images (left), we can observe appressorium formation over a plant stomata (pointed with a red arrow) penetrating the plant surface in the wild-type. However, in a similar scenario, with the appressorium developed over plant stomata, the pmt4 mutant is unable to penetrate the plant cuticle, producing hyphal outgrowing. This morphologic structure seems to be a consequence of its incapability to penetrate this surface as we can deduce from wild-type phenotype of the in vitro appressorium formation on the non-penetrable surface of parafilm M (right). The in vitro analysis was performed by fluorescence microscopy staining the cells with calcofluor white 15 hours after being sprayed on parafilm M (see Methods for additional details). Scale bar on the in vitro conditions represents 20 µm.
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ppat-1002563-g007: The pmt4 mutant appressorium shows outgrowing as a consequence of its incapability to penetrate the plant cuticle.Crosses of the sexually compatible strains FB1 and FB2, and FB1Δpmt4 and FB2Δpmt4 were inoculated into maize seedlings. Leaves were fixed and analysed by Scanning Electron Microscopy (SEM) one day post infection. In these images (left), we can observe appressorium formation over a plant stomata (pointed with a red arrow) penetrating the plant surface in the wild-type. However, in a similar scenario, with the appressorium developed over plant stomata, the pmt4 mutant is unable to penetrate the plant cuticle, producing hyphal outgrowing. This morphologic structure seems to be a consequence of its incapability to penetrate this surface as we can deduce from wild-type phenotype of the in vitro appressorium formation on the non-penetrable surface of parafilm M (right). The in vitro analysis was performed by fluorescence microscopy staining the cells with calcofluor white 15 hours after being sprayed on parafilm M (see Methods for additional details). Scale bar on the in vitro conditions represents 20 µm.

Mentions: We could previously observe that the penetration of Δpmt4 appressoria was arrested [11]. To find out more about this phenotype, we carried out an analysis of appressorium penetration of wild-type strains and pmt4 mutants using scanning electron microscopy (SEM). With this aim, we infected maize plants with a mixture of the compatible FB1 and FB2 as well as compatible FB1Δpmt4 and FB2Δpmt4 strains. 15 hours after infection maize leaves were fixed and visualized by SEM. In infections with compatible wild-type strains filaments differentiated into appressoria and penetrated inside the plant tissue. By contrast, the few appressoria formed by the Δpmt4 strain were unable to penetrate the cuticle (Figure 7), and instead the hyphae continued to grow on the plant surface. In order to verify that this aberrant morphology is a consequence but not the cause of the deficiency to penetrate the plant cuticle, we studied appressorium formation of FB1 and FB2, as well as FB1Δpmt4 and FB2Δpmt4 strains on a non-penetrable surface such as parafilm M. In the in vitro conditions post-appressorial outgrowth of hyphae was observed in both, pmt4 mutants and wild-type strains, resembling the phenotype of Δpmt4 strains on the plant surface (Figure 7). This suggests that the Δpmt4 appressoria are unable to penetrate the plant cuticle and as consequence continue to grow on the plant surface.


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)

The pmt4 mutant appressorium shows outgrowing as a consequence of its incapability to penetrate the plant cuticle.Crosses of the sexually compatible strains FB1 and FB2, and FB1Δpmt4 and FB2Δpmt4 were inoculated into maize seedlings. Leaves were fixed and analysed by Scanning Electron Microscopy (SEM) one day post infection. In these images (left), we can observe appressorium formation over a plant stomata (pointed with a red arrow) penetrating the plant surface in the wild-type. However, in a similar scenario, with the appressorium developed over plant stomata, the pmt4 mutant is unable to penetrate the plant cuticle, producing hyphal outgrowing. This morphologic structure seems to be a consequence of its incapability to penetrate this surface as we can deduce from wild-type phenotype of the in vitro appressorium formation on the non-penetrable surface of parafilm M (right). The in vitro analysis was performed by fluorescence microscopy staining the cells with calcofluor white 15 hours after being sprayed on parafilm M (see Methods for additional details). Scale bar on the in vitro conditions represents 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3295589&req=5

ppat-1002563-g007: The pmt4 mutant appressorium shows outgrowing as a consequence of its incapability to penetrate the plant cuticle.Crosses of the sexually compatible strains FB1 and FB2, and FB1Δpmt4 and FB2Δpmt4 were inoculated into maize seedlings. Leaves were fixed and analysed by Scanning Electron Microscopy (SEM) one day post infection. In these images (left), we can observe appressorium formation over a plant stomata (pointed with a red arrow) penetrating the plant surface in the wild-type. However, in a similar scenario, with the appressorium developed over plant stomata, the pmt4 mutant is unable to penetrate the plant cuticle, producing hyphal outgrowing. This morphologic structure seems to be a consequence of its incapability to penetrate this surface as we can deduce from wild-type phenotype of the in vitro appressorium formation on the non-penetrable surface of parafilm M (right). The in vitro analysis was performed by fluorescence microscopy staining the cells with calcofluor white 15 hours after being sprayed on parafilm M (see Methods for additional details). Scale bar on the in vitro conditions represents 20 µm.
Mentions: We could previously observe that the penetration of Δpmt4 appressoria was arrested [11]. To find out more about this phenotype, we carried out an analysis of appressorium penetration of wild-type strains and pmt4 mutants using scanning electron microscopy (SEM). With this aim, we infected maize plants with a mixture of the compatible FB1 and FB2 as well as compatible FB1Δpmt4 and FB2Δpmt4 strains. 15 hours after infection maize leaves were fixed and visualized by SEM. In infections with compatible wild-type strains filaments differentiated into appressoria and penetrated inside the plant tissue. By contrast, the few appressoria formed by the Δpmt4 strain were unable to penetrate the cuticle (Figure 7), and instead the hyphae continued to grow on the plant surface. In order to verify that this aberrant morphology is a consequence but not the cause of the deficiency to penetrate the plant cuticle, we studied appressorium formation of FB1 and FB2, as well as FB1Δpmt4 and FB2Δpmt4 strains on a non-penetrable surface such as parafilm M. In the in vitro conditions post-appressorial outgrowth of hyphae was observed in both, pmt4 mutants and wild-type strains, resembling the phenotype of Δpmt4 strains on the plant surface (Figure 7). This suggests that the Δpmt4 appressoria are unable to penetrate the plant cuticle and as consequence continue to grow on the plant surface.

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