Limits...
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.

Show MeSH

Related in: MedlinePlus

The STR region is required for Msb2 function in U. maydis.A. Schematic model of the protein variant Msb2ΔSTR-GFP compared to Msb2-HA-GFP. The deletion of the coding region of the Ser/Thr rich domain of Msb2 is shown. B. Cells of SG200Δmsb2/msb2-HA-GFP and SG200Δmsb2/msb2ΔSTR-GFP were grown to mid log phase in YEPSL and analyzed microscopically without addition (top row) or after addition of latrunculin A (bottom row). The fluorescent signal corresponding to GFP is shown. C. Total protein extracts of the indicated strains were subjected to western analysis with α-HA antibody (upper panel) and α-GFP antibody (middle panel). Tubulin served as loading control and was detected with α-tubulin antibody (bottom panel). The fusion proteins and processed fragments are indicated by arrowheads on the right. The molecular weight ruler is depicted on the left. D. Disease symptoms caused by SG200 (WT), SG200Δmsb2, and SG200Δmsb2 complemented with either Potef:msb2 or Potef:msb2ΔSTR were scored 12 dpi. N indicates the total number of plants evaluated in each case.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3295589&req=5

ppat-1002563-g003: The STR region is required for Msb2 function in U. maydis.A. Schematic model of the protein variant Msb2ΔSTR-GFP compared to Msb2-HA-GFP. The deletion of the coding region of the Ser/Thr rich domain of Msb2 is shown. B. Cells of SG200Δmsb2/msb2-HA-GFP and SG200Δmsb2/msb2ΔSTR-GFP were grown to mid log phase in YEPSL and analyzed microscopically without addition (top row) or after addition of latrunculin A (bottom row). The fluorescent signal corresponding to GFP is shown. C. Total protein extracts of the indicated strains were subjected to western analysis with α-HA antibody (upper panel) and α-GFP antibody (middle panel). Tubulin served as loading control and was detected with α-tubulin antibody (bottom panel). The fusion proteins and processed fragments are indicated by arrowheads on the right. The molecular weight ruler is depicted on the left. D. Disease symptoms caused by SG200 (WT), SG200Δmsb2, and SG200Δmsb2 complemented with either Potef:msb2 or Potef:msb2ΔSTR were scored 12 dpi. N indicates the total number of plants evaluated in each case.

Mentions: Considering that the extracellular domain of Msb2 is mannosylated by Pmt4, we assumed that this domain might be needed for the function of Msb2. We therefore deleted the coding region of the Ser/Thr rich domain of Msb2 and integrated the construct into the ip locus of SG200Δmsb2 generating the variant SG200Δmsb2/msb2ΔSTR-GFP (Figure 3A). The loss of this region did not alter the normal localization of Msb2 in the plasma membrane as revealed by treatment with the endocytosis inhibitor Latrunculin A [31] (Figure 3B). Western blot analysis revealed that processing of the truncated Msb2 protein still occurred since the C-terminal 65 kDa fragment could be detected for both full length Msb2-HA-GFP and truncated Msb2ΔSTR-GFP (Figure 3C). However, in plant infections no complementation was observed when the truncated Msb2 protein was expressed, while virulence could be completely restored when Msb2 was expressed as full-length protein (Figure 3D). This indicates that, contrary to the situation in S. cerevisiae, where the Ser/Thr rich region of Msb2p possesses a negative regulatory function [46], the mannosylated Ser/Thr-rich region of Msb2 in U. maydis has a positive role during virulence.


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 STR region is required for Msb2 function in U. maydis.A. Schematic model of the protein variant Msb2ΔSTR-GFP compared to Msb2-HA-GFP. The deletion of the coding region of the Ser/Thr rich domain of Msb2 is shown. B. Cells of SG200Δmsb2/msb2-HA-GFP and SG200Δmsb2/msb2ΔSTR-GFP were grown to mid log phase in YEPSL and analyzed microscopically without addition (top row) or after addition of latrunculin A (bottom row). The fluorescent signal corresponding to GFP is shown. C. Total protein extracts of the indicated strains were subjected to western analysis with α-HA antibody (upper panel) and α-GFP antibody (middle panel). Tubulin served as loading control and was detected with α-tubulin antibody (bottom panel). The fusion proteins and processed fragments are indicated by arrowheads on the right. The molecular weight ruler is depicted on the left. D. Disease symptoms caused by SG200 (WT), SG200Δmsb2, and SG200Δmsb2 complemented with either Potef:msb2 or Potef:msb2ΔSTR were scored 12 dpi. N indicates the total number of plants evaluated in each case.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002563-g003: The STR region is required for Msb2 function in U. maydis.A. Schematic model of the protein variant Msb2ΔSTR-GFP compared to Msb2-HA-GFP. The deletion of the coding region of the Ser/Thr rich domain of Msb2 is shown. B. Cells of SG200Δmsb2/msb2-HA-GFP and SG200Δmsb2/msb2ΔSTR-GFP were grown to mid log phase in YEPSL and analyzed microscopically without addition (top row) or after addition of latrunculin A (bottom row). The fluorescent signal corresponding to GFP is shown. C. Total protein extracts of the indicated strains were subjected to western analysis with α-HA antibody (upper panel) and α-GFP antibody (middle panel). Tubulin served as loading control and was detected with α-tubulin antibody (bottom panel). The fusion proteins and processed fragments are indicated by arrowheads on the right. The molecular weight ruler is depicted on the left. D. Disease symptoms caused by SG200 (WT), SG200Δmsb2, and SG200Δmsb2 complemented with either Potef:msb2 or Potef:msb2ΔSTR were scored 12 dpi. N indicates the total number of plants evaluated in each case.
Mentions: Considering that the extracellular domain of Msb2 is mannosylated by Pmt4, we assumed that this domain might be needed for the function of Msb2. We therefore deleted the coding region of the Ser/Thr rich domain of Msb2 and integrated the construct into the ip locus of SG200Δmsb2 generating the variant SG200Δmsb2/msb2ΔSTR-GFP (Figure 3A). The loss of this region did not alter the normal localization of Msb2 in the plasma membrane as revealed by treatment with the endocytosis inhibitor Latrunculin A [31] (Figure 3B). Western blot analysis revealed that processing of the truncated Msb2 protein still occurred since the C-terminal 65 kDa fragment could be detected for both full length Msb2-HA-GFP and truncated Msb2ΔSTR-GFP (Figure 3C). However, in plant infections no complementation was observed when the truncated Msb2 protein was expressed, while virulence could be completely restored when Msb2 was expressed as full-length protein (Figure 3D). This indicates that, contrary to the situation in S. cerevisiae, where the Ser/Thr rich region of Msb2p possesses a negative regulatory function [46], the mannosylated Ser/Thr-rich region of Msb2 in U. maydis has a positive role during virulence.

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