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, 19 (7), 2293-309

An Ustilago Maydis Gene Involved in H2O2 Detoxification Is Required for Virulence

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An Ustilago Maydis Gene Involved in H2O2 Detoxification Is Required for Virulence

Lázaro Molina et al. Plant Cell.

Abstract

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.

Figures

Figure 1.
Figure 1.
Domain Organization of Yap1. (A) Yap1p of S. cerevisiae and Yap1 of U. maydis are compared. The vertical gray lines indicate the position of Cys residues. NES (dark-gray bar), nuclear export sequence; aa, amino acids. (B) Alignment of the bZIP domain of AP-1–like proteins from U. maydis, Neurospora crassa, S .cerevisiae, S. pombe, C. albicans, and C. heterostrophus. Accession numbers for these proteins are in Methods. Uppercase letters indicate identity in all proteins compared, and lowercase letters indicate that three or more proteins carry this residue. (C) Alignment of the n-CRD domains of proteins listed in (A). Shading follows the scheme given in (A). (D) Alignment of the c-CRD domains of proteins listed in (A). Numbers give amino acid positions in the respective proteins.
Figure 2.
Figure 2.
yap1 from U. maydis Complements the Hydrogen Peroxide Sensitivity of an S. cerevisiae Δyap1 Strain. The growth of S. cerevisiae BY4742 (WT), S. cerevisiae BY4742ΔYML007W (Δyap1), and derivatives of this strain transformed with the empty vectors pYES2, pYESUst, and pYESSac was tested on SD plates with glucose (top left panel), SD plates with galactose and raffinose (top right panel), SD plates with glucose supplemented with 0.8 mM H2O2 (bottom left panel), and SD plates with glucose and raffinose supplemented with 0.8 mM H2O2 (bottom right panel). Serial 10-fold dilutions of cultures indicated on the left were spotted.
Figure 3.
Figure 3.
Sensitivity of U. maydis Wild-Type and yap1 Mutant Strains to Oxidative Stress. (A) Sensitivity of strains to H2O2 was assessed in an agar diffusion test in which a filter soaked with H2O2 (30% [v/v]) was placed on a CM-glucose agar plate seeded with the strains indicated on top. (B) Halo size was quantified for the strains given in (A). Error bars indicate standard deviations derived from three independent experiments consisting of three replicas each.
Figure 4.
Figure 4.
Subcellular Localization of Yap1 in the Presence of H2O2. Strain FB2yap1:3XeGFPcbx:pANNE3090 was cultured in liquid CM-glucose medium, exposed to the indicated concentrations of H2O2 for 1 h, and assayed microscopically for GFP and RFP fluorescence. FB1yap1:3XeGFPcbx:pANNE3090 displayed the same behavior (data not shown). Bars = 10 μm.
Figure 5.
Figure 5.
Pathogenicity of yap1 Mutant Strains. (A) Disease symptoms of compatible wild-type strains, compatible yap1 mutant strains, their complemented derivatives, and the solopathogenic strain SG200 and its Δyap1 derivative are depicted. Strains are listed under each column. For each strain or strain combination, three independent infections were performed, and the total number of infected plants is indicated above each column. Symptoms were scored 14 d after infection (see Methods for details). The color code for disease rating is given on the right. Numbers at the right of the bars indicate the average percentage in each disease category and includes the standard deviation calculated from the three independent experiments. (B) Tumor morphology of strain SG200 and its Δyap1 derivative. (C) Intracellular hyphae of SG200 and SG200Δyap1 strains 2 d after inoculation visualized by chlorazole black E staining. Bars = 50 μm.
Figure 6.
Figure 6.
Subcellular Localization of Yap1 during the Life Cycle of U. maydis. (A) Compatible strains FB1yap1:3XeGFP and FB2yap1:3XeGFP were inoculated in maize seedlings. Hyphae and appressoria on the leaf surface were visualized by calcofluor staining 16 h after infection. (B) GFP fluorescence of the same leaf area shown in (A). Hyphae that have not yet penetrated do not show fluorescent nuclei (left panel). Hyphae that have penetrated show fluorescent nuclei (arrowheads), and these could be visualized in a different focus plane, indicating that the appressoria shown in (A) had already penetrated. (C) Fluorescent nuclei (arrowheads) in intracellularly growing hyphae 2 d after infection. (D) Intracellular hyphae with diffuse fluorescence 6 d after infection. Bars = 10 μm.
Figure 7.
Figure 7.
Cys-399 and Cys-407 Are Important for Functionality of Yap1. (A) Strain FB2yap1C399A,C407:3XeGFP was cultured in liquid CM-glucose medium, exposed to 1 mM H2O2 for 1 h, and assayed microscopically for GFP fluorescence (right panel). In the left panel, cells are depicted without H2O2 treatment. Bars = 50 μm. (B) Proteins isolated from the strains indicated on top either treated with H2O2 or left untreated were separated by SDS-PAGE. Yap1:3XeGFP and Yap1C399A,C407A:3XeGFP were detected by protein gel blot analysis using monoclonal GFP IgG mouse antibody. (C) Disease rating of wild-type and yap1C399A,C407A mutant strains. The disease rating scheme is described in Figure 5.
Figure 8.
Figure 8.
H2O2 Accumulation during the Early Stages of Biotrophic Growth of U. maydis Δyap1 Strains. (A) Plant samples inoculated with either mixtures of FB1XFB2 (left panel) or FB1Δyap1XFB2Δyap1 strains (right panels) were stained with calcofluor and DAB 1 d after inoculation for visualization of hyphae and appressoria on the leaf surface by light microscopy using the 4‘,6’-diamidino-2-phenylindole filter. The closed arrowheads mark DAB precipitates. (B) Samples were stained with DAB only, and intracellular hyphae were visualized by light microscopy 1 d after infection. DAB precipitates are marked with closed arrowheads. Intracellularly growing hyphae not surrounded by DAB precipitate are marked with open arrowheads. (C) Samples were as in (B) 2 d after infection. Bars = 10 μm.
Figure 9.
Figure 9.
DPI Restores Virulence of U. maydis Δyap1 Mutants. (A) Maize seedlings were inoculated with mixtures of FB1XFB2 or FB1Δyap1XFB2Δyap1 to which DMSO or DPI dissolved in DMSO were added. Infected plant tissue samples were collected 2 d after infection and stained with DAB as indicated. DAB precipitates are marked with closed arrowheads, and hyphae without DAB precipitate in the vicinity are marked by open arrowheads. Bars = 10 μm. (B) Symptoms produced by the indicated U. maydis strain combinations were scored following the scheme described in Figure 5.
Figure 10.
Figure 10.
Yap1 Controls the Expression of Two Peroxidase Genes. RNA gel blot of U. maydis strains FB1, FB1Δyap1, SG200Δum01947, and SG200Δum02377. Strains were grown in CM-glucose medium with H2O2 added in the concentrations (μm) indicated on top. Ten micrograms of total RNA were loaded per lane on four separate gels. Individual membranes were hybridized with DIG-labeled fragments of the genes um01947, um02377, um10672, or um11067. The two mRNAs detected with the um02377 probe is likely to reflect two different start sites or polyadenylation sites. As loading control, one of the membranes was stained with methylene blue to visualize rRNA.
Figure 11.
Figure 11.
The Two Yap1-Regulated Peroxidases Affect Virulence. (A) Plants were infected with the strains indicated below each column. Disease rating followed the scheme described in Figure 5. (B) DAB staining of infected leaves 2 d after infection with the same strains assayed in (A). Intracellular hyphae are visualized by light microscopy and are marked by open arrowheads. DAB precipitates in the vicinity of the hyphae are marked by closed arrowheads. Bars = 10 μm.

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