Project description:The fungus Ustilago maydis is a biotrophic pathogen of corn. In its genome we have identified an ortholog of YAP1 from Saccharomyces cerevisae which regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H2O2 than wild type cells and their virulence was significantly reduced. U. maydis yap1 could partially complement the H2O2 sensitivity of a yap1 deletion mutant of S. cerevisiae and a Yap1-GFP fusion protein showed nuclear localization after H2O2 treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two cysteine residues prevented accumulation in the nucleus and the respective mutant strains showed the same virulence phenotype as Dyap1 mutants. DAB staining revealed an accumulation of H2O2 around yap1 mutant hyphae which was absent in 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 2-3 days 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 H2O2 detoxification system for coping with early plant defense responses. Keywords: Oxidative stress, yap1 dependent genes, Ustilago maydis
Project description:The fungus Ustilago maydis is a biotrophic pathogen of corn. In its genome we have identified an ortholog of YAP1 from Saccharomyces cerevisae which regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H2O2 than wild type cells and their virulence was significantly reduced. U. maydis yap1 could partially complement the H2O2 sensitivity of a yap1 deletion mutant of S. cerevisiae and a Yap1-GFP fusion protein showed nuclear localization after H2O2 treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two cysteine residues prevented accumulation in the nucleus and the respective mutant strains showed the same virulence phenotype as Dyap1 mutants. DAB staining revealed an accumulation of H2O2 around yap1 mutant hyphae which was absent in 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 2-3 days 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 H2O2 detoxification system for coping with early plant defense responses. Keywords: Oxidative stress, yap1 dependent genes, Ustilago maydis Two independent overnight cultures of U. mayidis FB1 and FB1Dyap1 grown in CM-glucose (OD600 0.8) were diluted in 100 ml of the same medium (OD600 0.2) and growth at 28° C until an OD600 0.6. The cultures were divided and one half was supplemented with 5mM H2O2. After one hour of exposition to H2O2, cells were harvested by centrifugation and frozen in liquid nitrogen. RNA extraction, purification, cDNA generation, purification and labeling were performed according to standard protocols (Affymetrix). DNA array analysis was performed performed on two biological replicates each, using custom-designed Affymetrix chips (MPIUstilagoA). Data were analysed using a GeneArray Scanner (Agilent/Affymetrix) and the GeneChip Expression Analysis software (GCOS)
Project description:Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host Zea mays. The biotrophic interaction is initiated upon host penetration, and involves expansion of the host plasma membrane around hyphae, which is thought to facilitate the exchange of nutrients and virulence factors. Transcriptional regulators involved in the establishment of an infectious dikaryon and penetration into the host have been identified, however, regulators involved in the post-penetration stages remained to be elucidated. In the study we report the identification of an Ustilago maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumour development in planta. Microarray analyses of Δfox1-infected plant tissue identified Fox1 as a transcriptional activator, involved in the expression of secreted effectors required for virulence.
Project description:Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host Zea mays. The biotrophic interaction is initiated upon host penetration, and involves expansion of the host plasma membrane around hyphae, which is thought to facilitate the exchange of nutrients and virulence factors. Transcriptional regulators involved in the establishment of an infectious dikaryon and penetration into the host have been identified, however, regulators involved in the post-penetration stages remained to be elucidated. In the study we report the identification of an Ustilago maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumour development in planta. Δfox1 hyphae induce plant defences including the overproduction and accumulation of H2O2 in and around infected cells. This oxidative burst acts as an intercellular signal, which elicits a specific host defence response phenotypically represented by the encasement of proliferating hyphae in extensions of the plant cell wall. Maize microarrays experiments were performed to identify genes involved in the observed plant defence responses on leaf tissue infected with U. maydis strain SG200∆fox1 4 dpi.
Project description:Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host Zea mays. The biotrophic interaction is initiated upon host penetration, and involves expansion of the host plasma membrane around hyphae, which is thought to facilitate the exchange of nutrients and virulence factors. Transcriptional regulators involved in the establishment of an infectious dikaryon and penetration into the host have been identified, however, regulators involved in the post-penetration stages remained to be elucidated. In the study we report the identification of an Ustilago maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumour development in planta. Microarray analyses of Δfox1-infected plant tissue identified Fox1 as a transcriptional activator, involved in the expression of secreted effectors required for virulence. Maize plants were infected with a mixture of either FB1 and FB2 (wild-type), or FB1∆fox1 and FB2∆fox1 crossings, to measure the impact of fox1 on pathogenic development. Early Golden Bantam maize plants were grown in a phytochamber in a 15h/9h light-dark cycle; light period started/ended with 1h ramping of light intensity. Maize plants were kept at 28°C (light) and 20° (dark). Plantlets were individually sown in pots with potting soil (Fruhstorfer Pikiererde) and infected 7 days after sowing, 1 h before end of the light period. Infected leaf tumor material from at least 10 plants was collected 5 days post infection, 1 h before the end of the light period and directly frozen in liquid nitrogen for RNA-extraction. RNA samples were extracted from infected leaf tissue 5 days after infection.
Project description:mRNAs comparison between Ustilago maydis wild type grown in diluted YEPS (control) and in cell-free supernatants of Ustilago maydis wild type treated with H202 in two different concentrations (0.4% and 0.7%).
Project description:Anthocyanin induction in plant is considered a general defense response against biotic and abiotic stresses. The infection by Ustilago maydis, the corn smut pathogen, is accompanied with anthocyanin induction in leaf tissue. We revealed that anthocyanin is intentionally induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with cytoplasmic maize protein kinase ZmTTK1. Tin2 masks an ubiquitin-proteasome degradation motif in ZmTTK1 leading to a more stable active kinase. Active ZmTTK1 controls transcriptional activation of genes in the anthocyanin biosynthesis pathway rerouting phenylalanine away from lignin biosynthesis. Therefore, we performed microarray analysis to understand how maize gene transcription in phenylpropanoid pathway is differentially changed after infection with Ustilago maydis SG200 (wild type) and SG200Dtin2 (anthocyanin-inducing effector mutant).
Project description:The fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize. Hallmarks of the disease are large plant tumors in which fungal proliferation occurs. Plants have developed various defense pathways to cope with pathogens. We used microarrays to detail the global programme of gene expression during the infection process of Ustilago maydis in its host plant to get insights into the defense programs and the metabolic reprogramming needed to supply the fungus with nutrients. Keywords: time course