Project description:Yap1 targets under normal and cobalt surplus growth conditions. Yeast strains (wild-type and yap1 mutant, BY4742 background) were grown until early log-phase and either untreated or exposed to 2mM of CoSO4 for 60 min. Changes in the transcriptome of yap1 mutant cells were then analyzed.
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)
