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Ustilago maydis rak1 mediated gene expression during exponential growth

ABSTRACT: In mammalian cells RACK1 serves as a scaffold protein that has a role in integrating inputs from different signaling pathways and affects translation through association with ribosomes. U. maydis contains a seven-WD40 repeat motif protein designated Rak1, which shows 68% identity to RACK1 and is homologous to Asc1p of Saccharomyces cerevisiae. The asc1 mutant could be complemented by introduction of U. maydis rak1. The deletion of rak1 affected cell growth, cell wall integrity and specifically attenuated cell fusion. This latter defect was caused by reduced expression of prf1 encoding the regulator for pheromone and pheromone-receptor genes. Rak1 interacts with a variety of ribosomal proteins and microarray analysis revealed that the deletion of rak1 led to severely reduced expression of rop1, an activator prf1. The constitutive expression of rop1 could rescue the defect of mfa1 expression as well as conjugation tube formation in response to pheromone induction in the rak1 mutant. Moreover, a solopathogenic rak1 mutant failed to respond to plant derived stimuli, resulting in attenuated filamentation and pathogenicity. This could be partially rescued by constitutive expression of the b heterodimer. These data suggest that rak1 is a regulator of rop1 expression with additional roles after cell fusion.

ORGANISM(S): Mycosarcoma maydis

PROVIDER: GSE30382 | GEO | 2011/07/06

SECONDARY ACCESSION(S): PRJNA143429

REPOSITORIES: GEO

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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 28Â° 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)

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Ustilago maydis rak1 mediated gene expression during exponential growth

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