| Ustilago maydis is a model organism used to study the molecular mechanisms controlling mating, morphological transition and pathogenicity of fungal pathogens. In this study, we isolated a Rho1 homologue from U. maydis and characterized its functions. Like other Rho type GTP binding proteins, this Rho1 homologue, named Uro1, is believed to have multiple functions in U. maydis: The functions of Uro1 in mating response, cell growth and cell morphology were examined. Overexpression of the uro1 gene or its constitutively active version (uro1 G14V ) reduced the mating efficiency of U. maydis, which was confirmed by a confrontation assay. In the confrontation assay, the mating filaments protruding from one mating type toward the opposite mating type were shortened when the uro1 gene was overexpressed. Disruption of the uro1 gene in haploid cells was not successful, suggesting this gene disruption might be lethal. One copy of the gene was disrupted in the U. maydis diploid strain d132. The mutant diploid cells tended to aggregate together in liquid culture compared to the even dispersal of wild type diploid cells. The mutant diploid lacked the ability to, form galls after infecting host plants, which suggests Uro1 might be required for pathogenicity of U. maydis. Since disruption of the uro1 gene in haploid cells was apparently lethal, we replaced the original promoter of the uro1 gene with a regulatable P crg promoter in the haploid strain 1/2 (a1b1). In medium that contains glucose, where the uro1 gene is turned off, the cells were not able to grow, but if they were put back into medium with arabinose, where the uro1 gene is turned on, the cells resumed growth. This suggests that Uro1 is required for normal cell growth. Through yeast-two-hybrid screening of a U. maydis cDNA library, we found several potential interaction partners of the Uro1 protein, including Cdc24(GEF, guanine-nucleotide exchange factor), PTEN (phosphatidylinositol 3,4,5-tri phosphate (PIP3) phosphatase) and Ump2 (U. maydis ammonium transporter). Several of the more interesting candidate proteins were tested directly by yeast two-hybrid analysis after switching the vectors for every pair of genes relative to the original screen. These interactions were also tested by co-immunoprecipitation. The results showed consistent and strong interaction between Uro1 and each of Cdc24, PTEN and 14-3-3, while the interaction between Uro1 and Smu1, and Ump2 were not consistent, as co-immumoprecipitation showed weak and inconclusive results. Based on the phenotype when uro1 was shut off, we propose that PTEN is a growth inhibitor for U. maydis, and Uro1 has a negative effect on PTEN activity. Hence, turning off the uro1 gene interferes with cell growth. The function of PTEN and its possible relationship with Uro1 needs to be further investigated. |
