| Ustilago maydis is a dimorphic, fungal plant pathogen. When grown in low ammonium the fungal cells produce a filamentous phenotype. There are two ammonium permeases present in this species, Ump1 and Ump2, which act in the transport of ammonium across the membrane; Ump2 also plays a role in signaling associated with low ammonium availability. In absence of the ump genes, especially ump2, the fungal cells fail to filament under conditions of low ammonium. However, like wild type U. maydis, the ump2 mutant cells produced filaments in response to low pH, indicating a separate regulatory pathway for which Ump2 was not required. The main aim of this research was to investigate if the Ump proteins function through the cAMP dependent PKA pathway and if they have any effect on the cAMP levels in the cells. U. maydis cells were grown in different media and the cell extracts used to assay the levels of cAMP via a competitive micro-immunoassay technique. According to the hypothesis, Ump2 proteins have some effect on the cAMP levels in the cell depending on the ammonium availability and Ump1 proteins also have some role in controlling the level of cAMP in the cell. Contrary to the hypothesis, no clear correlation was observed between Ump genotype and level of cAMP. However, cAMP levels determined for strains bearing site-directed Ump2 mutants provided data for an alternative hypothesis. From the results obtained with mutations of a putative phosphorylation target (a serine residue at 286 positions in the protein) in the Ump2 protein an apparent effect on cAMP levels was observed. From the data, phosphorylation state of the serine residue appears to affect the overall cAMP turnover rate in the cell, and that a feedback relationship may exist that couples phosphorylation of the Ump protein with transport of ammonium across the membrane. |
