Date on Master's Thesis/Doctoral Dissertation
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The fungus, Ustilago maydis, a plant pathogen of Zea mays, is capable of a dimorphic switch, e.g., a transition between a yeast-like form and a filamentous form. This switch is activated when U. maydis cells are starved for their preferred nitrogen source - ammonium. U. maydis has two important genes that play a role in this switch, ump1 and ump2. Each gene is responsible for the uptake of ammonium and the latter is responsible for the filamentous response in the absence of ammonium. Ump2 deletion mutants are unable to filament in response to low ammonium while ump1 deletion mutants do not show this phenotype. The Ump2 protein is responsible, not only for ammonium transport, by also for sensing the ammonium in the environment around the cell; in contrast, ump1 is solely a low-affinity, high-capacity transporter of ammonium and is not required for the filamentous response. Unexpectedly, single deletion strains and double deletion strains (i.e., Dump1 Dump2) are still able to grow in low ammonium conditions. The present study investigates the roles of ploidy (haploid vs. diploid strains), promoter type (native promoter vs. constitutive promoter), gene structure (genomic copy vs. cDNA), and gene location (native location vs. ectopic location) on transcription levels of both ammonium transporters under low ammonium conditions compared to high ammonium conditions. Each construct was normalized to their parent strains that served as the baseline of expression. Across the different ploidy constructs, I found that the diploid solopathogenic strain D132 had decreased expression of ump2 relative to the FB1 wild type strain under replete media conditions. D132 also increased its ump2 expression 8log2-fold when under low ammonium conditions as compared to itself under high ammonium conditions. The haploid solopathogenic strain SG200 showed an increase in ump2 expression compared to itself under low ammonium conditions and in high ammonium conditions in comparison to the FB1 wild type. An SG200 Δump2 mutant was used to test the roles of promoter type, gene structure, and gene location. Little difference in ammonium transporter transcription levels was observed compared to the FB1 wild type strain in replete media. Overexpression constructs had increased ump2 expression, as expected, and yielded a filamentous phenotype when under low ammonium conditions. SG200 Δump2 mutants complemented with the various promoters driving the ump2 expression reestablished levels of expression, demonstrating that function was restored at the transcriptional level. When tested for ability to filament on low ammonium, such complemented strains restored the filamentation ability comparable to that of SG200 wild type strain. However, none of the strains expressing the over-expression constructs regained the fuzz phenotype on charcoal media or the ability to cause disease on maize, demonstrating lack of complementation of those phenotypes of the SG200 Δump2 mutant.
Richardson, Kirsten, "Gene expression of ammonium transporters in Ustilago maydis and their role in pathogenicity and virulence." (2017). Electronic Theses and Dissertations. Paper 2798.