Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Perlin, Michael H.

Committee Co-Chair (if applicable)

Graham, James

Committee Member

Graham, James

Committee Member

Klinge, Carolyn

Committee Member

Schultz, David

Committee Member

Yoder-Himes, Deborah

Author's Keywords

ustilago maydis; mating type loci; low ammonium; haploid phenotypes

Abstract

Fungi that can switch from budding to a filamentous infectious state have evolved mating type loci. Ustilago maydis, the maize pathogen, must mate with compatible partners possessing different alleles at two mating type loci for successful host infection. The a locus encodes pheromones and receptors, while the b locus encodes subunits of a heterodimeric transcription factor that regulates expression of virulence genes. Mating is triggered by environmental signals, including nutrient deprivation. My goal was to determine the fate of nitrogen starved haploid cells without a compatible mating partner. On solid low ammonium media, wild-type U. maydis filaments. I examined the roles of the b mating locus, and Ump2, the high affinity ammonium transporter, in this phenotype. Expression of ump2 increases under low ammonium, while deletion of b or ump2 results in loss of filamentation. Deleting b did not affect the induction of ump2, but deletion of ump2 altered expression from the b locus. In my model of filamentation on low ammonium, ump2 senses nitrogen availability and, in a b-dependent manner, upregulates targets canonically involved in mating but, without a partner, functions in filamentation. The b locus contains two genes, bE and bW, but the heterodimer does not form in haploid cells. I found evidence that bE and bW function independently in haploids to regulate gene expression. Partial deletion of b also results in loss of filamentation in media depleted of ammonium. Although ump2 overexpression in a b deletion background rescues the loss of filamentous phenotype, this phenomenon is absent when bE remains. This suggests bW regulates transcription of mating and pathogenicity targets in a similar manner to ump2, while bE does so in the opposite direction. Finally, through biofluorescent labelling I visualized the rearrangement of the actin cytoskeleton in response to low ammonium. I also investigated via qRT-PCR several targets, including actin and its regulators, and cell wall remodeling enzymes. Although these targets showed dynamic expression levels in the mutants, no pattern emerged explaining the varied filamentous phenotypes. As such, changes of the actin cytoskeleton upon exposure to low ammonium are not regulated at the transcription level.

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