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Investigations into the gene expression of Neurospora crassa during mycelial contact with fungi of increasing phylogenetic distance

  • Author(s): Villalta, Christopher Francisco
  • Advisor(s): Taylor, John W.
  • et al.
Abstract

In the first chapter, using phylogenetic information about Neurospora, I designed a successful two restriction enzyme digest assay that separated PS 1-3 from the other known Neurospora species and found one new PS1, nine new PS2, and one new PS3. The topography of the phylogenetic tree did not change with the addition of the new PS isolate data and neither did the interspecific mating patterns between Neurospora. As a result of finding new PS isolates and successfully retesting the PSR concept, the PS 1-3 were properly named and described as N. hispaniola, N. metzenbergii, and N. perkinsii.

In the second chapter I observed changes in N. crassa gene expression during contact with the mycelia of other Neurospora. I framed the question from a phylogenetic perspective and collected mycelia from N. crassa during a self-self interaction, a intrapopulation interaction, a interpopulation interaction, and a intragenus interaction. After comparing RNAseq profiles of N. crassa interacting with the different fungi I found that the smallest change in gene expression occurred between the self-self interaction and the largest difference occurred in the interpopulation interaction. The intrapopulation and intragenus interactions shared the most in common. There was a large downregulation of metabolism in N. crassa when comparing N. crassa growing alone to N. crassa growing on a plate with another Neurospora, but before contact between mycelia. During contact with the mycelia of the other nonself Neurospora there was an upregulation of genes related to reactive oxygen species metabolism and melanin synthesis in N. crassa. In the interpopulation interaction there was visible production of melanin after mycelial contact between both N. crassa specimens. In interactions between nonself Neurospora there was a downregulation of genes involved in cell signaling and polar cell growth. Two genes, NCU01219 and NCU01074, were significantly upregulated in wild type N. crassa after contact between mycelia. Deletion mutants of both genes displayed reduced aerial mycelia in comparison to wild type N. crassa after contact. NCU01074 is an undescribed bzip transcription factor we found is closely related YAP bzip transcription factor family in S. cerevisiae and NCU01219 is a glutaredoxin.

In the third chapter I characterized N. crassa gene expression during growth with a young 24 hour P. chrysogenum colony that did not inhibit mycelial growth and an old 72 hour P. chrysogenum colony that inhibited mycelia growth with the anti fungal protein, PAF. I wanted to find the genes, functional categories, and pathways that were affected by PAF induced growth inhibition in N. crassa and gain better insight into how fungi behave in the wild. I compared expression data of N. crassa interacting with P. chrysogenum to the Neurospora interaction data from Chapter 2. N. crassa interacting with P. chrysogenum had very different expression profiles from the other interactions, but genes related to melanin synthesis were upregulated similar to interactions between nonself Neurospora. A large amount of gene upregulation occurred in N. crassa when grown with the 72 hour old P. chrysogenum colony while the smallest change in gene expression occurred in N. crassa when interacting with the 24 hour old P. chrysogenum colony in comparison to all interactions from Chapter 2 and 3. I found 19 genes that were significantly differentially expressed in N. crassa during PAF induced growth inhibition caused by the 72 hour old P. chrysogenum colony that would be interesting candidates for further study with gene deletion and over expression mutants. The genes were related to the cell wall, cell membrane, cross membrane transporters, Ca2+ dependent signaling, virulence, and transcriptional regulation.

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