Fusarium oxysporum is a filamentous fungus that is known to invade over a hundred different hosts and poses a major threat to the economy and food supply world-wide. Poly (Adenosine diphosphate-Ribose) Polymerase (PARP) is a family of regulatory proteins that affect change in the cell through transfer of ADP-Ribose moieties onto target molecules. The most well-studied PARP protein is the human PARP1, a PARylating nuclear protein that serves as our model PARP protein. F. oxysporum was found to contain a large expansion of PARP catalytic-domain-containing proteins compared to other filamentous fungi. We utilized in silico multiple sequence alignments and domain predictions to identify a human PARP1 homolog termed foPARP1 that was conserved within the core chromosomes in all three strains within our comparative system. Our in silico predictions also stated that only one strain, an Arabidopsis pathogen, Fo5176, contained several other predicted catalytically active PARP homologs within the accessory chromosome. To test the effect that foPARP1 knockout would have on DNA damage tolerance, we created a foParp1 knockout and found that only strains Fol4287 and Fo5176 had a significant reduction in tolerance upon being plated with methyl methanesulfonate (MMS), a DNA alkylating agent. To test how global PARylation trends would be affected by foParp1 knockout, we utilized immunodot-blotting with PAR antibodies to assess PARylation in total protein extracts. We found that all strains of the comparative system had the capacity to catalyze the synthesis of long PAR chains, while only Fo47 and Fo5176 had a significant PARylation increase when exposed to MMS, and no samples had a significant increase in PARylation within the foParp1 knockouts. Finally, we utilized RNA-Sequencing to determine the transcriptional impacts that foParp1 knockout would have and found aberrant DNA repair pathways and disruptions in stress responses. Taken together, we conclude that foPARP1 is in fact a functional PARP1 homolog and exhibits similar post-transcriptional modification and transcriptional impacts as its human counterpart. However, we were not able to correlate PARP copy number with DNA stress tolerance, and further research would be needed to assess the full function of the PARP expansion.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-2297 |
| Date | 28 October 2022 |
| Creators | Norment, Daniel |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
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