Research detailed in this thesis investigated the generation of Nitric Oxide (NO) and its role in the pathogenesis of the rice blast fungus Magnaporthe oryzae. Two putative nitric oxide synthase genes and single copy nitrate and nitrite reductase genes were identified as potential sources of NO in M. oryzae. Single and double gene disrupted mutants were generated and their phenotypes assessed. Detection of NO is problematic. Herein, a fluorescent plate reader assay was developed, exploiting the NO sensitive dye DAR-4M AM and the NO scavenger PTIO, to compare wildtype NO generation with the mutant strains. All strains were assessed for infection-related development on an artificial surface inductive to appressorium formation and maturation in the wildtype strain. Appressorium formation in the presence of PTIO and the NO donor DETANONOate was recorded for all strains on this surface. The pathogenicity of the wildtype and mutant strains were assessed, in terms of their ability to infect rice and barley plants. Finally, the capacity of each strain to metabolise nitrogen was evaluated to confirm the disruption of the nitrate and nitrite reductase genes. Collectively, the data demonstrates that the plate reader assay provides robust evidence for the generation of NO in M. oryzae. However, none of the various mutant strains showed a reduction in NO emission during germling morphogenesis. However, they exhibited significantly different infection-related development on an inductive artificial surface as compared with the wildtype strain. Moreover, exogenous application of PTIO to the wildtype strain provided evidence for NO and its involvement in germination and appressorium development. No significant differences in pathogenicity were observed between the wildtype and mutant strains, indicating the disrupted genes are dispensable for pathogenesis. The nitrate and nitrite reductase genes were found to be essential for nitrate assimilation. In summary, this work provides the most robust evidence for the generation of NO in fungi to- date, but the molecular mechanism underpinning the generation of NO remains elusive.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:556208 |
| Date | January 2011 |
| Creators | Johnson, Jasper |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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