Mannitol is purported to have role in fungi as a storage carbohydrate and has been shown to quench reactive oxygen species (ROS) both in vitro and in vivo. Mannitol metabolism in fungi is thought to occur through the mannitol cycle, which was proposed in the late 1970?s from studies of cell free extracts of the fungus Alternaria alternata. In this cycle, mannitol 1-phosphate 5-dehydrogenase (MPDH; EC 1.1.1.17) reduces fructose 6-phosphate into mannitol 1-phosphate, which is dephosphorylated by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in mannitol and inorganic phosphate. Mannitol also can be made through the enzyme mannitol dehydrogenase (MtDH; EC 1.1.1.138), which reduces fructose to mannitol. Here we report confirmation of these enzymes in the fungus A. alternata, the isolation of the genes, and the generation of strains mutated in MPDH, MtDH, or both genes. PCR confirmed gene replacement and enzyme assays using these mutants showed no activity for MtDH or MPDH. GC-MS analysis showed that double mutants did not produce mannitol, while single mutants had reduced mannitol production. Mannitol, as a quencher of ROS, may also have a role in host-pathogen interactions, by allowing the fungus to suppress ROS-mediated plant defense responses. To assess the contribution of mannitol in plant-pathogen interactions, wild type, single and double mutants were used in pathogenicity assays on tobacco plants. Severity of lesions caused by the MtDH disruptant was not significantly different from that of the wild type. By contrast, the MPDH disruptant and the double mutant caused significantly less disease. Microscopy analysis and histochemical staining for H2O2 showed that both the wild type strain and the double mutant were able to germinate, produced appressoria, and elicited a defense response from the host. Quantitative PCR studies showed that genes for both enzymes were upregulated in the presence of tobacco extracts, with MPDH having a stronger response. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for normal spore germination either in vitro or in planta or for initial infection.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-12282005-171313 |
| Date | 30 December 2005 |
| Creators | Vélez, Heriberto |
| Contributors | Marc A. Cubeta, Gary A. Payne, Margaret E. Daub, Greg G. Upchurch |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-12282005-171313/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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