Genomic Studies of the Rice Blast Fungus, Magnaporthe grisea

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dc.contributor.advisor Ralph A. Dean, Committee Chair en_US Jeong, Jun Seop en_US 2010-04-02T19:01:45Z 2010-04-02T19:01:45Z 2007-04-13 en_US
dc.identifier.other etd-03212006-230106 en_US
dc.description.abstract Magnaporthe grisea, the rice blast pathogen, is a model plant pathogenic fungus. Extensive genetic research and rich structural genomic studies on this fungus have opened the door into the era of functional genomics. Expression-based genomic technologies represent the core of functional genomics. cDNA libraries are central part of genomic studies for gene discovery and functional studies such as protein expression. We developed a novel and efficient method for the generation of normalized full-length enriched cDNA libraries by combining SMART with recombinational cloning. Sequence analysis indicated that the normalized cDNA library contained novel transcripts not found previously by large scale EST sequencing. Proteins that are secreted or presented on the outside of the fungal cell wall likely represent primary determinants of pathogenesis. Microarray analysis of microsome-associated and free cytosolic RNAs was performed to identify secreted and membrane-associated proteins. The data suggested that some proteins may be translocated to the secretory pathway post-translationally. Also, we observed differential enrichment of transcripts under different growth conditions, indicating that protein secretion might be a highly regulated process. Signal pathways play a central role in fungal-host interactions. 14-3-3 is a major regulator of signal transduction pathways; however, its function has not been clearly defined. Deletion of MgFTT1, a 14-3-3 homolog, in M. grisea resulted in reduced pathogenicity. Mutants also exhibited alteration in cell wall properties and extracellular carbohydrate hydrolase activities, consistent with the observation that deletion strains were defective in the ability to grow in planta. To improve the rate of homologous recombination in M. grisea, we developed a split-marker strategy for gene replacement. Split marker was at least 10 fold more efficient for deletion of a phytotoxic snodprot1 homolog, MgSPH1, than using whole construct-mediated transformation. The deletion mutant showed no obvious defect in appressorium formation. However, the mutants exhibited reduced pathogenicity, which at least in part was due to a defect in growth in planta. Application of purified MgSPH1p to wounded leaf tissue did not cause any apparent phytotoxic effects as has been shown for other snodprot1 homologs. In conclusion, we developed a variety of genomics tools and strategies to begin to functionally investigate the molecular basis of fungal pathogenicity. This work also contributes significantly to the functional annotation of the rice blast fungus genome. en_US
dc.rights 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. en_US
dc.subject cDNA library en_US
dc.subject secreted protein en_US
dc.subject secretion en_US
dc.subject 14-3-3 en_US
dc.subject microarray en_US
dc.subject genomics en_US
dc.subject rice blast en_US
dc.title Genomic Studies of the Rice Blast Fungus, Magnaporthe grisea en_US PhD en_US dissertation en_US Functional Genomics en_US

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