Genomic Studies of the Rice Blast Fungus, Magnaporthe grisea
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Date
2007-04-13
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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.
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cDNA library, secreted protein, secretion, 14-3-3, microarray, genomics, rice blast
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Degree
PhD
Discipline
Functional Genomics
