Progress 11/15/00 to 11/14/04
Outputs
The PMK1 (pathogenicity MAP kinase 1) gene is homologous to yeast FUS3/KSS1 and it is essential for appressoria formation and plant infection. Gene replacement mutants were generated for 12 genes involved in the PMK1 signaling pathway in Magnaporthe grisea, including the upstream MAPKK MST7 and MAPKKK MST11, the G-protein coupled receptor GPR1 and putative pheromone receptors MST2 and MST3, two Ras homologs, one putative adaptor protein MST50, two transcription factors MgTEC1 and MgEFG1, and the beta-subunit of trimeric G-protein MGB1. MST7 and MST11 are confirmed to be the upstream MAPKK and MAPKKK activating PMK1 in M. grisea. However, the PAK kinase genes and pheromone receptors are dispensable for appressorium formation, indicating that the upstream signaling of the PMK1 pathway is different from that of FUS3/KSS1 in yeast. Since MST11 had a Ras-association domain and expressing a dominant active MgRAS1 allele resulted in abnormal appressorium formation, Ras1
protein is likely involved in the activation of the PMK1 pathway in M. grisea. MST50 interacts with both MST7 and MST11 and may function as the scaffold protein for the PMK1 cascade. MST12 weakly interacts with PMK1 in yeast two-hybrid assays and likely to be one of the transcription factors regulated by PMK1. Further characterization indicated that MST12 may be also involved in the penetration peg formation. Three subtraction libraries (two appressorium stages, one 5 days after infection) enriched for genes regulated by PMK1 were constructed. Four genes identified in the appressorium stage libraries have been functionally characterized. Two of them, GAS1 and GAS2, are virulence factors specifically and highly expressed during appressorium formation. We have also constructed two new yeasts and two hybrid libraries and characterized two Pmk1-interacting genes. One of them appears to be involved in lipid metabolism in appressoria. The GFP-Pmk1 fusion was used to monitor the expression
and localization of PMK1 during different stages of appressorium formation. Nuclear localization of PMK1 is observed around 8 hr of appressorium formation.
Impacts
We have identified and characterized several upstream and downstream elements of the PMK1 pathway. Our data indicate that signal inputs and outputs and components of the PMK1 pathway are different from that of yeast FUS3/KSS1. This is likely to be true for many other fungal pathogens.
Publications
- Park, G., Bruno, K., Talbot, N., Staiger, C., and Xu, J. -R. 2004. Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus. Molecular Microbiology. 53: 1695-1707.
- Li, L., Xue, C. Y., Bruno, K., Nishimura, M., and Xu, J. -R. 2004. Two PAK kinase genes MST20 and CHM1 have distinct functions in Magnaporthe grisea. Molecular Plant-Microbe Interactions. 17: 547-556.
- Xue, C., Li, L., Seong, K, and Xu, J. -R. 2004. The Magnaporthe grisea-rice interaction: A model system for studying fungal-plant interactions. Pages 138-165, In Plant-Pathogen Interactions. (Ed. N. J. Talbot). Blackwell Scientific Publishers, UK.
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Progress 10/01/02 to 09/30/03
Outputs
The PMK1 (pathogenicity MAP kinase 1) and its homologs are essential for appressoria formation and plant infection in several pathogenic fungi. Gene replacement mutants were generated for 12 genes involved in the PMK1 signaling pathway in Magnaporthe grisea, including the upstream MAPKK MST7 and MAPKKK MST11, the G-protein coupled receptor GPR1 and putative pheromone receptors MST2 and MST3, two Ras homologs, one putative adaptor protein MST50, two transcription factors MgTEC1 and MgEFG1, and the beta-subunit of trimeric G-protein MGB1. The element upstream from MST11 was found to be the MgRAS1 instead of the PAK kinase genes, indicating that the PMK1 pathway is activated in M. grisea similar to the STY1 pathway in the fission yeast. Expression of a dominant active MgRAS1 allele resulted in abnormal appressorium formation and fluffy colony morphology. MST11 had a Ras-association domain and directly interacted with MgRAS1 in yeast two-hybrid assays. Similar to the
mst11 deletion mutant, the mst50 deletion mutant was defective in appressorium formation and infectious hyphal growth. MST50 also contained a Ras-association domain and it may form a complex with MST11 and MgRAS1 to activate the downstream MAP kinase cascade. MST12, one of the downstream effectors of PMK1, was found to be dispensable for appressorium turgor generation, but it regulated the penetration peg formation. It is likely that MST12 may form heterodimers with MgTEC1 or MgEFG1 to regulate different infection processes in M. grisea. The GFP-Pmk1 fusion protein was highly expressed in developing and mature appressoria, young conidia and infectious hyphae. Our preliminary data indicated that PMK1 may regulate the death of conidial cells after appressorium formation in M. grisea.
Impacts
We have identified and characterized several upstream and downstream elements of the PMK1 pathway. Our data indicated that MGB1 functions above both the cAMP signaling and the PMK1 pathway. Further characterization of the mst12 deletion mutant has led us to conclude that penetration peg formation is essential for the rice blast fungus to elicit localized plant defense responses in underlying plant cells.
Publications
- Nishimura, M., Park, G., and Xu, J. -R. 2003. The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea. Molecular Microbiology. 50: 231-243.
- Xue, C., Li, L., Seong, K, and Xu, J. -R. 2003. The Magnaporthe grisea - rice interaction: A model system for studying fungal-plant interactions. In Plant-Pathogen Interactions. (Ed. N. J. Talbot). Blackwell Scientific Publishers, UK.
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Progress 10/01/01 to 09/30/02
Outputs
The PMK1 (pathogenicity MAP kinase 1) and its homologs are essential for appressoria formation and plant infection in several pathogenic fungi. Gene replacement mutants were generated for nine genes involved in the PMK1 signaling pathway in Magnaporthe grisea. MST7 and MST11 are likely to be the upstream MAPKK and MAPKKK activating PMK1 in M. grisea because mst7 and mst11 have phenotypes similar to that of pmk1 mutants. However, the PAK kinase genes (CHM1 and MST20) and pheromone receptors (MgSTE2 and MgSTE3) are dispensable for appressorium formation, indicating that the upstream signaling of the PMK1 pathway is different from that of FUS3/KSS1 in yeast. The gbm1 mutants are defective in appressorium formation and infectious growth, but they are pleiotrophic and produce fluffy colonies. MST12 weakly interacts with PMK1 in yeast two-hybrid assays and likely to be one of the transcription factors regulated by PMK1. Further characterization indicated that MST12 may be
also involved in the cAMP signaling, and regulates penetration peg formation. We have also constructed two new yeast two hybrid libraries and characterized two Pmk1-interacting genes. One of them appears to be involved in lipid metabolism in appressoria. The GFP-Pmk1 fusion was used to monitor the expression and localization of PMK1 during different stages of appressorium formation. Nuclear localization of PMK1 is observed around 8 hr of appressorium formation.
Impacts
Our data indicate that signal inputs and outputs and components of the PMK1 pathway are different from that of yeast FUS3/KSS1. This is likely to be true for many fungal pathogens. Characterizations of GBM1, MST7, MST11 and MST12 improve our knowledge about the PMK1 MAP kinase pathway that is essential for fungal pathogenesis.
Publications
- Xue, C., Park, G., Choi, W., Zheng, L., Dean, R. A., and Xu, J. R. 2002. Two novel fungal virulence genes specifically expressed in appressoria of the rice blast fungus. The Plant Cell. 14: 2107-2119.
- Xu, J.R., and Xue, C. 2002. Time for a blast: Genomics of Magnaporthe grisea. Mol. Plant Path. 3: 173-176.
- Park, G., Xue, C., Zheng, L., Lam, S., and Xu, J. R. 2002. MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. Mol. Plant-Microbe Interact. 15: 183-192.
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Progress 10/01/00 to 09/30/01
Outputs
Gene replacement mutants were generated for several genes involved in the PMK1 signaling pathway in Magnaporthe grisea. Mutants with the beta subunit gene GBM1 of the trimeric GTP binding proteins deleted had phenotypes similar to that of the pmk1 mutants. Conidia from gbm1 mutants fail to form appressorium and penetrate rice leaves, indicating that the PMK1 pathway that may be activated by GBM1. We also generated and characterized M. grisea mutants with the transcription factor MST12 deleted. MST12 is homologous to yeast Ste12 and likely function downstream from PMK1. The mst12 deletion mutants still form appressoria but cannot infect healthy or wounded rice plants. In the yeast two-hybrid assays, MST12 weakly interacts with PMK1. Our data suggest that that MST12 is one of the transcription factors regulated by PMK1 for infectious hyphae growth in rice plants, but additional transcription factors must exist in M. grisea for regulating appressorium formation by PMK1.
In addition, we functionally characterize two genes that are specifically expressed during appressorium formation. These two genes were identified in a subtraction library enriched for genes regulated by PMK1 in M. grisea. Both of them encode glycine rich proteins with signal peptides and are not expressed in vegetative hyphae or conidia. Our preliminary data suggest that these two genes belonging to a novel class of virulence factors in filamentous fungal pathogens. Mutants deleted in a Ste20 like protein kinase were also generated and under phenotype characterization. Agrobactrium mediated transformation and double strand RNA silencing were tested in M. grisea for improving the efficiency of functional analysis.
Impacts
Characterizing the functions of GBM1 and MST12 further dissected the upstream and downstream components of the PMK1 signal transduction pathway that is essential for plant infection in the rice blast disease development. Two genes identified in this study may constitute a novel class of virulence factors in plant pathogenic fungi.
Publications
- Park, G., Xue, C., Zheng, L., and Xu, J. -R. 2001. MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. Molecular Plant-Microbe Interactions In press.
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