Progress 08/15/05 to 08/14/09
Outputs
OUTPUTS: The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in the rice blast fungus Magnaporthe grisea. The affinity purification approach was used to identify genes interacting with Mst50 or Mst11 in vivo. Among those genes identified by copurification, several of them had been confirmed by coimmunoprecipitation assays, including MGG13743, MGG02405, MGG05752, MGG08820, MGG00883, and MGG8015. MG13743 encodes a putative phosphatidylinositol phosphate kinase gene but it is dispensable for plant infection. MGG00883 encodes a MAP kinase kinase kinase gene that is required for the activation of Mps1 MAP kinase and plant infection, indicating that MST50 plays a role in the cross talking between the Pmk1 and Mps1 pathways. MGG05752 also physically interacts with MST11 and RAS2 in M. grisea. It is likely a novel component of the PMK1 MAP kianse pathway. Deletion of this gene resulted in a minor reduction in virulence in the laboratory conditions. Because RAS2 plays a critical role in the activation of the PMK1 pathway, we identified and characterized four putative Ras GEF genes, MG00371, MG00199, MG02419, and MG11784, named RGF1 to RGF4, respectively. Four putitive Ras GAP genes, MG03846, MG03700, MG08105, and Mg11425 named RGA1 to RGA4, also were characterized. The rgf1 mutant was nonpathogenic and the rgf3 mutant formed abnormal appressoria. The RGF2, RGF4, RGA1, and RGA4 genes were dispensable for plant infection and appressorium formation. Similar to the rgf3 mutant, the rga3 mutant formed appressoria with abnormal morphology. The RGA2 gene was essential. The rga2 silencing mutant displayed the multiple appressoria and branching germ tube phenotypes of the MST11 RA association domain deletion mutant. These results indicate that RGF1, RGF3, RGA2, and RGA3 are likely involved in regulating appressorium formation and plant infection processes. These Ras GEF and Ras GAP proteins may function upstream from the cAMP PKA and PMK1 MAP kinase pathways. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the rice blast fungus Magnaporthe grisea and other plant pathogenic fungi, the PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection. In this study, the proteomics and candidate gene approached were used to identify additional components of the PMK1 pathway. A number of novel components of the PMK1 pathway and several upstream genes important for PMK1 activation have been identified and characterized. Because the PMK1 pathway is well conserved in plant pathogenic fungi for regulating different plant infection processes, the identification and characterization of upstream components required for the activation of this pathway is important for understanding host pathogen interactions. Information gained from these experiments will be helpful to develop more effective fungicides or novel disease control strategies.
Publications
- Ding, S., Mehrabi, R., Koten, C., Kang, Z., Wei, Y., Seong, K., Kistler, H. C., and Xu, J. R. 2009. The transducin beta like gene FTL1 is essential for pathogenesis in Fusarium graminearum. Eukaryotic Cell. 8: 867 to 876.
- Seong, K.Y., Pasquali, M., Zhou, X., Song, J., Hilburn, K., McCormick, S.P., Dong, Y., Xu, J. R., and Kistler, H.C. 2009. Global gene regulation by Fusarium transcription factors Tri6 and Tri10 reveals adaptations for toxin biosynthesis. Molecular Microbiology. 72: 354 to 367.
- Mitchell, T., Dean, R., Xu, J. R., Zhu, H., Oh, Y. Y., and Rho, H. 2009. Protein Chips and Chromatin Immunoprecipitation emerging technologies to study molecule interactions in Magnaporthe grisea. pp. 73 to 82. In Advances in Genetics, Genomics and Control of Rice Blast Disease. Springer Publishing, Netherlands.
- Ding, S., Zhou, X., Zhao, X., and Xu, J. R. 2009. The PMK1 MAP kinase pathway and infection related morphogenesis in Magnaporthe grisea. pp. 13 to 21. In Advances in Genetics, Genomics and Control of Rice Blast Disease. Ed. G. Wang and B. Valent. Springer Publishing, Netherlands.
- Mehrabi, R., Zhao, X., Kim, Y., and Xu, J. R. 2009. The cAMP signaling and MAP kinase pathways in plant pathogenic fungi. In Plant Relationship (The Mycota V) Ed. H.B. Deising, Springer. Germany. pp. 157 to 172.
- Liu, W., Xie, S., Zhao, X., Chen, X., Yi, Y., Liu, S., Lu, G., Xu, J. R, Wang, Z. 2009. A homeodomain transcription factor is essential for asexual reproduction in a filamentous ascomycete. Molecular Plant Microbe Interactions. In press.
- Yang, J., Zhao, X., Sun, J., Kang, Z., Ding, S., Xu, J. R., Peng, Y. 2009. A novel protein Com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae. Molecular Plant Microbe Interactions. In press.
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in the rice blast fungus Magnaporthe grisea. Because MST50 and MST11 play critical roles in the activation of PMK1, we have used the TAP and 3Flag tagging approach to identify proteins interacting with these two genes in vivo. Several proteins that were copurified with Mst50 or Mst11, including MG13743, MG00083, MG04251, MG05752, MG8015, MG03663, MG03838, and MG08735, appear to be novel genes involved in MAP kinase signaling. The interaction of Mst50 with MG13743, MG05752, and MG8015 had been confirmed by coimmunoprecipitation assays. MG13743 encodes a putative Phosphatidylinositol phosphate kinase gene. We have identified putative gene deletion mutants of MG13743, MG05752, and MG8015. Phenotype characterization of these mutants is in progress. In addition, we have functionally characterized two RasGEF genes, RGF1 (MG00371) and RGF2 (MG00199), and three RasGAP genes, MG03846, MG11425, and MG03700. The rgf1 but not rgf2 deletion mutant was significantly reduced in appressorium formation and nonpathogenic. Deletion of MG03848 resulted in a spore morphological defect and reduction in virulence. MG03700 may be an essential gene. Silencing of MG03700 resulted in a phenotype similar to that of the MST11 RA association domain deletion mutant. RGF1 and MG03700 likely function upstream from Ras2 and are involved in the activation of the PMK1 pathway. PARTICIPANTS: JinRong Xu, principal investigator Xinhua Zhao, postdoctorate research associate studying MST50 and MST11 interacting genes and generating antibodies Xiaoying Zhou, a Ph.D student studying the Ras GEF and GAP proteins Ivan Borrero, a MS student studying genes regulated by PMK1 YoonE Choi, postdoctorate research associate studying MST50 and MST11 interacting genes Guotian Li, a Ph.D student studying the upstream components of the PMK1 pathway TARGET AUDIENCES: Molecular biologists PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Results from our previous studies have indicated that the activation of the PMK1 MAP kinase pathway in the rice blast fungus differs significantly from those the model, nonpathogenic fungal systems. We used the protein copurification approach to identify novel components of this important pathogens. A number of proteins that interact with MST50 and MST11 and two genes that play key roles in the activation of the PMK1 pathway have been identified and characterized in this study. Because the PMK1 pathway is well conserved in plant pathogenic fungi for regulating different plant infection processes, the identification and characterization of upstream components required for the activation of this pathway is important for understanding host pathogen interactions and developing new strategies to protect plants from fungal infection.
Publications
- Seong, K, Zhao, X., Xu, J. R., Guldener, U., and Kistler, H. C. 2008. Conidial germination in the filamentous fungus Fusarium graminearum. Fungal Genetics and Biology. 45: 389 399.
- Barhoom, S., Kupiec, M., Zhao, X., Xu, J. R., and Sharon, A. 2008. Functional characterization of cgCTR2, a vacuole copper transporter that is involved in germination and pathogenicity of Colletotrichum gloeosporioides. Eukaryotic Cell. 7: 1098 1108.
- Mehrabi, R., Ding, S., and Xu, J. R. 2008. The MADSbox transcription factor Mig1 is required for infectious growth in Magnaporthe grisea. Eukaryotic Cells. 7: 791 799.
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Progress 08/15/06 to 08/14/07
Outputs
OUTPUTS: The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressoria formation and plant infection in the rice blast fungus Magnaporthe grisea. Because MST50 and MST11 play critical roles in the activation of PMK1, we have used the TAP and 3Flag tagging approach to identify proteins interacting with these two genes in vivo. Several proteins that copurified with Mst50 or Mst11 appear to be novel and have not been implicated in this MAP kinase pathway. One of them encodes a putative histidine kinase. Another one is homologous to a yeast gene known to be associated with Ste11. We have developed a polyclonal antibody against Mst50 and in the process of generating Mst11 antibody. We also have proved the direction interaction of Pmk1 with Mst7 during appressorium formation by the coimmunoprecipitation and BiFC approaches. In addition, we have identified two putative GEF and GAP proteins that may be functionally related to Ras1 and Ras2 in M. grisea. Their functions in the
activation of the cAMP PKA and PMK1 MAP kinase pathways are under investigation.
PARTICIPANTS: JinRong Xu, principal investigator Shengli Ding, postdoctorate research associate studying the TBL1 gene and MST12 transcription factor Xinhua Zhao, postdoctorate research associate studying MST50 and MST11 interacting genes and generating antibodies Xiaoying Zhou, a Ph.D student studying the Ras GEF and GAP proteins Ivan Borrero, a MS student studying genes regulated by PMK1
TARGET AUDIENCES: Plant Pathologists
Impacts
The activation of the PMK1 MAP kinase pathway in the rice blast fungus is different from that of the model fungi. To identify novel components of the Pmk1 pathway, we used the copurification approach to identify proteins that interact with MST50 and MST11, two genes that play key roles in the activation of PMK1. Functional characterization of these MST50 or MST11 interacting genes will be helpful to understand molecular mechanisms regulating plant infection process in the rice blast fungus and other pathogens.
Publications
- Zhao, X., Mehrabi, R., and Xu, J. R. 2007. MAP kinase pathways and fungal pathogenesis. Eukaryotic Cell. 10: 1701 1714.
- Betts, M. F., Tucker, S. L., Galadima, N., Meng, Y., Patel, G., Li, L., Donofrio, N. M., Floyd, A., Nolin, S., Brown, D., Mandel, M. A., Mitchell, T. K., Xu, J. R., Dean, R. A., Farman, M. L., Orbach, M. J. 2007. Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae. Fungal Genetics and Biology. 44: 1035 1049.
- Meng, Y., Patel, G., Heist, M., Betts, M., Tucker, S. L., Donofrio, N. M., Brown, D., Mitchell, T. K., Li, L., Xu, J. R., Orbach, M. J., Thon, M., Dean, R. A., and Farman, M. L. 2007. A systematic analysis of T DNA insertion events in Magnaporthe oryzae. Fungal Genetics and Biology. 44: 1050 1064.
- Li, L., Ding, S., Orbach, M., Sharon, A., and Xu, J. R. 2007. A novel nuclear protein MIR1 is highly upregulated during infectious hyphal growth in the rice blast fungus. Molecular Plant Microbe Interactions. 20: 448 458.
- Zhao, X., and Xu, J. R. 2007. A highlyconserved MAPKdocking site in Mst7 is essential for Pmk1 activation in Magnaporthe grisea. Molecular Microbiology. 63: 881 894.
- Xu, J. R., Zhao, X., and Dean, R. A. 2007. From genes to genomes; a new paradigm for studying fungal pathogenesis in Magnaporthe oryzae. In Advances in Genetics Fungal Genomics 57: 175 218.
- Park, G., Xue, C., Zhao, X., Kim, Y., Orbach, M., and Xu, J. R. 2006. Multiple upstream signals converge on an adaptor protein Mst50 to activate the PMK1 pathway in Magnaporthe grisea. The Plant Cell. 18: 2822 2835.
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Progress 08/15/05 to 08/15/06
Outputs
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressoria formation and plant infection in Magnaporthe grisea. To further characterize the upstream and downstream components of the PMK1 pathway in M. grisea, we have determined the function of the adaptor protein MST50 and its interaction with MST7, MST11, MGB1, and RAS2. Our data suggest that multiple upstream signals are converged at MST50 for regulating appressorium formation and plant infection. The direct interaction between MST50 and MST11 and the association of MST11 with the activated form of RAS2 are involved in the activation of MST11-MST7-PMK1 MAP kinase cascade. For the putative downstream transcription factor genes MST12 and PTH12, we have examined their interactions with CPKA and PMK1 in yeast two-hybrid assays and determined the effects of overexpressing MST12 and PTH12 in the pmk1 mutant. We also have generated the TAP and 3xflag tagged constructs of MST50, MST11, MST7, and MST12.
Transformants expressing these constructs will be used to identify additional components of the PMK1 MAP kinase pathway by the coimmunoprecipitation approach.
Impacts
Results from our experiment indicated that signal inputs and outputs of the PMK1 MAP kinase pathway in the rice blast fungus are different from what have been characterized in model fungi. MST50 functions as an adaptor protein for the PMK1 pathway. Multiple signals may be converged on MST50 to activate downstream appressorium formation and infectious growth. The activation of MST11 may involve its association with MST50 and the activated form of RAS2. The PMK1 pathway is well conserves in plant pathogenic fungi. Further characterization of MST50 and MST11 will be helpful to understand molecular mechanisms regulating plant infection process and develop novel disease control strategies.
Publications
- Soderlund, C., Pampanwar, V., Haller, K., Ebbole, D., Farman, M., Mitchel, T., Orbach, M., Wang, G., Wing, R., Xu, J. R., and Dean, R. 2006. MGOS: a resource for studying Magnaporthe grisea and Oryza sativa interactions. Molecular Plant-Micrcobe Interactions 19: 1055-1061.
- Xu, J. R., Peng, Y., Dickman, M. B., and Sharon, A. 2006.The dawn of fungal pathogen genomics. Annual Reviews of Phytopathology 44: 337-366.
- Park, G., Xue, C., Zhao, X., Kim, Y., Orbach, M., and Xu, J. R. 2006. Multiple upstream signals converge on an adaptor protein Mst50 to activate the PMK1 pathway in Magnaporthe grisea. The Plant Cell. In press (October issue of 2006)
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Progress 10/01/04 to 09/30/05
Outputs
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressoria formation and plant infection in Magnaporthe grisea. To further characterize the upstream and downstream components of the PMK1 (pathogenicity MAP kinase 1) signal transduction pathway in Magnaporthe grisea, we have determined the interaction between the MST7 MAP kinase kinase and PMK1. A MAP kianse docking site of MST7 and a corresponding association region on PMK1 have been identified. The MST7-PMK1 interaction during appressorium formation is examined with the BIFC technique. In addition, we have conducted detailed analyses of two RAS homologs, RAS1 and RAS2. While RAS1 is dispensable for fungal pathogenicity, RAS2 is an essential gene. Expressing a dominant active RAS2 allele causes improper regulation of appressorium formation and results in loss of pathogenicity. We have also found that the N-terminal portion of MST11 directly binds with its kinase domain. Activation of MST11 involves the
release of this self-inhibitory binding.
Impacts
This is a new project developed on the basis of a previous grant from NRI. Our data indicate that signal inputs and outputs and components of the PMK1 pathway are different from that of the yeast pheromone pathway. RAS proteins may play important roles in activation of PMK1. In addition, our characterization of MST11 self-inhibitory binding and MST7-PMK1 binding sites will be important for illustrating molecular mechanisms activating this important pathway for plant infection.
Publications
- Dean, R., Talbot, N., Ebbole, D., Farman, M., Mitchell, T., Orbach, M., Thon, M., Kulkarni, R., Xu, J. R., Pan, H., Read, N., Lee, Y., Carbone, I., Brown, D., Soanes, D., Djonovic, S., Kolomiets, E., Rehmeyer, C., Li, W., Harding, M, Kim, S., Lebrun, M., Bohnert, H., Butler, J., Calvo, S., Ma, L., Nicol, R., Purcell, S., Nusbaum, C., Galagan, J., and Birren, B. 2005. Analysis of the genome sequence of the plant pathogenic fungus Magnaporthe grisea, the causal agent of rice blast disease. Nature. 434: 980-986.
- Zhao, X., Kim, Y., Park, G., and Xu, J. R. 2005. A MAP kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea. The Plant Cell. 17 (4): 1317-1329.
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