Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Graduate and undergraduate students who took the Biology of Fungi and Pathogens of Plants courses I taught. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Xue Zhang, Ph.D student Yang Li, Ph.D student Cong Jiang, Postdoc research associate How have the results been disseminated to communities of interest? Publications and presentations at meeting and seminars What do you plan to do during the next reporting period to accomplish the goals? One of the research goals is to determine the specificities of WSC1, WSC2, and WSC3 in ligand recognition. We are also interested in in further characterize the regulatory function of Mcm1 in phialide formation and the role of microconidia in rice blast.
Impacts
What was accomplished under these goals?
In the rice blast fungus Magnaporthe oryzae, the Mps1 MAP kinase pathway regulates cell wall integrity and is important for appressorium penetration, invasive growth, and conidiation. We have identified and characterized three putative upstream receptor genes, WSC1, WSC2, and WSC3, that may be involved in recognizing different environmental signals. Deletion of WSC2 or WSC3 had no detectable phenotypes but deletion of WSC3 resulted minor reductions in penetration efficiency and virulence. However, the wsc1 wsc2 wsc3 triple mutants were defective in plant penetration and non-pathogenic, suggesting that these receptor genes have overlapping functions in M. oryzae. In an earlier study, we showed that the Mcm1 transcription factor related to the PMK1 pathway is essential for phialide formation and microconidium production. We further showed that its ortholog in the wheat scab fungus Fusarium graminearum is also important for phialide formation. The FgMCM1 deletion mutant was significantly reduced in mycotoxin production and virulence. In addition, we showed that approximately 10% of microconidia could germinate in M. oryzae. When inoculated through wounds, microconidia could infect and colonize plant tissues although at a relatively low efficiency in comparison with macroconidia.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhang, H. L., Wu, Z. S., Li, Y., Wang, C. F., and Xu, J. R. 2014. Germination and infectivity of microconidia in the rice blast fungus Magnaporthe oryzae. Nature Communications. 5: 4518.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhou, X. Y., Zhao, X. H., Xue, Z. Y., and Xu, J.-R. 2014. Formation of aerial appressoria without surface attachment in Magnaporthe oryzae by overactive Ras signaling. Mol. Plant Microbe Interactions. 27: 996-1004.
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Graduate and undergraduate students who took the Biology of Fungi and Pathogens of Plants courses I taught in 2013. Research results from my lab were included in lectures. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two postdocs, three PhD students, and two visiting scholars participated in this project were trained in molecular biology and fungal genetics. How have the results been disseminated to communities of interest? Publications in scientific journals and presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals? Finish up and publish results from ongoing projects related to the transcription factors and other components of the Pmk1 and cAMP/PKA signaling pathways.
Impacts
What was accomplished under these goals?
Transcription factors that are functionally related to the PMK1 and cAMP/PKA pathways, including Mst12, Mcm1, and Sfl1, have been characterized in details. The putative Mst12 binding site has been identified. Several Mst12 interacting genes were functionally characterized. For the PMK1 cascade, several interacting genes have been identified as putative novel components of this important signaling pathway. Further characterization of these genes are in progress. For the cAMP/PKA pathway, we have generated the cpkA cpk2 double mutant. Phenotype characterization of this double mutant is in progress. Furthermore, we have identified a number of spontaneous suppressors of this double mutant, which is significantly reduced in growth and conidiation. Identification of suppressor mutations in these suppressors is in progress.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Li, G. T., Zhou, X. Y., and Xu, J. R. 2012. Genetic control of infection-related development in Magnaporthe oryzae. Current Opinion in Microbiology. 15: 678-684
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kong, L. A., Li., G. T., Zhang, S. J., Yang, J., Zhou, X. Y., Peng, Y. L., and Xu, J. R. 2013. Characterization of the differences between appressorium formation and hyphopodium development in Magnaporthe oryzae. Fungal Genetics and Biology. 56: 33-41.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Xu, J. R. 2013. Invited presentation at the 6th International Congress of Plant Pathology. Beijing, China. August 25-30. 2013.
|
Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: In the rice blast fungus Magnaporthe oryzae, the PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation, penetration, and invasive growth. We have functionally characterized Sfl1, one of the transcription factors interacting with Pmk1 in M. oryzae. Mst12 is another transcription factor that functions downstream from Pmk1. One of the Mst12interacting genes identified by affinity purification is MoMCM1, which was found to be important for appressorium penetration and virulence. MoMcm1 may interact with Mst12 and MatA1 to regulate germ tube identity and male fertility, respectively in M. oryzae. Its ortholog in Fusarium graminearum is required for sexual reproduction and proper regulation of hyphal growth. We also have used the RNA sequencing and microarray analysis approaches to identify genes that have altered expression levels in the pmk1 and mst12 mutants. Functional characterization of selected genes and putative promoter elements are in progress. Several putative receptor genes for the Pmk1 and Mps1 MAP kinase pathways also have been identified and characterized, including the Msb2, Sho1, and Mid1 genes. The role of Msb2 in surface sensing and pathogenesis was determined. Its functional relationship with Pth11 and Cbp1, other two putative receptors known in M. oryzae, is under investigation. In addition, the affinity purification and proteomics approaches have been used to identify proteins that interact with over 50 known pathogenicity factors, including components of the cAMP PKA, Pmk1, Mps1, Osm1, and Cmk1 signaling pathways. Protein protein interaction maps have been established for these pathogenicity factors based on their interaction proteins, and compared with the interaction network of their orthologs in the budding yeast. Over 20 of these interactions have been confirmed by coimmunoprecipitation assays. PARTICIPANTS: JinRong Xu, principal investigator Xiaoying Zhou, Ph.D student Guotian Li, Ph.D student Ynag Li, Ph.D student TARGET AUDIENCES: Plant pathologists examining genetic aspects of plant diseases. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in Magnaporthe oryzae and other plant pathogens. We have functionally characterized several upstream and downstream components of this important signal transduction pathway, including the MST12, MCM1, and MSB2 genes. Many plant pathogenic fungi may use similar molecular mechanisms to sense and respond to plant surface signals as the rice blast fungus. Results from this study will improve our understanding of molecular mechanisms regulate plant infection processes. We also have used the proteomics approached to identified conserved and novel elements of the cAMP signaling, Pmk1, and Mps1 MAP kinase pathways. In addition, protein protein interaction maps have been established for over 50 known pathogenicity or virulence factors based on their interacting proteins, and compared with the interaction network of their orthologs in the budding yeast. Information gained from this study will be helpful to develop more effective fungicides or novel disease management strategies to control the rice blast and other fungal diseases.
Publications
- Zhou, X., Zhang, H., Li, G., Shaw, B., and Xu, J. R. 2012. The cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae. PLoS Pathogens. 9: e1002911.
- Xue, M. F., Yang, J., Li, Z., Hu, S., Yao, N., Dean, R. A., Zhao, W., Shen, M., Zhang, H., Li, C., Liu, L., Cao, L., Xu, X., Xing, Y., Hsiang, T., Zhang, Z., Xu, J. R., and Peng, Y. L. 2012. Comparative analysis of the genomes of two field isolates of the rice blast fungus Magnaporthe oryzae. PLoS Genetics. 8: e1002869. (Xu and Peng are co-corresponding authors)
- Wang, G. H., Zheng, Q., Wang C. F., Zhou, X. Y., and Xu, J. R. 2012. The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum. PLoS One. 7: e38324.
- Kong, L., Yang, J., Li, G., Qi, L., Zhang, Y., Zhao, W., Zhang, Y., Xu, J.R., and Peng, Y. L. 2012. Systematic characterization of chitin synthase genes in the rice blast fungus Magnaporthe oryzae. PLoS Pathogens. 8: e1002526. (Xu and Peng are co-corresponding authors)
|
Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: In the rice blast fungus Magnaporthe oryzae, the PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation, penetration, and invasive growth. Mst12 is a transcription factor that functions downstream from Pmk1 for regulating appressorial penetration and invasive growth. One of the Mst12interacting genes identified by affinity purification was MoMCM1, which encodes a MADS box protein orthologous to yeast Mcm1. MoMcm1 interacts with both Mst12 and a mating type gene MatA1 in yeast two hybrid assays. Deletion of the MoMCM1 gene resulted in the loss of male fertility and microconidium production. The Momcm1 mutant was defective in appressorium penetration and formed narrower invasive hyphae in plant cells. It was significantly reduced in virulence. The Momcm1 mst12 double mutant was normal in appressorium formation but defective in penetration and nonpathogenic. On hydrophilic surfaces, the double mutant formed curved germ tubes that formed appressoria at the 20% frequency. Under the same conditions, the Momcm1 or mst12 mutant did not form appressoria on hydrophilic surfaces. These results indicate that MoMCM1 and MST12 have overlapping functions to suppress appressorium formation under nonconducive conditions. MoMcm1 may interact with Mst12 and MatA1 to regulate germ tube identity and male fertility, respectively. The MCM1 ortholog in Fusarium graminearum and F. verticillioides also have been characterized. In F. graminearum, Mcm1 is required for sexual reproduction and proper regulation of hyphal growth. In F. verticillioides, Mcm1 is important for conidiation, conidium germination. For the downstream targets of the PMK1 MAP kinase cascade, we have used the deep sequencing approach to identify genes that have altered expression levels in the pmk1 and mst12 mutants. Among these genes that had the most significant reduction in transcription are the GAS1 and GAS2 genes. Functional characterization of other genes identified in this study is in progress. In addition, the affinity purification and proteomics approaches have been used to identify proteins that interact with known pathogenicity factors, including components of the cAMP PKA, Pmk1, Mps1, Osm1, and Cmk1 signaling pathways. To date, 3xFLAG and S tag constructs and transformants have been generated over a total of 65 genes. Affinity purification data have been generated for over 40 of them. Our goal is to establish protein protein interaction maps will be established for these pathogenicity factors. PARTICIPANTS: JinRong Xu, principal investigator Xiaoying Zhou, Ph.D student Guotian Li, Ph.D student TARGET AUDIENCES: Plant pathologists and molecular biologists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in Magnaporthe grisea and other important plant pathogens. We have functionally characterized the MCM1 gene that is associated with MST12, a main target of Pmk1 for regulating appressorium penetration and plant infection. Functional characterization of its orthologs in Fusarium graminearum and F. verticillioides indicated that this well conserved transcription factor gene is required for proper regulation of conidiation and hyphal growth. We also have used the proteomics approached to identified conserved and novel elements of the cAMP signaling, Pmk1, and Mps1 MAP kinase pathways. Understanding the functions of these genes will be helpful to understand the signal inputs and outputs of these conserved MAP kinase cascades in fungal pathogenesis.
Publications
- Zhou, X., Liu, W., Wang, C., Xu, Q., Wang, Y., Ding, S., and Xu, J. R. 2011. A MADS box transcription factor Mmc1 is required for male fertility and virulence in Magnaporthe oryzae. Molecular Microbiology. 80: 33 to 53.
- Li, Y., Wang, C., Liu, W., Wang, G., Kang, Z., Kistler, H. C., and Xu, J. R. 2011. Systematic Characterization of Type II HDAC Genes in Fusarium graminearum. Molecular Plant Microbe Interactions. 24: 487 to 496.
- Liu, W., Zhou, X., Li, G., Li, L., Kong, L., Wang, C., and Xu, J. R. 2011. Multiple plant surface signals are sensed by different mechanisms in the rice blast fungus. PLoS Pathogens. 7: e1001261. doi:10.1371.
- Zhang, Y. P., Choi, Y., and Xu, J. R. 2011. The FvMK1 mitogen activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides. Fungal Genetics and Biology. 48: 71 to 79.
- Zhang, H., Wang, C., Cheng, Y., Wang, X., Li, F., Han, Q., Xu, J. R., Chen, X., Huang, L., Wei, G., Kang, Z. S. 2011. Histological and molecular studies of the nonhost interaction between wheat and Uromyces fabae. Planta. 234: 979 to 991.
- Guo, J., Dai, X., Xu, J. R., Wang, Y., Bai, P., Liu, F., Duan, Y., Zhang, H., Huang, L., and Kang, Z. S. 2011. Molecular characterization of a Fus3 Kss1 type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1. PLoS ONE. 6: e21895.
- Zhang, H., Xue, C., Kong, L., Li, G., and Xu, J. R. 2011. A Pmk1 interacting gene is involved in appressorium differentiation and plant infection in Magnaporthe Oryzae. Eukaryotic Cell. 10: 1062 to 1070.
- Li, G., Zhou, X., Kong, L., Wang, Y., Zhang, H., Zhu, H., Mitchell, T., Dean, R. A., and Xu, J. R. 2011. MoSFL1 Encodes a transcription factor important for virulence and heat sensitivity in Magnaporthe oryzae. PLoS One 6(5):e19951. doi:10.1371.
|
Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: In the rice blast fungus Magnaporthe oryzae, the PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection. We have identified two upstream receptor genes MoMSB2 and MoSHO1 that function upstream from the PMK1 pathway. The Momsb2 mutant was significantly, but the Mosho1 mutant was only slightly, reduced in appressorium formation and virulence. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols, but not paraffin waxes and alkanes, stimulated appressorium formation in the Mosho1 Momsb2 mutant but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. In addition, the affinity purification and proteomics approaches have been used to identify proteins that interact with known pathogenicity factors, including components of three important signaling pathways. We have generated 3xFLAG tagged constructs and transformants for 35 genes, including all the key components of the cAMP signaling and three MAP kinase pathways. For the following 27 genes, we have conducted affinity purification and proteomics analyses: PMK1, MST50, MST11, MST7, RAS2, RASGAP1, MCM1, CAP1, MST12, MAC1, MAC1, TCO89, SIN1, LST8, MIP1, SCH9, BEM1, SFL1, CPKA, CON1, COM1, MMK2, OSM1, MPS1, MIR1, BCK1, and TIG1. For six putative MST50 interacting genes, we have generated 3xFLAG fusion constructs and transformants. Four of them were confirmed by coimmunoprecipitation analysis to be physically associated with MST50 in M. oryzae. For three of them, we have generated gene replacement mutants. One of them is essential for plant infection. PARTICIPANTS: JinRong Xu, principal investigator Xiaoying Zhou, Ph.D student Guotian Li, Ph.D student Wende liu, postdoc research associate Alisa Ponomarenko, Ph.D student TARGET AUDIENCES: Moleculear biologists conducting research on rice fungus PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in Magnaporthe grisea and other important plant pathogens. We have identified two upstream receptor genes, MoMSB2 and MoSHO1, that have overlapping functions in recognizing various surface signals for Pmk1 activation. In addition, primary alcohols, a major component of epicuticular leaf waxes in grasses, were found to be recognized by M. oryzae as signals for appressorium formation. We have also identified a number of putative novel components of the cAMP signaling and MAP kinase pathways by the proteomics and candidate gene approaches. Functional characterization of these genes will be important to understand the signal inputs and outputs of conserved signal transduction pathways that are essential for plant infection in various plant pathogenic fungi.
Publications
- Ding, S., Liu, W., Iliuk, A., Ribot, C., Vallet, J., Wang, Y., Tao, A., Lebrun, M., and Xu, J. R. 2010. The TIG1 HDAC complex regulates infectious growth in the rice blast fungus Magnaporthe oryzae. The Plant Cell. 22: 2495 to 2508.
- Ma, L., Rep, M., Borkovich, K. A., Coleman, J. J., Daboussi, M., DiPietro, A., Dufresne, M., Freitag, M., Grabherr, M., Henrissat, B., Kang, S., Park, J., Shim, W., Woloshuk, C. Xie, X., Xu, J. R., Antoniw, J., Baker, S., Brown, D., Chapman, S., Coulson, R., Coutinho, P. M., Danchin, E., G. J., Diener, A., Gale, L., Goff, S., Kodira, C. D., Hammond Kosack, K., Hua Van, A., Hilburn, K., Jonkers, W., Li, L., Koehrsen, M., Miranda Saavedra, D., Leary, S., Park, G., Proctor, R., Regev, A., Ruiz Roldan, C. M., Sain, D., Sykes, S., Wapinski, I., Schwartz, D. C., Turgeon, G., Yoder, O., Young, S., Zeng, Q., Zhou, S., Galagan, J., Birren, B. W., Cuomo, C. A., and Kistler, H. C. 2010. Fusarium comparative genomics reveals pathogenicity related lineage specific genome expansion. Nature. 464: 367 to 373.
- Liu, W., Xie, S., Zhao, X., Chen, X., Yi, Y., Liu, S., Lu, G., Xu, J. R, Wang, Z. 2010. A homeodomain transcription factor is essential for asexual reproduction in a filamentous ascomycete. Molecular Plant Microbe Interactions. 23: 366 to 375.
- Yang, J., Zhao, X., Sun, J., Kang, Z., Ding, S., Xu, J. R., Peng, Y. 2010. A novel protein Com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae. Molecular Plant Microbe Interactions. 23: 112 to 123.
- Choi, Y. E. and Xu, J. R. 2010. The cAMP signaling pathway in Fusarium verticillioides is important for conidiation, plant infection, and stress responses but not fumonisin production. Molecular Plant Microbe Interactions. 23: 522 to 533.
- Zhou, X., Heyer, C., Choi, Y., Mehrabi, R., and Xu, J. R. CID1 is important for plant infection in Fusarium verticillioides. 2010. Fungal Genetics and Biology. 47: 143 to 151.
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Magnaporthe grisea is pathogenic to economically important crops such as rice, barley, and wheat. 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, including3, MGG02405 and MGG05752. 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. Because RAS2 plays a critical role in the activation of the PMK1 pathway, we identified and characterized four putative Ras GEF genes named RGF1 to RGF4. Four putitive Ras GAP genes named RGA1 to RGA4 also were characterized. The rgf1 mutant was nonpathogenic and the rgf3 mutant formed abnormal appressoria. 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. In addition, we have further characterized the Tbl1 HDAC complex that is known to regulate infectious growth after penetration. Several components of the Tbl1 complex, include MgSET3, MgSNT1, MHD1, MHD2, MgHST1, and CPR1, have been identified by affinity purification and confirmed by coimmunoprecipitation or yeast two hybrid assays. Their expression and localization also were examined with GFP as the reporter gene. Parallel studies in the wheat scab fungus Fusarium graminearum indicated that a similar HDAC complex also is important for fungal pathogenicity, indicating that the TBL1 HDAC complex may be well conserved in plant pathogens for regulating plant infection processes. PARTICIPANTS: JinRong Xu, principal investigator Xiaoying Zhou, a Ph.D student studying the Ras GEF and GAP proteins 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 Wende liu, a postdoc studying TBL1 interacting genes and putative HDAC genes. TARGET AUDIENCES: Plant Pathologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in Magnaporthe grisea and other important plant pathogens. The proteomics and candidate gene approached were used to identify several putative novel components of the Pmk1 signal transduction pathway. A number of novel components of the PMK1 pathway and several upstream genes important for PMK1 activation of 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. In addition, our results have proved that TBL1 and its interacting genes form a protein complex that localizes to the nucleus in M. grisea. Further characterization indicated that he TBL1 HDAC complex is well conserved for regulating plant infection processes or fungal plant interactions in M. grisea and Fusarium graminearum. Information gained from these experiments will be helpful to develop more effective fungicides or novel disease control strategies.
Publications
- 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.
- 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.
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We used the TAP and 3Flag tagging approach to identify proteins interacting with MST50 and MST11, two genes playing a critical role in the activation of PMK1. 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. We also have functionally characterized two RasGEF genes, RGF1 (MG00371) and RGF2 (MG00199), and three RasGAP genes, MG03846, MG11425, and MG03700. RGF1 and MG03700 likely function upstream from Ras2 and are involved in the activation of the PMK1 pathway. In addition, we have further characterized the transducin beta like gene TBL1 and genes interacting with TBL1 in M. grisea. For the MgSET3, MgSNT1, MHD1, and MHD2 genes that are associated with TBL1 in vivo, gene replacement mutants have identified. The Mgset3, Mgsnt1, and mhd2 deletion mutants had similar defects in conidiation and plant infection as the tbl1 mutant. In comparison with the wild type strain, these mutants were significantly reduced in HDAC activities and increased histone acetylation. The tbl1 mutant also had enhanced sensitivities to hydrogen peroxiside, MsDef1, and osmotin. For the wheat scab fungus Fusarium graminearum, we have found that the type C like cyclin encoded by the CID1 gene plays an important role in plant infection. CID1 is homologous to SSN8 of Saccharomyces cerevisiae and the FCC1 gene of F. verticilloides, which is required for regulating fumonisin production on infected corn kernels. In F. graminearum, the cid1 mutant was enhanced in the production of a reddish pigment on V8 agar plates and liquid cultures. In infection assays with flowering wheat heads and corn stalks, the cid1 deletion mutant was significantly reduced in virulence. Only a very low amount of DON and 15ADON was detected in wheat kernels colonized by the mutant. The expression level of the trichodiene synthase gene TRI5 was reduced in the cid1 mutant. Reintroduction of the wildtype CID1 allele into the cid1 mutant complemented all its defects. These data suggest that CID1 may function as a regulatory factor in DON synthesis. Another project that was recently initiated is on comparative functional genomics of plant pathogenic Fusarium species in collaboration with Drs. H. Corby Kistler and Lijun Ma. We aim to develop a GeneChip microarray containing all predicted ORFs of four sequenced Fusarium species and conduct comparative microarray analysis to identify genes required for pathogenesis, host specificity, sexual and asexual reproduction processes in these Fusairum species. 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 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: Nothing significant to report during this reporting period.
Impacts
The PMK1 (pathogenicity MAP kinase 1) gene is essential for appressorium formation and plant infection in the rice blast fungus Magnaporthe grisea. In this study, we used the proteomics and candidate gene approach to identify upstream components involved in the PMK1 pathway. A number of proteins that interact with MST50 and MST11 and two genes that play key roles in the activation of PMK1 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. Although appressorium formation and penetration are well characterized, there are only limited studies on infectious hyphal growth in the rice blast fungus. Our results have indicated that TBL1 may encode a component of a well conserved regulatory complex that has been evolved to regulate the development and growth of infectious hyphae during plant infection. Information gained from our studies with the PMK1 pathway and the TBL1 complex may lead to the development of more effective fungicides or disease control strategies.
Publications
- 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.
- 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.
|
Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: To further characterize the PMK1 (pathogenicity MAP kinase 1) pathway that is essential for appressoria formation and plant infection in Magnaporthe grisea, 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 also have developed a polyclonal antibody against Mst50 and in the process of generating Mst11 antibody. In addition, we have identified two putative GEF and GAP proteins that may be functionally related to Ras1 and Ras2 in M. grisea. To identify downstream genes regulated by the PMK1 pathway, we have compared the microarray data with the pmk1 and mst12 mutants of M. grisea and the fmk1 and fst12 mutants of Fusarium graminearum. Several genes identified in
the comparative microarray analysis have been functionally characterized, including one putative zinc finger transcription factor and one putative PTH11 like receptor. We also have used the GST MST12 fusion to identify oligonucleotides that bind to MST12. Recently, the transducin beta like gene TBL1 was found to be essential for infectious growth. TBL1 may be a component of this well conserved regulatory complex that has been evolved to regulate the development and growth of infectious hyphae in M. grisea. For the wheat scab fungus Fusarium graminearum, in comparison with the wild type strain, a total of 333 and 155 genes were down and up regulated, respectively, in the fmk1 mutant. Functional classification of the probe sets revealed multiple processes were affected by the deletion of FMK1. Many of these genes were unique to F. graminearum. Forty four of them encoded putative transcription factors with DNA binding motifs. We selected 12 genes with altered expression levels in the
pmk1 for verification by qRT PCR. Four of the genes verified by qRT PCR were functionally characterized. While two other genes appeared to be dispensable for growth and pathogenesis in F. graminearum, deletion of the ATG8 homolog and a putative Zn2Cys6 transcription factor significantly reduced its virulence on flowering wheat heads.
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 proteomics and candidate gene approached were used to identify several putative novel components of the Pmk1 signal transduction pathway that regulates the key plant infection processes in the rice blast fungus and other plant pathogens. Downstream target genes regulated by the PMK1 pathway were identified by microarray analysis. Functional characterization of these genes will be helpful to understand molecular mechanisms regulating fungal pathogenesis. Our results also indicate that the transducin beta like gene TBL1 may be a component of a well conserved regulatory complex that controls infectious growth in fungal pathogens. Information gained from these experiments will be helpful to develop more effective fungicides or novel disease control strategies.
Publications
- Zhao, X., Mehrabi, R., and Xu, J. R. 2007. MAP kinase pathways and fungal pathogenesis. Eukaryotic Cell. 10: 1701 1714.
- Cuomo, C., Guldener, U., Xu, J. R, Trail, F., Turgeon, B. G., et al., and Kistler, H. C. 2007. The genome sequence of Fusarium graminearum reveals localized diversity and pathogen specialization. Science 317: 1402 1405.
- 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 TDNA insertion events in Magnaporthe oryzae. Fungal Genetics and Biology. 44: 1050 1064.
- Bluhm, B. H., Zhao, Z., Flaherty, J., Xu, J. R., and Dunkle, L. D. 2007. RAS1 regulates growth and pathogenesis in Fusarium graminearum. Molecular PlantMicrobe Interactions. 20: 627 636.
- 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 PlantMicrobe Interactions. 20: 448 458.
- Ramamoorthy, V., Zhao, X., Snyder, A. K., Xu, J. R., and Shah, D. M. 2007. Two Mitogenactivated protein kinase signaling cascades regulate sensitivity to antifungal plant defensins in Fusarium graminearum. Cellular Microbiology. 9: 1491 1506.
- 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.
|
Progress 10/01/05 to 09/30/06
Outputs
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 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. We also have further characterized the self-inhibitory interaction between the N-terminal region and the kinase domain of MST11. 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 MST11. Transformants expressing these constructs will be used to identify additional components of the PMK1 MAP kinase pathway by the coimmunoprecipitation approach. For the wheat scab fungus Fusarium graminearum, we have generated and characterized gene replacement mutants of the adenylate cyclase gene and the catalytic subunit of PKA. Our data indicate that the cAMP signaling pathway is essential for pathogenesis and female fertility in F. graminearum. For the transducin beta subunit like gene TBL1, we found that the LisH domain is essential for its function. Mutants deleted of the TBL1 orthologs in M. grisea and Neurospora crassa also have been generated. The tbl1 mutants appear to be normal in the initial penetration but defective in establish infectious growth. In addition, we have found that mutants defective in the GPMK1 and MGV1 MAP kinase pathways are hypersensitive to a plant defensin AtDef1. Both GPMK1 and MGV1 may be
involved in overcoming plant defensive responses.
Impacts
Results from our experiment indicated that signal inputs and outputs of the PMK1 signal transduction pathway in the rice blast fungus are different from what have been characterized in model fungi. MST50 functions as an adaptor protein and multiple signals may be converged on MST50 to regulate various plant infection processes. For the wheat scab fungus, we found the well conserved cAMP signaling pathway is ssential for plant infection and sexual reproduction. Spores from sexual reproduction are the primary inoculum for the initial infection of flowering wheat heads. Theses results are helpful to understand molecular mechanisms regulating fungal-plant interactions and may lead to the development of more effective fungicides or 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.
- Goswami, R. S., Xu, J. R., Trail, F., Hilburn, K., and Kistler, H. C. 2006. Genomic analysis of host-pathogen interaction between Fusarium graminearum and wheat during early stages of disease development. Microbiology 6: 1877-1890.
- Guldener, U., Seong, K., Boddu, J., Cho, S., Trail, F., Xu, J. R., Adam, G., Mewes, H., Muehlbauer, G. J., and Kistler, H. C. 2006. Development of a Fusarium graminearum Affymetrix GeneChip for profiling fungal gene expression in vitro and in planta. Fungal Genetics and Biology 43: 316-325.
- Seong, K., Li, L., Hou, Z., Kistler, H. C., and Xu, J. R. 2006. Cryptic promoter activity of the HMR1 coding region in the wheat scab fungus Fusarium graminearum. Fungal Genetics and Biology 43: 34-41.
|
Progress 10/01/04 to 09/30/05
Outputs
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. In intracellular level of cAMP is not significantly altered. However, deletion of the Ras-association domain in MST50 or MST11 reduced the efficiency of appressorium formation or penetration and causes a reduction in virulence on rice seedlings. For the
wheat scab fungus Fusarium graminearum, we have identified genes disrupted in six REMI mutants. Two of them, the b-ZIP transcription factor and beta-subunit of transducin like genes have been functionally characterized in details, including their expression, localization, domain structure, and overexpression. We also have generated targeted deletion mutants for several candidate pathogenicity factor genes, including the MST12 and PTH12 homologs. In addition, we have used the F. graminearum whole-genome microarray to study genes with different expression patterns in mycelia grown under different nutritional conditions and in the fmk1 and tbl1 mutants.
Impacts
Our data on upstream and downstream elements of the PMK1 pathway indicated that signal inputs of this important MAP kinase cascade in M. grisea are different from what have been characterized in model fungi. Functional characterization of RAS proteins indicated that two RAS proteins have distinct functions and RAS2 is critical for appressorium formation and plant infection. The transient interaction between PMK1 and MST7 is mediated by the docking and association sites. In addition to novel fungal virulence identified in F. graminearum by insertional or targeted deletion mutagenesis, microarray analysis has been used to understand molecular events underlying Fusarium pathogenesis.
Publications
- Seong, K., Hou, Z., Kistler, H. C., and Xu, J. R. 2005. Random Insertional Mutagenesis Identifies Genes Associated with Virulence in the Wheat Scab Fungus Fusarium graminearum. Phytopathology. 95 (7): 744-750.
- 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.
|
Progress 10/01/03 to 09/29/04
Outputs
To further characterize the upstream and downstream components of the PMK1 (pathogenicity MAP kinase 1) signal transduction pathway in Magnaporthe grisea, we have conducted detailed analyses of one putative adaptor protein MST50 and one zinc finger transcription factor MST12. The mst50 deletion mutant is defective in appressorium formation and infectious hyphal growth. The Mst50 protein contains a Ras-association domain at the C-terminus and a SAM domain at the N-terminus, and possibly is involved in both the cAMP and PMK1 signaling pathways. It physically interacts with Mst11 and Mst7 in yeast two-hybrid assays. The SAM domain is required for its association with Mst11 and its biological function. MST12, one of the downstream effectors of PMK1, was found to be dispensable for appressorium turgor generation, but it regulated the penetration peg formation. Using GFP-tagged tubulin and actin, we have determined that the mst12 mutant is defected in maintaining normal
cytoskeleton structures in mature appressoria. Aggregation of actin at the penetration site and formation of vertical microtubules may involve genes regulated by MST12. For the wheat scab fungus Fusarium graminearum, we have identified genes disrupted in four REMI mutants. Two of them, the b-ZIP transcription factor and beta-subunit of transducin like genes disrupted in mutants M7 and M75, respectively, are novel fungal virulence factors. We also have generated targeted deletion mutants for over 10 candidate genes, including the MAP kinase kinase and MEK kinase genes activating the FMK1 MAP kinase. In addition, a whole-genome microarray has been developed in collaboration with Affemetrix.
Impacts
Our data on upstream and downstream elements of the PMK1 pathway indicated that signal inputs of this important MAP kinase cascade in M. grisea are different from what have been characterized in model fungi. Functional characterization of MST50 showed that MST50 plays an important role in activating the PMK1 cascade. Novel fungal virulence factors identified in our study and the whole-genome microarray of F. graminearum will be very helpful to better understanding pathogenesis of this important pathogen.
Publications
- Zhao, X., Xue, C., Kim, Y., and Xu, J. -R. 2004. A Ligation-PCR approach for generating gene replacement constructs in Magnaporthe grisea. Fungal Genetics Newsletter. 51: 17-18.
- 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.
- Park, G., and Xu, J. -R. 2004. Mechanisms of Infection-Imperfect Fungi. pp.694-696 in The Encyclopedia of Plant and Crop Science (ISBN: 0-8247-4268-0). Ed. Robert Goodman, Marcel Dekker, Inc. 270 Madison Ave. New York, USA.
- 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.
|
Progress 10/01/02 to 09/30/03
Outputs
Genetic, biochemical, and cell biological approaches were used to further characterize the PMK1 (pathogenicity MAP kinase 1) signal transduction pathway, which regulates appressoria formation and pathogenic growth in Magnaporthe grisea. 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. 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. For the wheat scab fungus Fusarium graminearum, we have generated over 7000 restriction-enzyme mediated integration mutants. Over 10 mutants with reduced virulence have been identified. In addition, targeted deletion mutants
have been identified for two polyketide synthase genes, two novel proteins with nucleus localization signal, and two putative virulence factor genes. In 2003, we have generated 10x coverages of the F. graminearum genome in collaboration with the Whitehead Genome Research Institute. The second release of the assembly and automated annotation of the F. graminearum genome are now available on-line to the public.
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. The REMI mutant collection and the genome sequence of F. graminearum will be very helpful to pathogenesis studies in this important pathogen.
Publications
- Trail, F., Xu, J. -R., San Miguel, P., Halgren, R. G., and Kistler, H. C. 2003. Analysis of Expressed Sequence Tags from Gibberella zeae (anamorph Fusarium graminearum). Fungal Genetics and Biology 38: 187-197.
- 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.
|
Progress 10/01/01 to 09/30/02
Outputs
The candidate gene, subtractive hybridization, and yeast two-hybrid approaches were used to characterize the PMK1 (pathogenicity MAP kinase 1) signal transduction pathway, which regulates appressoria formation and pathogenic growth in Magnaporthe grisea. Gene replacement mutants were generated for nine genes involved in the PMK1 pathway in Magnaporthe grisea. While the PAK kinase and pheromone receptor genes may be not involved, MST7 and MST11 are likely to be the MAPKK and MAPKKK activating PMK1 in M. grisea because mst7 and mst11 have phenotypes similar to that of pmk1 mutants. MST12 weakly interacts with PMK1 in yeast two-hybrid assays and likely to be one of the transcription factors regulated by PMK1. 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. For the wheat scab fungus Fusarium graminearum, we have constructed and sequenced two subtraction libraries enriched for genes expressed during plant infection. We also sequenced 2000 ESTs from a cDNA library constructed with RNA isolated from mycelia grown on complete medium. Mutants with targeted deletion of three selected genes have been generated. The mgv1 mutants are reduced in growth, deoxynivanol production, and plant infection.
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. MGV1 is the second pathogenicity gene characterized in F. graminearum.
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.
- Hou, Z., Xue, C., Katan, T., Peng, Y., Kistler, H. C., and Xu, J. -R. 2002. A MAP kinase gene (MGV1) for hyphal growth and plant infection in Fusarium graminearum. Molecular Plant-Microbe Interactions. 15: 1119-1127.
|
Progress 10/01/00 to 09/30/01
Outputs
Mutants with the beta subunit gene GBM1 of the trimeric GTP binding proteins deleted were identified and characterized in the rice blast fungus Magnaporthe grisea. Phenotypically, these mutants are similar to the pmk1 mutants and fail to form appressorium and infect rice leaves, indicating that the PMK1 pathway that regulating the infection process may be activated by GBM1. We also generated and characterized 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. MST12 weakly interacts with PMK1 in the yeast two hybrid assays. Most likely, MST12 is one of the transcription factors regulated by PMK1 for infectious hyphae growth in rice plants. However, our data suggest that 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 in M. grisea. These two genes are secreted and glycine rich proteins that have no homologues from organisms other than filamentous fungi. Our preliminary data suggest that these genes belonging to a novel class of virulence factors in filamentous fungal pathogens. We also generated about 10,000 ESTs and 5,000 REMI transformants for the wheat scab fungus Fusarium graminearum. One mutant dramatically reduced in wheat head infection was isolated and characterized. A cosmid library and BAC library were also constructed for future genomics studies. Finally, we constructed two subtraction libraries enriched for genes specifically or highly expressed during plant infections in the gray mold fungus Botrytis cinerea. A condition that enhances microconidia production in B. cinerea was developed.
Impacts
Characterizing the functions of GBM1 and MST12 further dissected the PMK1 signal transduction pathway that is essential for plant infection in the rice blast disease development. The EST database and random mutants of the wheat scab fungus generated in this study will be used to identify virulence factors in this important pathogen.
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.
|
Progress 10/01/99 to 09/30/00
Outputs
The beta subunit gene (GBM1) of the trimeric G protein was isolated from the rice blast fungus Magnaporthe grisea. It encodes a 358 amino-acid peptide and is highly homologous to G beta genes identified in other filamentous fungi. A GBM1 gene replacement vector was constructed and transformed into M. grisea. One putative GBM1 knockout mutant was identified after screening over 100 transformants. The phenotype of the gbm1 mutant is being characterized to determine the role of G protein on activating the PMK1 pathway that regulates the plant infection processes in M. grisea. A transcription factor homologous to yeast Ste12 has also been isolated from M. grisea. This transcription factor (MST12) is likely the major target of the PMK1 MAP kinase, and it may regulate the expression of many genes involved in appressorium formation and pathogenic growth. Functionally characterizing the MST12 gene is in the process. In addition, a forward subtractive cDNA library was
constructed using RNAs isolated wild type appressoria and pmk1 mutant germ tubes. Over 96 clones have been sequenced from this library enriched for genes regulated by PMK1 during appressorium formation. For the wheat scab fungus Fusarium graminearum, two different cDNA libraries and one cosmid library were constructed. Over 2500 expressed sequence tags (ESTs) have been sequenced. These ESTs will be used for microarray analyses to identify genes expressed in different growth and infection stages. Choline is a plant compound present in wheat anthers that enhances F. graminearum infection and virulence. To illustrate the molecular mechanism of choline's stimulatory activity on F. graminearum, one putative choline transporter gene has been isolated and will be functionally characterized.
Impacts
The isolation and characterization of GBM1 and MST12 will help us better understand the PMK1 MAP kinase pathway that is highly conserved and important for plant infection in many fungal pathogens. Generating the EST database and characterizing the choline transporter in F. graminearum will be beneficial to determine fungal pathogenesis of this important pathogen.
Publications
- Zheng, L., Campbell, M., Murray, J., Lam, S., and Xu, J. -R. 2000. The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea. Mol. Plant-Microbe Interact. 13: 724-732.
- Xu, J. -R. 2000. MAP kinases in fungal pathogens. Fungal Genet. Biol. In press.
|
|