Source: PURDUE UNIVERSITY submitted to NRP
THE INTERACTOME OF PATHOGENICITY FACTORS IN THE RICE BLAST FUNGUS MAGNAPORTHE ORYZAE.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0220498
Grant No.
2010-65110-20439
Cumulative Award Amt.
(N/A)
Proposal No.
2009-03028
Multistate No.
(N/A)
Project Start Date
Jan 15, 2010
Project End Date
Jan 14, 2014
Grant Year
2010
Program Code
[91312]- Microbial Genomics Functional Genomics
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Department of Botany and Plant Pathology
Non Technical Summary
Rice blast is an important disease and is a model system for studying plant pathogen interactions. Although over 100 plant infection related genes have been identified in the rice blast fungus, there is only limited knowledge about their interactions. The goal of this study to characterize the interaction and relationship among these plant infection related proteins. First proteins that interact with known pathogenicity factors in the rice blast fungus will be identified by the affinity purification and proteomics approaches. Protein protein interaction maps will be established. The same approach will then be used to identify interacting proteins of selected genes that are expressed in appressoria and invasive hyphae. Pathogenicity factor-associated genes identified in these studies will be analyzed bioinformatically and further characterized to determine their functions in disease development. All the data generated in this study will be available to the scientific community in a web based database. Overall, proposed experiments represent the first study of protein-protein interaction networks of pathogenicity factors in plant pathogenic fungi. Results from this study will improve our understanding of the interactions and biological functions of pathogenicity factors.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240201040100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Rice blast is a disease of significant economic impact worldwide and a model system for studying fungal plant interactions. To date, over 100 pathogenicity factors have been identified in Magnaporthe oryzae. However, there is only limited knowledge about their interactions. To better understand molecular mechanisms regulating plant infection processes, it is critical to identify protein-protein interaction networks important for pathogenesis. The goal of this study is to characterize the interactome of pathogenicity-related proteins. Objective 1 is to use the affinity purification and proteomics approaches to identify proteins that interact with known pathogenicity factors, including components of three important signaling pathways. Protein protein interaction maps will be established for these pathogenicity factors. Results from this study will improve our understanding of their relationship and functions. Objective 2 is to identify interacting proteins of selected genes that are expressed in appressoria and invasive hyphae. Experimental procedures developed in this study will have a significant impact on studying appressoria and fungal-plant interactions. Objective 3 is to functionally characterized pathogenicity factor-associated genes identified in objectives 1 and 2. Some of them may encode novel pathogenicity factors. Objective 4 is to generate a database and protein-protein interaction networks with data from this study. Overall, proposed experiments represent the first study of protein-protein interaction networks of pathogenicity factors in phytopathogenic fungi. Results from this study will improve our understanding of the interactions and biological functions of pathogenicity factors.
Project Methods
The wild-type strain for proposed experiments will be 70-15. Rice cultivars Nipponbare will be used for spray or injection or root infection assays. Gene replacement constructs will be generated by the ligation-PCR method. Fungal transformation will conducted by treatment of protoplasts with PEG. Appressorium formation will be assayed on plastic coverslips or hydrophobic and hydrophilic sides of GelBond membranes. Penetration and infectious hyphal growth will be assayed with onion or rice leaf sheath epidermal cells. Standard molecular biology procedures will be followed for manipulation of DNA, RNA, and proteins. GFP-fusions will be constructed by the gap repair approach and examined with a Nikon E800 epifluorescence microscope. The Quickchange II site-direct mutagenesis kit will be used to generate point mutations and small deletions. The 3xFlag tag constructs will be generated with the yeast in vivo recombination approach and introduced into strain 70-15. The anti-flag antibody will be used to purify proteins that associate with tagged pathogenicity factors in vivo. Mass spectrometry will be used to identify co-purified proteins. Protein-protein interaction network will be established as a web-based database.

Progress 01/15/10 to 01/14/14

Outputs
Target Audience: Results were presented at the Annual Phytopathological Society Meeting, Asilomar Fungal Genetics Conference, Golden Conference on Molecular and Cellular Mycology, Annual meeting of the Asian Mycological Society, International Congress of Plant Protection, and International Rice Blast Conference. Scientists attended these meetings include plant pathogogists, fungal geneticists, plant breeders, and molecular biologists from the US and other countries. The strains used for co-immunoprecipitation assays developed in this project were used in one lab exercise for the fungal course taught by the PI at Purdue. This course had 13 graduate students from Department of Botany and Plant Pathology and Department of Agriculture Engineering. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate students and postdoc research associates participated in this project were trained in multi-disciplinary research approaches to identify interacting proteins for different pathogenicity and effector proteins. This project provides training opportunities on molecular cloning, protein isolation and purification, mass spectromety analysis, phosphoproteomics analysis, co-immunoprecipitation, and fungal-plant interactions. Participating graduate students and postdocs include: Xiaoying Zhou Guotian Li Yang Li Anto Iliuk Keerthi Jayasundera Lingfei Zeng Martha Giraldo How have the results been disseminated to communities of interest? Results were presented at the Annual Phytopathological Society Meeting, Asilomar Fungal Genetics Conference, Golden Conference on Molecular and Cellular Mycology, Annual meeting of the Asian Mycological Society, International Congress of Plant Protection, and International Rice Blast Conference. Scientists attended these meetings include plant pathogogists, fungal geneticists, plant breeders, and molecular biologists from the US and other countries. In addtion to meeting presentations, results from this project have been published in multiple book chapters and journal articles, which should reach a broad ranger of readers with different research interests. What do you plan to do during the next reporting period to accomplish the goals? This is the final report. We are preparing two manuscripts and expect to submit them in next two months. The resulting database will be released to the public when these two publications were submitted.

Impacts
What was accomplished under these goals? To identify protein-protein interaction networks important for known pathogenicity or virulence factors, we generated fusion constructs fused with the S-Tag or 3xFLAG tag for 94 bait proteins of Magnaporthe oryzae. These 94 genes iinvoved in diverse biological functions were selected on the basis of published results showing their important for plant infection in the rice blast fungus. We then selected 22 genes of interest based on affinity purification data from these initial 94 genes to conduct the second round of affinity purification. Most of them were related to the major signal transduction pathways. In total, we generated 116 tagged-proteins expression constructs in M. oryzae. The expected fusion proteins were reliably detected in 71 of them in vegetative hyphae or appressoria. Proteins assoicated these pathogenicity factors were then isolated by affinity purification and identified by mass spectrometry analysis. A data-processing pipeline was developed to remove low quality peptides and non-specific background proteins. In total, high quality data were obtained from a total of 1,106 purificaiton experiments. The resulting affinity purificaiton data for individual genes were plotted together to establish the interaction network. In comparison with the yeast interaction network data, 10% of the interactions identified in M. orzyae have their yeast orthologs interacting with each other. For the three MAPK pathways, several novel components were identified for each of them, including MGG13806 and MGG03838. Further characterization of these two genes indicated that they are important for virulence in M. oryzae. MGG03838 may encode a phosphatase involved in the Pmk1 pathway. In addition, we selected 25 interactions identified in this study for verification by co-immunoprecipitation or BiFC assays. For the ones confirmed by co-IP or BiFC assays, gene replacement mutants were generated. A total of 10 mutants, including mutants deletected of the rack1 ortholog, have detectable phenotypes in infection-related morhogenesis or virulence. A database with all the interacting data and predicted protein-protein interacting network have been developed. We expect that it will be released to the public when this manuscript is published later this summer.

Publications

  • Type: Book Chapters Status: Published Year Published: 2011 Citation: Zhou, X., Li, G., and Xu, J. R. 2011. Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi. Pages 199-212. In: Fungal Genomics: Methods and Protocols 722 (eds. Jin-Rong Xu and Burt Bluhm). Humana Press, Springer Science+Business Media, LLC. Heidelberg, Germany. ISSN 1064-3745.
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Zhang, H. L., Wu, Z. S., Li, Y., Wang, C. F., and Xu, J.-R. 2014. Germination. 2014. Germination and infectivity of microconidia in the rice blast fungus Magnaporthe oryzae. Nature Communications. Under Revision.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, S. J. and Xu, J.-R. 2014. Effectors and effector delivery in Magnaporthe oryzae. PLoS Pathogens. 10(1): e1003826.
  • 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: 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: 2012 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: 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(2) e1002526.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: 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: 10621070.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: 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: 33-53.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: 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.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Chen, X. L., Shi, T., Yang, J., Chen, D., Xu, X. W., Xu, J. R., Talbot, N. J., and Peng, Y. L. 2014. N-Glycosylation of effector proteins by an alpha-1,3-mannosyltransferase is required to evade host innate immunity by the rice blast fungus. The Plant Cell. In Press.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Li, G. and Xu, J. R. The involvement of Mst50 in multiple signaling pathways in Magnaporthe oryzae. Golden Research Conferences on Cellular & Molecular Fungal Biology. June 17-22, 2012. Holderness, NH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Zhou, XY and Xu, J.-R. Identification and characterization of RasGAPs and RasGEFs functioning upstream from the Pmk1 MAPK pathway. Golden Research Conferences on Cellular & Molecular Fungal Biology. June 17-22, 2012. Holderness, NH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Xu, J. R. Signaling Pathways Regulating Appressorium Morphogenesis in Magnaporthe oryzae. The Annual Asian Mycological Congress, Beijing, China., August 19-24. 2013
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Iliuk AB, Tao WA. Is phosphoproteomics ready for clinical research? Clin Chim Acta. 2013 May;420:23-7.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Xue L, Wang W, Hu L, Iliuk A, Geahlen RL, Tao WA. Sensitive kinase assay linked with phosphoproteomics for identifying kinase substrates. 60th ASMS Conference, Vancouver, BC. Canada. May 20-24, 2012.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Xue L and Tao WA. Developing proteomic toolbox to study plant phosphoproteomics. American Chemical Society (ACS) Annual Conference. Indianapolis. Sep 09, 2013.


Progress 01/15/10 to 01/14/11

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Jiang Cong, Postdoc Yang Li, PhD Student Xue Zhang, PhD student How have the results been disseminated to communities of interest? Meeting presentations and journal articles (manuscripts) What do you plan to do during the next reporting period to accomplish the goals? Finish up and publish all the publishable results

Impacts
What was accomplished under these goals? In the rice blast fungus, over a hundred of pathogenicity or virulence factors have been identified in various independent genetic studies. However, for most of those infection related genes, their functional relationships during plant infection are not clear. In this study, we used the affinity purification and proteomics approaches to identify proteins that interact with 60 known pathogenicity factors, including components of three important 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. One manuscript is in preparation to publish the overall interactome results. We are also preparing two separate manuscripts on proteins interacting with CpkA and Ras2. In addition, we have identified several rice proteins that putatively interact with fungal effectors. Functional characterizaiton of these rice proteins are in progress. Among the genes identified by affinity purification to interact with MST50, over a dozen have been examined by coimmunoprecipitation assays. Further functionally characterization of five selected MST50 interacting genes showed that this scaffold protein is involved in multiple signaling in Magnaporthe oryzae that are related to development and pathogenesis, including the cell wall integrity and osmoregulation pathways. One of the MST12 interacting genes is MCM1, which has been characterized and published in Molecular Microbiology. One of the MAC1 interacting genes is CAP1, which was found to be important for conidiation, appressorium formation, and invasive growth and publisehd in PLoS Pathogens.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Li, Y., Zhou, X., Jayasundera, K. B., Iliuk, A., Tao, A., and Xu, J. R. 2013. The interactome of pathogenicity factors in the rice blast fungus Magnaporthe oryzae. APS-MSA Joint Meeting Location: Austin, TX Date: AUG 10-14, 2013. Phytopathology 103 (6S2): 81-81.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Giraldo M. C., Dalby, M., Zhou, X. Y., Jayasundera, K. B., Tao, A., Xu, J. R., and Valent, B. 2013. Identification of Rice Proteins Interacting with Magnaporthe oryzae Effector Proteins. The 6th International Rice Blast Conference. Korea. August 20-14, 2013.
  • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Zhou, X. Y., Zhao, X. H., Xue, Z. Y., and Xu, J.R. 2013. Formation of aerial appressoria without surface attachment in Magnaporthe oryzae by overactive Ras signaling. Submitted to PloS Pathogens