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Localization of Secreted Proteins During Penetration and Iinvasive Growth of the Rice Blast Fungus Magnaporthe oryzae
M. Farman, M. M. Goodin, B. Valent, C. Soderlund
Department of Plant Pathology
Many fungi that cause diseases of plants are biotrophs that develop specialized infection hyphae within living plant cells. Interestingly, biotrophic fungi do not breach the host cell's plasma membrane. Instead, the membrane expands to accommodate the hyphae as they grow within the cell lumen. How the fungus is able to promote the required proliferation of plant membranes without triggering a defensive response is unknown. There is increasing evidence that proteins secreted by the fungus play critical roles in the establishment and maintenance of biotrophy. Some secreted proteins appear to be translocated into the plant cell cytoplasm, where they function to suppress defense responses and modify the host cell's metabolism. Others are deposited in the interface between the fungal cell wall and the plant membrane, where they may serve structural/protective roles, or function in the transfer of nutrients from the plant to the fungus.
To gain a comprehensive insight into the functions of secreted proteins in plant pathogenesis, we plan to use Magnaporthe oryzae infection of rice as a model pathosystem to study the many (>900) secreted proteins that are predicted to be encoded in the M. oryzae genome. To do this, we will fuse each secreted protein gene to another gene that codes for a fluorescent protein. The resulting fusion proteins will fluoresence under UV light. This will allow us to see when each proteins is expressed, and where it is distributed as the fungus grows inside rice cells. In this manner, we will classify secreted proteins based on their in planta localization patterns.
We expect to identify secreted proteins that are involved in the following processes: penetration into host cells, establishment of biotrophic hyphae inside the cells, formation of the interface between pathogen and host, passage through host cell walls (via plasmodemata), and, eventually, destruction of host tissues. Successful completion of this study will yield unique insights into the roles of secreted proteins in the development of the biotrophic interface. The formation of this interface is a crucial step in the pathogenic process and, therefore, a better understanding of this unique structure could lead to the development of improved strategies for controlling fungal diseases of plants.
2009 Project Description
1. To facilitate the design of PCR primers for amplification of secreted protein genes, we developed a web-based bioinformatic tool that accepts a list of genes and automatically designs the 3' primer. The tool then provides the sequence of each gene's promoter, so that the researcher can pick a suitable region for designing the 5' primer. This tool is adaptable to other genome projects and should be of broad utility for other functional genomics projects in fungi.
2. We have developed and tested a series of vectors for high throughput production of autofluorescent protein fusions in fungi. The vectors include a hygromycin B resistance gene driven by the Aspergillus nidulans TrpC promoter and can be used for Agrobacterium-mediated transformation of a wide range of fungi. Preliminary results indicate that protein localization patterns are unaffected by the presence of the Gateway vector-derived linker between the protein of interest and the fluorescent moiety.
3. To date, we have amplified over 100 secreted protein genes and have made fusion constructs for approximately 30 of them. These 30 have also been introduced into Magnaporthe and protein localization experiments are ongoing. So far, most of the proteins we have studied are expressed and localized in appressoria, including one that reveals a novel structure within the appressorium.
4. We have developed a prototype database and GU interface for uploading and storage of protein localization images, as well as relevant transformant data. The image database will connect to MGOSdb for dissemination of data to the public.
5. We have initiated a collaboration with Dr. Gou-Liang Wang at the Ohio State University who is performing functional analyses of secreted proteins through expression in rice protoplasts. Guo-Liang has provided us with data from his Serial Analysis of Gene Expression studies of Magnaporthe infection in rice plants. Specifically, he has provided us with a list of secreted protein genes that are expressed during infection. We are using this list to prioritize our protein localization experiments. In return, we are localizing proteins that he has shown to cause cell death in the protoplast assay. To date, we have generated fusions for six of his proteins. These have been transformed into Magnaporthe and we have preliminary localization data for two of them.
To our knowledge, the vectors developed in this project are the first ones that allow for high-throughput protein localization in fungi. The work describing the utility of these vectors is currently being written up in a manuscript to be submitted for publication. We expect there to be great demand for these vectors among the fungal research community.
Even though our protein localization studies are just starting, we already have identified a novel subcellular structure that we believe to be important for appressorium function.