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Genes Controlling Invasive Growth in the Rice Blast Fungus Magnaporthe oryzae
Department of Plant Pathology
It is not known how fungi colonize host plant cells without triggering the plants' innate defenses. The availability of fungal mutants that are deficient in host cell colonization will allow identification of genes that control this process. Current evidence suggests that fungi deliver proteins into plant cells, and it is believed that these proteins modify the plant cell's metabolism. However, the identities of these proteins, and the mechanism by which they enter the host cell, remain elusive. The first goal of this project is to identify genes that control the ability of Magnaporthe oryzae to colonize rice cells. Functional characterization of these genes will provide clues as to their roles in the pathogenic process. The second goal is to develop a reporter system to detect when a fungal protein enters the host cell cytoplasm. This system will then be used to screen the Magnaporthe genome to identify all of the proteins that are secreted into host cells.
2011 Project Description
Activities: We continued studies on the localization of Magnaporthe secreted proteins during host penetration and invasive growth. We expanded the number of secreted proteins studied to more than 400. We developed plasmid constructs which allow for simultaneous labeling of two proteins with different colors. We used confocal microscopy to study the expression/localization of certain proteins in more detail. We generated gene knockouts for a fungal protein that accumulates in the nuclei of the invaded host cells and tested the effect of pathogenicity.
Events: All project participants attended the Fungal Genetics Conference at the Asilomar Conference Grounds, Pacific Grove, CA.
Services: The Farman lab provided plasmid constructs to numerous researchers from around the globe. A website and database allowing gene knockout/gene fusion primer design was generated for the Colletotrichum graminicola research community.
Products. Five plasmid vectors with broad utility for the fungal research community.
Dissemination (outreach): Results from the project were presented in poster sessions at the Fungal Genetics Conference.
We discovered that Magnaporthe appressoria retain two nuclei throughout the biotrophic stages of infection. Coupled with our earlier discovery that appressoria accumulate certain proteins long after the fungus has gained entry into the host tissues, this leads us to propose that appressoria continue to serve a function post-penetration.
Deletion of a gene for a secreted protein that accumulates in the nuclei of invaded host cells had no effect on pathogenicity.
We found that wild-type Magnaporthe produces fluorescent compounds in the appressorial walls; and on the walls of hyphae where they emerge from the spore.
We identified a novel appressorial structure that is labeled by a histone 2B:GFP fusion, as well as two different secreted protein:RFP fusions.
Change in actions: We adopted a simplified Agrobacterium-based method of introducing protein:fusion constructs into Magnaporthe spores.