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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.
2010 Project Description
During the past year we have focused our efforts on studying the role of a G-protein gamma subunit in phytopathogenesis. We previously identified a T-DNA tagged mutant with an insertion in Magnaporthe gene MGG 10193 which is predicted to code for a G-protein gamma subunit. The original mutant was identified as one that did not form appressoria and which germinated to form aerial hyphae. The mutant is also unable to infect rice. Previously, we had confirmed that the T-DNA insertion is responsible for the mutant phenotype, we recreated the insertion in the wild-type strain and performed a clean knock-out of the entire open reading frame. As a final confirmation of the gene's role in pathogenesis, we tried to complement the original T-DNA insertion mutant with an intact copy of the MGG 10193 gene. This had been unsuccessful due to an inability to re-transform the mutant. In the current review period, we sought to circumvent this problem by trying to recreate the original T-DNA insertion in a different (more easily transformed) Magnaporthe strain.
To develop a sensitive reporter system to detect delivery of fungal proteins into the cytoplasm of a plant cell. Due to difficulties in transforming rice plants with the reporter construct, we are using an alternative and more direct approach to detect translocation of fungal proteins. We have fused a large number of fungal secreted proteins to a red fluorescent protein tag. These constructs were introduced into Magnaporthe via Agrobacterium-mediated transformation. The transformants were inoculated onto rice plants and the fusion proteins were then visualized using epifluorescence microscopy of infection sites.
Due to problems with the generation of transgenic rice plants, we have abandoned Objective 3.
Events and Dissemination: Farman and other lab members presented data from this project at several conferences/events: Plant and Animal Genome Conference, San Diego, CA; KY Innovation and Entrepreneurship conference, Lexington, KY; Mycological Society of America-International Symposium on Fungal Endophytes of Grasses Conference, Lexington, KY; International Rice Blast Meeting, Little Rock, AR; USDA Functional Genomic of Microbes awardees' workshop. Plant Pathology seminar series, Ohio State University; Plant Pathology seminar series, Kansas State University.
Services: Farman taught a graduate level class on Critical Methods in Plant Microbe Interactions and mentored 4 undergraduate students who were conducting independent research projects in the lab.
G-protein signaling is known to be involved in developmental processes required for plant pathogenesis - in particular it is important for appressorium formation and function. Genes for G-protein alpha and beta subunits have been extensively characterized in a number of plant pathogens. However, there has been no research on the function(s) of the gamma subunit, at least in pathogenic filamentous fungi.
Research on this particular gene promises to provide new insights into the G-protein signaling cascade and, in particular, how the gamma subunit modulates the inputs and outputs of the cascade to affect different processes required for pathogenic success (i.e. surface sensing, appressorium formation/function, invasive growth and sporulation). This new information on the cellular mechanisms that allow fungi to infect plants ultimately will facilitate the development of rational, knowledge-based approaches to controlling disease.