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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.
2009 Project Description
Objective 1. To characterize a collection of Magnaporthe oryzae non-pathogenic mutants to identify strains deficient in biotrophic growth. a. 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. To confirm that the T-DNA insertion is responsible for the mutant phenotype, we recreated the insertion in the wild-type strain and also performed a clean knock-out of the entire open reading frame.
This year we have performed a quantitative phenotypic analysis as well as molecular characterization of the knock-out strains, as well as a number of control transformants with ectopic insertions (non knock-outs). This has identified a potential new phenotype associated with the MGG 10193 null phenotype, that being a dramatic reduction in sporulation.
b. As a final confirmation of the gene's role in pathogenesis, we attempted to complement the original T-DNA insertion mutant with an intact copy of the MGG 10193 gene. This was unsuccessful due to an inability to re-transform the mutant. Therefore, we are currently attempting to re-create the T-DNA insertion in a new strain that is highly transformable. Large numbers of transformants have been generated and we are currently characterizing the transformants to identify ones that carry the desired mutation.
Objective 2. To develop a sensitive reporter system to detect delivery of fungal proteins into the cytoplasm of a plant cell. Initial attempts to express a translocation reporter protein in rice plants were unsuccessful due to an inability to obtain stable transformants.
Objective 3. To develop rice plants with fluorescently tagged cell compartments for cytological studies of fungus:plant interactions. a. Several vectors for that have been used for protein localization in maize were obtained from the Jackson laboratory (Cold Spring Harbor). Plasmid DNAs were prepared form these constructs and sequenced to confirm their veracity. Preliminary attempts to express the constructs in rice leaves following particle bombardment were unsuccessful.
Services: Farman taught a graduate level class on Critical Methods in Plant Microbe Interactions and gave a total of four guest lectures in other classes. Thornbury had a leading role in a Building Emergency Action Plan within the College of Agriculture.
Dissemination: Farman presented work on fungal protein localization during plant infection at the USDA Functional Genomic of Microbes awardees' workshop.
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.