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Cellular and Molecular Biology of Plant Rhabdoviruses
Department of Plant Pathology
The need to prevent viral epidemics has never been greater. Globalization and rapidly increasing international trade of horticultural products make the spread of highly destructive viruses, and/or their insect vectors a real and present danger to U.S. and world agriculture.
Therefore the research objectives of this proposal involve utilizing N. benthamiana, the most widely used plant model in virology and pharmaceutical research to identify candidate genes for engineering plants to combat a variety of devastating viruses. Given the genetic relatedness of N. benthamiana to many crops of agronomic importance it is expected that the results of the proposed research can be translated to crop plants, which are far less tractable in terms of the genetics and molecular manipulations required to identify novel sources of resistance to viruses.
This project is built upon high-resolution yeast two-hybrid (Y2H) screens to identify N. benthamiana proteins that interact with either the movement or replicase proteins of plant-infecting rhabdoviruses. The rationale for the experimental design is based on the fact that the genomes of plant viruses typically encode movement proteins (MPs) that facilitate the transfer of viral replication complexes or mature virus particles from infected cells to adjacent cells where the infection process can continue. In order to function, MPs must interact with cellular factors that link adjacent cells. Identification of such factors and subsequent engineering to prevent their association with MPs, would provide an effective means to inhibit the systemic infection of plants and thus prevent disease. A second effective strategy would be to similarly identify and inhibit plant factors that are essential for function of viral replicase proteins that are required for increasing the copy number of viral genomes.
In addition the two hybrid screens, the proposed research seeks to determine how viral proteins interact with plant membranes and how they are imported into the nucleus. This research will provide fundamental insight into nuclear transport in plants, which is still poorly understood.
The importance of this research lies in the fact that the nucleus is the principal regulator of cellular functions, therefore and understanding of how proteins enter into and alter nuclear structure and function are essential for understanding how the physiology of cells is regulated. Taken together, the proposed research will offer novel insight into the molecular basis of virus-plant interactions, which could lead to novel strategies for engineering plants that are resistant to virus infections. This has critical importance for agriculture, as viruses are a major threat to food and fiber production in the United States and the world.
2009 Project Description
We have developed a new combination of plant expression and transformation vectors for studying intracellular protein interaction, localization and movement in conjunction with our transgenic marker lines constitutes powerful tools for the plant biology community. Information regarding these vectors has been disseminated internationally via one publication, conference proceedings and seminars at the local, national and international levels.
We reported construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in selection of vectors. We also determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs) and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. We showed that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker.
In addition to testing protein interactions, we demonstrated that transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization.
Martin, K., K. Kopperud, R. Chakrabarty, R. Banerjee. R. Brooks, and M. M. Goodin. (2009).Transient expression in Nicotiana benthamiana fluorescent marker line provides enhanced definition of protein localization, movement and interactions in planta. Plant J. 59:150-162.