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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.
2010 Project Description
Viruses pose one of the major threats to human health, directly as human pathogens or indirectly by affecting food and fiber production. To meet the challenges of developing stable resistance against viruses that limit the sustainable production of food amid the era of global warming, an ever more sophisticated understanding of the molecular details underlying virus-plant interactions is required. Implicit in this understanding is the requirement to identify the host factors that are common or unique to genetically diverse viruses that infect a common host. Finally, not only is it necessary to identify host proteins that interact with viral proteins, but elucidation of mechanism(s) by which these protein function is required.
Thus, this research will (i) generate protein interaction and localization maps for plant viruses, with particular emphasis on plant-adapted rhabdoviruses, (ii) characterize novel or understudied proteins in model plant species, (iii) will provide novel insight into the mechanism by which plant-adapted rhabdoviruses move cell-to-cell and (iv) will provided candidate genes for engineering virus resistance in a number of economically important crops.
To identify host factors that play critical roles in processes including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution N. benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. BiFC and biochemical assays were used to validate two-hybrid interactions in detail. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the ER. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals.
We have also conducted a two-hybrid screen to identify factors that interact with the CI protein of Tobacco etch virus, one of the most agronomically important plant viruses. The results of these projects have been disseminated in a peer-reviewed journal, as well as at the poster and oral presentations at two national scientific meetings.
We reported a model for the mechanism by which SYNV moves from an infected cell into adjacent non-infected cells. This model incorporated protein interaction and localization data as well as information obtained by electron microscopy. It is, the date, the most comprehensive assessment of the way plant rhabdoviruses move through plants.
To generate the model we considered steady-state localization studies using autofluorescent protein fusions to full-length clones of plant proteins that interact with SYNV proteins. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 protein interactor, sc4i21, localized to microtubules, thus providing the "highway" transport system for the viral movement complex. The N protein interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum, which presumably the source of membranes to envelope the movement complex. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor.
Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm. This is the first such report implicating such transcription factors in biological processes other than gene regulation.
Min, B-E., Martin, K., Wang, R., Tafelmeyer, P., Bridges, M., Goodin, M. (2010) A Host-Factor Interaction and Localization Map for a Plant-Adapted Rhabdovirus Implicates Cytoplasm-Tethered Transcription Activators in Cell-to-Cell Movement. Mol Plant-Microbe Interact. 23:1420-1432.