Browsing by Author "Steven Spiker, Committee Co-Chair"

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    Localization of Red Clover Necrotic Mosaic Virus Polymerase Proteins at the Endoplasmic Reticulum of Living Plant Cells
    (2002-11-07) Turner, Katherine Anne; Steven Spiker, Committee Co-Chair; Steven A Lommel, Committee Co-Chair
    The replication of positive strand RNA viruses requires association with host membranes and frequently results in membrane proliferation and rearrangement. The fact that widely divergent virus families share form and function of replication strategies is likely due to common evolutionary origin and suggests that elucidating the reproduction strategies of one virus provides insight to virus life cycles in general. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus in the Dianthovirus genus, Tombusviridae family. RCNMV encodes N-terminally overlapping proteins of 27 kDa and 88 kDa (p27 and p88). p88 contains motifs characteristic of RNA-dependent RNA polymerases, and while the function of p27 is unclear, it is very likely to be part of the RCNMV replication complex. I have investigated the localization of the RCNMV replicase proteins in plants using green fluorescent protein (GFP) fusions observed in live cells using confocal microscopy. The GFP:p27 and GFP:p88 fusions were expressed in N. benthamiana epidermal cells and protoplasts. GFP:p27 consistently localized to the endoplasmic reticulum (ER) and caused membrane restructuring and proliferation. Fractionation of virus-inoculated leaves demonstrated the association of p27 with isolated ER membranes. Additionally, GFP:p27 co-localized with ER-targeted YFP. GFP:p88 also localized to the ER and co-localized with GFP:p27. In addition, GFP:p27 and GFP:p88 were associated with invaginations of the nuclear membrane. These grooves and channels of ER crossed the nucleus, giving the appearance of fluorescence within the nucleus, while the signal from GFP:p27/p88 was actually associated with ER. GFP:p88 co-localized with GFP:p27 in two expression systems. This and the fact that GFP:p27 localized to the cortical ER, the nuclear envelope, and cytoplasmic ER domains in the presence of viral genomes as well as in the absence of any viral proteins suggests that the two proteins co-localize to the ER during infection. Microscopic observations suggest that the virus remodels ER structure to create membranous bodies containing active viral replication complexes. The fact that both proteins independently accumulated in the ER and perturbed the ER morphology suggests that the proteins function together, as part of a larger replication complex. This is the first report of a Tombusvirus replicating in association with the ER.
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    Mapping Replication and Silencing Suppression Elements in the RCNMV Genome
    (2009-06-16) Powers, Jason Gannon; Steven A. Lommel, Committee Chair; Robert G. Franks, Committee Member; Steven Spiker, Committee Co-Chair; Dominique Robertson, Committee Member
    Viruses infect all Kingdoms of life on Earth. Their life cycle represents a constant struggle for survival in hostile cellular environments. To survive the virus must both avoid the host response and replicate in a timely manner. The following dissertation includes investigations into both of these aspects of viral infection focusing on plant-infecting viruses. When infected by viruses, plants respond by initiating defense pathways. One of these pathways is known as RNA silencing. In this pathway host proteins target double-stranded viral RNA intermediates for cleavage. The product of this cleavage, a short-interfering RNA (siRNA), is incorporated into a protein complex that guides sequence specific cleavage of viral RNA. To cause a productive infection, the virus must devise countermeasures to this targeting. They accomplish this by suppressing the RNA silencing pathway by encoding proteins known as viral suppressors of RNA silencing (VSRs). Identifying these VSRs is critical to the understanding of how any plant virus survives in its host. Commonly used assays for identifying VSRs all use reporters expressed in the nucleus. These nuclear-based DNA reporters are being used to assay for the RNA silencing suppression activity of RNA virus proteins. In this thesis I describe the development of a new VSR identification assay that uses a disarmed RNA virus as a reporter, which should be an accurate predictor for VSR activity of other RNA viruses. The reporter is the plant-infecting virus Turnip crinkle virus (TCV) with its previously characterized VSR, the coat protein, replaced with sGFP resulting in a construct termed TCV-sGFP. After validating the use of TCV-sGFP as a reporter for RNA silencing suppression activity TCV-sGFP was used to identify a previously uncharacterized VSR for the plant-infecting virus Red clover necrotic mosaic virus (RCNMV). RCNMV’s replication complex was previously implicated in the suppression of RNA silencing, while the new TCV-sGFP assay allowed us to detect a second suppressor, a protein known to be involved in viral movement, known simply as the movement protein (MP). Domains of MP were analyzed and it was found that the amino acid residues between positions 122 and 277 were required for suppression activity. The RNA silencing pathway has two principal components, siRNAs and host proteins. To disrupt the pathway the MP of RCNMV must interfere with one or both of these components. In this thesis the possibility that the MP suppresses RNA silencing by binding to siRNAs is addressed by employing electrophoretic mobility shift assays to examine MP’s ability to bind to siRNAs. MP was found to have no siRNA binding capability, indicating that it’s mode of action likely relies on binding to or modifiying host proteins. Finally, initial studies into the temporal regulation of RCNMV replication were undertaken. As mentioned, for a productive infection to take place the virus must go beyond evading the host defense response, it must also properly regulate the timing of expression for its many genes. Using real time PCR, the timing of various replication events in the RCNMV life cycle were mapped to more fully understand how RCNMV replicates inside the cell. These time course studies have revealed that virion formation has little effect on RCNMV’s ability to replicate, and that RNA-1 replicates to a slightly higher degree in the absence of RNA-2. Additionally several uncharacterized negative sense replication intermediates that exist during a wildtype infection were identified. This work forms the foundation for future studies into the temporal regulation of RCNMV replication.
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    The Use of Flow Cytometry to Investigate the Effects of Matrix Attachment Regions on Transgene Expression in Plant Cells.
    (2005-02-14) Halweg, Christopher Jay; Arthur Weissinger, Committee Member; William Thompson, Committee Co-Chair; Steven Spiker, Committee Co-Chair; Dominique Robertson, Committee Member
    Many studies in both plant and animal systems have shown that Matrix Attachment Regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. MARs are AT-rich sequences of DNA that bind in vitro to the proteinacous filament-like structure within the nucleus called the nuclear matrix. In our investigation of transgenic Nicotiana tabacum NT-1 cells in culture, we have observed that transgene expression is often variegated. In other words, some cells in an isogenic population do not express the transgene, and/or other cells within the same population express the transgene at varying levels. The question was raised: Do MARs increase transgene expression by altering variegation? More specifically, do MARs increase the percentage of cells expressing the transgene, increase the magnitude of expression in expressing cells, or both? In order to address these questions, it was necessary to quantitate transgene expression variegation at the resolution of individual cells. We chose to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells by flow cytometry. In order to analyze individual cells and because NT-1 cells in culture grow as filaments, it is necessary to prepare protoplasts that can pass one at a time through the flow cell of the flow cytometer. We found that current flow cytometry methods for measuring GFP expression in plants were susceptible to debris resulting from protoplast preparations. We observed that when the plasma membrane of protoplasts is breached, GFP diffuses out into the medium, and flow cytometric measurements of these non-viable protoplasts imply they do not express GFP. This debris can overestimate the proportion of non-expressing cells in the population. In order to correct this problem, we used an approach called a dye exclusion test. Because propidium iodide enters protoplasts to stain nuclei only when the plasma membrane is breached, debris that stains with this dye can be removed from our analysis. Using this approach we were able to quantitate GFP expression in individual cells without complications from debris. We used flow cytometry to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells from lines transformed by Agrobacterium. We find that in this system the Rb7 MAR increases GFP expression 2-4 fold. This increase is caused by both an increase in the percentage of expressing cells and an increase in the magnitude of expression in expressing cells. Cell lines transformed with MAR-containing vectors averaged 27-39% more cells expressing GFP and these cells expressed GFP at 2-3 fold higher levels than cells transformed with control constructs. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from a population of cell lines obtained by liquid culture of entire Agrobacterium co-cultivation plates. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression. Our results indicate that the Rb7 MAR increases the frequency of transgene expression, presumably by reducing gene silencing, while also increasing the levels of expression in expressing cells, perhaps through an enhancer-like activity.