Browsing by Subject "Tomato bushy stunt virus"
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Item A Study of Nicotiana Benthamiana Protein Interactions with Tomato Bushy Stunt Virus(2013-04-29) McLachlan, JuanitaTwo Tomato bushy stunt virus (TBSV) proteins, P19 and P22, have been found to interact with the Nicotiana benthamiana host proteins Hin19 and HFi22 in yeast two,hybrid assays. To determine functional roles of these interacting host proteins, viral induced gene silencing (VIGS) was employed to knock,down their expression. TBSV has been demonstrated to activate a virus,specific antiviral response pathway in N. benthamiana. To characterize this pathway, the antiviral RNAi induced silencing complex (RISC) was isolated from TBSV-infected plants. Additionally, putative RISC-associated proteins were identified in silico and suggested roles for these have been identified through literature and database searches. A further aim was the identification of proteins that coimmunoprecipitate with the TBSV-induced RISC following RISC isolation. A primary aim of this investigation was to identify functional roles for host proteins that interact with the two TBSV 3-terminal encoded proteins, P22 and P19. Each of these has functional roles in viral movement and pathogenicity. In yeast two-hybrid assays, P22 has been shown to interact with HFi22 while P19 interacts with Hin19. VIGS was utilized in attempts to silence the expression of these two host proteins in order to determine their functional roles. VIGS-mediated suppression of the TBSV-interacting proteins Hin19 and HFi22 has not been accomplished. Despite multiple attempts and multiple approaches, these proteins have not been amenable to silencing. In light of this finding, it is proposed that rather than utilizing VIGS to down-regulate protein levels for Hin19 and HFi22, other approaches should be utilized. To characterize the TBSV-mediated RNAi pathway, functionally active antiviral RISC was purified from TBSV-infected N. benthamiana plants using ion-exchange chromatography. This RISC was found to be active only in the degradation of TBSV transcripts, indicating the specificity expected from a programmed RISC. Characterization and identification of proteins that copurify with RISC has not yet been accomplished, though in silico analysis has yielded over 150 putative RISC-associated proteins. Of these, a subset has been identified as highly likely candidates based upon function and/or homology to RISC-associated proteins in non-plant organisms, and a model for the TBSV-induced antiviral pathway has been proposed.Item Host factors involved in viral movement through plants(2009-05-15) Seaberg, Bonnie LeeTomato bushy stunt virus (TBSV) is a positive-sense single-stranded RNA virus. It encodes five open reading frames (ORFs), including two nested genes, expressing movement-associated proteins. One of these proteins, P22, interacts with a host transcription factor containing a homeodomain leucine-zipper motif, known as HFi22. Similar proteins of this type traffic their RNA from cell-to-cell, suggesting the possiblity that HFi22 is involved in the cell-to-cell movement of TBSV RNA. To further characterize the nature of the interaction between P22 and HFi22 on the cellular level, cellular fractionation experiments were conducted. To investigate the functional role of HFi22 in viral movement I attempted to inactivate its expression using a virus induced gene silencing system with a Tobacco rattle virus (TRV) vector. A final objective was based on the notion that different hosts can impact the stability of viruses used to express foreign genes of biotechnological interest. To compare virus stability in different hosts, TBSV expressing the green fluorescent protein (GFP) was inoculated onto various TBSV hosts, and infected leaf tissue was then used as inoculum to be rubbed onto a local lesion host. This technique made it possible to quantify the number of fluorescent foci versus total lesions. Results obtained for the first objective indicate that P22 and HFi22 co-fractionate in nucleus and membrane-enriched samples. In addition, it was found that HFi22 is largely conserved through a wide variety of plant species but not in lettuce, which was found to be compromised for effective virus spread. Control experiments for the second objective showed that plants were successfully silenced with TRV carrying the phytoene desaturase (PDS) gene resulting in photobleaching, however attempts to silence HFi22 have not yielded conclusive results. The results obtained for the third objective indicate there is a difference in how efficiently a foreign gene insert is maintained by TBSV in different host plants. In summary, the overall results of this research showed that host factors influence the host-virus interaction but their exact contributions remain elusive.