Browsing by Subject "TBSV"
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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 PROPERTIES OF THE TOMBUSVIRUS MOVEMENT PROTEIN AND RNAi SUPPRESSOR THAT INFLUENCE PATHOGENESIS(2010-01-16) Hsieh, Yi-ChengTomato bushy stunt virus (TBSV) provides a good model system to investigate molecular virus-host interactions in plants. P22 and P19 proteins encoded by TBSV contribute to multiple invasion-associated functions. Green fluorescence-mediated visualization of TBSV invasion in this study suggests that virus exit from inoculated epidermal cells is a crucial event. Close examination of one P22 mutant showed that it had lost the capacity to move between epidermis and mesophyll which was possibly due to an altered subcellular localization. P19 is a potent suppressor of RNA interference (RNAi) in various systems by forming dimers that bind 21-nucleotide (nt) duplex siRNAs (short interfering RNAs), to affect the programming of the RNA-induced silencing complex (RISC). P19 is attractive for biotechnological and research purposes to prevent RNAi of certain value-added genes in plants. To obtain a good plant-based expression platform, a suppression-active mutant P19 was expressed in transgenic N. benthamiana lines. This is the first example of P19 accumulating to detectable levels in a transgenic plant and initial results suggest it is actively suppressing RNAi. Furthermore, to investigate the correlation between siRNA binding of P19 and its various biological roles, predicted siRNA-interacting sites of TBSV P19 were modified, and the corresponding TBSV mutants were used to inoculate plants. Substitutions on siRNA-contact sites on the central domain of P19 resulted in more severe symptoms in N. benthamiana compared to those affecting peripheral regions. All tested combinations of siRNA-binding mutations were associated with reduced accumulation of total TBSV-derived siRNAs, and loss of siRNA sequestration by P19. Additionally, some modifications were found to cause RNAi-mediated disappearance of viral and host materials in N. benthamiana but not in spinach. In conclusion, exit out of epidermal cells is a key host range determinant for TBSV and particular amino acids on P22 may influence this by regulating the proper subcellular localization. Mutant P19 transgenic plants were successfully established with minor physiological effects to be applied as a platform to study RNAi and to over-express proteins. Finally, a compromised P19-siRNA binding impacts symptom development, systemic invasion, integrity of virus plus host RNA and proteins, and that all in a hostdependent manner.