| Summary: | 碩士 === 國立中興大學 === 植物病理學系所 === 104 === The RNA silencing suppressor (RSS) is the essential protein of a virus to overcome the plant major antiviral mechanism, RNA silencing. The pathogenicity of Watermelon silver mottle virus (WSMoV) relied on its non-structural protein (NSs) protein functioning as RSS. The identified novel motifs of WSMoV NSs protein responsible for silencing suppression are: - H113 on its conserved epitope (109KFTMHNQIF117, nss) for Asia type tospovirus, and Y398 (397IYFL400) at the C-terminal β-sheet motif. Self-interaction of NSs protein depended on the 8th putative α-helix at its C-terminal region proved to be vital to its silencing suppression ability as well. In this study, the dsRNA binding ability of the respective mutated NSs proteins, H113A, Y398A and Y338A/H350A/F353A (TPM) on the 8th α-helix expressed by Escherichia coli was examined via gel shift assay by radiolabeling of short (siRNA) and long synthesized dsRNA. Our results showed that only the H113A protein and epitope deleted NSs protein (∆nss) loss their ability to bind to siRNA. Furthermore, when the monoclonal antibody (MAb) targeting nss epitope as competitor against siRNAs, wild type NSs protein binding to siRNA was diminished. Our results also indicated that the WSMoV NSs protein is unable to bind long dsRNA. Pathogenicity of the wild type NSs and mutated NSs proteins was also studied here by employing the mild potyviral vector, Turnip mosaic virus (TuMV) TuK-GFP with reduced HC-Pro function (mutation on R182K). When replacing GFP reporter gene between NIa and coat protein of a heterologous TuK-GFP vector with wild type NSs and its respective mutated NSs proteins studied above, only the wild type NSs protein complemented the mutated HC-Pro function. Taken together, we concluded that the common nss epitope of Asia type tospoviruses is responsible for siRNA sequestration, thus resulted in RSS. On the other hand, the mutated Y398A protein and TPM protein that affects RSS by alter protein stability and self-interaction do not affect its siRNA binding ability.
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