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Structural and Subcellular Distribution of Vpg Protein Encoded by Wheat Yellow Mosaic Virus

Author: BianZuo
Tutor: ChenJianPing
School: Zhejiang Normal University
Course: Botany
Keywords: wheat yellow mosaic virus VPg protein NLS NES prokaryoticexpression co-expression protein interaction
CLC: S435.121.4
Type: Master's thesis
Year: 2013
Downloads: 28
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Abstract


Wheat yellow mosaic disease is one of main virus diseases endangering winter wheat in China. The disease is caused by wheat yellow mosaic virus (WYMV), spreaded by Polymyxa graminis Ledingham. WYMV is a member of the genus Bymovirus in the family of Potyviridae. Its genome has two positive sense (+) RNAs and each RNA encodes a single polyprotein. RNA1encodes eight proteins:P3,7K, CI,14K, NIa-Vpg, NIa-Pro, NIb and CP, while RNA2encodes two proteins:P1and P2. This study focused on a viral Genome-linked Protein (VPg) encoded by RNA1. VPg protein is a multifunctional protein involved in viral replication, viral translation, viral movement and other activities of life. Taking advange of modern bio informatics, we analysed the VPg protein characteristic that contained a nuclear localization signal (NLS) and a nuclear export signal (NES). Using cell fractionation, immunogold electron miscroscopy and fluorescence localization, we realized that eGFP-VPg was exclusively localed in nucleus when expressed alone in absence of other viral proteins, in contrast, VPg protein was detected in both of cytoplasm and nucleus when we fractionated nucleus and cytoplasm from virus infected leaves of wheat plant cells. Translocation of VPg in cytoplasm and nucleus is associated with interaction with WYMV capsid protein. To investigate the regions of the VPg domain controlling protein nuclear localization, we constructed a series of deletion mutants and found that the NLS at N-terminal region of WYMV VPg regulates protein nuclear localization.In order to analyze the interaction between VPg and protein coded by WYMV, we prepared a fused protein GST-VPg as bait to capture VPg associated factors. Two viral proteins, CP and NIa, were found to bind to GST-VPg fusion protein. The further immunoprecipation and BiFC assay revealed that CP physically interacted with VPg. In order to examine the CP binding domain on VPg, a series of VPg mutants were introduced into BiFC plasmids and BiFC assay was performed to examine its interaction with CP. The results showed that C-termini (96-187aa) of VPg was responsible for VPg binding to CP. To investigate whether VPg nucleocytoplasmic shuttling is associated with CP binding, eGFP tagged VPg and its mutants were co-expressed with pBIN-CP in Nicotiana benthamiana leaves. The results revealed that CP was contributed to VPg nucleocytoplasmic shuttling and the C-terminal portion of VPg is important for its subcellular translocation. Further experiments indicated that NES region localized on C-terminal of VPg is essential for VPg subcellular translocation.In this study, we also demonstrated that VPg binded to virus particle with two experiments. First, we purified WYMV particle and examined the presence of the VPg in purified virus particle preparation by specific antibody, indicating VPg is co-purified with virus and binds to virus particles directly. Secondly, we confirm the same result by immunogold electron microscopy to analyse VPg localization at virus particle in WYMV-infected wheat plant cells.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Cereal crop pests and diseases > Wheat pests and diseases > Disease > Transgression ( pass ) an infectious agent harmful
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