Study of host factors interacting with the capsid protein of Odontoglossum ringspot virus
Date Issued
2014
Date
2014
Author(s)
Chen, Po-Yen
Abstract
Orchid is one of the most important export crops in the floral industry in Taiwan. However, the quality and yield of orchid frequently reduce due to the threat of Odontoglossum ringspot virus (ORSV). ORSV belongs to the genus Tobamovirus and requires capsid protein (CP) for systemic movement, but not for cell-to-cell movement in Nicotiana benthamiana. Nevertheless, the detailed movement mechanism of ORSV is still needed to be further studied. In our previous studies, when glutamic acid (E) at the 100th amino acid of ORSV CP mutated to glycine (G) or alanine (A), the mutant virus lost its ability to systemically infect N. benthamiana. Co- immunoprecipitation (co-IP) assay was tried previously to identify host factors involved in the movement of ORSV or host defense. In co-IP assay, we identified a putative proteinase inhibitor, NbPI, which was highly accumulated in CPE100A co-IP products. In this strudy, we first conducted 5’ RACE assay, and the 5’ untranslated region (5’ UTR) of NbPI mRNA was proved to be 33 nucleotides. For further confirmation of the interaction of NbPI and ORSV capsid proteins, we utilized Escherichia coli to express proteins above. NbPI co-precipitated with ORCPE100A in the in vitro pull down assay. Moreover, we transiently expressed NbPI and ORSV capsid proteins in N. benthamiana. NbPI co-precipitated with ORSV capsid proteins (CPWT and CPE100A) in planta as well. In nucleotide level, Northern blot assay showed that the mRNA level of NbPI gene was rapidly elevated after buffer and ORSV inoculation in N. benthamiana. To study the role of NbPI during ORSV infection, we established a virus-induced gene silencing (VIGS) system in N. benthamiana. After inoculating ORSV to NbPI-silenced plants for several days, ELISA assays were performed to detect ORSV. The result showed that the accumulation levels of ORSV capsid protein in the systemic leaves of NbPI-silenced plants were higher than those of control one. Accordingly, these results suggested that NbPI gene expression level was rapidly elevated after ORSV infection, and then NbPI proteins might be associated with ORSV capsid proteins. As to the result of VIGS, NbPI might participate in N. benthamiana defense response and involve in retarding the systemic movement of ORSV.
Subjects
齒舌蘭輪斑病毒
鞘蛋白
酵母菌雙雜合系統
病毒誘導基因靜默
免疫共沉澱
系統性移動
Type
thesis
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