- 學位論文
探討齒舌蘭輪斑病毒之系統移動機制以及鑑定出與鞘蛋白具交互作用之寄主蛋白
Studying the role of capsid protein of Odontoglossum ringspot virus in virus systemic movement and identifying a novel CP- interacting tobacco protein, p26/proteinase inhibitor
摘要
植物病毒Tobamovirus屬的鞘蛋白(capsid protein, CP)為此類病毒於植物中系統性移動之關鍵因數，而其分子機制如寄主蛋白之參與等皆尚未明瞭。前人研究顯示，齒舌蘭輪斑病毒(Odotoglossum ringspot virus, ORSV) 之鞘蛋白上麩胺酸 (E100) 之突變將會造成此病毒無法系統感染菸草Nicotiana benthamiana。為進一步瞭解E100於病毒系統感染所扮演之角色和參與的寄主因數及機制等，本研究將ORSV野生型感染性選殖株(pORSV-7)之E100突變成A100，並加入一限制酵素切位做為區分之標記，創造出突變株pORSV-7E100A。由感染力測試結果再次證實E100之突變將造成病毒無法在N. benthamiana上系統性移動。我們以生體外蛋白交互作用分析，穿透式電子顯微鏡觀察以及分子篩選層析法(size-exclusion chromatography)證實了E100A是經由影響鞘蛋白間交互作用，導致無法組裝成完整病毒顆粒，此為E100A突變株失去系統性移動的原因之一。而利用農桿菌表現蛋白間交互作用較佳的CPWT，以及蛋白間交互作用較差的CPE100A於植物中時，因CPWT干擾病毒脫鞘能力較佳而具有較好的交叉保護抗性。以ORSV CP專一性之免疫球蛋白G (IgG)將ORSV CP及與其具有交互作用之植物蛋白共沉澱(co-immunoprecipitation, co-IP)，並大規模以質譜分析，鑑定出一些可能和ORSV CP有交互作用，並且可能與病毒系統性移動相關之植物蛋白。其中一p26/proteinase inhibitor在蛋白電泳以及質譜分析結果中非常顯著地只和CPE100A緊密結合。顯示p26/proteinase inhibitor可能為ORSV所誘導之植物防禦機制相關蛋白，我們並預測其作用之原理。本研究為植物蛋白p26/proteinase inhibitor與ORSV CP結合並可能干擾病毒系統感染之首次報導，加上大規模鑑定出與CP有交互作用之植物蛋白，將有助於對病毒感染機制以及病毒與寄主間交互作用機制之瞭解，期望因此設計出嶄新的抗病策略。
並列摘要
The capsid protein (CP) of tobamoviruses has been demonstrated to involve in virus systemic movement. However, the CP-mediated systemic movement mechanism is unclear. Our previous results indicated that E100 in the CP (CPE100) of Odotoglossum ringspot virus (ORSV) plays an important role in virus long-distance movement in Nicotiana benthamiana plant. In order to study the effect of E100 on CP-mediated function, this amino acid was mutated to A100 in an ORSV infectious clone (pORSV-7) to create a mutant clone (pORSV-7E100A) with a distinguishable RFLP marker. The results indicated that ORSVE100A could be detected in the infected protoplasts and the inoculated leaves of N. benthamiana and Chenopodium quinoa but lost its systemic infectivity in N. benthamiana plant. The data of in vitro protein binding assay, observation through transmission electron microscopy and size-exclusion chromatography (SEC) showed that the CP produced by pORSV-7E100A (CPE100A) might affect the CP-CP interaction and resulted in viral particle assembly deficiency in planta. Moreover, the differential CP-CP interaction ability between CPWT and CPE100A could explain the good protection to ORSV infection by transient-expressing the CPWT in N. benthamiana, whereas poor protection on the CPE100A-expressing plants. ORSV CP-specific IgG was used for immunoprecipitation of the CP to identify the CP-interacting proteins. Here, several putative host proteins were identified to interact with ORSV CP through mass spectrometry analysis. Among them, a defense-related p26/proteinase inhibitor of N. benthamiana was proved to be highly associated with CP by in vitro pull-down assay. This is the first report of plant p26/proteinase inhibitor interacting with ORSV CP and might interfere with virus systemic movement. The viral particle assembly and the ORSV CP-interacting host proteins will help us to understand the virus movement in plant and also the plant-virus interaction.
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