Study of host factors interacting with the capsid protein of Odontoglossum ringspot virus

蘭花為我國重要之外銷花卉作物之一，其在栽培過程中易遭受齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)之侵染而造成經濟損失。ORSV為 Tobamovirus屬病毒，其在寄主中的系統性移動需要鞘蛋白(capsid protein)的協助，然而詳細的移動機制仍尚待釐清。本實驗室先前發現將ORSV鞘蛋白的第100個胺基酸glutamic acid (E)置換為glycine (G)，或點突變為alanine (A)，則ORSV鞘蛋白突變株CPE100G和CPE100A會喪失於圓葉菸草(Nicotiana benthamiana)中系統性感染的能力。欲尋找可能參與病毒移動，或啟動植物防禦反應的寄主分子，本實驗室之前進行了免疫共沉澱法(co- immunoprecipitation, co-IP)，找出與鞘蛋白有交互作用的寄主因子。接著經由後續質譜分析，找到了一個與鞘蛋白突變株CPE100A有較強交互作用的寄主蛋白，其為一putative proteinase inhibitor，因此將其命名為NbPI。本研究首先對此基因進行了5’ RACE，確認其mRNA的5’ UTR共有33個核苷酸。為了進一步確認NbPI與ORSV鞘蛋白之間的交互作用，本研究以大腸桿菌系統表現NbPI與ORSV鞘蛋白後，進行生體外蛋白結合實驗(in vitro pull down)，結果發現NbPI與ORSV-CPE100A具有交互作用。此外，以圓葉菸草短暫共同表現NbPI及ORSV鞘蛋白，結果發現在植物體內(in planta)，ORSV鞘蛋白(CPWT與CPE100A)與NbPI也具有交互作用。在核酸層次，為了解NbPI在ORSV侵染過程中是否受到影響，本研究以北方雜合法偵測NbPI的表現量，結果圓葉菸草在接種緩衝液和ORSV後，NbPI表現量皆顯著地上升。最後，為了探討NbPI在ORSV感染圓葉菸草的過程中的重要性，本研究在圓葉菸草建立了病毒誘導基因靜默(virus-induced gene silencing, VIGS)系統。當NbPI產生基因靜默之後，先接種ORSV，數日後以ELISA偵測ORSV；結果發現ORSV在NbPI基因靜默植物的上位葉的累積量相較於對照組顯著地增加。依據上述研究結果，圓葉菸草受到ORSV侵染時，NbPI基因被快速誘導表現，並且NbPI蛋白可能會與ORSV鞘蛋白結合。而VIGS實驗結果顯示NbPI可能參與在圓葉菸草的抗病反應中，或許具有減緩ORSV系統性移動的能力。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.中文摘要 i 英文摘要 iii 壹、前言 1 一、 蘭花與齒舌蘭輪班病毒 1 二、 Tobamovirus屬病毒的移動 2 三、 寄主蛋白影響病毒移動 2 四、 尋找與病毒有交互作用的寄主蛋白 2 五、 前人研究 4 六、 研究動機及策略 5 貳、材料與方法 6 一、 植物材料與生長條件 6 二、 以yeast two-hybrid在菸草中尋找可能與ORSV-CP 有交互作用的寄主蛋白 6 三、 以5’ RACE確認NbPI基因mRNA之5’端UTR序列 9 四、 目標基因之多序列併列分析 14 五、 接種ORSV之菸草NbPI表現情形 14 六、 in vitro protein-protein結合測試 15 七、 in planta protein-protein結合測試 18 八、 菸草NbPI 基因靜默與分析 22 九、 ORSV病毒累積量之測定 24 叄、結果 26 一、 以yeast two-hybrid並未找到菸草中與ORCPE100A有交互作用 的寄主蛋白 26 二、 以5’ RACE確認NbPI基因mRNA的5’ UTR及前端序列 27 三、 NbPI基因與其他相似基因之多序列併列分析 27 四、 buffer及ORSV接種菸草可迅速誘導NbPI mRNA的表現 28 五、 以yeast two-hybrid無法證明NbPI與ORSV CPwt 及CPE100A 或TuMV CP有交互作用 28 六、 以in vitro coprecipitation證明NbPI與ORSV CPE100A 有交互作用 29 七、 以in planta coimmunoprecipitation證明在植物體中NbPI 與ORSV-CPwt、ORSV-CPE100A有交互作用 30 八、 以TRV病毒載體在菸草中進行NbPI 基因靜默條件之建構 31 九、 菸草NbPI基因靜默後提升ORSV的系統性移動速率 32 肆、 討論 34 一、 以酵母菌雙雜合系統並未找到與ORCPE100A有交互作用 的菸草蛋白 34 二、 NbPI在植物中可能的功能 34 三、 NbPI與ORCP結合的情形 36 四、 NbPI會阻礙ORSV系統性移動 37 五、 NbPI阻礙ORSV系統性移動之可能機制 38 六、 結語 39 伍、參考文獻 40 陸、附表 44 柒、附圖 45 捌、補充資料 5