於圓葉菸草中探討蕙蘭嵌紋病毒與齒舌蘭輪斑病毒之交互作用

Interactions between Cymbidium mosaic virus and Odontoglossum ringspot virus in Nicotiana benthamiana

郭尚明(Shang-Ming Guo)

指導教授 ： 張雅君

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703539

摘要

植物病毒共同感染於相同寄主時，常會產生交互作用。齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)與蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)複合感染至蘭花時，往往會造成較單獨感染更為嚴重的病徵，此病徵加劇的現象稱為協力作用(synergistic effect)。本實驗室於圓葉菸草(Nicotiana benthamiana)系統中發現，ORSV與CymMV複合感染於菸草原生質體時，仍保有協力作用，且ORSV似乎透過具有基因靜默抑制功能之p126蛋白調控與CymMV的協力作用。本研究延續兩病毒於圓葉菸草中之試驗，以農桿菌接種法接種CymMV與ORSV之其他蛋白，發現除了p126外，鞘蛋白也會使CymMV的累積量上升，但機制仍不清楚。ORSV p126的四個功能區單獨存在時，已被證明均不具有基因靜默抑制能力；而本研究構築出不同功能區組合的p126蛋白，發現缺少任一功能區者，亦無法抑制植物之基因靜默。將ORSV與CymMV複合接種於菸草中，發現在接種葉的病毒累積量上無法觀察到兩者有協力作用產生；但透過罹病組織之汁液接種，在接種葉與系統葉均可觀察到較明顯且嚴重的病徵。利用間接式酵素免疫吸附法(I-ELISA)測定，發現原本無法移動至系統葉之CymMV可成功感染至系統葉，顯示雖然在病毒累積量上無協力現象，但在移動能力上ORSV仍可協助CymMV。若將CymMV與失去系統性移動能力之ORSVE100A複合感染，則此協力現象消失，顯示CymMV之系統性移動可能倚賴ORSV之CP或ORSV之感染過程。欲了解CymMV受ORSV協助的情形是否具有專一性，嘗試共表現CymMV與Turnip mosaic virus (TuMV) HCPro、Cucumber mosaic virus (CMV) 2b、Potato virus X (PVX) p25及Tomato bushy stunt virus (TBSV) p19等已知的病毒基因靜默抑制子。結果顯示，除PVX p25幫助CymMV的效應較不明顯外，其餘基因靜默抑制子均可顯著地提升CymMV累積量，顯示在病毒累積量上p126協助CymMV的情形並不具有專一性。然而在系統性移動上，TuMV、PVX及隸屬於Tobamovirus屬之Tomato mild green mosaic virus (TMGMV)均可協助CymMV移動至系統葉。在TuMV+CymMV與PVX+CymMV複合感染時，分別有57%及50%的植株在系統葉上偵測到CymMV；而在TMGMV+CymMV複合感染植株中，CymMV均可系統性感染。最後，本實驗構築出可表現明顯綠色螢光之CymMV-eGFP螢光病毒株，並利用該材料驗證在病毒累積量與系統性移動上所觀察到的現象。

關鍵字

齒舌蘭輪斑病毒 ； 蕙蘭嵌紋病毒 ； 協力作用 ； 基因靜默抑制 ； GFP 螢光病毒株

並列摘要

Mixed infection of plant viruses usually leads to intrahost virus-virus interactions. Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) commonly co-infect orchid plants and cause more severe symptoms, which is defined as synergistic effect. Recently, we found that the synergistic effect between ORSV and CymMV did exist on Nicotiana benthamiana protoplasts. This interaction seems to be regulated by the silencing suppression activity of ORSV p126. In this study, we continued to explore the interactions between ORSV and CymMV on N. benthamiana. In addition to p126, transiently expressed ORSV capsid protein (CP) facilitated CymMV accumulation on the inoculated leaves of N. benthamiana, but ORSV movement protein did not. The mechanism under this phenomenon remains unknown. Individual domains of ORSV p126 were proved without RNA silencing suppression ability and could not improve CymMV accumulation. In this study, we constructed five different domain combination of p126 and found that all four domains are necessary for RNA silencing suppression. Surprisingly, viral RNA and CP accumulation of both ORSV and CymMV had no significant difference between singly and doubly inoculated leaves of N. benthamiana plants through agroinoculation. However, by means of sap inoculation, more severe symptoms on both inoculated and systemic leaves of doubly infected plants were observed compared to singly infected ones. Next, we detected the viruses in systemic leaves of ORSV and CymMV doubly infected plants by indirect-ELISA, and found that the systemic movement-deficient CymMV could systemically infect N. benthamiana. These results suggested that although mixed infection of ORSV and CymMV did not exhibit synergistic interaction on inoculated leaves, ORSV still facilitated CymMV in other mechanism, probably on movement. Interestingly, facilitation on CymMV systemic movement disappeared when CymMV was co-inoculated with systemic movement-deficient ORSV (ORSVE100), which suggested the systemic movement of CymMV may rely on ORSV CP or ORSV infection processes. To understand the specificity of ORSV-CymMV synergism, we co-expressed CymMV with some well-known RNA silencing suppressors (RSSs), e.g. Turnip mosaic virus (TuMV) HCPro, Cucumber mosaic virus 2b, Potato virus X (PVX) p25 and Tomato bushy stunt virus p19. Except for PVX p25, all RSSs could significantly increase the accumulation of CymMV, which indicated that p126-mediated enhancement of CymMV accumulation probably can be replaced by other RSSs. Furthermore, we were curious about whether CymMV can systemically infect N. benthamiana with the aid of other ORSV-related or ORSV-unrelated viruses. For mixed infection of TuMV+CymMV, and PVX+CymMV, about 57% and 50% infected plants showed systemic CymMV infection. Co-infection of CymMV and Tomato mild green mosaic virus (TMGMV), a tobamovirus, facilitated systemic movement of CymMV on all tested plants. Finally, we constructed three eGFP-expressing CymMV clones and used one of them to confirm the experimental results.

並列關鍵字

Odontoglossum ringspot virus ； Cymbidium mosaic virus ； RNA silencing suppression ； GFP-expressing virus

參考文獻

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