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研究生(外文):Yung-Jen Hsiao
論文名稱(外文):The study of the role of autophagy in the infection cycle of Bamboo mosaic virus
外文關鍵詞:Bamboo mosaic virusBaMVinfection cycleautophagy
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細胞自噬是真核細胞中高度保留的主要自我降解現象。它利用在細胞質中產生一雙層膜構造將欲降解之物質或胞器包裹形成自噬體(autophagosome),再與胞內具分解力之胞器(如:酵母菌及植物中的液胞或動物中的溶小體)結合進行降解,並將物質分解或清除受損胞器以循環再利用。因此該現象被認為在細胞生長、遭遇逆境或病原入侵時扮演重要角色。在植物中,細胞自噬可協助其生長、對抗逆境及衰老的調控,以維持其正常生理功能及生存。在先前研究中已發現細胞自噬現象參與菸草嵌紋病毒(Tobacco mosaic virus, TMV)感染具NN抗性基因之菸草,以限制過敏性反應(hypersensitive reaction, HR)之訊號,達到控制病毒入侵之目的,並被認為在植物的先天性免疫反應中具有重要調控功能。在此研究中,由於竹嵌紋病毒(Bamboo mosaic virus, BaMV)在感染菸草(Nicotiana benthamiana)時僅產生輕微病徵且不引發過敏性反應。因此我們自菸草中找出細胞自噬過程中的調控蛋白ATG8之同源基因NbLC3。發現NbLC3之表現在受BaMV感染之原生質體高於未接種病毒之原生質體,而病毒鞘蛋白之累積量在NbLC3-knockdown植物上低於健康植物。另一方面我們以螢光標定的方式表現T7-YFP-NbLC3來監測竹嵌紋病毒感染期間,細胞自噬的現象。為了要進一步了解BaMV的感染和細胞自噬的關係,我們使用兩種不同的抑制劑以干擾自噬現象及自噬體的形成。並且接種不同病毒,如:胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)、菸草嵌紋病毒及蕃茄嵌紋病毒 (Tomato mosaic virus, ToMV)。其結果發現,在加入抑制劑後竹嵌紋病毒與胡瓜嵌紋病毒的複制受到影響,而其他兩種病毒的複制對細胞自噬受到抑制並沒有明顯的改變。綜合以上結果,自噬作用可能在竹嵌紋病毒的感染週期中提供了某種幫助並且在感染週期裡扮演重要角色。

Autophagy is a highly conserved major self-degradation process in eukaryotic cell. During autophagy process, a double-membrane vesicle structure, named autophagosome, appears in the cytoplasm to encapsulate proteins or organelles target for degrading or recycling. The targeted proteins or organelles in the autophagosome will be delivered to lytic compartments, such as vacuole in yeast and plant or lysosome in mammal, by fusion for degradation. Therefore, the autophagy process is thought to play an important role in development or pathological situation. In plant, autophagy is active in its development, starvation, environmental stress, and senescence. From previous study, it has been demonstrated that autophagy is involved in the infection of Tobacco mosaic virus (TMV) and restricting the size expansion of hypersensitive response (HR) lesion to contain the virus inside the lesion in NN Nicotiana benthamiana. They assume that autophagy may regulate plant innate immune response. During Bamboo mosaic virus (BaMV) infection in N. benthamiana plant, it only causes a mosaic symptom but not induces a HR. We identified the ATG8 homologous in the N. benthaminana, named NbLC3 which is a regulatory protein in the autophagosome formation. We found that the expression level of NbLC3 was higher in protoplast of N. benthamiana during BaMV infection and the viral accumulation was decreased in NbLC3-knockdown plant. We also traced the autophagy and autophagsomes in plants during BaMV infection by overexpressed T7-YFP-NbLC3. To understand the possible roles of autophagy in BaMV infection cycle, we blocked autophagic process by inhibitors, Wortmanin and 3-methyladenine (3-MA), and infected with different viruses, BaMV, Cucumber Mosaic Virus (CMV), TMV, and Tomato Mosaic Virus (ToMV). Only BaMV and CMV showed susceptible to the interference of autophagy and the others showed that their replication was not affect by inhibitors. Finally, these result suggested that autophagy may play an important role during BaMV infection.


中文摘要 i
Abstract ii
Introduction 1
Bamboo mosaic virus, BaMV 1
Autopahgy 1
Viral interaction with autophagy 2
Autophagy may participate in the life cycle of plant virus 3
Materials and Methods 5
Identification of NbLC3 gene 5
Plasmid construction 5
Transient expression of NbLC3 6
Immunoprecipitation 6
Protoplast isolation and inoculation 7
Protein extraction and western blot 8
Plant RNA extraction 8
Reverse transcription 9
Real-time PCR 9
Confocal microscopy 10
Results 11
The identification of ATG8 homolog, NbLC3, in Nicotiana benthamiana 11
NbLC3 might be involved in BaMV infection cycle 11
YFP-NbLC3 could successfully localiz to autophagosome in N. benthamiana 12
Autophagy could help the accumulation of BaMV 13
Discussion 15
Conclusion 17
Figures 18
References 33

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