菸草蛋白與齒舌蘭輪斑病毒鞘蛋白交互作用之研究

蘭花為我國重要的外銷花卉作物，菸草嵌紋病毒屬(Tobamovirus)中的齒舌蘭輪斑病毒(Odontoglossum mosaic virus, ORSV)為常感染蘭花的病毒之一，在蘭花栽培過程中可引起嚴重的損失。病毒鞘蛋白(capsid protein, CP)的功用在於保護病毒基因體，使其不易被外在物質所分解；部分病毒可藉由其鞘蛋白與寄主特定蛋白的辨識結合，進而得以完成感染寄主的過程。本實驗室先前發現當ORSV 鞘蛋白的第100個胺基酸由glutamic acid突變成glycine時，ORSV喪失在菸草(Nicotiana benthamiana)中系統性移動的能力。因此本研究希望藉由酵母菌雙雜合系統尋找和ORSV鞘蛋白有交互作用之植物因子，並探討此植物因子在病毒感染植株時所扮演的角色。首先，利用酵母菌雙雜合系統(yeast two hybrid)篩選被ORSV感染之菸草的cDNA library，尋找可能與ORSV鞘蛋白有交互作用之植物因子。經由選擇性培養基和β-galactosidase assay的雙重篩選下，找到一個尚未被研究的菸草基因，命名為S-13。經由序列分析發現S-13蛋白屬於AT-hook motif一類的蛋白，對於此類蛋白的研究目前尚不清楚。南方雜合分析結果顯示，S-13在菸草中有一個以上的同源性基因；經由RT-PCR從菸草中增幅出與S-13非常相似的序列，並命名為S-13-2。利用酵母菌雙雜合系統分析發現S-13-2蛋白也會與ORSV CP有交互作用。北方雜合分析結果顯示，S-13 mRNA在一般情況下就會表現，且其表現量不因ORSV感染或植物接種受傷而有所改變。利用PCR的方式進行S-13片段缺失的突變，分析S-13與ORSV CP交互作用的位置，結果顯示S-13至少需保留第70到332個胺基酸才能與ORSV CP產生交互作用。此外，也利用酵母菌雙雜合系統測試其他種病毒鞘蛋白與S-13的交互作用，結果顯示S-13蛋白只會專一性的與ORSV CP有交互作用。接著利用in vitro及in planta pull-down的方式，進一步驗證S-13和ORSV鞘蛋白之間的交互作用，並發現同種類蛋白之間的交互作用比S-13和ORSV鞘蛋白之間的交互作用強。除此之外，也利用雙分子螢光互補法(bimolecular fluorescence complementation, BiFC)確認其兩者交互作用位置位於植物細胞核中。在植物葉片中短暫過量表現S-13，可以於兩天後觀察到明顯的H2O2累積及壞疽現象。在S-13基因靜默的菸草原生質體與植株上接種ORSV，發現ORSV在原生質體中的複製量及植株上的系統性移動不受影響。此外，構築了一個經由p35S promotor控制的ORSV感染性選殖株，且發現在ORSV的3’-UTR是否有ribozyme會影響到ORSV對植物的感染力，代表正確的ORSV 3’-UTR序列在ORSV的感染中扮演著很重要的角色。

關鍵字

齒舌蘭輪斑病毒；鞘蛋白；菸草蛋白；酵母菌雙雜合系統

並列摘要

Orchids are important export floral plants in Taiwan. Odontoglossum ringspot virus (ORSV), a member of the genus Tobamovirus, is one of the common virus affecting cultivated orchids and results in severe loss. The main function of capsid protein (CP) is to protect virus genome from degradation. Some of viral CPs can be recognized by specific host factors, and this interaction leads to successful infection in planta. In our previous studies, we found that ORSV could not infect N. benthamiana systemically when the 100th amino acid of CP was mutated from glutamic acid to glycine. Based on this result, the aims of this study are to screen the host factors which can interact with ORSV CP and to study the function of the host factors. To find the host factor interacting with ORSV CP, we screened the cDNA library of ORSV-infected N. benthamiana by yeast two hybrid using ORSV CP as bait. An unknown host factor, named S-13, which could interact with ORSV CP was identified by selective medium and β-galactosidase assay. Based on the sequence analysis, S-13 belongs to AT-hook motif family proteins which are still not well known. From the result of Southern hybridization assay, we found there are more than one homologous genes of S-13 in N. benthamiana. We cloned an S-13-like gene by RT-PCR and named S-13-2. S-13-2 protein could also interact with ORSV CP in yeast two-hybrid system. According to Northern hybridization assay, S-13 mRNA constantly expressed in N. benthamiana and its expression level was not altered by ORSV infection or woumding. To analyze the interaction region of S-13, three S-13 deletion mutants was constructed by PCR. The result of yeast two-hybrid assay indicated that S-13 interacted with ORSV CP through the region of 70th-332nd amino acid. The interaction of S-13 and other viral CPs were also tested and the result showed that S-13 only interacted with ORSV CP specifically. We further confirmed the interaction between S-13 and ORSV CP by in vitro and in planta pull-down assays, and found the interactions between homologous proteins are stronger than heterologous proteins. In addition, bimolecular fluorescence complementation (BiFC) assays demonstrated S-13 and ORSV CP interacted in the plant nucleus. The transient overexpression of S-13 resulted in H2O2 accumulation and necrosis formation on the leaves 2 days after agroinfiltration. The replication level of ORSV was not changed in the S-13-silenced protoplasts. S-13-silenced plants would not affect ORSV systemic movement. To obtain a stable and low-cost ORSV inoculum, p35S-driven ORSV infectious clones with or without ribozyme were constructed. The experimental results revealed the ribozyme fused with ORSV 3’-UTR could help ORSV infect plants successfully. This means a correct 3’-UTR sequence is very important for ORSV infection.

並列關鍵字

Odontoglossum ringspot virus ； capsid protein ； tobacco protein ； yeast two hybrid

參考文獻

李淑娟. 2008. 兩種感染蘭科植物重要病毒之檢測法開發及齒舌蘭輪斑病毒感染性選殖株之構築與特性分析.

Google Scholar

林品均. 2011. 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.

Google Scholar

Adams, M. J., Antoniw, J. F., and Kreuze, J. 2009. Virgaviridae: a new family of rod-shaped plant viruses. Arch. Virol. 154:1967–1972

Google Scholar

Aravind, L., and Landsman, D. 1998. AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 26:4413–4421

Google Scholar

Asurmendi, S., Berg, R. H., Koo, J. C., and Beachy, R. N. 2004. Coat protein regulates formation of replication complexes during tobacco mosaic virus infection. Proc. Natl. Acad. Sci. U. S. A. 101:1415–1420

Google Scholar

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菸草蛋白與齒舌蘭輪斑病毒鞘蛋白交互作用之研究

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