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標題: 木瓜輪點病毒超強株系P1和HC-Pro基因在基因沉寂抑制作用之交互功能
Possible roles of P1 and HC-Pro genes of the super virulent strain of Papaya ringspot virus in gene silencing suppression
作者: 張仲萍
Chang, Chung-Ping
關鍵字: 木瓜輪點病毒
Papaya ringspot virus (PRSV)
post-transcriptional gene silencing (PTGS)
transgenic papaya
super virulent strain
gene silencing suppression
出版社: 植物病理學系所
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Cell. 101: 25-33.
摘要: Papaya ringspot virus (PRSV) is the major limiting factor for papaya production worldwide. Current strategy for control relies on generation of coat protein (CP)-transgenic papaya lines through the mechanism of post-transcriptional gene silencing (PTGS). However, during field trials, we encountered a highly virulent strain of PRSV, designated PRSV 5-19, which can overcome the resistance of PRSV YK (a common mosaic strain of Taiwan) CP-3''UTR transgenic papaya lines. The CP gene and the contiguous 3''UTR of the super-strain PRSV 5-19 share high levels of homology with those of PRSV YK, suggesting that PRSV YK CP-transgenic resistance is overcome in a sequence homology-independent manner. In this study, we investigated the possible roles of PRSV 5-19 P1 and HC-Pro responsible for the homology-independent breakdown of the transgenic resistance. The antiserum to the 5-19 HC-Pro protein purified from leaf tissue of infected Cucumis metuliferus plants and P1 protein expressed from bacteria were used to prepare antisera from rabbits for detecting the corresponding antigens. The optimal reactions of HC-Pro antiserum are recommended at 1/5000 dilution, and P1 antiserum at 1/2000 dilution. The involvement and relative strength of P1 and HC-Pro proteins of 5-19 and YK in PTGS suppression were analyzed by their transient expression along with a GFP expresser and a hairpin silencing inducer, by agro-infiltration into leaves of Nicotiana benthamiana plants. Our results indicated that (i) P1 protein was not functional for gene silencing suppression; (ii) in cis or in trans P1 protein reduce the silencing suppression activity of HC-Pro; and (iii) gene silencing suppressor activity of PRSV 5-19 HC-Pro is stronger than that of PRSV YK. The stability analysis of PRSV 5-19 P1 showed that P1 can not be detected by western blotting. When in the presence of HC-Pro in cis, the mRNA of P1 accumulated in higher a levels, and the accumulation of P1 siRNA was lower, suggesting that P1 was not stable, both at protein and mRNA levels. In addition, we found other new PRSV super virulent isolates from transgenic papaya test field. These isolates all broke down the resistance of transgenic papaya after inoculation. Sequencing analysis of their HC-Pros indicated that few amino acid changes in the central region of HC-Pro may enhance the PTGS suppression ability of HC-Pro. Here, we have illustrated the molecular mechanism for the emergence of super virulent virus stain that contain a stronger silencing suppressor, which is selected out by CP-transgenic resistance in a sequence homology independent manner under field conditions. These super virulent virus strains represent a severe threat to CP-transgenic crops. When they are extensively cultivated, they may cause severe damage to other non-transgenic crops.
木瓜輪點病毒 (Papaya ringspot virus, PRSV) 是木瓜生產上最重要的限制因子,利用轉基因抗病毒的方式可以有效的防治木瓜輪點病。本實驗室先前根據後轉錄基因沉寂 (post transcriptional gene silencing, PTGS) 的機制發展出一種對抗台灣嵌紋PRSV YK株系之鞘蛋白 (coat protein, CP) 轉基因木瓜,於隔離田間試驗中發現其抗性會被另一PRSV分離株擊垮,此病毒被命名為PRSV 5-19 超強病毒株系 (super strain)。前人研究中發現此病毒株系擊垮木瓜轉基因抗性的特性與CP序列同源性不相關,根據實驗結果推測可能與病毒本身CP除外的HC-Pro蛋白有關係。HC-Pro是病毒RNA沉寂的抑制因子,因此本研究進一步探討PRSV 5-19 P1和HC-Pro兩蛋白在基因沉寂抑制作用之交互功能,及其在擊垮CP轉基因抗性的可能作用。為了研究之需要,首先由感染5-19的Cucumis metuliferus 植物葉片組織純化PRSV 5-19 HC-Pro 蛋白,以及純化由細菌表現之P1蛋白,分別製備其兔子抗血清。經過分析評估後,HC-Pro血清之最佳使用稀釋倍數訂定為1/5000倍,P1抗血清為1/2000倍。為了探討普通台灣嵌紋株系PRSV YK和強系5-19之P1和HC-Pro於基因沉寂抑制上強度的比較,利用在菸草植物中以農桿菌短暫表現蛋白方式,共同注射會表現綠色螢光和誘發綠色螢光沉寂作用之二元載體於菸草葉片,注射後第三天在藍光下觀察葉片綠色螢光,並分析其蛋白表現。我們得到的結果為(1) PRSV P1不具有抑制基因沉寂的功能; (2) PRSV P1在in cis 或in trans狀態顯示皆不會幫助HC-Pro抑制基因沉寂反應; (3) 5-19 HC-Pro之抑制基因沉寂能力比YK HC-Pro強。另外在分析PRSV 5-19 P1穩定性上,西方轉漬分析的結果顯示,利用PRSV 5-19 P1抗體無法偵測植物中P1蛋白表現;北方轉漬分析結果顯示,當存在有HC-Pro基因時,其P1之訊息RNA的表現是比較強的,P1之 siRNA的累積亦有比較少的情形;從結果可以發現PRSV 5-19 P1蛋白不穩定。另外在轉基因木瓜試驗田發現新的木瓜輪點病毒株,在非轉基因木瓜上會產生嵌紋病徵,亦會擊垮YK鞘蛋白轉基因木瓜抗性,將其HC-Pro胺基酸序列與YK和5-19株系比對,發現少數胺基酸之差異就可能會產生較強抑制基因沉寂作用之HC-Pro蛋白。由實驗結果可以推論當廣泛種植CP轉基因作物時可能會選擇出超強的病毒株系,並且這種病毒株系在擊垮轉基因抗性機制上和CP序列同源性不相關,而是和基因沉寂抑制因子HC-Pro之較強抑制PTGS作用相關。
其他識別: U0005-0508201320252200
Appears in Collections:植物病理學系



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