蕪菁嵌紋病毒鞘蛋白的第五個胺基酸決定其在單斑寄主長距離移行及在系統性寄主蚜傳之性狀

蔡叡奇

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蕪菁嵌紋病毒鞘蛋白的第五個胺基酸決定其在單斑寄主長距離移行及在系統性寄主蚜傳之性狀

The fifth residue of the coat protein of Turnip mosaic virus is responsible for long distance movement in a local-lesion host and aphid transmissibility in a systemic host

蔡叡奇 , Masters　　Advisor：葉錫東　　

英文

馬鈴薯Y群病毒屬；蕪菁嵌紋病毒；長距離移動；蚜蟲傳播； Potyvirus ； Turnip mosaic virus ； long-distance movement ； aphid transmission

Abstract │ Reference (116) │ Altmetrics

Abstract 〈TOP〉

過去有報導指出馬鈴薯 Y 群病毒屬病毒之 HC-Pro 以及 CP 能夠促進病毒的細胞間移行，並同時參與病毒的系統性移動。此外，HC-Pro 以及 CP 之間的交互作用也被證實是病毒被蚜蟲傳播時所必要的。過去在我們實驗室構築了蕪菁嵌紋病毒(Turnip mosaic virus, TuMV)的具感染力轉殖株 pYC5D。然而其所衍生的病毒卻無法在單斑寄主葵藜(Chenopodium quinoa)上進行系統性移動，並喪失了在系統性寄主圓葉菸草(Nicotiana benthamiana)上被蚜傳之能力。在這篇報告中，我們自當初分離之野生型蕪菁嵌紋病毒YC5 增幅了 HC-Pro 以及 CP 區段之 cDNA，並置換到 pYC5D 的對應區段。我們的結果發現來自野生型蕪菁嵌紋病毒的 CP 區段具有回復病毒在葵藜上行系統性移動的能力。此外，較高之溫度亦無法回復 YC5D 無法進行系統性移動之現象，因此我們推論 YC5D並非一種對溫度敏感之突變株。經過分析 pYC5D 以及野生型 YC5 病毒之 CP 序列後，我們發現 pYC5D 的 CP 區段之 N 端及 C 端各有一處點突變，分別位於第 5 個胺基酸，由原本的亮胺酸(leucine, L)突變為脯氨酸(proline, P) (L5P)；另一處則位在第 262 個胺基酸，由原本的蘇胺酸(threonine, T)突變為丙胺酸(alanine, A) (T262A)。值得注意的是，點突變 L5P 的發生位置就緊鄰於 DAG 區段，而該區段曾被報導和馬鈴薯 Y 群病毒屬之病毒被蚜蟲傳播之性狀有緊密的關聯。經由點突變之手法分析後，我們發現將第 5 個胺基酸由脯氨酸回復至亮胺酸後，可以修復 YC5D 病毒在葵藜上系統移行的能力。為了追蹤蕪菁嵌紋病毒在葵藜中移行的路徑，我們將 GFP 或者 GUS 基因標定至 pYC5D-P5L以及 pYC5D-A262T 上。經由螢光顯微鏡的鏡檢，我們發現 YC5D-P5L-GFP 的螢光可在接種葉及系統葉被觀察到；然而YC5D-A262T-GFP的螢光則只被侷限於接種葉之葉柄。另外，透過 GUS 之染色手法，YC5D-A262T-GUS 的訊號被發現在葵藜接種葉的葉柄與主莖連接處被阻擋。綜合上述結果，我們發現 L5P 之點突變會使得病毒被阻擋在接種葉葉柄之維管束而無法進入葵藜的主莖。而藉由蚜蟲傳播能力試驗，我們發現 P5L 能夠回復 pYC5D 的蚜傳能力缺失。總結這些結果，我們證明蕪菁嵌紋病毒鞘蛋白上的第五個胺基酸，負責該病毒在其單斑寄主上進行系統移行以及在系統性寄主上被蚜蟲傳播之能力。然而此二現象其背後的機制連結還需要更多的實驗證據來加以證實。

Parallel Abstract 〈TOP〉

Previous studies revealed that HC-Pro and CP genes of a potyvirus facilitate cell-to-cell movement and involve in systemic movement of the viruses. In addition, the interaction between HC-Pro and CP is mandatory for aphid transmission. Previously, we constructed an in vivo Turnip mosaic virus (TuMV) infectious clone pYC5D, however, its derived virus cannot move systemically in Chenopodium quinoa plants and loses aphid transmissibility in Nicotiana benthamiana plants. In this study, the HC-Pro and CP regions of pYC5D infectious clone were replaced with the corresponding cDNA fragments amplified from the original virus isolate YC5. Our results showed that the newly replaced wild type CP region restored the ability for systemic movement in inoculated C. quinoa plants. In addition, YC5D is not a temperature-sensitive mutant since higher temperature failed to restore the ability of YC5D for systemic movement. Sequencing analysis indicated that leucine (L) at position 5 and threonine (T) at position 262 of the wild type CP were changed to proline (P) and alanine (A), respectively, in YC5D virus. Interestingly, the L5P mutation was found adjacent to the 6DAG8 motif, which is involved in aphid transmissibility. Analysis by site-directed mutagenesis, our results indicated that P5L, but not A262T, is critical to permit systemic spread of the YC5 virus in C. quinoa plants. GFP-tagged or GUS-tagged TuMVs with or without changes in the two aa residues were constructed for investigation of long distance movement of TuMV within C. quinoa plants. The results of fluorescence microscopy revealed that GFP signal can be observed in petioles of inoculated leaves and upper leaves of C. quinoa plants infected by YC5D-P5L-GFP, while that of YC5D-A262T-GFP was observed only in petioles of inoculated leaves. In addition, visualization of the GUS activity revealed that the GUS signal of YC5D-A262T-GUS was blocked at the basal part of the petiole connecting to the main stem of the inoculated C. quinoa plants. Taken together, our results suggest that amino acid substitution from leucine to proline (L5P) in the CP of TuMV hampers the virus to enter the vascular tissue of the main stem of C. quinoa plants. Also, aphid transmission test of YC5D-P5L recombinant virus showed that its aphid transmissibility is restored. Taken our results together, we conclude that a single amino acid, the residue L5 at the N-terminal region, adjacent to the 6DAG8 motif, is important for the systemic translocation ability in a local-lesion host and aphid transmissibility in a systemic host. However, the exact correlation between the two phenotypes remains to be further investigated.

Reference ( 116 ) 〈TOP〉

Abdul‐Razzak, A., Guiraud, T., Peypelut, M., Walter, J., Houvenaghel, M.C., Candresse, T., Le Gall, O., and German‐Retana, S. 2009. Involvement of the cylindrical inclusion (CI) protein in the overcoming of an eIF4E‐mediated resistance against Lettuce mosaic potyvirus. Molecular Plant Pathology 10:109-113.
連結：
Adams, M.J., Antoniw, J.F., and Beaudoin, F. 2005. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Molecular Plant Pathology 6:471-487.
連結：
Andersen, K., and Johansen, I.E. 1998. A single conserved amino acid in the coat protein gene of Pea seed-borne mosaic potyvirus modulates the ability of the virus to move systemically in Chenopodium quinoa. Virology 241:304-311.
連結：
Atreya, C.D., Raccah, B., and Pirone, T.P. 1990. A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus. Virology 178:161-165.
連結：
Atreya, P.L., Atreya, C.D., and Pirone, T.P. 1991. Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proceedings of the National Academy of Sciences 88:7887-7891.
連結：

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