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標題: 番茄黃化捲葉泰國病毒移動蛋白基因定點突變對機械接種特性之影響
Study of the point mutations on movement protein genes for mechanical transmissibility of tomato yellow leaf curl Thailand virus
作者: 康昀婷
Yun-Ting Kang
關鍵字: 雙生病毒;番茄黃化捲葉泰國病毒;機械接種;寄主因子;Geminiviridae;tomato yellow leaf curl Thailand virus;mechanical transmission;host factor
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雙生病毒科 (Geminiviridae) 是植物病毒中最大的一科,Begomovirus是其中最重要的病毒屬,具有單條或兩條單股環狀 DNA 兩種基因體形式,主要是以銀葉粉蝨作為傳播媒介,鮮少可藉由機械摩擦或汁液接種方式傳播。本研究室先前的研究,經由番茄捲葉新德里病毒 (tomato leaf curl New Delhi virus, ToLCNDV) 可機械接種之東方型甜瓜分離株 (ToLCNDV-OM) 及無法機械傳播之胡瓜分離株 (ToLCNDV-CB) 基因互換相對照下已直接證實雙基因體 (bipartite) 的ToLCNDV 其機械傳播能力決定於 DNA-B 上的移動蛋白 (movement protein, MP) 基因,此研究為全球首例解開雙生病毒可以被機械接種之迷的成果。亞蔬中心自1998至2009年間於臺灣所做的番茄捲葉病害田間調查研究中顯示,2005年才出現在臺灣的可機械傳播之番茄黃化捲葉泰國病毒 (tomato yellow leaf curl Thailand virus, TYLCTHV) 於 2007年的田間發生率已超過無法機械傳播的番茄捲葉臺灣病毒 (tomato leaf curl Taiwan virus, ToLCTV),造成TYLCTHV成為優勢病毒的關鍵性因素在於原普遍存在田間之 ToLCTV 僅能經由粉蝨傳播,而TYCTHV除了利用粉蝨傳播,還可藉由機械方式感染植物。因此,本計畫將以目前在臺灣感染番茄嚴重,且同樣具機械傳播特性、bipartite 的番茄黃化捲葉泰國病毒TYLCTHV為主要研究對象,去了解TYLCTHV的機械傳播特性的決定因子是在哪一個基因上及寄主植物之協力因子。根據實驗室前人對於ToLCNDV的實驗結果,先從 TYLCTHV MP (MPTYLCTHV) 進行點突變及接種測試,研究結果顯示其MPTYLCTHV的第17個及第23個原為帶負電荷之 Glutamate (E),當此兩個胺基酸任一突變為不帶電荷之 Alanine (A) 後,將失去機械機種能力,故 MP 亦是 TYLCTHV 的機械傳播能力的關鍵主因,目前我們能嘗試以互補試驗給予MPTYLCTHV (E17A) 及MPTYLCTHV (E23A) 兩病毒突變株之原始MPTYLCTHV,來回復其機械接種能力。後續將試圖找出協助 MP 蛋白參與機械接種過程的寄主因子,以了解begomoviruses可以被機械接種的分子機制。


Geminiviridae is the largest family of plant viruses. The genus Begomovirus, which is the most important genus of geminiviridae, has one or two circular single stranded- DNA genome. Begomoviruses are usually transmitted by whitefly (Bemisia tabaci). Very few begomoviruses are mechanically transmissible by sap-inoculation on leaves. Previous studies in our lab have shown that tomato leaf curl New Delhi virus oriental melon isolate (ToLCNDV-OM) is an isolate of bipartite begomovirus and can infect cucurbits via mechanical transmission. Genome reassortment assay of ToLCNDV-OM and a non-mechanically transmissible ToLCNDV-CB isolate showed that the movement protein (MP) of ToLCNDV-OM plays the key role in mechanical transmission. This is the first identification of a specific geminiviral movement protein as a determinant of mechanical transmissibility. Tomato leaf curl Taiwan virus (ToLCTV) was first detected in Taiwan in 1981 and was endemic throughout the island by the early 1990's. tomato yellow leaf curl Thailand virus (TYLCTHV), which was first detected in 2005, is now widespread throughout Taiwan. TYLCTHV is a bipartite and mechanically transmissible begomovirus, whereas ToLCTV is a monopartite and not mechanically transmissible. Through monitoring the begomoviruses in Taiwan during 1998-2009, it becomes apparent that the introduced TYLCTHV is now the predominant strain in the most tomato production areas. Based on the result of ToLCNDV study, we first focused on the MP of TYLCTHV and constructed four mutants, each carrying a point mutation within MP. When the 17th or 23rd amino acid (Glutamate, E) was mutated to Alanine (A), the mutants cannot be mechanically transmitted to Nicotiana benthamiana. This result showed that TYLCTHV MP is also the critical viral factor for mechanical transmissibility. Future studies will focus on the identification and isolation of host factors that may play crucial roles in the viral infection process by interacting with MP and assisting mechanical transmissibility of TYLCTHV.
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