Functional Study of the Nuclear Shuttle Protein of Tomato Leaf Curl New Delhi Begomovirus and Its Relationship to Mechanical Transmissibility and Host Range
本研究室在2007 年於田間表現嵌紋、葉片捲曲變形的罹病東方甜瓜上，分離到一個新德里番茄捲葉病毒分離株，並將其命名為新德里番茄捲葉病毒東方甜瓜分離株(Tomato leaf curl New Delhi virus -oriental melon isolate, ToLCNDV-OM)。ToLCNDV-OM是一個可以被機械接種傳播至多種瓜類寄主的新德里番茄捲葉病毒分離株，但是在機械接種的實驗中卻發現其無法感染番茄。序列分析結果顯示，ToLCNDV-OM NSP(nuclear shuttle protein) 基因的N 端序列，與同樣被報導指出可以被機械接種傳播回原寄主的馬鈴薯分離株 (ToLCNDV-Pot; NCBI accession number: AY158080) 較為相似，而與無法機械接種回原寄主的番茄分離株 (ToLCNDV-Svr; NCBI accession number:U15017)、胡瓜分離株 (ToLCNDV-Cuc; NCBI accession number: AB330080) 有明顯差異。過去的研究指出NSP 的功能與協助病毒基因體穿透核膜運送、協助病毒基因體在細胞內定位與運輸、病徵表現、病源性及病毒的系統性感染相關。依據本研究室先前的研究結果，我們提出了NSP 基因可能與病毒的寄主範圍及可機械接種傳播特性有關的假設性看法，為了進一步了解其間的關聯性，我們研擬了這次的研究計畫。首先，我們會進行ToLCNDV-OM NSP 基因N 端修飾，使其擁有番茄或胡瓜病毒分離株N 端多出來的序列，並分析重組病毒的病源性、感染後病徵發展，及其寄主範圍。另一方面，我們將與亞洲蔬菜中心合作，取得不同作物來源、且以機械接種及粉蝨接種方式分析過寄主範圍的ToLCNDV 分離株，分析這些病毒分離株的NSP 基因序列，並選殖其中表現出不同感染特性的分離株之NSP 基因，用以取代ToLCNDV-OM 分離株DNA-B 上的NSP 基因產生重組病毒株，同樣地進一步分析這些重組病毒株的病源性、感染後病徵發展，以及其寄主範圍。我們預期研究的結果將能闡明ToLCNDV 的NSP基因在病毒寄主範圍及機械接種特性上所扮演的角色。In 2007, a virus culture was isolated from a diseased oriental melon plant showingsymptoms of mosaic, leaf curl and puckering by our Lab and was designated as Tomato leafcurl New Delhi virus oriental melon isolate (ToLCNDV-OM). ToLCNDV-OM is amechanically transmissible isolate of ToLCNDV which can infect cucurbitaceous plants butcannot infect tomato plant with mechanical transmission. Sequence analysis showed that theN terminal sequence of the nuclear shuttle protein (NSP) of ToLCNDV-OM is similar inlength to the NSP of a mechanically transmissible potato isolate (ToLCNDV-Pot; NCBIaccession number: AY158080) of ToLCNDV but is different from those of a tomato(ToLCNDV-Svr; NCBI accession number: U15017) and a cucumber (ToLCNDV-Cuc; NCBIaccession number: AB330080) isolate. Previous, various studies have discussed on thefunction of NSP, viz. transport viral genomes across the nuclear membrane, intracellulartranslocation of viral genomes, cell-to-cell movement, symptom development, pathogenicity,and virus systemic infection. In our study, we hypothesize that the NSP may also relate to thehost range and the mechanical transmissibility of ToLCNDV. To investigate the relationshipbetween the NSP and the host range or the mechanical transmissibility, we design severalexperiments in this project. First, the N terminus NSP of ToLCNDV-OM will be modified asthose of ToLCNDV-Svr or ToLCNDV-Cuc, and the recombinant viruses will be analyzed forpathogenicity, symptom development and host range with mechanical or whiteflytransmission. On the other hand, we will cooperate with AVRDC for getting differentisolates of ToLCNDV and the host range of these ToLCNDV isolates will be confirmed withthe mechanical and whitefly transmission. The NSP genes from ToLCNDV isolates thatshowed differences on the host range and mechanical transmissibility will be cloned forgenerating recombinant viruses. These recombinant viruses will also be analyzed forpathogenicity, symptom development and host range. We expect that the results willelucidate the role of the ToLCNDV NSP gene on host range and mechanical transmissibility
