Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism

Background: The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP). Results: Following inoculation with BaMV, N. benthamiana displayed differential gene expression in response to the infection. Isolation, cloning, and sequencing analysis using cDNA-AFLP furnished 90 cDNA fragments with eight pairs of selective primers. Fifteen randomly selected genes were used for a combined virus-induced gene silencing (VIGS) knockdown experiment, using BaMV infection to investigate the roles played by these genes during viral infection, specifically addressing the means by which these genes influence the accumulation of BaMV protein. Nine of the 15 genes showed either a positive or a negative influence on the accumulation of BaMV protein. Six knockdown plants showed an increase in the accumulation of BaMV, suggesting that they played a role in the resistance to viral infection, while three plants showed a reduction in coat protein, indicating a positive influence on the accumulation of BaMV in plants. An interesting observation was that eight of the nine plants showing an increase in BaMV coat protein were associated with cell rescue, defense, death, aging, signal transduction, and energy production. Conclusions: This study reports an efficient and straightforward method for the identification of host genes involved in viral infection. We succeeded in establishing a cDNA-AFLP system to help track changes in gene expression patterns in N. benthamiana plants when infected with BaMV. The combination of both DNA-AFLP and VIGS methodologies made it possible to screen a large number of genes and identify those associated with infections of plant viruses. In this report, 9 of the 15 analyzed genes exhibited either a positive or a negative influence on the accumulation of BaMV in N. benthamiana plants

94年度大學學術追求卓越發展延續計畫成果發表會(植物病毒前瞻性的研究)

Our research team started a project, Frontier Research in Plant Virology, under theprogram for promoting academic excellence of universities in April 94. According tothe program assessment set for this program, we are planning an internationalsymposium titled on Frontir Researches in Plant Virology held on Sep, 98. Thissymposium aims to publicize our research breakthroughs and major achievements andinteracts with the researchers in the fields. Four well-known plant virologists in theworld will be invited to attend this meeting. We are requesting 300,000 NTD to partlycover this symposium.本研究團隊94年度起執行大學學術追求卓越發展延續計畫「植物病毒前瞻性的研究」，本計畫將於98年3月執行四年期滿，依據「大學學術追求卓越發展延續計畫考核評估作業方案」第三條第五項，「各計畫全程執行期滿六個月內，應舉行學術成果發表會」，是以本計畫規劃辦理「94年度大學學術追求卓越發展延續計畫成果發表會」，將邀請國內產官學界代表參加，以加強計畫成果之交流與推廣，於98年舉行成果發表會暨成果海報展、DV展，預定參與人員約120人，所需經費統計30萬元。成果發表會將另邀請四位國際病毒學家，以植物病毒前瞻性研究國際研討會型式舉行

Differentiation of Cucumber Mosaic Virus Subgroups in Taiwan

此本計畫擬建立台灣地區胡瓜嵌紋病毒株系亞群歸屬及分佈之資料庫，並研發對單一亞群或全部亞群中株系具高度專一性及敏感性之診測技術，以作為植物防疫工作之重要基本工具，本年度擬進行之重要工作如下1. 持續採集台灣地區胡瓜嵌紋病毒株系。2. 建立胡瓜嵌紋病毒不同亞群之分子鑑別分析系統。3. 建立台灣地區各胡瓜嵌紋病毒株系亞群歸屬之基本資料庫，及針對胡瓜嵌紋病毒之植物防疫生物技術。本計畫預期可建立台灣地區胡瓜嵌紋病毒株系亞群歸屬與分布之相關資料庫，並利用生物技術發展針對胡瓜嵌紋病毒單一亞群或全部亞群之核酸探針及寡核引子對等診測技術。可提供作為植物防疫等工作上之重要參考及基本工具。Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus in the family Bromoviridae, is an economically important pathogen of more than one thousand species of agricultural and horticultural corps in Taiwan and worldwide. Based on the serological relationships and nucleotide sequence homologies, CMV strains can be further classified into two distinct subgroups, designated subgroups I and II. This divergence of CMV strains complicated the work for disease indexing and resistance breeding against CMV, since the detection probes and disease-resistance against strains in one subgroup usually works poorly against strains in the other subgroup. To our knowledge, there are no information concerning the sub-classification and distribution of CMV strains into different subgroups in Taiwan. Therefore, this study intends to establish such database regarding the sub-grouping and distribution of CMV stains in various regions of Taiwan, and to develop species- or subgroup-specific molecular differentiation system for the quarantine of the diseases in Taiwan. The specific aims for the year 2002 include the followings: 1) to continuously collect different CMV strains from various geographical regions of Taiwan; 2) to examine the nucleic acid contents of the virus strains by double-stranded RNA analysis; 3) to develop molecular differentiation system for CMV subgroups; 4) to verify the accuracy of the differentiation system; and 5) to establish quarantine system for CMV diseases in Taiwan

Development of Cucumber Mosaic Virus-Based Vector for Gene Expression in Plants(III)

傳統上在植物體表現外源基因，必須經過穩定的轉形，建立轉殖植株，此工作不僅需植物基因轉殖的技術（包括轉殖及組織培養再生），還需耗費大量人力及時間。植物病毒感染，在植物細胞內有自動複製及系統性轉移的特性，而且病毒增殖的濃度遠超過轉殖植物所能表現的量，因此提供了一個理想的外源基因表現系統。近年來，對RNA 病毒複製的瞭解，及全長具感染力的cDNA 構築，使得利用 RNA 病毒為植物載體的可行性大為增加。本計劃擬利用廣寄主性的胡瓜嵌紋病毒（Cucumber Mosaic Virus，CMV），發展成表現外來基因的載體。CMV 為一球形之植物病毒，其基因體包括三段大小不同的單鏈、正股 RNAs，RNA1及2分別決定 1a(110Kd)及2a(94Kd)蛋白，為RNA複製酵素的組成分子；RNA3決定病毒移動蛋白3a及外鞘蛋白。上年度已完成構築對應各個RNA可在試管中轉錄的全長、具感染力的cDNA。並改良此構築添加一個核甘酸G於其 5’ 端，則可使轉錄產物大幅增加，且此改良之轉錄體仍具有感染力。將水母綠色螢光蛋白基因置入RNA3的外鞘蛋白基因區，或於3a與外鞘蛋白基因區間重複置入外鞘蛋白基因的啟動子及綠色螢光蛋白基因，構築成攜帶外來基因的載體。本年度將分析各種載體在植物上的表現情形，並測試此載體系統的實用性