Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in Nicotiana benthamiana
Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in Nicotiana benthamiana
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DOI：
10.1128/jvi.77.24.13315-13322.2003
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发表时间：
2003-12-01
影响因子：
5.4
通讯作者：
van Wezel, R
中科院分区：
文献类型：
--
作者：
Hong, YG;Stanley, J;van Wezel, R
利用非洲木薯花叶病毒（ACMV）的复制起点和马铃薯X病毒的基因表达载体，构建了转基因烟草（Nicotiana benthamiana）pOri-2中双生病毒复制相关蛋白（Rep）的植物体内表达系统。该细胞系包含ACMV复制起点的串联重复序列的整合拷贝，该复制起点侧翼的非病毒序列可以作为附加型复制子被Rep动员和复制。Rep-GFP融合蛋白还可以移动和扩增复制子，促进植物中的Rep检测。利用该系统可以有效地研究Rep及其突变体的活性、Rep介导的宿主反应以及Rep细胞内定位与生物学功能的相关性。我们的结果表明，氨基酸残基R-2，R-5，R-7和K-11或H-56，L-57和H-58的修饰阻止复制中的Rep功能。这种缺陷与Rep核定位的可能丧失以及不能触发类似于过敏反应的宿主防御机制相关。
The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellullar localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R-2, R-5, R-7 and K-11 or H-56, L-57 and H-58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.