Hong Yiguo, Stanley John, van Wezel Rene
Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, United Kingdom.
J Virol. 2003 Dec;77(24):13315-22. doi: 10.1128/jvi.77.24.13315-13322.2003.
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 intracellular 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.
利用非洲木薯花叶病毒（ACMV）的复制起点和基于马铃薯X病毒的基因表达载体，设计了一种在植物体内对转基因本氏烟草pOri - 2系中的双生病毒复制相关蛋白（Rep）进行功能分析的系统。该品系含有ACMV复制起点串联重复序列的整合拷贝，其侧翼为非病毒序列，可被Rep作为游离复制子进行动员和复制。Rep - GFP融合蛋白也能动员和扩增复制子，便于在植物体内检测Rep。利用该植物体内系统可以有效评估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之间的相关性。我们的结果表明，对氨基酸残基R(2)、R(5)、R(7)和K(11)或H(56)、L(57)和H(58)进行修饰会阻止Rep在复制中的功能。这种缺陷与Rep核定位的可能丧失以及无法触发类似过敏反应的宿主防御机制相关。
