Jin Mingfei, Li Chunyang, Shi Yan, Ryabov Eugene, Huang Jing, Wu Zirong, Fan Zaifeng, Hong Yiguo
School of Life Science, East China Normal University, Shanghai 200062, PR China.
Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK.
J Gen Virol. 2008 Oct;89(Pt 10):2636-2641. doi: 10.1099/vir.0.2008/001966-0.
We have devised an in planta system for functional analysis of the replication-associated protein (Rep) of African cassava mosaic virus (ACMV). Using this assay and PCR-based random mutagenesis, we have identified an ACMV Rep mutant that failed to trigger the hypersensitive response (HR), but had an enhanced ability to initiate DNA replication. The mutant Rep-green fluorescent protein (GFP) fusion protein was localized to the nucleus. Sequence analysis showed that the mutated Rep gene had three nucleotide changes (A6-->T, T375-->G and G852-->A); only the A6-->T transversion resulted in an amino acid substitution (Arg to Ser), which is at the second residue in the 358 amino acid ACMV Rep protein. Our results indicate that a single amino acid can alter the differential ability of ACMV Rep to trigger the host-mediated HR defence mechanism and to initiate viral DNA replication. The implications of this finding are discussed in the context of plant-virus interactions.
我们设计了一种用于非洲木薯花叶病毒（ACMV）复制相关蛋白（Rep）功能分析的植物体内系统。利用该检测方法和基于PCR的随机诱变技术，我们鉴定出了一种ACMV Rep突变体，该突变体未能引发过敏反应（HR），但启动DNA复制的能力增强。突变的Rep-绿色荧光蛋白（GFP）融合蛋白定位于细胞核。序列分析表明，突变的Rep基因有三个核苷酸变化（A6→T、T375→G和G852→A）；只有A6→T颠换导致了氨基酸替换（Arg变为Ser），该替换位于358个氨基酸的ACMV Rep蛋白的第二个残基处。我们的结果表明，单个氨基酸可以改变ACMV Rep触发宿主介导的HR防御机制和启动病毒DNA复制的不同能力。在植物-病毒相互作用的背景下讨论了这一发现的意义。
