Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1
Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1
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DOI：
10.1007/s11262-013-0896-0
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发表时间：
2013-06-01
期刊：
影响因子：
1.6
通讯作者：
Kim, Kook-Hyung
中科院分区：
文献类型：
--
作者：
Park, Mi-Ri;Kim, Kook-Hyung
利用酵母双杂交技术和本氏烟草cDNA文库，我们先前鉴定了一个烟草N.本塞姆氏菌蛋白NbPCIP 1与马铃薯X病毒（PVX）外壳蛋白（CP）相互作用。我们先前也确定NbPCIP 1增强植物中PVX的复制。为了确定PVX CP和NbPCIP 1相互作用所需的结构域和/或氨基酸残基，我们使用酵母双杂交和β-半乳糖苷酶过滤测定来测试缺失和定点突变对相互作用的影响。截断分析表明，PVX CP的N-末端区域与NbPCIP 1相互作用。为了鉴定哪些N-末端区域PVX CP氨基酸与NbPCIP 1相互作用，我们将PVX CP N-末端区域上的12个带电荷的氨基酸取代为丙氨酸。酵母双杂交，β-半乳糖苷酶过滤，和双分子荧光互补（BiFC）测定证实，12个丙氨酸取代的突变中的10个阻断了与NbPCIP 1的相互作用。结果表明PVX CP的N-末端区域包括其螺旋结构对于与NbPCIP 1的相互作用是重要的。
Using yeast two-hybrid assays and a Nicotiana benthamiana cDNA library, we previously identified an N. benthamiana protein, NbPCIP1, that interacts with Potato virus X (PVX) coat protein (CP). We also previously determined that NbPCIP1 enhances PVX replication in plants. To determine the domains and/or amino acid residues required for PVX CP and NbPCIP1 interaction, here we used yeast two-hybrid and beta-galactosidase filter assays to test the effects of deletion and site-directed mutations on the interaction. Truncation analysis revealed that the N-terminal region of PVX CP interacts with NbPCIP1. To identify which N-terminal region PVX CP amino acid(s) interact with NbPCIP1, we substituted the 12 charged amino acids on the PVX CP N-terminal region to alanine. Yeast two-hybrid, beta-galactosidase filter, and bimolecular fluorescence complementation (BiFC) assays confirmed that ten of the 12 alanine-substituted mutations blocked the interaction with NbPCIP1. The results suggest that the N-terminal region of PVX CP including its helical structure is important for interaction with NbPCIP1.