Two-hybrid protein-protein interaction analysis in Arabidopsis protoplasts:: establishment of a heterodimerization map of group C and group S bZIP transcription factors
Two-hybrid protein-protein interaction analysis in Arabidopsis protoplasts:: establishment of a heterodimerization map of group C and group S bZIP transcription factors
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DOI：
10.1111/j.1365-313x.2006.02731.x
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发表时间：
2006-06-01
期刊：
影响因子：
7.2
通讯作者：
Droege-Laser, Wolfgang
中科院分区：
文献类型：
--
作者：
Ehlert, Andrea;Weltmeier, Fridtjof;Droege-Laser, Wolfgang
体内蛋白质-蛋白质相互作用经常通过酵母双杂交分析来研究。然而，在酵母中检测到的相互作用在植物系统中可能有很大不同。基于GAL4 DNA结合（BD）和激活域（AD），我们建立了拟南芥原生质体双杂交（P2H）系统。使用 Gateway((R)) 兼容载体能够对植物细胞中蛋白质-蛋白质相互作用进行高通量筛选。通过检查碱性亮氨酸拉链 (bZIP) 转录因子的同二聚体和异二聚体特性来测试系统的效率。通过比较酵母和原生质体双杂交实验的结果，生成了拟南芥C组和S组bZIP转录因子的综合异二聚化矩阵。令人惊讶的是，几乎没有检测到同二聚化，而是检测到特异性和选择性异二聚化。异二聚体优先在C组成员(AtbZIP9、-10、-25、-63)和S1组成员(AtbZIP1、-2、-11、-44、-53)之间形成。此外，在 S1、S2 或 C AtbZIP 组内分别检测到显着但亲和力较低的相互作用。作为一种定量方法，P2H 鉴定了在酵母系统中未检测到的弱异二聚化事件。因此，除了细胞生物学技术之外，P2H 也是研究活植物细胞中蛋白质-蛋白质相互作用的重要工具。
In vivo protein-protein interactions are frequently studied by means of yeast two-hybrid analysis. However, interactions detected in yeast might differ considerably in the plant system. Based on GAL4 DNA-binding (BD) and activation domains (AD) we established an Arabidopsis protoplast two-hybrid (P2H) system. The use of Gateway((R))-compatible vectors enables the high-throughput screening of protein-protein interactions in plant cells. The efficiency of the system was tested by examining the homo- and heterodimerization properties of basic leucine zipper (bZIP) transcription factors. A comprehensive heterodimerization matrix of Arabidopsis thaliana group C and group S bZIP transcription factors was generated by comparing the results of yeast and protoplast two-hybrid experiments. Surprisingly, almost no homodimerization but rather specific and selective heterodimerization was detected. Heterodimers were preferentially formed between group C members (AtbZIP9, -10, -25, -63) and members of group S1 (AtbZIP1, -2, -11, -44, -53). In addition, significant but low-affinity interactions were detected inside group S1, S2 or C AtbZIPs, respectively. As a quantitative approach, P2H identified weak heterodimerization events which were not detected in the yeast system. Thus, in addition to cell biological techniques, P2H is a valuable tool for studying protein-protein interaction in living plant cells.