Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings
Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings
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DOI：
10.1111/j.1365-313x.2008.03596.x
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发表时间：
2008-10-01
期刊：
影响因子：
7.2
通讯作者：
Faure, Jean-Denis
中科院分区：
文献类型：
--
作者：
Marion, Jessica;Bach, Lien;Faure, Jean-Denis
功能基因组学方法需要系统分析蛋白质亚细胞分布和相互作用网络，最好是通过优化实验的简单性和生理意义。在这里，我们提出了一个有效的植物瞬时转化系统，允许单个或多个表达的构建体含有各种荧光蛋白标签在拟南芥子叶。优化的方案是基于农杆菌直接真空渗透到年轻的拟南芥幼苗。我们证明，拟南芥表皮细胞显示出类似于烟草表皮细胞中的参考标记的亚细胞分布，并可用于共定位或双分子荧光互补研究。然后，我们使用这个新系统来研究参与鞘脂代谢的酶的亚细胞分布。与使用烟草表皮细胞或培养的拟南芥细胞的转化系统相反，我们的系统提供了利用拟南芥中可用的突变体和转基因株系的广泛集合的机会。该测定使用常规二元载体和常规农杆菌菌株，并且与多种荧光标签相容的事实，使其成为稳定转化前构建体筛选和表征的通用工具。因此，在拟南芥幼苗中的瞬时表达是一种快速简单的方法，其需要最少的处理，并且可能允许在全植物细胞环境中对携带荧光标签的融合蛋白进行中高通量分析。
The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co-localization or bi-molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium- to high-throughput analyses of fusion proteins harboring fluorescent tags in a whole-plant cellular context.