Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings
Article first published online: 4 JUL 2008
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd
The Plant Journal
Volume 56, Issue 1, pages 169–179, October 2008
How to Cite
Marion, J., Bach, L., Bellec, Y., Meyer, C., Gissot, L. and Faure, J.-D. (2008), Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. The Plant Journal, 56: 169–179. doi: 10.1111/j.1365-313X.2008.03596.x
- Issue published online: 25 SEP 2008
- Article first published online: 4 JUL 2008
- Received 18 April 2008; revised 15 May 2008; accepted 23 May 2008; published online 29 July 2008.
- transient expression;
- bi-molecular fluorescence complementation;
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.