Gene Functional Analysis Using Protoplast Transient Assays
The protoplast transient assay system has been widely used for rapid functional analyses of genes using cellular and biochemical approaches. This system has been increasingly employed for functional genetic studies using double-stranded (ds) RNA interference (RNAi). Here, we describe a modified procedure for the isolation of protoplasts from leaf mesophyll cells of 14-day-old Arabidopsis thaliana. This modification significantly simplifies and speeds up functional studies without compromising the yield and the viability of protoplasts. We also present the procedure for the isolation and transfection of protoplasts from mesophyll cells of an emerging model grass species, Brachypodium distachyon. Further, we detail procedures for RNAi-based functional studies of genes using transient expression of in vitro synthesized dsRNA in protoplasts.
Key wordsProtoplasts Arabidopsis Brachypodium Transfection RNAi Synthetic dsRNAs
This work was supported by the USDA National Institute of Food and Agriculture, Hatch projects NYC -125433, NYC-125485 and MRF S1041 NYC 125853, awarded to O.K.V. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the National Institute of Food and Agriculture (NIFA) or the US Department of Agriculture (USDA).
- 11.Li J-F, Zhang D (2014) Quantitative analysis of protein-protein interactions by Split Firefly Luciferase complementation in plant protoplasts. Curr Protoc Mol Biol 107:20.9.1-20.9.8, © 2014 by John Wiley & Sons, IncGoogle Scholar
- 20.Zhai Z, Jung HI, Vatamaniuk OK (2009) Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. J Vis Exp 30Google Scholar