Protoplast for Gene Functional Analysis in Arabidopsis.

Protoplast, a plant cell without cell wall, can be readily transfected by exogenous macromolecules (DNA, RNA, protein) and therefore offer a versatile single cell-based functional analysis system to rapidly assess these exogenous macromolecules' functions. Properly prepared Arabidopsis leaf mesophyll protoplasts exhibit similar responses as intact plants to diverse abiotic and biotic stress signals as well as different hormones and nutrients, based on well-established reporter and marker gene assays. The protoplast transient expression system has been proven to be a vital and reliable tool for elucidation of the activities of transcription factors and protein kinases, protein subcellular localization and trafficking, protein-protein interaction, and protein stabilities in various signal transduction pathways. Moreover, protoplast also offers a platform for single cell-based plant regeneration, gene silencing, and genome editing. Healthy protoplasts isolated from plant tissues and the high transfection efficiency are key steps for successful use of the protoplast transient expression system. In this chapter, we describe the detailed methods of the protoplast transient expression system in Arabidopsis, including plant material preparation, high-quality maxi-plasmid DNA extraction, non-stressed protoplast isolation, highly efficient PEG-calcium transfection of plasmid DNA, and protoplast culture and harvest. We also provide several examples of gene functional analysis using this protoplast transient expression system.