title: Transient expression assays for quantifying signaling output.
abstract: The protoplast transient expression system has become a powerful and popular tool for studying molecular mechanisms underlying various plant signal transduction pathways. Arabidopsis mesophyll protoplasts display intact and active physiological responses and are easy to isolate and transfect, which facilitate high-throughput screening and systematic and genome-wide characterization of gene functions. The system is suitable for most Arabidopsis accessions and mutant plants. Genetic complementation of mutant defective in sensor functions, gene expression, enzymatic activities, protein interactions, and protein trafficking can be easily designed and explored in cell-based assays. Here, we describe the detailed protocols for protoplast isolation, polyethylene glycol-calcium transfection, and different assays for quantifying the output of various signaling pathways.
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA. niu@molbio.mgh.harvard.edu
authors.forename: Yajie
authors.initials: Y
authors.lastname: Niu
authors.forename: Jen
authors.initials: J
authors.lastname: Sheen
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection