The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development. INTRODUCTIONPlant protoplasts without cell walls offer a versatile cell-based experimental system. Macromolecules such as DNA, RNA and proteins can be delivered into protoplasts using various methods, e.g., PEG-calcium fusion, electroporation and microinjection. Signal transduction and metabolic pathways as well as transcription and translation machineries can be transiently manipulated to investigate cell-autonomous regulation and responses. The method is especially powerful in functional genomics analysis when combined with rich genetic resources, such as insertion libraries, available in the model plant Arabidopsis thaliana. We describe here a protocol for a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) and provide troubleshooting advice and detailed information on useful reporter constructs. Applications, advantages and limitations of the TEAMP assay are also discussed. More general information about plant protoplasts can be found in a review 1 and a live performance of the TEAMP procedure can be seen at the Sheen lab website (http://genetics. mgh.harvard.edu/sheenweb/protocols_reg.html).

There is remarkable conservation in the recognition of pathogen-associated molecular patterns (PAMPs) by innate immune responses of plants, insects and mammals. We developed an Arabidopsis thaliana leaf cell system based on the induction of early-defence gene transcription by flagellin, a highly conserved component of bacterial flagella that functions as a PAMP in plants and mammals. Here we identify a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/WRKY29 transcription factors that function downstream of the flagellin receptor FLS2, a leucine-rich-repeat (LRR) receptor kinase. Activation of this MAPK cascade confers resistance to both bacterial and fungal pathogens, suggesting that signalling events initiated by diverse pathogens converge into a conserved MAPK cascade.