The use of plant protoplast transient expression system has facilitated the discovery
and dissection of many signal transduction pathways in response to hormones, metabolites,
and stresses. Recently, Arabidopsis protoplasts also have been used successfully to
study plant innate immune responses triggered by pathogen-derived elicitors. Here, we
describe the detailed protocols for studying innate immune responses, including cell death
and early defense gene regulation activated by two types of elicitors, pathogen-associated
molecular patterns and bacterial type III effectors in Arabidopsis protoplasts. This cellbased
system simplifies the complex pathogen–plant interactions to pure individual signals
and synchronized cell-autonomous responses. The application of this novel approach
provides high temporal and spatial resolution to enhance our understanding of the distinct
and overlapping signaling events in pathogen-associated molecular pattern- and bacterial
type III effector-activated immune responses at the molecular and cellular level.