Cell-surface-localized plant immune receptors, such as FLS2, detect pathogen-associated molecular patterns (PAMPs) and initiate PAMP-triggered immunity (PTI) through poorly understood signal-transduction pathways. The pathogenic Pseudomonas syringae effector AvrPphB, a cysteine protease, cleaves the Arabidopsis receptor-like cytoplasmic kinase PBS1 to trigger cytoplasmic immune receptor RPS5-specified effector-triggered immunity (ETI). Analyzing the function of AvrPphB in plants lacking RPS5, we find that AvrPphB can inhibit PTI by cleaving additional PBS1-like (PBL) kinases, including BIK1, PBL1, and PBL2. In unstimulated plants, BIK1 and PBL1 interact with FLS2 and are rapidly phosphorylated upon FLS2 activation by its ligand flg22. Genetic and molecular analyses indicate that BIK1, and possibly PBL1, PBL2, and PBS1, integrate immune signaling from multiple immune receptors. Whereas AvrPphB-mediated degradation of one of these kinases, PBS1, is monitored by RPS5 to initiate ETI, this pathogenic effector targets other PBL kinases for PTI inhibition.

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.Noncovalent interactions among proteins are vital for all aspects of cellular processes. Thus, the identification and characterization of interacting proteins are key to our understanding of protein functions. A plethora of techniques have been developed to detect protein-protein interactions in vitro and in vivo (Piehler, 2005). The most widely used among these techniques is the yeast two-hybrid assay, which is ideal for largescale screening for interacting proteins and the construction of protein interactomes (Fields and Song, 1989;Li et al., 2004). However, the yeast two-hybrid assay detects protein-protein interactions under heterologous conditions, and results must be validated by assays under physiological conditions. Examination of protein-protein interactions under physiological conditions is often technically demanding and requires tedious procedures. For example, the co-immunoprecipitation assay requires specific antibodies; lengthy procedures that are influenced by parameters such as schemes for protein extraction, binding, and washing; and expertise of individuals performing the experiment. Thus, the results are often variable from laboratory to laboratory. Tandem affinity purification represents a more advanced technique primarily designed to identify new proteins in a protein complex in a native state (Puig et al., 2001;Rohila et al., 2006).The development of reporter-based in vivo proteinprotein interaction assays, such as fluorescence resonance energy transfer (FRET;Ha et al., 1996;Heim and Tsien, 1996;Mahajan et al., 1998), the ...

Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. Biochemical mechanisms underlying plant NLR activation have until now remained poorly understood. We reconstituted an active complex containing the Arabidopsis coiled-coil NLR ZAR1, the pseudokinase RKS1, uridylated protein kinase PBL2, and 2′-deoxyadenosine 5′-triphosphate (dATP), demonstrating the oligomerization of the complex during immune activation. The cryo–electron microscopy structure reveals a wheel-like pentameric ZAR1 resistosome. Besides the nucleotide-binding domain, the coiled-coil domain of ZAR1 also contributes to resistosome pentamerization by forming an α-helical barrel that interacts with the leucine-rich repeat and winged-helix domains. Structural remodeling and fold switching during activation release the very N-terminal amphipathic α helix of ZAR1 to form a funnel-shaped structure that is required for the plasma membrane association, cell death triggering, and disease resistance, offering clues to the biochemical function of a plant resistosome.