Analysis of the ZAR1 Immune Complex Reveals Determinants for Immunity and Molecular Interactions

Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary function of most T3SEs is to suppress immunity; however, the plant can evolve nucleotide-binding domain-leucine-rich repeat domain-containing proteins to recognize specific T3SEs. The AtZAR1 NLR induces strong defense responses against P. syringae and X. campestris. The P. syringae T3SE HopZ1a is an acetyltransferase that acetylates the pseudokinase AtZED1 and triggers recognition by AtZAR1. However, little is known about the molecular mechanisms that lead to AtZAR1-induced immunity in response to HopZ1a. We established a transient expression system in Nicotiana benthamiana to study detailed interactions among HopZ1a, AtZED1, and AtZAR1. We show that the AtZAR1 immune pathway is conserved in N. benthamiana and identify AtZAR1 domains, and residues in AtZAR1 and AtZED1, that are important for immunity and protein-protein interactions in planta and in yeast (Saccharomyces cerevisiae). We show that the coiled-coil domain of AtZAR1 oligomerizes, and this domain acts as a signal to induce immunity. This detailed analysis of the AtZAR1-AtZED1 protein complex provides a better understanding of the immune signaling hub controlled by AtZAR1.