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

Abstract

Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like <i>Pseudomonas syringae</i> and <i>Xanthomonas campestris</i>, 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 <i>P. syringae</i> and <i>X. campestris</i> The <i>P. syringae</i> 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 <i>Nicotiana benthamiana</i> to study detailed interactions among HopZ1a, AtZED1, and AtZAR1. We show that the AtZAR1 immune pathway is conserved in <i>N. benthamiana</i> and identify AtZAR1 domains, and residues in AtZAR1 and AtZED1, that are important for immunity and protein-protein interactions in planta and in yeast (<i>Saccharomyces cerevisiae</i>). 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.