The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a

Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1–resistance complex, resulting in ETI activation.The plant immune system can be divided into two major branches that share commonalities with animal innate immunity. Pattern recognition receptor (PRR)-triggered immunity (PTI) is activated by the recognition of conserved microbial molecules called microbe-associated molecular patterns (MAMPs) by extracellular PRRs (1), similar to the recognition of MAMPs by animal Toll-like receptors (2). Effector-triggered immunity (ETI) is activated by the recognition of pathogen-derived effector proteins by intracellular NB-LRR proteins that share structural features with animal nucleotide-binding domain, leucine-rich repeat containing (NLR) proteins (3). The ETI response overlaps significantly with PTI but is accelerated, amplified, and often accompanied by the hypersensitive cell death response (HR; refs. 4 and 5). Recognition of effector proteins can occur directly where the effector protein binds directly to the NB-LRR protein, or indirectly, in which case both the effector and NB-LRR proteins bind to a common host protein. In the latter case, ETI is initiated by the NB-LRR protein in response to an effector-induced modification to the host protein (6). In some cases, the host protein monitored by the NB-LRR may represent a true virulence target of the effector protein, whereas in other cases, this protein may be a nonfunctional decoy of the true virulence target maintained by the host for the purposes of pathogen surveillance (7).The continual arms race between host and pathogen has directed the coevolution of host innate immunity with bacterial virulence strategies. The type III secretion system (T3SS) is a predominant virulence mechanism used by many Gram-negative bacterial pathogens to cause disease in eukaryotic hosts (8). The T3SS delivers type III secreted effector (T3SE) proteins into host cells where they can promote the infection process by suppressing host immunity and/or participating in the pathogen life cycle.The YopJ/HopZ/AvrRxv T3SE superfamily is evolutionary diverse and found in both animal and plant pathogens (9, 10). Yersinia pestis YopJ, the archetypal member of this superfamily, acetylates serine or threonine residues in the activation loops of members of the mitogen-activated protein kinase kinase (MAPKK) and MAP kinase kinase kinase superfamilies, thereby suppressing innate immunity (11–15). In the plant pathogen Pseudomonas syringae, the HopZ1 subfamily is represented by three closely related alleles, HopZ1a, HopZ1b, and HopZ1c, that diversified under pressure from the host immune system (10). The most phylogenetically basal representative of this subfamily, HopZ1a, is recognized in Arabidopsis by the ZAR1 NB-LRR protein (16, 17) as well as by unidentified proteins in rice, Nicotiana benthamiana, sesame, and soybean (10). Like YopJ, HopZ1a is an acetyltransferase and can promote pathogen growth in Arabidopsis plants lacking the ZAR1 NB-LRR protein. The virulence and immune-eliciting functions of HopZ1a both require the cysteine residue in the acetyltransferase catalytic triad (16, 17). It is likely that ZAR1 evolved to recognize an ancestral virulence activity of HopZ1a; however, the molecular relationship between virulence and immunity-eliciting functions remain to be established (17, 18).Here, we describe a forward genetic screen designed to characterize the genetic requirements of ZAR1-mediated immunity by identifying Arabidopsis mutant plants that lacked a HopZ1a-induced HR response. We named these mutants hopZ-ETI deficient (zed) and mapped zed1 to At3g57750 by Illumina-based next-generation sequencing. Different zed1 point mutants identified from our mutant screen or a tDNA insertion line in At3g57750 all lack recognition of HopZ1a. PTI and basal defenses are unaffected in zed1; however, zed1 exhibits a loss of HopZ1a-mediated ETI. We show that ZED1 interacts directly with HopZ1a, as well as the N-terminal coiled-coil (CC) domain of ZAR1. ZED1 is an uncharacterized pseudokinase and is acetylated on threonines 125 and 177 by HopZ1a. We hypothesize that ZAR1 is activated in response to ZED1 acetylation by HopZ1a and speculate that ZED1 may be a decoy of the true HopZ1a virulence target because HopZ1a retains its virulence function in zed1Arabidopsis plants.