Molecular dissection of the ZED1 pseudokinase enables expansion of HopZ1a recognition to Solanum lycopersicum ZAR1

Authors: DIPLOCK, NATHAN - University Of California Berkeley; BAUDIN, MAEL - University Of California Berkeley; XIANG, DEREK - University Of California Berkeley; LIANG, LUNG-YU - University Of Melbourne; DAI, WEIWEN - University Of Melbourne; MURPHY, JAMES - Walter & Eliza Hall Institute; LUCET, ISABELLE - Walter & Eliza Hall Institute; HASSAN, JANA - University Of California Berkeley; Lewis, Jennifer

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Plant diseases significantly reduce crop productivity and yield. While the innate immune system of plants defends them against different pathogens, plants evolve over long time frames to recognize pathogens. This makes it very challenging for plants to recognize a new pathogen. Immune receptors commonly monitor critical host proteins for modification by plant pathogens. Immune receptors serve as a mousetrap, to initiate immune responses when the mouse (a pathogen) nibbles on the cheese (critical host proteins such as kinases). The ZAR1 immune receptor is found in most plant species, making it a good candidate to help protect plants from pathogens. We found that mutations in one kinase affect the ability of the ZAR1 immune receptor to detect modification by a pathogen. We identified changes to the kinase that enabled recognition by ZAR1 immune receptors from different plant species, including tomato. This work will help design better immune receptors as more effective mousetraps for pathogens.

Technical Abstract: The highly conserved angiosperm immune receptor ZAR1 is a bacterial pathogen recognition hub, which mediates resistance by guarding host kinases for modification by pathogen effectors. The pseudokinase ZED1 is the only known ZAR1-guarded protein that interacts directly with a pathogen effector, HopZ1a from the bacterial pathogen Pseudomonas syringae, making it a promising system for rational design of effector recognition for plant immunity. Here, we conduct the most in-depth molecular analysis of ZED1 to date. We generated a library of 164 random ZED1 mutants and identified 50 mutants that were unable to recognize the effector HopZ1a when transiently expressed in Nicotiana benthamiana. Based on our random mutants, we generated a library of 27 point mutants and found evidence of minor functional divergence between A. thaliana and N. benthamiana ZAR1 orthologs. We leveraged our point mutant library to identify regions in ZED1 that are critical for ZAR1 and HopZ1a interactions, and identified two likely ZED1-HopZ1a binding conformations. We explored ZED1 nucleotide and cation binding activity, and showed that ZED1 is a catalytically dead pseudokinase, functioning solely as an allosteric regulator upon effector recognition. We used our library of ZED1 point mutants to identify the ZED1 activation loop regions as the most likely cause of interspecies ZAR1-ZED1 incompatibility. Finally, we identified a mutation that abolished ZAR1-ZED1 interspecies incompatibility while retaining the ability to mediate HopZ1a recognition, which enabled recognition of HopZ1a through Solanum lycopersicum ZAR1. This is the first example of expanded effector recognition through a ZAR1 ortholog from a non-model species.