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

The angiosperm immune receptor ZAR1 and pseudokinase ZED1 recognize Pseudomonas syringae pathogen effector HopZ1a. To identify functional constraints in engineering ZAR1 for broader immunity, we randomly mutagenized ZED1 and identified 27 mutants lacking HopZ1a recognition. We characterized mutants for impaired ZAR1/HopZ1a interactions or autoactive immunity. ZED1 is catalytically inactive, and likely acts as an allosteric regulator for effector recognition. ZED1 activation loop regions are the most likely cause of interspecies ZAR1-ZED1 incompatibility. One mutation eliminated interspecies incompatibility while retaining HopZ1a recognition with tomato ZAR1, marking the first instance of effector recognition through a ZAR1 ortholog from a non-model species.