Molecular dissection of the pseudokinase ZED1 expands effector recognition to the tomato immune receptor ZAR1.

The highly conserved angiosperm immune receptor HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) is a bacterial pathogen recognition hub that mediates resistance by guarding host kinases for modification by pathogen effectors. The pseudokinase HOPZ-ETI DEFICIENT 1 (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 conducted an in-depth molecular analysis of ZED1. We generated a library of 164 random ZED1 mutants and identified 50 mutants that could not 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 Arabidopsis (Arabidopsis thaliana) and N. benthamiana ZAR1 orthologs. We leveraged our point mutant library to identify regions in ZED1 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 tomato (Solanum lycopersicum) ZAR1. This provides an example of expanded effector recognition through a ZAR1 ortholog from a non-model species.

Co-clustering of EphB6 and ephrinB1 in trans restrains cancer cell invasion.

EphB6 is an understudied ephrin receptor tyrosine pseudokinase that is downregulated in multiple types of metastatic cancers. Unlike its kinase-active counterparts which autophosphorylate and transmit signals upon intercellular interaction, little is known about how EphB6 functions in the absence of intrinsic kinase activity. Here, we unveil a molecular mechanism of cell-cell interaction driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and show that in trans interaction of EphB6 with ephrinB1 on neighboring cells leads to the formation of large co-clusters at the plasma membrane. These co-clusters exhibit a decreased propensity towards endocytosis, suggesting a unique characteristic for this type of cell-cell interaction. Using lattice light-sheet microscopy, 3D structured illumination microscopy and cryo-electron tomography techniques, we show that co-clustering of EphB6 and ephrinB1 promotes the formation of double-membrane tubular structures between cells. Importantly, we also demonstrate that these intercellular structures stabilize cell-cell adhesion, leading to a reduction in the invasive behavior of cancer cells. Our findings rationalize a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans.