Kireeva, I. | Search bioRxiv

bioRxiv2019

Perturbations of a Kinase-Pseudokinase Module Activate Plant Immunity

The Pseudomonas syringae acetyltransferase HopZ1a is delivered directly into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the ZED1 pseudokinase. Here we demonstrate that HopZ1a can acetylate members of a family of receptor-like cytoplasmic kinases (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1/ZED1/PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger immunity in planta, all independently of HopZ1a. Our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family and activating ZAR1-mediated ETI. AUTHOR SUMMARYAll plants must ward off potentially infectious microbes, and those grown in large-scale crop operations are especially vulnerable to the rapid spread of disease by successful pathogens. Although many bacteria and fungi can supress plant immune responses by producing specialized virulence proteins called effectors, these effectors can also trigger immune responses that render plants resistant to infection. We studied the molecular mechanisms underlying one such effector-triggered immune response elicited by the bacterial effector HopZ1a in the model plant host Arabidopsis thaliana. We have shown that HopZ1a promotes binding between a ZED1, a pseudokinase required for HopZ1a-triggered immunity, and several true kinases (known as PBLs) that are likely targets of HopZ1a activity in planta. HopZ1a-induced ZED1-PBL interactions also recruit ZAR1, an Arabidopsis resistance protein previously implicated in HopZ1a-triggered immunity. Importantly, ZED1 mutants that restore degenerate kinase motifs can bridge interactions between PBLs and ZAR1 (independently of HopZ1a) and trigger immunity in planta. Our results suggest that equilibria between active and inactive kinase domain conformations regulate ZED1-PBL interactions and formation of ternary complexes with ZAR1. Improved models describing molecular interactions between immunity determinants, effectors and effector targets will inform efforts to exploit natural diversity for development of crops with enhanced disease resistance.

molecular biology↗

bioRxiv2018

Yeast Genomic Screens Identify Kinesins as Potential Targets of the Pseudomonas syringae Type III Effector, HopZ1a

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as promiscuous individual T3SEs that can have multiple host targets. To overcome these challenges, we conducted heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. We screened 75 T3SEs from the plant pathogen Pseudomonas syringae and identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media, including the acetyltransferase HopZ1a. We focused our further analysis on HopZ1a, which interacts with plant tubulin and alters microtubule networks. We first performed a Pathogenic Genetic Array (PGA) screen of HopZ1a against ~4400 yeast carrying non-essential mutations and found 95 and 10 deletion mutants which reduced or enhanced HopZ1a toxicity, respectively. To uncover putative HopZ1a host targets, we interrogated both the genetic- and physical-interaction profiles of HopZ1a by identifying yeast genes with PGA profiles most similar (i.e. congruent) to that of HopZ1a, performing a functional enrichment analysis of these HopZ1a-congruent genes, and by analyzing previously described HopZ physical interaction datasets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1.\n\nARTICLE SUMMARYBacterial pathogens utilize secretion systems to directly deliver effector proteins into host cells, with the ultimate goal of promoting pathogen fitness. Despite the central role that effectors play in infection, the molecular function and host targets of most effectors remain uncharacterized. We used yeast genomics and protein interaction data to identify putative virulence targets of the effector HopZ1a from the plant pathogen Pseudomonas syringae. HopZ1a is an acetyltransferase that induces plant microtubule destruction. We showed that HopZ1a acetylated plant kinesin proteins known to regulate microtubule networks. Our study emphasizes the power of yeast functional genomic screens to characterize effector functions.

microbiology↗