Analysis of the ZAR1 Immune Complex Reveals Determinants for Immunity and Molecular Interactions

Baudin, Maël; Hassan, Jana A.; Schreiber, Karl J.; Lewis, Jennifer D.

doi:10.1104/pp.17.00441

Cited by 76 publications

(195 citation statements)

References 88 publications

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“…We therefore tested the ability of ZED1 mutants described above to cause HR in N. benthamiana using transient transformations. As described previously [38], macroscopic HR-like cell-death was apparent by 8-24 h post-induction of protein expression in N. benthamiana tissue co-transformed with ZED1 wt and HopZ1a, but not in tissue transformed with either ZED1 wt or HopZ1a alone (S15 Fig). In contrast, ZED1 ‘DT’ and ZED1 V212T were able to cause HR in N. benthamiana even in the absence of HopZ1a (S15 Fig, panel A).…”

Section: Resultssupporting

confidence: 78%

“…Nicotiana benthamiana has two ZAR1 homologues ( Nb ZAR1 and Nb ZAR2) that are ∼57% identical to Arabidopsis ZAR1 [38]. Despite lacking closely-related homologues of ZED1 and ZRKs (S14 Fig), ZAR1-dependent immune responses triggered by HopZ1a can be recapitulated in N. benthamiana when complemented with Arabidopsis ZED1 [38]. Cell death resulting from an HR is observed as early as 8 hours following induction of transgene expression in N. benthamiana leaves co-transformed with ZED1 and HopZ1a, but not in leaves transformed with either ZED1 or HopZ1a alone [38].…”

Section: Resultsmentioning

confidence: 99%

“…Transgene expression was induced by spraying whole leaves with 20 μM dexamethasone in water with 0.01% surfactant Silwet-L77 (8 - 24h following infiltrations). Gene silencing was performed as described previously [38–40], using Agrobacterium strains generously provided by Maël Baudin and Jennifer D. Lewis (University of California, Berkeley).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Perturbations of a Kinase-Pseudokinase Module Activate Plant Immunity

Bastedo

Seto

Martel

et al. 2019

Preprint

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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.

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Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Perturbations of a Kinase-Pseudokinase Module Activate Plant Immunity

Bastedo

Seto

Martel

et al. 2019

Preprint

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“…). By contrast, transient expression of AtZAR1 in zar1‐1 was able to complement the immune response triggered by coexpression of ZED1 and HopZ1a, which is consistent with previously reported VIGS results (Baudin et al ., ). The inability of AtZAR1 to complement the XopJ4 perception defect in zar1‐1 plants indicates a partial functional divergence between NbZAR1 and AtZAR1.…”

Section: Resultsmentioning

confidence: 97%

Using forward genetics in Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of the Xanthomonas perforans effector XopJ4

Schultink

Bally

et al. 2018

New Phytologist

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The immune pathway responsible for perception of the Xanthomonas perforans effector XopJ4 was identified in the plant Nicotiana benthamiana. This pathogen causes significant yield loss in commercial tomato cultivation. Genetic mapping and viral-induced gene silencing were used to identify immune signaling components of the XopJ4 perception pathway in N. benthamiana. Transient complementation assays were performed to determine the functionality of gene variants and co-immunoprecipitation assays were used to gain insight into the molecular mechanism of the pathway. Two N. benthamiana ethyl methanesulfonate (EMS) mutants deficient for XopJ4 perception were identified as having loss-of-function mutations in the gene encoding the nucleotide binding, leucine-rich repeat (NLR) protein NbZAR1. Silencing of a receptor-like cytoplasmic kinase family XII gene, subsequently named XOPJ4 IMMUNITY 2 (JIM2), blocks perception of XopJ4. This study demonstrates the feasibility of conducting mutant screens in N. benthamiana to investigate the genetic basis of the plant immune system and other processes. The identification of NbZAR1 and JIM2 as mediating XopJ4 perception in N. benthamiana supports the model of ZAR1 being involved in the perception of many different pathogen effector proteins with specificity dictated by associated receptor-like cytoplasmic kinases.

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“…A more complex NLR model was reported as an 'NLR network', primarily in 38 Solanaceous plants. Helper NLRs, known as NLR-REQUIRED FOR CELL DEATH (NRC), 39 are associated with phylogenetically linked sensor NLRs and exhibit functional redundancy 40 for conferring resistance to a diverse array of pathogens (16). 41 NLR proteins are generally composed of three major domains: a variable N-terminus, a 42 central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR) 43 domain.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR cluster in seed plants

Lee

Mang

Choi

et al. 2019

Preprint

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1 Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding 2 domain and leucine-rich repeat (NLR) proteins. Autoactive NLRs, in some cases a specific 3 NLR domain, induce plant cell death in the absence of pathogen infection. In this study, we 4 identified a group of NLRs (G10) carrying autoactive coiled-coil (CC) domains in pepper 5 (Capsicum annuum L. cv. CM334) by genome-wide transient expression analysis. The G10-6 CC-mediated cell death mimics hypersensitive response (HR) cell death triggered by 7 interaction between NLR and effectors derived from pathogens. Sequence alignment and 8 mutagenesis analyses revealed that the intact α 1 helix of G10-CCs is critical for both G10-9 CC-and R gene-mediated HR cell death. The cell death induced by G10-CCs does not 10 require known helper NLRs, suggesting G10-NLRs are novel singleton NLRs. We also found 11 that G10-CCs localize in the plasma membrane as Arabidopsis singleton NLR ZAR1. 12 Extended studies revealed that autoactive G10-CCs are well conserved in other Solanaceae 13 plants, including tomato, potato, and tobacco, as well as a monocot plant, rice. Furthermore, 14 G10-NLR is an ancient form of NLR that present in all tested seed plants (spermatophytes). 15Our studies not only uncover the autonomous NLR cluster in plants but also provide powerful 16 resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in 17 plants.18 1 1 131 CNL clades (34). Taken together, these data suggest that G10-NLRs have features specific to 132 other CNLs and they may play unique a role(s) in plant immunity.

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Analysis of the ZAR1 Immune Complex Reveals Determinants for Immunity and Molecular Interactions

Cited by 76 publications

References 88 publications

Perturbations of a Kinase-Pseudokinase Module Activate Plant Immunity

Perturbations of a Kinase-Pseudokinase Module Activate Plant Immunity

Using forward genetics in Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of the Xanthomonas perforans effector XopJ4

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR cluster in seed plants

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