Research Article

Reconstitution and structure of a plant NLR resistosome conferring immunity

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Science  05 Apr 2019:
Vol. 364, Issue 6435, eaav5870
DOI: 10.1126/science.aav5870

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The plant resistosome comes into focus

Nucleotide-binding, leucine-rich repeat receptors (NLRs) initiate immune responses when they sense a pathogen-associated effector. In animals, oligomerization of NLRs upon binding their effectors is key to downstream activity, but plant systems differ in many ways and their activation mechanisms have been less clear. In two papers, Wang et al. studied the composition and structure of an NLR called ZAR1 in the small mustard plant Arabidopsis (see the Perspective by Dangl and Jones). They determined cryo–electron microscopy structures that illustrate differences between inactive and intermediate states. The active, intermediate state of ZAR1 forms a wheel-like pentamer, called the resistosome. In this activated complex, a set of helices come together to form a funnel-shaped structure required for immune responsiveness and association with the plasma membrane.

Science, this issue p. eaav5868, p. eaav5870; see also p. 31

Structured Abstract


Nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) mediate plant immunity by directly or indirectly sensing pathogen effector proteins delivered into plant cells. The activation of plant NLRs stops pathogen proliferation through the induction of a variety of defenses, including the hypersensitive response, a form of programmed cell death. In the small mustard plant Arabidopsis thaliana, the coiled-coil (CC)–NLR HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) exists in a preformed complex with resistance-related kinase 1 (RKS1) to sense the uridylyltransferase effector AvrAC from the microbial pathogen Xanthomonas campestris pv. campestris (Xcc). AvrAC uridylates the PBS1-like protein 2 (PBL2) kinase to produce PBL2UMP, which is recruited to ZAR1-RKS1. As members of the adenosine triphosphatases associated with diverse cellular activities, NLRs are hypothesized to function through oligomerization. Evidence for this model is provided by studies of animal NLRs. However, whether plant NLRs oligomerize after activation into large protein complexes like NLR inflammasomes remains unknown. Furthermore, little is known about the biochemical functions of plant NLRs.


In an accompanying paper, we show that the ZAR1-RKS1-PBL2UMP complex in the absence of deoxyadenosine triphosphate (dATP) or adenosine triphosphate (ATP) is in a primed state. Gel filtration and cryo–electron microscopy (cryo-EM) were used to investigate whether the primed complex oligomerizes in the presence of dATP or ATP. We verified the biological relevance of the oligomerized ZAR1-RKS1-PBL2UMP complex induced by dATP or ATP with biochemical, cell-based, and functional assays.


Gel filtration analysis showed that ZAR1-RKS1 and PBL2UMP formed a high-order oligomeric complex with a molecular mass of ~900 kDa in the presence of dATP or ATP. We termed the complex the ZAR1 resistosome. A cryo-EM structure of the ZAR1 resistosome determined at a resolution of 3.4 Å revealed that it formed a wheel-like pentamer, the assembly of which is mediated by ZAR1. All the structural domains of ZAR1, including the CC domain, NB domain (NBD), helical domain 1 (HD1), winged-helix domain (WHD), and LRR domain, are involved in the pentamerization of the ZAR1 resistosome, which is further stabilized by dATP. Mutagenesis analyses and functional studies indicate that the resistosome activates the defensive hypersensitive cell death response and contributes to resistance to Xcc.

The ZAR1 CC domain (ZAR1CC) contributes to the oligomerization of the ZAR1 resistosome by forming an α-helical barrel. ZAR1CC undergoes fold switching during ZAR1 activation, in addition to the structural remodeling of ZAR1WHD-ZAR1LRR relative to ZAR1NBD-ZAR1HD1. The very N-terminal α helix (α1) buried in the inactive ZAR1 becomes exposed in the ZAR1 resistosome. The five exposed α1 helices in the ZAR1 resistosome form a funnel-shaped structure projecting out of the wheel-defined plane. Biochemical and functional data showed that this structure is required for AvrAC-induced ZAR1 plasma membrane (PM) association, cell death, and resistance to Xcc. Simultaneous mutation of two negatively charged residues at its inner surface did not affect ZAR1 binding to the PM but did abolish cell death and disease resistance, suggesting that the ZAR1 resistosome function requires the inner surface of the funnel structure.


Our study revealed the oligomerization of ZAR1, a plant NLR protein; clarified its activation mechanism; and provided insights into its biochemical functions.

PBL2UMP-induced assembly of the ZAR1 resistosome.

Interaction of PBL2UMP (blue) with the preformed ZAR1-RKS1 complex (inactive ZAR1-RKS1) triggers conformational changes in ZAR1NBD and adenosine diphosphate (ADP) release, allowing the complex to bind dATP or ATP. dATP or ATP binding induces structural remodeling and fold switching of ZAR1, resulting in full activation of ZAR1 (activated ZAR1-RKS1-PBL2UMP) and formation of the pentameric ZAR1 resistosome (shown in two orientations). The very N-terminal α helix (α1) (red) of ZAR1 buried in the inactive ZAR1-RKS1 complex becomes solvent-exposed in the activated ZAR1-RKS1-PBL2UMP complex and forms a funnel-shaped structure (highlighted within the purple frame) in the ZAR1 resistosome that is required for ZAR1 PM association, cell death triggering, and disease resistance.


Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. Biochemical mechanisms underlying plant NLR activation have until now remained poorly understood. We reconstituted an active complex containing the Arabidopsis coiled-coil NLR ZAR1, the pseudokinase RKS1, uridylated protein kinase PBL2, and 2′-deoxyadenosine 5′-triphosphate (dATP), demonstrating the oligomerization of the complex during immune activation. The cryo–electron microscopy structure reveals a wheel-like pentameric ZAR1 resistosome. Besides the nucleotide-binding domain, the coiled-coil domain of ZAR1 also contributes to resistosome pentamerization by forming an α-helical barrel that interacts with the leucine-rich repeat and winged-helix domains. Structural remodeling and fold switching during activation release the very N-terminal amphipathic α helix of ZAR1 to form a funnel-shaped structure that is required for the plasma membrane association, cell death triggering, and disease resistance, offering clues to the biochemical function of a plant resistosome.

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