Research Article

Ligand-triggered allosteric ADP release primes a plant NLR complex

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Science  05 Apr 2019:
Vol. 364, Issue 6435, eaav5868
DOI: 10.1126/science.aav5868

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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

INTRODUCTION

Nucleotide-binding (NB), leucine-rich repeat (LRR) receptor (NLR) proteins constitute a family of intracellular immune receptors in both animals and plants that detect the presence of pathogen molecules or host-derived signals. NLRs share a conserved tripartite domain structure with a conserved central NB and oligomerization domain (NOD), a C-terminal LRR domain, and a variable N-terminal domain. The NOD module can be further divided into an NB domain (NBD), a helical domain (HD1), and a winged-helix domain (WHD). In plants, direct or indirect recognition of pathogen effectors by NLRs induces numerous defenses, including programmed cell death called hypersensitive response, and restricts pathogens to the infection site. For instance, the coiled-coil (CC)–NLR HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) of the small mustard plant Arabidopsis thaliana forms a preactivation complex with resistance-related kinase 1 (RKS1, a pseudokinase belonging to receptor-like cytoplasmic kinase subfamily XII-2) and recognizes the uridylyltransferase effector AvrAC from the pathogen Xanthomonas campestris pv. campestris that is responsible for the black rot disease of crucifiers. AvrAC uridylates a number of host protein kinases, including the PBS1-like protein 2 (PBL2) kinase. PBL2UMP, the version of the Arabidopsis protein uridylated by AvrAC, then acts as a ligand of the preformed ZAR1-RKS1 complex. NLRs are believed to function as a nucleotide [adenosine diphosphate (ADP) or adenosine triphosphate (ATP)]–operated molecular switch, with ADP- and ATP-bound forms corresponding to the “off” and “on” states, respectively, but the mechanism of how ADP is released from an NLR for exchange with ATP remains elusive. Structural elucidation of a full-length plant NLR protein and its recognition of modified self is lacking.

RATONALE

We reconstituted a ZAR1-RKS1 and a ZAR1-RKS1-PBL2UMP complex and determined their cryo–electron microscopy (cryo-EM) structures at resolutions of 3.7 and 4.3 Å, respectively. The structures were verified by biochemical, cell-based, and functional data. We determined how PBL2UMP affects the ADP-binding activity of the ZAR1-RKS1 complex by radiolabeled assays. Structural comparison of the ZAR1-RKS1 and ZAR1-RKS1-PBL2UMP complexes was used to probe the mechanism of PBL2UMP-induced ADP release from ZAR1, which was further validated by biochemical assays.

RESULTS

The cryo-EM structure of the ZAR1-RKS1 complex revealed that intramolecular interactions within ZAR1 maintain the NLR protein in an inactive state. The inactive state is further stabilized by an ADP. The LRR domain of ZAR1 (ZAR1LRR) is positioned differently from LRR domains of animal NLRs but functions similarly to sequester ZAR1 in a monomeric state. ZAR1CC appears to be kept in an inactive state via contacts with ZAR1LRR, ZAR1HD1, and ZAR1WHD. This contrasts with the flexible N-terminal domain of inactive apoptotic protease-activating factor 1 (Apaf-1). ZAR1LRR mediates interaction with RKS1 in the preformed ZAR1-RKS1 complex. The ZAR1-RKS1-PBL2UMP structure shows that RKS1 is exclusively responsible for the binding of PBL2UMP. The two uridylyl moieties of PBL2UMP interact with and consequently stabilize the activation segment of RKS1. Comparison of the two cryo-EM structures shows that the stabilized activation segment of RKS1 sterically clashes with the ADP-bound ZAR1NBD from the ZAR1-RKS1 complex, resulting in conformational changes in the NBD but not other domains of ZAR1: ZAR1NBD is rotated outward about 60° compared with that from the inactive ZAR1. Thus, PBL2UMP allosterically induces release of ADP from the ZAR1-RKS1-PBL2UMP complex. Indeed, radiolabeling assays showed that PBL2UMP, but not PBL2, reduced the ADP-binding activity of the ZAR1-RKS1 complex.

CONCLUSION

Our study revealed the mechanisms of PBL2UMP recognition by ZAR1-RKS1 and PBL2UMP-induced priming of ZAR1, providing a structural template for understanding NLR proteins.

PBL2UMP-induced ADP release from ZAR1.

ZAR1 is maintained in an inactive state through contacts of multiple domains and an ADP molecule (in stick representation). ZAR1LRR mediates ZAR1 interaction with RKS1. The AvrAC-uridylated PBL2 (PBL2UMP, blue) as a ligand is exclusively recognized by the ZAR1-bound RKS1. The activation segment of RKS1, which is flexible in the inactive ZAR1-RKS1 complex (red mesh), becomes stabilized (red surface) after interaction with the two uridylyl moieties (in sphere representation) of PBL2UMP and clashes with ZAR1NBD. The steric interference then causes ZAR1NBD to rotate outward and, consequently, ADP release. The ZAR1-RKS1-PBL2UMP complex thus represents an intermediate state.

Abstract

Pathogen recognition by nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) plays roles in plant immunity. The Xanthomonas campestris pv. campestris effector AvrAC uridylylates the Arabidopsis PBL2 kinase, and the latter (PBL2UMP) acts as a ligand to activate the NLR ZAR1 precomplexed with the RKS1 pseudokinase. Here we report the cryo–electron microscopy structures of ZAR1-RKS1 and ZAR1-RKS1-PBL2UMP in an inactive and intermediate state, respectively. The ZAR1LRR domain, compared with animal NLRLRR domains, is differently positioned to sequester ZAR1 in an inactive state. Recognition of PBL2UMP is exclusively through RKS1, which interacts with ZAR1LRR. PBL2UMP binding stabilizes the RKS1 activation segment, which sterically blocks ZAR1 adenosine diphosphate (ADP) binding. This engenders a more flexible NB domain without conformational changes in the other ZAR1 domains. Our study provides a structural template for understanding plant NLRs.

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