Research Article

Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme

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Science  04 Dec 2020:
Vol. 370, Issue 6521, eabe3069
DOI: 10.1126/science.abe3069

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Tetrameric immune receptors

Nucleotide-binding/leucine-rich repeat (NLR) immune receptors detect pathogen effectors and trigger a plant's immune response. Two groups have now defined the structures of two NLRs that carry Toll-like interleukin-1 receptor (TIR) domains (TIR-NLRs) (see the Perspective by Tian and Li). Ma et al. studied the Arabidopsis thaliana TIR-NLR RPP1 (recognition of Peronospora parasitica 1) and its response to effectors from an oomycete pathogen. Martin et al. studied the Nicotiana benthamiana TIR-NLR ROQ1 (recognition of XopQ 1) and its response to the Xanthomonas effector. Both groups found that these TIR-NLRs formed tetramers that, when activated by binding to the pathogen effector, exposed the active site of a nicotinamide adenine dinucleoside (NAD) hydrolase. Thus, recognition of the pathogen effector initiates NAD hydrolysis and begins the immune response.

Science, this issue p. eabe3069, p. eabd9993; see also p. 1163

Structured Abstract

INTRODUCTION

Discrimination of self from nonself is pivotal for cellular organisms, as it allows the perception of pathogenic invaders that might otherwise multiply unchecked and cause disease in the host. To recognize nonself and to repel intruders, multicellular organisms deploy complex immune systems, in which repertoires of dedicated immune receptors play a central role. Innate immunity is an evolutionarily ancient arm of immunity in plants and animals that relies on structurally related, germline-encoded receptors. One class of these immune receptors inside cells, called the NLR protein family, shares a nucleotide-binding domain and leucine-rich repeats (LRRs). Plant sensor NLRs are classified into two main groups that are defined by different N-terminal domains: a coiled-coil (CC) domain in CC-NLRs (CNLs) and a Toll–interleukin-1 receptor (TIR) domain in TIR-NLRs (TNLs). A deeper understanding of the principles that govern nonself recognition by NLRs and their activation of innate immune responses necessitates protein structure–based approaches and reconstitution of signaling-active receptor complexes.

RATIONALE

Host-adapted plant pathogens secrete numerous effectors into the host extracellular spaces or inside cells. These effectors promote virulence, often by interfering with defense responses. Plant NLRs typically detect strain-specific pathogen virulence factors (effectors) delivered into host cells. This triggers immune responses that curtail pathogen proliferation and often culminate in localized host cell death. During plant host-pathogen coevolution, positive selection of random mutations in effector genes that abrogate NLR recognition drives the diversification of NLR repertoires at the population level. A well-studied coevolved pathosystem involves Arabidopsis thaliana and the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). The A. thaliana TNL receptor RPP1 confers strain-specific immunity through recognition of Hpa effector ATR1. Specific allelic variants of ATR1 in Hpa populations activate only certain RPP1 variants in particular A. thaliana accessions. Previous work detected a nicotinamide adenine dinucleotide (NAD+)–consuming enzymatic activity mediated by the N-terminal TIR domains of TNLs. How the TIR-associated NAD+ hydrolase (NADase) activity and downstream signaling is enabled by TNL effector recognition is unknown.

RESULTS

We coexpressed a naturally occurring A. thaliana RPP1 receptor variant with its matching Hpa effector ATR1 in insect cells. Protein purification revealed an oligomeric protein complex of ~600 kD consisting of RPP1 and ATR1, which we term the “RPP1 resistosome.” Biochemical assays showed that the RPP1 resistosome displays much higher Mg2+/Ca2+-dependent NADase activity than RPP1 alone. Using cryo–electron microscopy, we resolved a structure of the oligomeric complex that contains four RPP1 and four ATR1 molecules and reveals a tetrameric assembly mediated entirely by RPP1 subdomains. In contrast to other adenosine triphosphate (ATP)–bound NLRs in their active forms, RPP1 in the resistosome is adenosine diphosphate–bound, probably because of the lack of a motif required for ATP binding. The structure also reveals direct binding of ATR1 to a C-terminal jelly roll/Ig-like domain (C-JID) and the LRRs of the RPP1 receptor. Protein sequences corresponding to contact regions between the receptor and the pathogen effector are polymorphic in naturally occurring RPP1 and ATR1 variants, explaining why only certain RPP1 variants can detect strain-specific ATR1 molecules. The sequence-diversified RPP1C-JID is shared by many other TNLs, but not CNLs, in diverse plant species and might serve a role in the detection of other pathogen effectors. Receptor tetramerization creates two potential NADase active sites, each formed by an asymmetric TIR homodimer. Structure-guided substitutions of residues at this homodimeric TIR interface abolished ATR1-induced cell death in planta, supporting an essential role of the TIR-TIR interface in RPP1 function. Our combined biochemical and in planta assays show that assembly of two asymmetric TIR homodimers by the tetrameric receptor complex is responsible for NAD+ hydrolysis and RPP1-mediated immune signaling.

CONCLUSION

Our findings indicate that the RPP1 resistosome acts as a pathogen effector–inducible holoenzyme for NAD+ hydrolysis. The tetrameric RPP1 oligomeric structure provides an example of direct pathogen effector recognition by a plant NLR receptor and uncovers the mechanism of strain-specific recognition leading to NLR conformational activation. Our work suggests a multilayered regulation of RPP1 tetramerization, including ATR1 binding, RPP1 oligomerization driven by interactions among nucleotide-binding domains, and RPP1TIR self-association. The analysis provides a structural insight to induced NAD+ hydrolysis mediated by the RPP1 holoenzyme and a framework for TNL receptor signaling. As a holoenzyme, the RPP1 resistosome bears similarity to the animal apoptosome and inflammasome, which form holoenzymes after the recruitment of procaspases.

Pathogen activation of an NLR holoenzyme.

Recognition of pathogen effector ATR1 via the C-JID and LRR domains of the plant TNL receptor RPP1 triggers the assembly of a tetrameric receptor complex with two asymmetric N-terminal TIR domain homodimers. This tetramer-induced TIR asymmetry creates, via two centrally located BB-loops, active sites for NAD+ hydrolysis, which is essential for RPP1 signaling leading to host cell death.

Abstract

Direct or indirect recognition of pathogen-derived effectors by plant nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) initiates innate immune responses. The Hyaloperonospora arabidopsidis effector ATR1 activates the N-terminal Toll–interleukin-1 receptor (TIR) domain of Arabidopsis NLR RPP1. We report a cryo–electron microscopy structure of RPP1 bound by ATR1. The structure reveals a C-terminal jelly roll/Ig-like domain (C-JID) for specific ATR1 recognition. Biochemical and functional analyses show that ATR1 binds to the C-JID and the LRRs to induce an RPP1 tetrameric assembly required for nicotinamide adenine dinucleotide hydrolase (NADase) activity. RPP1 tetramerization creates two potential active sites, each formed by an asymmetric TIR homodimer. Our data define the mechanism of direct effector recognition by a plant NLR leading to formation of a signaling-active holoenzyme.

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