A wheat resistosome defines common principles of immune receptor channels

Abstract

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2–4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35–AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

The N-terminal executioner domains of NLR immune receptors are functionally conserved across major plant lineages

“…Fungal and metazoan N-terminal 4 helix bundle domains cause cell death in an analogous fashion to plant CC domains 12,[46][47][48] . Intriguingly, CC domain mechanisms may be widely transferable between kingdoms as cell death was reported in metazoan cells expressing angiosperm CC-NLRs 18,19 . While this remains to be explored in further detail, these findings suggest that fundamental biochemical mechanisms may underpin the function of CC-like 4 helix bundles across the tree of life.…”

“…Upon pathogen virulence factor-dependent activation, the Arabidopsis CC-NLR receptor ZAR1 (hopZ1-Activated Resistance1) forms higher order oligomer complexes ('resistosome') in which the primary helix of each CC domain 4-helix bundle assembles into a funnel-shaped structure predicted to form pores within the plasma membrane 15,16 . In support of this idea, activated Arabidopsis ZAR1 or wheat Sr35 (Stem rust resistance 35) pentamers were shown to accumulate at lipid bilayers and act as non-selective cation channels in vitro 17,18 . This paradigm was further confirmed for the CCRPW8 domains of Arabidopsis NRG1 and ADR1, which exhibited oligomerization-dependent ion channel activity requiring the N-terminal region of the CCRPW8 domain 19 .…”

Majority of the highly variable NLRs in maize share genomic location and contain additional target-binding domains

“…In all four hvNLR clades we observed that the surface of the LRR domains contained a cluster of variable residues (Figure 3). This pattern of sequence variation suggests a direct recognition mechanism similar to that of RPP1, Roq1, and Sr35, the direct-recognition NLRs with structures presently available in the PDB (Zhao et al, 2022;Martin et al, 2020;Ma et al, 2020;Förderer et al, 2022). In the Int3480 clade, there were two such clusters on the opposite side of the LRR horseshoe that both faced the central cavity, suggesting a bipartite binding site in this receptor (Figure 3A).…”

“…These specificity-determining residues characteristically cluster on the concave side of the LRR domain. Targeted modification of these residues might lead to derivation of novel immune specificities in the native maize NLRs, as demonstrated for wheat disease resistance genes Sr33, Sr50 (Tamborski et al, 2022), and Sr35 (Förderer et al, 2022) thus overcoming the relative lack of natural diversity. Despite the differences in exact positions of the candidate binding residues among hvNLRs, our results agree with the inference by Förderer et al that target binding on the inside of the LRR favors an open NB-ARC conformation in an activation mechanism shared among distantly related direct-recognition NLRs.…”

“…NLR signaling is dependent on NB-ARC multimerization and usually is achieved by N-terminal RPW8, Coiled Coil (CC), or TIR domains (Song et al, 2021). Recent structures of NLRs in inactive monomer (Wang et al, 2019b) and signaling-competent multimeric conformations (Wang et al, 2019a; Ma et al, 2020; Martin et al, 2020; Zhao et al, 2022; Förderer et al, 2022) have revealed the structural basis for NLR activation and signaling. They also revealed a novel non-LRR C-terminal domain present in some TIR domain-containing NLRs that contributed to target binding (Martin et al, 2020; Ma et al, 2020).…”