Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ

Abstract

Plants and animals detect pathogen infection using intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Here we describe the 3.8-angstrom-resolution cryo-electron microscopy structure of the activated ROQ1 (recognition of XopQ 1), an NLR native to Nicotiana benthamiana with a Toll-like interleukin-1 receptor (TIR) domain bound to the Xanthomonas euvesicatoria effector XopQ (Xanthomonas outer protein Q). ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain.

Fig. 1.. Overall structure of the ROQ1-XopQ complex.

(A) Schematic representations of ROQ1 and XopQ with color-coded domain architecture: TIR, yellow; NB-ARC NDB, HD1, and WHD, light green, green, and dark green, respectively; LRR, violet; C-JID (or PL domain), light blue; and XopQ, salmon. (B and C) Composite density map of the ROQ1-XopQ complex from three cryo-EM reconstructions (B) and corresponding atomic model (C) shown in three orthogonal views. Colors are according to the nomenclature in (A).

Fig. 2.. Structure of the ROQ1 LRR and C-JID (PL domain) binding to XopQ.

(A) Surface contacts between the N-terminal region of the LRR, shown with a violet ribbon, and XopQ, represented by its Coulombic surface potential. (B) Surface contacts made by the loop between β-strands 7 and 8 of the C-JID domain (light blue) and XopQ. (C) The elongated LRR between repeats 23 and 24 (violet) interacting with XopQ (salmon). (D) Interactions between the NR loop (light blue) and active-site residues of XopQ required for ADPR binding. Catalytic Ca²⁺ is shown in gold. (E) Left: Structure of XopQ in the open conformation built from our cryo-EM density, with the NR loop inserted into the active-site cleft. The position of ADPR (green arrow) from the close state of XopQ (PDB: 4P5F) is modeled to show its overlapping position with the NR loop. Right: ADPR-bound, closed state of XopQ. The NR loop is modeled to demonstrate the clashes that would occur upon XopQ closure. (F) Residue conservation of the C-JID. Regions where too few sequences aligned to calculate a reliable conservation score are colored in gray (labeled “Insuff.”).

Fig. 3.. Oligomerization interfaces between NB-ARC domains.

(A) ATP modeled in the cryo-EM density (4.8σ) near an oligomerization interface, showing the side chains of residues involved in ATP and Mg²⁺ (magenta) binding. (B) Interface between two NB-ARC domains of neighboring protomers. (C) Left: Contacts between the WHD and HD1. Right: Contacts between neighboring NBDs. Colors are according to the nomenclature in Fig. 1.

Fig. 4.. TIR domain interfaces and conformational rearrangement of the BB-loop.

(A) Top view of ROQ1 displaying the four TIR domains organized as a dimer of dimers (each symmetric dimer shown in distinct yellow and orange). The two interfaces are marked with black dotted lines; the AE interface is formed between TIR domains shown in the same color. (B) Orthogonal view from (A) of the AE interface. (C) Orthogonal view from (A) of the BE interface marking the BB-loop positioned under the αD₃ to αE₁ helices. The proposed paths of the protein chain linking the TIR domain to the NBD are shown with purple dotted lines. (D) Top: NADase active site of a TIR domain for which the BB-loop is not interfacing with the DE surface. Bottom: Conformational rearrangement in the BB-loop bound to the DE surface. The side chain of the putative catalytic glutamate (E86) is shown in stick representation. (E) Hypothetical mechanism of TIR oligomerization with the position of the BB-loop in red. (1) Individual TIR domains are brought in close proximity. (2) TIR domains recognize each other at the AE and BE interface. (3) Assembly causes the conformational rearrangement in the BB-loop that opens the NADase active site. A Gaussian filter was applied to the map in (A) to (D) (width 1.5 Å) to reduce noise.