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A conserved juxtamembrane motif in plant NFR5 receptors is essential for root nodule symbiosis
Saturday, 2024/11/16 | 06:50:47

Simon Boje HansenThi Bich LuuKira GyselDamiano LironiChristina KrönauerHenriette RübsamIngeborg Bitsch JensenMagdalini TsitsikliThea Gramkov BirkefeldtAlen TrgovcevicJens StougaardSimona Radutoiu, and Kasper Røjkjær Andersen

 

PNAS; November 4, 2024; 121 (46) e2405671121; https://doi.org/10.1073/pnas.2405671121

Significance

Understanding the molecular mechanisms that enable nitrogen-fixing symbiosis is crucial for ongoing efforts to engineer this trait into nonlegume crops. The plant LysM receptor kinase NFR5 is required for perception of bacterial Nod factor and facilitates signal transduction leading to root nodule symbiosis in legumes. Here, we describe the structure and function of the Lotus NFR5 intracellular part and provide unique insights into the sparsely explored area of pseudokinase biology by identifying a conserved juxtamembrane αA and αA′ motif that mediates protein–protein interaction and is important for the signaling function of NFR5.

Abstract

Establishment of root nodule symbiosis is initiated by the perception of bacterial Nod factor ligands by the plant LysM receptor kinases NFR1 and NFR5. Receptor signaling initiating the symbiotic pathway depends on the kinase activity of NFR1, while the signaling mechanism of the catalytically inactive NFR5 pseudokinase is unknown. Here, we present the crystal structure of the signaling-competent Lotus japonicus NFR5 intracellular domain, comprising the juxtamembrane region and pseudokinase domain. The juxtamembrane region is structurally well defined and forms two α-helices, αA and αA′, which contain an exposed hydrophobic motif. We demonstrate that this “juxtamembrane motif” promotes NFR5–NFR5 and NFR1–NFR5 interactions and is essential for symbiotic signaling. Conservation analysis reveals that the juxtamembrane motif is present throughout NFR5-type receptors and is required for symbiosis signaling from barley RLK10, suggesting a conserved and broader function for this motif in plant–microbe symbioses.

 

See https://www.pnas.org/doi/10.1073/pnas.2405671121

 

Figure 1. Crystal structure of the signaling-competent NFR5 intracellular domain. (A) Phosphorylation assay showing catalytic activity of the NFR1 kinase and no activity of the NFR5 pseudokinase. (B) nfr5 complementation assay showing that the kinase domain of NFR5 is required for root nodule symbiosis. Number of nodules per plant on hairy roots expressing the indicated NFR5 constructs from the native Nfr5 promoter and terminator in nfr5 mutant plants, 6 wk after inoculation with M. loti R7A rhizobia. Transformation marker (tYFP) was used as the negative control. (C) NFR5-Nb200 structure model. N-lobe and C-lobe of NFR5 are highlighted. Nb200 binds to the C-lobe αG helix (see details in SI Appendix, Fig. S2 F and G). (D) Overview of the NFR5 intracellular domain. Secondary structure elements are highlighted. The juxtamembrane αA and αA′ helices precede the pseudokinase domain with the N- and C-lobe marked. An ATP molecule from the structure of PKA [PDB: 1ATP (41)] can be docked by superposition into the ATP binding pocket of NFR5 without steric clashes. The K339-E349 salt bridge, diverged DFG to “NFA” and HRD motifs are indicated. (E) The juxtamembrane region is marked along with the truncated glycine-rich and activation loops. The catalytic and regulatory spines are highlighted in transparent surface representations showing that the C-spine is broken due to the unoccupied ATP pocket, while the R-spine is fully formed. Highlighted residues A337, L440 in the C-spine and L353 in the R-spine were targeted in mutagenesis experiments in SI Appendix, Fig. S3. (F) Fusion of Nb200 as a C-terminal tag to the NFR1 receptor forces an artificial NFR1–NFR5 receptor complex that induces spontaneous nodulation in Lotus nfr1 mutant plants. Number of nodules per plant on hairy roots expressing the indicated NFR1 constructs from the native Nfr1 promoter and terminator in nfr1 mutant plants, 9 wk after transformation. Transformation marker (tYFP) was used as the negative control. (B and F) Lowercase letters indicate significant differences between samples [ANOVA (Kruskal–Wallis) and post hoc analysis (Dunn’s test), P < 0.05]. Circles represent individual plants, and the number of nodulating plants out of total plants is indicated below boxplots.

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