Functional analysis of NFR1 delineates a conserved residue and a region of juxtamembrane domain essential for nodulation in chickpea

Root nodule symbiosis signaling is initiated by the perception of the rhizobial Nod Factors (NFs) by two plant Lysin motif (LysM) containing nod factor receptors (NFR1 and NFR5). Here, we identified the NFR1 homologue from chickpea and functionally established its role by a gene silencing approach. To achieve valuable insights into the molecular mechanisms of CaNFR1, we evaluated a highly conserved residue in its activation loop and a specific region of the juxtamembrane (ΔJM) domain. The substitution of threonine with alanine at position 476 has significant implications, causing the loss of a phosphorylation event and disrupting the interaction with NFR5. The elimination of ΔJM domain of CaNFR1 also weakens the interaction strength with CaNFR5. Based on molecular docking and simulation studies, these structural alterations align with our yeast-based and in planta protein-protein interaction data, as well as a significant reduction in nodule number, size, and infection cell abundance after overexpression of CaNFR1 variants. Cross-species genetic complementation in lyk3 mutant of Medicago truncatula highlights the critical role of the evolutionarily conserved T476 in the activation loop and ΔJM domain of NFR1, in its interaction with NFR5, underscoring a crucial step in the receptor-mediated activation mechanism leading to root nodulation in legumes.