Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

Proc Natl Acad Sci U S A. 2012 Aug 21;109(34):13859-64. doi: 10.1073/pnas.1205171109. Epub 2012 Aug 2.


Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man(3)XylFucGlcNAc(4), were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor-ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The K(d) values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Binding Sites
  • Fabaceae / metabolism*
  • Fabaceae / microbiology
  • Kinetics
  • Ligands
  • Mass Spectrometry / methods
  • Models, Biological
  • Mucoproteins / chemistry
  • Oligosaccharides / chemistry*
  • Phosphorylation
  • Plant Proteins / metabolism
  • Plants / microbiology
  • Polysaccharides / chemistry
  • Protein Binding
  • Rhizobium / metabolism*
  • Symbiosis


  • Ligands
  • Mucoproteins
  • Oligosaccharides
  • Plant Proteins
  • Polysaccharides
  • lysin, gastropoda