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

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  • Angelique Broghammer, Denmark
  • Lene Krusell, Denmark
  • Mickaël Blaise, Denmark
  • Jørgen Sauer, Denmark
  • John T Sullivan
  • ,
  • Nicolai Nareth Maolanon, LUKKET: 2012 Nanobiovidenskab, Denmark
  • Maria Vinther
  • Andrea Maria Lorentzen, Institut for Biokemi og Molekylær Biologi, Denmark
  • Esben B Madsen, Denmark
  • Knud J Jensen, Denmark
  • Peter Roepstorff, Institut for Biokemi og Molekylær Biologi, Denmark
  • Søren Skou Thirup
  • Clive W Ronson
  • ,
  • Mikkel B Thygesen, Denmark
  • Jens Stougaard
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.
Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Pages (from-to)13859-13864
Number of pages6
Publication statusPublished - 21 Aug 2012

    Research areas

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

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