title: Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding.
abstract: 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.
authors.affiliation: Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics,  Aarhus University, DK-8000 Aarhus,  Denmark.
authors.forename: Angelique
authors.initials: A
authors.lastname: Broghammer
authors.forename: Lene
authors.initials: L
authors.lastname: Krusell
authors.forename: Mickaël
authors.initials: M
authors.lastname: Blaise
authors.forename: Jørgen
authors.initials: J
authors.lastname: Sauer
authors.forename: John T
authors.initials: JT
authors.lastname: Sullivan
authors.forename: Nicolai
authors.initials: N
authors.lastname: Maolanon
authors.forename: Maria
authors.initials: M
authors.lastname: Vinther
authors.forename: Andrea
authors.initials: A
authors.lastname: Lorentzen
authors.forename: Esben B
authors.initials: EB
authors.lastname: Madsen
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.forename: Søren
authors.initials: S
authors.lastname: Thirup
authors.forename: Clive W
authors.initials: CW
authors.lastname: Ronson
authors.forename: Mikkel B
authors.initials: MB
authors.lastname: Thygesen
authors.forename: Jens
authors.initials: J
authors.lastname: Stougaard
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D007887:Fabaceae / Q000378:metabolism* / Q000382:microbiology; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D009088:Mucoproteins / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000382:microbiology; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D012231:Rhizobium / Q000378:metabolism*; D013559:Symbiosis
chemical_list: D008024:Ligands; D009088:Mucoproteins; D009844:Oligosaccharides; D010940:Plant Proteins; D011134:Polysaccharides; C010393:lysin, gastropoda