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

TY - JOUR

T1 - Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

AU - Broghammer, Angelique

AU - Krusell, Lene

AU - Blaise, Mickaël

AU - Sauer, Jørgen

AU - Sullivan, John T

AU - Maolanon, Nicolai Nareth

AU - Vinther, Maria

AU - Lorentzen, Andrea Maria

AU - Madsen, Esben B

AU - Jensen, Knud J

AU - Roepstorff, Peter

AU - Thirup, Søren Skou

AU - Ronson, Clive W

AU - Thygesen, Mikkel B

AU - Stougaard, Jens

PY - 2012/8/21

Y1 - 2012/8/21

N2 - 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.

AB - 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.

KW - Amino Acid Motifs

KW - Binding Sites

KW - Fabaceae

KW - Kinetics

KW - Ligands

KW - Mass Spectrometry

KW - Models, Biological

KW - Mucoproteins

KW - Oligosaccharides

KW - Phosphorylation

KW - Plant Proteins

KW - Plants

KW - Polysaccharides

KW - Protein Binding

KW - Rhizobium

KW - Symbiosis

U2 - 10.1073/pnas.1205171109

DO - 10.1073/pnas.1205171109

M3 - Journal article

C2 - 22859506

SN - 0027-8424

VL - 109

SP - 13859

EP - 13864

JO - Proceedings of the National Academy of Sciences

JF - Proceedings of the National Academy of Sciences

IS - 34

ER -