Angelique Broghammer, Lene Krusell, Mickaël Blaise, Jørgen Sauer, John T. Sullivan, Nicolai Maolanon, Maria Vinther, Andrea Lorentzen, Esben B. Madsen, Knud J. Jensen, Peter Roepstorff, Søren Thirup, Clive W. Ronson, Mikkel B. Thygesen, and Jens Stougaard
Proceedings of the National Academy of Sciences of the United States of America
2012 vol: 109(34) pp: 13859-13864 doi: 10.1073/pnas.1205171109
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.
Topics: Lysin motif proteins, Plant–microbe interactions, Symbiotic signalling, Lysin motif receptor-like kinase, Non-self recognition, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications