Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures

Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involve d in host-range recognition for the establishment of the symbiosis with leg uminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medica go plants contains four or five N-acetylglucosamines, O-acetylated and N-ac ylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulf ated on the reducing sugar. In this paper, the ligand specificity of a high -affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elem ents, important for symbiotic activities, as recognition motifs for NFBS2 w as determined. The results show that the substitutions on the nonreducing s ugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl gro up on the C-4 Of the sugar) are determinants for high-affinity binding to N FBS2, In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2 However, the reducin g sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the nu mber of N-acetyl glucosamine residues. These results suggest that the recog nition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.