Structures of Exopolysaccharides Involved in Receptor-mediated Perception of Mesorhizobium loti by Lotus japonicus

Abstract

In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Recently, it was shown that a plant receptor-like kinase, EPR3, binds low molecular mass exopolysaccharide from strain R7A to regulate bacterial passage through the plant's epidermal cell layer (Kawaharada, Y., Kelly, S., Nielsen, M. W., Hjuler, C. T., Gysel, K., Muszyński, A., Carlson, R. W., Thygesen, M. B., Sandal, N., Asmussen, M. H., Vinther, M., Andersen, S. U., Krusell, L., Thirup, S., Jensen, K. J., et al. (2015) Nature 523, 308-312). In this work, we define the structure of both high and low molecular mass exopolysaccharide from R7A. The low molecular mass exopolysaccharide produced by R7A is a monomer unit of the acetylated octasaccharide with the structure (2,3/3-OAc)β-d-RibfA-(1→4)-α-d-GlcpA-(1→4)-β-d-Glcp-(1→6)-(3OAc)β-d-Glcp-(1→6)-*[(2OAc)β-d-Glcp-(1→4)-(2/3OAc)β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-Galp]. We propose it is a biosynthetic constituent of high molecular mass EPS polymer. Every new repeating unit is attached via its reducing-end β-d-Galp to C-4 of the fourth glucose (asterisked above) of the octasaccharide, forming a branch. The O-acetylation occurs on the four glycosyl residues in a non-stoichiometric ratio, and each octasaccharide subunit is on average substituted with three O-acetyl groups. The availability of these structures will facilitate studies of EPR3 receptor binding of symbiotically compatible and incompatible EPS and the positive or negative consequences on infection by the M. loti exo mutants synthesizing such EPS variants.

Keywords: Mesorhizobium loti; acetylation; carbohydrate structure; exopolysaccharide; octasaccharide; oligosaccharide; polysaccharide; rhizobia; riburonic acid; symbiosis.