Strain-ecotype specificity in Sinorhizobium meliloti-Medicago truncatula symbiosis is correlated to succinoglycan oligosaccharide structure

Sinorhizobium meliloti and Medicago sativa (alfalfa) enter into a nitrogen-fixing symbiosis, and the initiation of a functional symbiosis involves an exchange of signal molecules. Plant-derived flavonoids activate the transcription of bacterial nodulation (nod) genes, resulting in the biosynthesis of N-acylated chito-oligosaccharides (Nod factors). The Nod factors then elicit root hair curling and nodule initiation in the plant (11, 14). The subsequent infection of the nodule by the microsymbiont results in the development of nitrogen-fixing nodules (Fix+) containing physiologically distinct bacteroids. Sinorhizobium spp. lipopolysaccharides, capsular polysaccharides (K antigens), and exopolysaccharides (succinoglycan or galactoglucan) are involved in the infection process, and specific polysaccharide mutants yield nonfunctional “pseudonodules” and a Fix− phenotype (9, 26, 29, 36). Succinoglycan (exo) mutants of S. meliloti Rm1021 elicit Fix− nodules due to a lack of infection thread development (10, 31, 38). Succinoglycan is composed of octasaccharide repeat units (one galactosyl and seven glucosyl residues) substituted with pyruvyl (1-carboxyethylidene), acetyl, and succinyl groups (2, 23, 24, 43). The functional oligosaccharin, an oligosaccharide with signal activity (1), consists of three repeats (5, 53), and the presence of the succinyl groups is essential for activity (30). S. meliloti also produces K antigens (46), and the KR5 antigen of S. meliloti Rm41 exoB functionally replaces succinoglycan in the infection of alfalfa (8, 41, 42, 45, 48, 54). Unlike exopolysaccharides, however, K antigen composition and structure varies significantly between Sinorhizobium spp. strains (15-17, 37, 45-47, 49), and not all K antigens can promote Fix+ symbiosis on alfalfa (6). In addition to succinoglycan, S. meliloti Rm1021 expR101 also produces a second exopolysaccharide, galactoglucan, consisting of acetylated, pyruvylated disaccharide repeating units (one galactosyl and one glucosyl residue), which can promote infection thread development on alfalfa (18, 22, 32, 56). Purified galactoglucan added to the inoculum of S. meliloti Rm1021 exopolysaccharide mutants was shown to promote infection at levels as low as 7 × 10−12 M (21). In contrast to alfalfa, galactoglucan and K antigens cannot functionally replace succinoglycan in the S. meliloti-M. truncatula symbiosis (18; this report), and although all three bacterial polysaccharides may function in the nodulation of alfalfa, there are differences in the plant response to each (38). Succinoglycan, galactoglucan, and K antigen production are genetically distinct: the majority of the succinoglycan biosynthetic genes (exo/exs) are located in a 27-kb cluster on pSYMb (20, 44), a megaplasmid that carries many of the genes involved in symbiosis, whereas galactoglucan production is directed by the exp genes located in a distinct 23-kb cluster on pSYMb (7). Capsule expression in S. meliloti involves, at least, three separate gene regions genes located on pSYMb and the chromosome (25, 27, 28, 40). M. truncatula is a model legume that is closely related to alfalfa (3), and although there is no evidence for host specificity in S. meliloti-M. sativa symbiosis, host specificity in S. meliloti-M. truncatula interactions has been demonstrated (52). This report shows that wild-type (WT) S. meliloti strains Rm41 and NRG247 are Fix+ (compatible) with M. truncatula A20 but are Fix− (incompatible) with M. truncatula A17, that the phenotypes are reversed with S. meliloti strains NRG185 and NRG34, and that the incompatible phenotype on M. truncatula is similar to that of an exopolysaccharide/K antigen mutant on alfalfa. This study examined the biochemical basis for this ecotype-strain specificity (compatibility versus incompatibility) in M. truncatula-S. meliloti interactions.