3c). These differences were not the consequence of different growth rates as both strains showed similar growth curves in minimal medium (data not shown). The M. loti triple mutant also showed a significantly lower competitive ability when co-inoculated with the rhcN mutant strain (Fig. 3a). Different independent experiments (Fig. 3a) indicated a positive role for the protein encoded in mlr6316 in the symbiotic competitiveness on Lo. tenuis cv. Esmeralda: The wt strain showed a slightly higher competitiveness than the mlr6316 mutant, and the same difference was observed when the double mutant mlr6358/mlr6361 was co-inoculated with the triple mutant. The comparison between the results obtained when the wt strain

was co-inoculated with the mlr6316 mutant and those obtained when the wt strain was co-inoculated with the triple mutant indicates CX-5461 molecular weight that the triple mutation affects competitiveness more drastically than the single mutation in mlr6316 (Fig. 3a and b). This suggests the possibility that the protein encoded in mlr6358 and/or the protein encoded in mlr6361 play a positive role in the symbiotic

competitiveness. Consistent with this, the double mutant mlr6358/mlr6361 was less competitive than the wt strain (Fig. 3a). The triple mutation in mlr6358, mlr6361, and mlr6316 also GSK-3 beta phosphorylation caused a more drastic effect on competitiveness than the combined mlr6316/mlr6361 mutation (Fig. 3a and b). This indirectly indicates that Mlr6358 has a positive effect on competitiveness on Lo. tenuis. No statistically significant differences were observed in competitiveness on Lo. tenuis cv. Esmeralda between the wt and the mlr6361 mutant or between the wt and the double mutant mlr6331/mlr6361 (Fig. 3a). However, the mutant affected in both Mlr6361

and Mlr6331 showed decreased competitiveness compared with the wt strain on Lo. japonicus selleck chemicals llc MG-20 (Fig. 3c). To determine which of the two proteins are responsible for the positive effect on this plant, co-inoculation assays of the double mutant with each of the single mutants were performed. Results indicate that the double mutant was less competitive than the single mutant affected in mlr6361 but more competitive than the single mutant affected in mlr6331 (Fig. 3c). This indicates that Mlr6331 has a positive effect and that Mlr6361 has a negative effect on the competitiveness on Lo. japonicus MG-20. We determined the nodulation kinetics for the M. loti wt, the rhcN mutant, and the triple mutant on Lo. tenuis cv. Esmeralda (Fig. 4). Although the rhcN mutant showed greater competitive ability on this plant (Fig. 3a), its nodulation kinetics was negatively affected when compared with the wt strain. On the other hand, in concordance with the competitiveness results, the M. loti triple mutant presented a kinetic phenotype significantly negatively affected compared with the wt strain and a delayed nodulation kinetics compared with the rhcN mutant strain (Fig. 4).