We here demonstrated the positive involvement of Mlr6316 in the symbiotic competitive capacity. It has been previously described that the msi059 mutant (affected in the mlr6316 homolog in M. loti R7A) shows a delayed nodulation on Lo. corniculatus (Hubber et al., 2004). From results, we also indirectly conclude a positive participation of Mlr6358 in the bacterial competitiveness on Lo. tenuis cv. Esmeralda. No effect on competitiveness was demonstrated for Mlr6361 in co-inoculation experiments on Lo. tenuis. However, it could not be discarded that this protein exerts certain effect.

In fact, although a Z-VAD-FMK ic50 positive effect was indirectly demonstrated for Mlr6358, no direct evidence was obtained for this, co-inoculating strains that differed only in the presence of Mlr6358. In

general, the functional analysis of type III secreted effectors in phytopathogenic bacteria is hindered by the fact that mutation of effectors genes frequently has very small or no effect on the bacterial phenotype in the interaction with the plant (Grant et al., 2006). A possible reason is the existence of effectors with redundant functions. A functional redundancy for the putative T3SS effectors described in M. loti is possible as Mlr6331 is 68% similar to the 2360-aa C-terminal of Mlr6361, and Mlr6358 is 54% similar to the 684-aa N-terminal of Mlr6361 (Sánchez et al., 2009). In spite of the positive effects attributed to some check details of the proteins, individually or in combination, on the symbiotic competitiveness on the two lotus species assayed, a mutation in the rhcN gene had either no effect SB-3CT or a significant positive effect on this phenotype, depending on the legume examined. The rhcN mutant is affected in the protein secretion through T3SS. Our results suggest that some

pili components or T3SS-secreted proteins could negatively affect bacterial competitiveness on these plants. The balance between positive and negative effects may determine the role of T3SS in the symbiotic process on the respective legumes (negative on Lo. tenuis cv. Esmeralda and no effect on Lo. japonicus MG-20). The results obtained for Mlr6331 and Mlr6361 on Lo. japonicus MG-20 also indicate a positive effect for Mlr6331 and a negative effect for Mlr6361. This negative effect was evident only in the absence of Mlr6331. As this condition, was not assayed on Lo. tenuis cv. Esmeralda, it could not be discarded that Mlr6361 also has a negative effect on this plant. The fact that the double mutant mlr6331/mlr6361 was less competitive than the wt strain on Lo. japonicus MG-20 seems to indicate that the positive effect of Mlr6331 is stronger than the negative effect of Mlr6361 and that the net balance result is thus positive.