The secretion was more efficient in induction media in the absence of calcium. In animal pathogenic bacteria, Smad inhibitor a decrease in calcium concentrations has been proposed as one of the signals that trigger T3SS secretion of T3SS effectors (Lee et al., 2001; Deng et al., 2005). Although no canonical T3SS signal sequence is present in Mlr6316, we demonstrated that its N-terminal region (160 aa) directs secretion in a T3SS-dependent manner. The homologous Mlr6316 protein expressed by M. loti R7A is encoded
by the msi059 gene and is translocated into the host cell through a type IV secretion system (T4SS) (Hubber et al., 2004). It has been suggested that an RxR motif in the C-terminal region forms part of the T4SS signal (Hubber et al., 2004). Mlr6316 and the protein encoded by msi059 (Msi059) share 88% of amino acid identity, and very few differences have been observed between their respective N-terminal regions. Both Msi059 and Mlr6316 also have an RxR
motif in their C-terminal region. It is possible that the two proteins conserve the capacity to be secreted both by T3SS and T4SS. The case of mlr6331 is similar to that of mlr6316 as it does not have the characteristic amino acid pattern present in T3SS substrates. learn more However, Yang et al. (2010) applied a computational prediction of type III secreted proteins in Gram-negative bacteria and found that the protein encoded by mlr6331 is a putative T3SS substrate. Competitive experiments were carried out to analyze the participation of M. loti T3SS or putative M. loti T3SS effectors in the symbiotic process. Competitive assays have been used in several works to analyze the changes in the symbiotic phenotype (Lagares et al., 1992; Vinuesa et al., 2002; Hubber et al., 2004). This method
has the advantage that the symbiotic capacities of two bacterial strains are compared on the same plant, and this could improve the sensitivity for the detection of a subtly altered phenotype. The results presented here demonstrate that symbiotic competitiveness on Lo. tenuis cv. Esmeralda was negatively affected by a functional T3SS. To determine which proteins were responsible for this effect P-type ATPase and taking into account that a particular T3SS effector is often only partially responsible for the overall effect of the T3SS (Kambara et al., 2009), we went on to analyze the nodulation competitiveness phenotype on Lo. tenuis cv. Esmeralda using single, double, and triple mutants affected in the potentially secreted M. loti T3SS proteins described. Surprisingly, we observed a significantly diminished competitiveness associated with the triple mutant compared to the wt strain. The same phenotype was observed on Lo. japonicus MG-20. The results of the nodulation kinetic test indicate that the triple mutant also induced a lower number of nodules than the wt strain on Lo. tenuis cv. Esmeralda.