Evidences for an involvement of the bacterial type IV secretion system (T4SS) in the symbiotic relationship between rhizobia and legumes have been pointed out by several recent studies. However, information regarding this secretion system in Mesorhizobium is still very scarce. The aim of the present study was to investigate the phylogeny and expression of the traG gene, which encodes a substrate receptor of the T4SS. In addition, the occurrence and genomic context of this and other T4SS genes, namely, genes from tra/trb and virB/virD4 complexes, were also analyzed in order to unveil the structural and functional organization of T4SS in mesorhizobia. The location of the T4SS genes in the symbiotic region of the analyzed rhizobial genomes, along with the traG phylogeny, suggests that T4SS genes could be horizontally transferred together with the symbiosis genes. Regarding the T4SS structural organization in Mesorhizobium, the virB/virD4 genes were absent in all chickpea (Cicer arietinum L.) microsymbionts and in the Lotus symbiont Mesorhizobium japonicum MAFF303099T. Interestingly, the presence of genes belonging to another secretion system (T3SS) was restricted to these strains lacking the virB/virD4 genes. The traG gene expression was detected in M. mediterraneum Ca36T and M. ciceri LMS-1 strains when exposed to chickpea root exudates and also in the early nodules formed by M. mediterraneum Ca36T, but not in older nodules. This study contributes to a better understanding of the importance of T4SS in mutualistic symbiotic bacteria.
Aims: The aim of this study was to investigate whether the overexpression of NifA and NodD regulators contribute to the symbiotic improvement of chickpea mesorhizobia. Methods: The native strains V-15b, ST-2 and PMI-6 were transformed with extra copies of nifA or nodD genes and several plants trial were performed. Results: Plant growth assays showed that nifA overexpression was able to improve the symbiotic effectiveness of V-15b, while nodD overexpression lead to the improvement of ST-2 and PMI-6. Hydroponic assays showed that plants inoculated with V15bnifA+ and PMI6nodD+ started developing nodules earlier than those inoculated with the corresponding control strains. In addition, the number of nodules was always higher in plants inoculated with the strains overexpressing the symbiotic genes. Analysis of histological sections of nodules formed by V15bnifA+ showed a more developed fixation zone when compared to control. On the other hand, nodules induced by PMI6nodD+ did not show a senescent zone, which was observed in nodules from plants inoculated with the control strain. Plants inoculated with PMI6nodD+ and ST2nodD+ showed a higher number of infection threads than the corresponding control inoculations. Conclusion: These results indicate that overexpressing nifA and nodD may be an important tool to achieve the improvement of the symbiotic performance of mesorhizobia.
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