The microbiomes of rhizocompartments (nodule endophytes, root endophytes, rhizosphere and root zone) in soya bean and alfalfa were analysed using high-throughput sequencing to investigate the interactions among legume species, microorganisms and soil types. A clear hierarchical filtration of microbiota by plants was observed in the four rhizocompartments - the nodule endosphere, root endosphere, rhizosphere and root zone - as demonstrated by significant variations in the composition of the microbial community in the different compartments. The rhizosphere and root zone microbial communities were largely influenced by soil type, and the nodule and root endophytes were primarily determined by plant species. Diverse microbes inhabited the root nodule endosphere, and the corresponding dominant symbiotic rhizobia belonged to Ensifer for alfalfa and Ensifer-Bradyrhizobium for soya bean. The nonsymbiotic nodule endophytes were mainly Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The variation in root microbial communities was also affected by the plant growth stage. In summary, this study demonstrated that the enrichment process of nodule endophytes follows a hierarchical filtration and that the bacterial communities in nodule endophytes vary according to the plant species.

Rhizobia and legume plants are famous mutualistic symbiosis partners who provide nitrogen nutrition to the natural environment. Rhizobial type III secretion systems (T3SSs) deliver effectors that manipulate the metabolism of eukaryotic host cells. Mesorhizobium amorphae CCNWGS0123 (GS0123) contains two T3SS gene clusters, T3SS-I and T3SS-II. T3SS-I contains all the basal components for an integrated T3SS, and the expression of T3SS-I genes is up-regulated in the presence of flavonoids. In contrast, T3SS-II lacks the primary extracellular elements of T3SSs, and the expression of T3SS-II genes is down-regulated in the presence of flavonoids. Inoculation tests on Robinia pseudoacacia displayed considerable differences in gene expression patterns and levels among roots inoculated with GS0123 and T3SS-deficient mutant (GS0123ΔrhcN1 (GS0123ΔT1), GS0123ΔrhcN2 (GS0123ΔT2) and GS0123ΔrhcN1ΔrhcN2 (GS0123ΔS)). Compared with the GS0123-inoculated plants, GS0123ΔT1-inoculated roots formed very few infection threads and effective nodules, while GS0123ΔT2-inoculated roots formed a little fewer infection threads and effective nodules with increased numbers of bacteroids enclosed in one symbiosome. Moreover, almost no infection threads or effective nodules were observed in GS0123ΔS-inoculated roots. In addition to evaluations of plant immunity signals, we observed that the coexistence of T3SS-I and T3SS-II promoted infection by suppressing host defense response in the reactive oxygen species defense response pathway. Future studies should focus on identifying rhizobial T3SS effectors and their host target proteins.

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Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuS pSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuS pSym mutant strain. The balance of defence reaction was also impaired in ΔhmuS pSym strain-infected root nodules. hmuS pSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuS pSym protein could bind to heme but not degrade it. Inactivation of hmuS pSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuS pSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.