Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

Abstract

Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance.

Keywords: 16S; Lotus japonicus; microbiota; nitrogen fixation; symbiosis.

Fig. 1.

Images depicting L. japonicus WT (A) and nodule symbiosis-deficient mutant plants lhk1-1 (B), nfr5-3 (C), and nin-2 (D) following harvest. (A and B, Insets) For nodulating genotypes, close-up views of nodules are shown. (Scale bars: 1 cm.)

Fig. 2.

(A) Constrained PCoA plot of Bray–Curtis distances between samples including only the WT constrained by compartment (19.97% of variance, P > 0.001; n = 94). (B) Constrained PCoA plot of Bray–Curtis distances constrained by genotype (9.82% of variance explained, P < 0.001; n = 164). Each point corresponds to a different sample colored by compartment, and each host genotype is represented by a different shape. The percentage of variation indicated in each axis corresponds to the fraction of the total variance explained by the projection. Corresponding unconstrained PCoA plots for each soil batch are shown in SI Appendix, Fig. S3.

Fig. 3.

(A) Ternary plot depicting compartment RAs of all OTUs (>5 ‰) for WT root, rhizosphere, and nodule samples (n = 67) across three soil batches (CAS8–CAS10). CAS, Cologne agricultural soil. Each point corresponds to an OTU. Its position represents its RA with respect to each compartment, and its size represents the average across all three compartments. Colored circles represent OTUs enriched in one compartment compared with the others (green in root, orange in rhizosphere, and red in nodule samples), whereas gray circles represent OTUs that are not significantly enriched in a specific compartment. (B) Rank abundance plot depicting RAs aggregated to the order taxonomic level for the top abundant taxa found in the WT nodule samples (n = 21). (C) Comparison of abundances between Mesorhizobium and other Rhizobiales genera in WT roots (n = 48), mutant roots (n = 100), and WT nodule samples (n = 21).

Fig. 4.

Ternary plots depicting compartment RA of all OTUs (>5 ‰) for WT samples (A; WT; n = 73) and mutant samples (B; nfr5-2, nfr5-3, nin-2, and lhk1-1; n = 118) across three soil batches (CAS8–CAS10). Each point corresponds to an OTU. Its position represents its RA with respect to each compartment, and its size represents the average across all three compartments. Colored circles represent OTUs enriched in one compartment compared with the others (green in root, orange in rhizosphere, and brown in root samples). Aggregated RAs of each group of enriched OTUs (root-, rhizosphere- and soil-enriched OTUs) in each compartment for the WT samples (C; WT; n = 73) and mutant samples (D; nfr5-2, nfr5-3, nin-2, lhk1-1; n = 118) are shown. In each compartment, the difference from 100% RA is explained by OTUs that are not significantly enriched in a specific compartment.

Fig. 5.

Manhattan plots showing root-enriched OTUs in WT (A) or in the mutants (B) with respect to rhizosphere and rhizosphere-enriched OTUs in WT (C) or in the mutants (D) with respect to root. OTUs that are significantly enriched (also with respect to soil) are depicted as full circles. The dashed line corresponds to the false discovery rate-corrected P value threshold of significance (α = 0.05). The color of each dot represents the different taxonomic affiliation of the OTUs (order level), and the size corresponds to their RAs in the respective samples [WT root samples (A), mutant root samples (B), WT rhizosphere samples (C), and mutant rhizosphere samples (D)]. Gray boxes are used to denote the different taxonomic groups (order level).

Fig. 6.

(A) PCoA plot of Bray–Curtis distances for root and soil samples showing a clear separation between the roots of all Lotus genotypes (circles) compared with the roots of Arabidopsis and relative species (hollow shapes) grown in Cologne soil and sequenced using the same primer set. RAs aggregated to the phylum (B) and order taxonomic levels (C, 10 most abundant orders) showing a comparison between A. thaliana (n = 26) and L. japonicus root samples (n = 74).