Lotus japonicus Symbiosis Genes Impact Microbial Interactions between Symbionts and Multikingdom Commensal Communities

Abstract

The wild legume Lotus japonicus engages in mutualistic symbiotic relationships with arbuscular mycorrhiza (AM) fungi and nitrogen-fixing rhizobia. Using plants grown in natural soil and community profiling of bacterial 16S rRNA genes and fungal internal transcribed spacers (ITSs), we examined the role of the Lotus symbiosis genes RAM1, NFR5, SYMRK, and CCaMK in structuring bacterial and fungal root-associated communities. We found host genotype-dependent community shifts in the root and rhizosphere compartments that were mainly confined to bacteria in nfr5 or fungi in ram1 mutants, while symrk and ccamk plants displayed major changes across both microbial kingdoms. We observed in all AM mutant roots an almost complete depletion of a large number of Glomeromycota taxa that was accompanied by a concomitant enrichment of Helotiales and Nectriaceae fungi, suggesting compensatory niche replacement within the fungal community. A subset of Glomeromycota whose colonization is strictly dependent on the common symbiosis pathway was retained in ram1 mutants, indicating that RAM1 is dispensable for intraradical colonization by some Glomeromycota fungi. However, intraradical colonization by bacteria belonging to the Burkholderiaceae and Anaeroplasmataceae is dependent on AM root infection, revealing a microbial interkingdom interaction. Despite the overall robustness of the bacterial root microbiota against major changes in the composition of root-associated fungal assemblages, bacterial and fungal cooccurrence network analysis demonstrates that simultaneous disruption of AM and rhizobium symbiosis increases the connectivity among taxa of the bacterial root microbiota. Our findings imply a broad role for Lotus symbiosis genes in structuring the root microbiota and identify unexpected microbial interkingdom interactions between root symbionts and commensal communities.IMPORTANCE Studies on symbiosis genes in plants typically focus on binary interactions between roots and soilborne nitrogen-fixing rhizobia or mycorrhizal fungi in laboratory environments. We utilized wild type and symbiosis mutants of a model legume, grown in natural soil, in which bacterial, fungal, or both symbioses are impaired to examine potential interactions between the symbionts and commensal microorganisms of the root microbiota when grown in natural soil. This revealed microbial interkingdom interactions between the root symbionts and fungal as well as bacterial commensal communities. Nevertheless, the bacterial root microbiota remains largely robust when fungal symbiosis is impaired. Our work implies a broad role for host symbiosis genes in structuring the root microbiota of legumes.

Keywords: microbiome; plant-microbe interactions; symbiosis.

FIG 1

Bacterial and fungal community profiles for different root fractions of L. japonicus. (A) Cartoon showing the length of the three different root fractions. (B) Community profile showing the relative abundances of bacterial (top) and fungal (bottom) families across compartments and fractions (only samples with >5,000 reads [bacteria] or >1,000 reads [fungi] are shown, and taxa having an average RA of <0.1 [bacteria] or <0.15 [fungi] across all samples are aggregated as low-abundance taxa). (C) Ternary plots showing bacterial OTUs that are enriched in the endosphere of specific root fractions, compared to the soil samples. (D) Ternary plots showing fungal OTUs that are enriched in the endosphere of specific root fractions, compared to the soil samples. The circle size corresponds to the RA across all fractions. Dark-gray circles denote OTUs that are enriched in soil, and light-gray circles always represent OTUs that are not enriched in any of the fractions.

FIG 2

Phenotypes of WT and mutant plants. (A to E) Images depicting L. japonicus wild type (A) and ram1 AMS-deficient (B), nfr5 RNS-deficient (C), symrk AMS- and RNS-deficient (D), and ccamk AMS- and RNS-deficient (E) mutant plants. Insets show closeup views of nodules. Bars, 1 cm. (F) Box plots displaying the shoot length for the same set of genotypes as the one presented panels A to E. (G) Box plots displaying the shoot fresh mass. Letters above plots correspond to groups based on Tukey’s HSD test (P < 0.05). Numbers of samples are indicated in parentheses.

FIG 3

Constrained PCoA showing the effect of genotype on microbial communities. (A and B) Constrained PCoA plots for bacterial data sets showing rhizosphere samples (n = 100) (A) and root samples (n = 100) (B). (C and D) Constrained PCoA plots for fungal data sets showing only rhizosphere samples (n = 124) (C) and root samples (n = 122) (D) from ram1 AMS-deficient, nfr5 RNS-deficient, symrk AMS- and RNS-deficient, and ccamk AMS- and RNS-deficient plants.

FIG 4

Relative abundances of the main microbial taxa across plant compartments and genotypes. (A) RAs for bacterial families in rhizosphere (left) and root (right) compartments. (B) RAs for fungal families in rhizosphere (left) and root (right) compartments. Taxa are sorted in decreasing order according to their average RA in WT plants (only the first 10 most abundant taxonomic groups are shown). RAs in the WT as well as the respective mutants are displayed. Significant differences compared to the WT are marked with an asterisk in the color of the mutant (P < 0.05 by a Kruskal-Wallis test). Families that include known symbionts are marked in red (Phyllobacteriaceae for bacteria and Glomeromycetes for fungi). For some fungal taxa, the next-highest rank is shown when no family-level information was available. Data for ram1 AMS-deficient, nfr5 RNS-deficient, symrk AMS- and RNS-deficient, and ccamk AMS- and RNS-deficient plants are shown.

FIG 5

Differential abundance analysis of root-associated OTUs. (A) Dendrogram of bacterial OTUs that are differentially abundant in the roots of mutants compared to WT roots. (B) Dendrogram of fungal OTUs that are differentially abundant in the roots of mutants compared to WT roots. Only OTUs that have an average RA of >0.1% across all root samples, including mutants, are considered here. The dendrogram is based on hierarchical clustering. For each OTU, the fold change in RA from the WT to mutants is indicated (P < 0.05 by a Kruskal-Wallis test). Next to each OTU, the RA in WT roots is indicated. Phylum and family associations (if available) are given for each OTU. Abbreviations of bacterial phyla: Del, Deltaproteobacteria; Gem, Gemm-1; Chl, Chloroflexi; Bet, Betaproteobacteria; Alp, Alphaproteobacteria; Gam, Gammaproteobacteria; Cyt, Cytophagia; Sap, Saprospiria; Ped, Pedosphaera; Sph, Sphingobacteria; Mol, Mollicutes. Abbreviations of fungal phyla: Sor, Sordariomycetes; Dot, Dothideomycetes; Mic, Microbotryomycetes; Ust, Ustilaginomycetes; Eur, Eurotiomycetes; Leo, Leotiomycetes; Aga, Agaricomycetes; Glo, Glomeromycetes; Pez, Pezizomycotina; Muc, Mucoromycotina. Data for ram1 AMS-deficient, nfr5 RNS-deficient, symrk AMS- and RNS-deficient, and ccamk AMS- and RNS-deficient plants are shown.