Soil origin and plant genotype structure distinct microbiome compartments in the model legume Medicago truncatula

Abstract

Background: Understanding the genetic and environmental factors that structure plant microbiomes is necessary for leveraging these interactions to address critical needs in agriculture, conservation, and sustainability. Legumes, which form root nodule symbioses with nitrogen-fixing rhizobia, have served as model plants for understanding the genetics and evolution of beneficial plant-microbe interactions for decades, and thus have added value as models of plant-microbiome interactions. Here we use a common garden experiment with 16S rRNA gene amplicon and shotgun metagenomic sequencing to study the drivers of microbiome diversity and composition in three genotypes of the model legume Medicago truncatula grown in two native soil communities.

Results: Bacterial diversity decreased between external (rhizosphere) and internal plant compartments (root endosphere, nodule endosphere, and leaf endosphere). Community composition was shaped by strong compartment × soil origin and compartment × plant genotype interactions, driven by significant soil origin effects in the rhizosphere and significant plant genotype effects in the root endosphere. Nevertheless, all compartments were dominated by Ensifer, the genus of rhizobia that forms root nodule symbiosis with M. truncatula, and additional shotgun metagenomic sequencing suggests that the nodulating Ensifer were not genetically distinguishable from those elsewhere in the plant. We also identify a handful of OTUs that are common in nodule tissues, which are likely colonized from the root endosphere.

Conclusions: Our results demonstrate strong host filtering effects, with rhizospheres driven by soil origin and internal plant compartments driven by host genetics, and identify several key nodule-inhabiting taxa that coexist with rhizobia in the native range. Our results set the stage for future functional genetic experiments aimed at expanding our pairwise understanding of legume-rhizobium symbiosis toward a more mechanistic understanding of plant microbiomes. Video Abstract.

Keywords: Common garden; Evolution; Genetic variation; Mutualism; Nodule microbiome.

Fig. 1

Bacterial diversity metrics (a) and abundance (mean ± SE) of the most abundant bacterial OTUs (b) for the four compartments studied. All estimates are based on iterative subsampling (1500 sequences per iteration at 1000 iterations). Letters represent significant differences (Tukey HSD) across compartments

Fig. 2

Bacterial communities in the root endosphere respond to plant genotype. a Nonmetric multidimensional scaling plot (Bray-Curtis) of bacterial root endophytes plotted by genotype, with insert showing Axis 2 loading scores (explains 48.36% of community variation) across genotypes (ANOVA). b Average bacteria Bray-Curtis dissimilarity values between paired rhizosphere and root endosphere samples (samples are paired by plant)

Fig. 3

Paired effect size analysis of the 20 most abundant OTUs comparing relative abundances of each OTU within the same plant between root and nodule (a), rhizosphere and root (b), and root and leaf (c) compartments. Bacterial genera are on the left and OTU number presented parenthetically. Presented are quintiles (minimum, 25%, median, 75%, maximum) of paired effect size [e.g., nodule − root/(nodule + root)] where a value of 1.0 indicates this OTU is only found in the nodules where as a value of − 1.0 means the genus was only found in the Root. Tests for significant enrichment of OTUs between compartments using Wilcoxon signed-rank tests are presented where significant and indicated with tests statistics and p values

Fig. 4

Genomic content of Ensifer from root versus nodule endosphere compartments from shotgun metagenomic sequencing data, shown as the median proportion of reads mapping to the symbiotic plasmid (pSymB in black, pSymA in white) relative to the chromosome. Lower and upper bounds of each box depict the first and third quartiles, respectively, with whiskers representing the range of observed values