Dysfunction in the arbuscular mycorrhizal symbiosis has consistent but small effects on the establishment of the fungal microbiota in Lotus japonicus

Abstract

Most land plants establish mutualistic interactions with arbuscular mycorrhizal (AM) fungi. Intracellular accommodation of AM fungal symbionts remodels important host traits like root morphology and nutrient acquisition. How mycorrhizal colonization impacts plant microbiota is unclear. To understand the impact of AM symbiosis on fungal microbiota, ten Lotus japonicus mutants impaired at different stages of AM formation were grown in non-sterile natural soil and their root-associated fungal communities were studied. Plant mutants lacking the capacity to form mature arbuscules (arb^- ) exhibited limited growth performance associated with altered phosphorus (P) acquisition and reduction-oxidation (redox) processes. Furthermore, arb^- plants assembled moderately but consistently different root-associated fungal microbiota, characterized by the depletion of Glomeromycota and the concomitant enrichment of Ascomycota, including Dactylonectria torresensis. Single and co-inoculation experiments showed a strong reduction of root colonization by D. torresensis in the presence of AM fungus Rhizophagus irregularis, particularly in arbuscule-forming plants. Our results suggest that impairment of central symbiotic functions in AM host plants leads to specific changes in root microbiomes and in tripartite interactions between the host plant, AM and non-AM fungi. This lays the foundation for mechanistic studies on microbe-microbe and microbe-host interactions in AM symbiosis of the model L. japonicus.

Keywords: RNA-seq; arbuscular mycorrhizal (AM) fungi; fungal community; legume; natural soil; symbiosis.

Figure 1

Fungal colonization and LjPT4 expression in the roots of Lotus japonicus lines growing in an agricultural (NPK) soil for 6 wk. (a) Percentage of plant roots with observable fungal colonization (Hyphae) or with well‐developed arbuscules (Arbuscules). ‘Colonization’ indicates the sum of ‘Hyphae’ plus ‘Arbuscules’ representing the overall percentage of plant roots colonized by fungal structures. No arbuscules or aberrant arbuscules were observed in arb⁻ lines. Error bars represent + SD (n = 5). (b) and (c) Root expression of mycorrhiza‐induced phosphate transporter gene LjPT4 and fatty acid biosynthesis gene LjBCCP2, respectively. In both panels different letters designate significant differences between the plant lines (ANOVA followed by Tukey's HSD test, P < 0.05, n = 4 or 5). Both analyses were conducted in three independent experiments with similar results. Results from Expt 1 are shown.

Figure 2

Transcriptome profiles of arbuscular mycorrhizal (AM) symbiosis defective mutants grown in NPK soil. (a) Principal components analysis of the normalized RNA‐seq counts of wild type Gifu B‐129 (WT), ram1‐2 and str‐2. RNA from root samples collected from Expt 1 was used, n = 3. (b) Venn diagram with the overlap among differentially expressed genes (DEG) from str‐2 or ram1‐2 compared with WT and reference gene sets for AM symbiosis (Rhizophagus irregularis) and P starvation response (PSR) (log₂ fold‐change ≥ 2 or ≤ −2, adjusted P ≤ 0.05). (c) Heatmap of the intersect of 369 AM symbiosis‐regulated DEG. Average of log₁₀ (cpm + 1) was used. (d) Heatmap of the intersect of 87 PSR‐regulated DEG. (e) Heatmap of −log₁₀ P‐values of GO terms enriched in the remaining 996 common DEG.

Figure 3

Roots of Lotus japonicus mycorrhizal mutants establish altered root fungal communities. (a) Relative abundance of fungal orders in bulk soil (Bs), rhizosphere and roots of L. japonicus. Plant lines are color‐coded with green shades for arbuscule‐forming lines (arb⁺) and blue/purple shades for lines without arbuscules (arb⁻). The three independent experiments (Experiment) are symbol‐coded. Arrowheads indicate fungal orders depleted (green) or enriched (purple) in the rhizosphere and roots of arb⁻ lines (t‐test, P < 0.01). (b) Principal coordinates analysis on Bray‐Curtis distances. Samples are color‐ and symbol‐coded according to the legend in (a). (c) P‐values and percentage of variance explained by mycorrhizal status from pairwise comparisons of root fungal communities between plant lines. Bold numbers indicate significant differences between lines (PerMANOVA, P < 0.05), the grey scale indicates P‐values < 0.05, asterisks indicate P‐values still significant at 0.05 after FDR correction. Results from three independent experiments are shown in (a) and (b), data from Expt 3 was excluded for the analysis in (c) because of incomplete representation of all plant lines.

Figure 4

Roots of Lotus japonicus mycorrhizal mutants are depleted in Glomeromycota and enriched for several taxa from the Ascomycota. Heatmap showing fungal operational taxonomic units (OTUs) enriched (cluster C1) or depleted (cluster C2) in the roots of arb⁻ plant lines. OTUs were identified by differential abundance analysis (in DESeq2; P < 0.05), ‘P‐value’ indicates results from subsequent pairwise tests comparing OTUs relative abundances in arb⁺ and arb⁻ lines (Wilcoxon's test). Arbuscules or nodule‐forming lines are indicated with black squares.

Figure 5

Root colonization by the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis inhibits colonization by a Dactylonectria torresensis isolate. (a) Relative abundance of D. torresensis OTU00003 in the root endosphere of arb⁺ and arb⁻ Lotus japonicus plant lines across three independent experiments. The values are inferred from fungal ITS2 sequencing data. The P‐value indicates the higher relative abundance in arb⁻ roots (Wilcoxon's test). In the box‐plots, horizontal lines represent the first, second (median) and third quartiles while whiskers depict the dispersion of the data (1.5 × interquartile range). (b) CLS microscopy images of wild type (WT) roots colonized by D. torresensis. Plant cells were stained with propidium iodide and fungal structures with WGA‐Alexa 488. Bars: 50 μm (upper panels); 20 μm (lower panels). (c) qPCR quantification of root colonization by D. torresensis isolate 107 and R. irregularis in single and co‐inoculation in a simplified sand/soil system. Data was analyzed by the 2^−ΔΔCt method using L. japonicus Ubiquitin as reference gene and ‘Single inoculation GifuB‐129’ as reference treatment. Different letters indicate significant differences between treatments and experiments (Kruskal‐Wallis followed by Dunn's test, P < 0.05) and asterisks indicate significant differences between plant lines in the co‐inoculation experiment (Wilcoxon test: **, P < 0.01; ***, P < 0.001). Three independent experiments including four independent biological replicates were conducted (n = 12) with similar results (results from independent experiments are shown in Supporting information Fig. S7c,d). In the box‐plots, points represent biological replicates, horizontal lines represent the first, second (median) and third quartiles while whiskers depict the dispersion of the data (1.5 × interquartile range).