The Defective in Autoregulation (DAR) gene of Medicago truncatula encodes a protein involved in regulating nodulation and arbuscular mycorrhiza

Abstract

Background: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood.

Results: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root.

Conclusions: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

Keywords: Medicago truncatula; AOM; AON; Autoregulation of mycorrhizal symbiosis; Autoregulation of nodulation; DAR; Hypernodulation.

Fig. 1

The dar-1 mutant has a hypernodulation phenotype. A Nodule counts at 10 dpi for dar-1 compared to wild type A17 and hypernodulating mutants sunn-1 and lss. dar1 and lss display higher nodule numbers than wildtype and the hypernodulating sunn-1 mutant (Kruskal–Wallis test, p = 6.613 × 10^–07; different letters denote significant differences (p < 0.05) in pairwise comparisons using Dunn’s post-hoc test; n = 10 plants per genotype.) B Nodules at 18 dpi in dar-1 (upper panel) and A17 (lower panel)

Fig. 2

dar-1 acts in the root to control nodule number. Nodule counts on grafted plants 21 days after inoculation with Sinorhizobium medicae ABS7. Grafted plants with dar-1 roots nodulated significantly more than wild type self-grafts, while plants with dar-1 only in the shoots were similar to wild type (ANOVA p = 3.28 × 10^–07, different letters denote significant differences (p < 0.05) in pairwise comparisons using Tukey’s HSD test; n = 7 to 12 plants per grafting combination)

Fig. 3

dar-1 is not suppressed by CLE peptide expression. Wild type R108 and dar-1 plants with roots carrying control (EV), p35S::CLE12, or p35S::CLE13 plasmids were inoculated with Sinorhizobium medicae and assessed for nodulation after 21 days (n = 7 to 9 plants per condition). Nodulation was suppressed by CLE12 or CLE13 expression in the wild type (Kruskall-Wallis test p < 0.05) but not in dar-1 (Kruskal–Wallis test p > 0.05). Different letters denote significant differences (p < 0.05) in pairwise comparisons for each genotype using Dunn’s post-hoc test

Fig. 4

Identification of a gene deletion in a region associated with the dar-1 phenotype. Analysis of progeny of a mapping cross between the dar-1 mutant and ecotype A20 revealed linkage of the dar-1 lesion to a 6 Mb region between nucleotide positions 22 and 28 Mb on chromosome 2 (in red). A deletion of between 8 and 17 kb was identified in dar-1 near nucleotide position 23.1 Mb which included a single gene, MtrunA17_Chr2g0304631

Fig. 5

Rescue of dar-1 and identification of dar-2 (A) Ectopic expression of MtrunA17_Chr2g0304631 in roots of the dar-1 mutant restores nodule number regulation. The dar-1 plants with the empty vector were significantly different from wild type, while dar-1 plants with the MtrunA17_Chr2g0304631 expression construct were similar to wild type (Kruskal–Wallis test p = 4.799 × 10^–06; different letters denote significant differences between samples (p < 0.05) in pairwise comparisons using Dunn’s post-hoc test). For each condition, 10 to 11 plants were analyzed. B A line derived from NF2117 (dar-2) carrying a Tnt1 insertion in MtrunA17_Chr2g0304631 showed an increased number of nodules compared to its parental wild type line, R108. (*, p < 5 × 10^–4, Student’s t-test; n = 4 to 23 plants)

Fig. 6

dar-1 mutants show elevated root length colonization with the AM fungus Rhizophagus irregularis relative to the A17 wildtype. Colonization levels in dar-1 and sunn-4 mutants were indistinguishable. For each line, 10 colonized root systems were analyzed. Statistical analysis was performed using Kruskal–Wallis test followed by Dunn’s test for pairwise comparisons. Different letters denote significant differences (p < 0.05)

Fig. 7

Expression of DAR1 in M. truncatula root tissues in response to inoculation with arbuscular mycorrhiza and rhizobia. A Normalized expression (Log2TMM) of DAR expression in mock- vs R. irregularis-inoculated roots in A17 or sunn-4 roots harvested 21 post days inoculation. Data from [17]. B Normalized expression (Log2TMM) of DAR expression in roots inoculated with Rhizophagus irregularis (Ri) and/or Sinorhizobium meliloti (Sm) individually or in combination; roots harvested 7 weeks post inoculation. Data from [43]. ANOVA was used to compare means, followed by Tukey's HSD post-hoc analysis. C Expression of DAR normalized by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) in roots over 72 h post inoculation with Sinorhizobium medicae (+ R) in A17, sunn-4, and rdn1-2. Data from [44]. D Schema for displaying tissue expression using default parameters at https://bar.utoronto.ca/eplant_medicago/. Data are compiled from three independent replicates and colors are painted on an entire tissue regardless of which cells within that tissue expressed the gene. E Relative expression of DAR in each tissue at indicated hours post inoculation

Fig. 8

DAR1 related proteins in M. truncatula, Arabidopsis, rice, and soybean. A Neighbor-Joining tree of DAR and related proteins from Medicago truncatula, Glycine max, Arabidopsis thaliana, and Oryza sativa. The bootstrap consensus tree was inferred from 500 replicates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test shown next to the branches. A family member in M. truncatula annotated as Medtr4g116290 in v4.1 was split into two transcripts in v5.0 (MtrunA17_Chr4g0065031 and MtrunA17_Chr4g0065021); the coding sequence predicted from the v4.1 transcript was used for this analysis

Fig. 9

Protein structure of DAR (A) The transmembrane topology of DAR. The predicted structure includes five transmembrane spanning domains. Cytoplasmic links between consecutive TM domains are short, while on the side of the membrane annotated as non-cytoplasmic by Phytozome there are regions of 78 to 185 amino acids. B Displayed is the AlphaFold predicted structure of the DAR protein and corresponding confidence metrics. The left panel displays the predicted 3D structure of the DAR protein with the respective confidence level for each region of the DAR. * indicates the start of the protein. Blue: Very high confidence (pLDDT > 90), Cyan: Confident (90 > pLDDT > 70), Yellow: Low confidence (70 > pLDDT > 50), Orange: Very low confidence (pLDDT < 50). The right panel shows the predicted alignment error (PAE) heatmap that represents the expected position error (in Ångströms) for each pair of residues in the predicted structure, where darker and lighter green indicate the lower and higher expected error rate respectively. The diagonal represents self-alignment (perfect prediction). The color scale at the bottom shows the range of expected position errors