The Full-Size ABCG Transporter of Medicago truncatula Is Involved in Strigolactone Secretion, Affecting Arbuscular Mycorrhiza

Abstract

Strigolactones (SLs) are plant-derived signaling molecules that stimulate the hyphal branching of arbuscular mycorrhizal fungi (AMF), and consequently promote symbiotic interaction between the fungus and the plant. Currently, our knowledge on the molecular mechanism of SL transport is restricted to the Solanaceae family. In the Solanaceae family, SL translocation toward the rhizosphere occurs through the exodermis via hypodermal passage cells and involves a member of the G subfamily, of the ATP-binding cassette (ABC) membrane transporters. Most Fabaceae species, including those that are agriculturally important, have a different root anatomy compared to most angiosperm plants (i.e., lacking an exodermis). Thus, we have investigated how SL transport occurs in the model legume Medicago truncatula. Here, we show that overexpression of a SL transporter from petunia (PaPDR1) enhances AMF colonization rates in M. truncatula. This result demonstrates the importance of ABCG proteins for the translocation of orobanchol-type molecules to facilitate arbuscular mycorrhiza, regardless of root anatomy and phylogenetic relationships. Moreover, our research has led to the identification of Medicago ABCG59, a close homologue of Petunia PDR1, that exhibits root specific expression and is up-regulated by phosphate starvation as well as in the presence of rac-GR24, a synthetic SL. Its promoter is active in cortical cells, root tips, and the meristematic zone of nodules. The mtabcg59 loss-of-function mutant displayed a reduced level of mycorrhization compared to the WT plants but had no impact on the number of nodules after Sinorhizobium meliloti inoculation. The reduced mycorrhization indicates that less SLs are secreted by the mutant plants, which is in line with the observation that mtabcg59 exudates exhibit a reduced stimulatory effect on the germination of the parasitic plant Phelipanche ramosa compared to the corresponding wild type.

Keywords: ABC transporters; Medicago truncatula; arbuscular mycorrhiza; exodermis; strigolactones; symbioses.

Figure 1

Root anatomical structure. (A) Scheme of Nicotiana tabacum (Solanaceae) and Medicago truncatula (Fabaceae) root layers. (B) Deposition of suberin lamellae in roots of 6-week-old N. tabacum and M. truncatula (representative images from 10 roots). The cross-sections of the roots comes from the regions located 3 cm above the root tip. Fluoral yellow 088 fluorescence (upper panel), fluorescence images overlayed with corresponding bright-field images (lower panel).

Figure 2

Phylogenetic analysis of full-size ABCG (Pleiotropic drug resistance - PDR) proteins in various plants. Neighbor-joining phylogenetic tree (bootstraps: 1000) was conducted using MEGA X software (Kumar et al., 2018) based on the amino acid sequences generated after multiple sequence alignments with MUSCLE. The highlighted PaPDR1 and NtPDR6, as well as their homologues from Medicago truncatula belong to Cluster I (Crouzet et al., 2013).

Figure 3

Expression profiles of Medicago truncatula ABCG59, ABCG43, and ABCG44. (A, B) Quantitative Real-Time PCR expression analysis of MtABCG59, MtABCG43, and MtABCG44 in roots under phosphate-limiting conditions (A) or treated with the synthetic strigolactone analogue rac-GR24 (B). The transcript levels were normalized to the actin gene from Medicago truncatula. Data represent the mean ± SD of three and four independent biological experiments, respectively, and three technical repeats. Significant differences from the control plants determined by Student's t-test are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Expression pattern and subcellular localization of MtABCG59. (A) GUS staining of 2-week-old transgenic Medicago truncatula expressing ProMtABCG59:GUS and quantitative PCR expression analysis of MtABCG59 in different M. truncatula organs (three-month-old plants) revealed the MtABCG59 transcript accumulation exclusively in the roots and nodules. The transcript levels were normalized to the actin gene from M. truncatula. Data represent the mean ± SD of three independent biological experiments and three technical repeats. (B) X-Gluc staining of ProMtABCG59:GUS reporter line in root cortex (upper panel) and in root meristem (middle panel), X-Gluc staining of root cross-sections (bottom panel). (C) Fluorescence signal of ProMtABCG59:NLS-GFP reporter line in root cortex (upper panel) and in root meristem (bottom panel). (D) Arabidopsis mesophyll protoplast expressing the fusion gene Pro35S:GFP-MtABCG59. The GFP signal was observed in the plasma membrane (left panel). Control Arabidopsis protoplast expressing free cytoplasmic GFP (right panel). The red color represents chlorophyll autofluorescence.

Figure 5

Phenotypic characterization of the mtabcg59 mutant in mycorrhization contexts. (A) Mycorrhizal colonization of Medicago truncatula control (WT) and mtabcg59-1 mutant roots 3 and 5 weeks after inoculation with Rhizophagus irregularis. The percentage of the root length colonized by the mycorrhizal fungi is shown. Data represent the means ± SE of five independent biological experiments (i.e. 5 pools of 3 plants each). Significant differences from the control plants were determined by Student's t-test and are indicated by: *P < 0.05. (B) Arbuscules formed in the WT and mtabcg59-1 mutant were morphologically similar. Ink-staining of fungal structures (left panel), WGA-AlexaFluor 488 staining of arbuscules (right panel), scale bar, 10 µm. (C) Transcript accumulation of AM-related marker genes MtBCP1 (left panel) and MtPT4 (right panel) in mtabcg59 mutants and control (WT) roots measured by quantitative real-time PCR. The data represents the mean ± SD of four independent biological experiments and two technical repeats. Significant differences from the control plants were determined by Student's t-test and are indicated by: * P < 0.05. (D) Relative expression of MtABCG43 and MtABCG44 in WT and mtabcg59 mutant plants. Values represent the mean of three biological replicates ± SD. Significant differences in genes expression between WT and mtabcg59 mutants were determined by an ANOVA test and Tukey's multiple comparison test, and are as follows: ns, not significant; *P < 0.05, **P < 0.01.

Figure 6

Germination rates of Phelipanche ramosa seeds exposed to WT and mtabcg59-1 exudates. Root attached (seeds placed directly in root surface); off root (seeds placed 1 to 3 mm aside). The data represent the mean ± SE of six to seven independent biological experiments (approx. 150 seeds screened for each biological replicate). Significant differences from the control plants were determined by Student's t-test and are indicated by: ns, not significant; **P < 0.005.

Figure 7

MtABCG59 affects the SL-biosynthetic pathway. Relative expression of SL-biosynthesis gene (MtCCD7 and MtCCD8) in WT and mtabcg59 mutant plants. Values represent the mean of three biological replicates ± SD. Significant differences in genes expression between WT and mtabcg59 mutants were determined by an ANOVA test and Tukey's multiple comparison test, and are as follows: ns, not significant; **P < 0.01, ***P < 0.001.

Figure 8

Phenotypic characterization of mtabcg59 in nodulation context. (A) Expression of ProMtABCG59:GUS in nodules. (B) Average nodule number per plant in WT and mtabcg59 plants. Six-day-old seedlings were inoculated with Sinorhizobium meliloti and grown on modified Fahraeus (-N) medium. At 14- and 21-days post-inoculation (dpi), the nodules were quantified. The data represent the mean ± SD of (N = 5, n = 9), per line. (C) WT and mtabcg59 nodules morphology.

Figure 9

Comparison of strigolactone (SL) exudation into the soil and SL-dependent guidance of arbuscular mycorrhizal fungi to the host root between Petunia axillaris and Medicago truncatula. In P. axillaris, SLs are secreted into the rhizosphere by PaPDR1 that is localized in the hypodermal passage cells (HPCs) within the exodermis. A steep concentration gradient of SLs, created by PaPDR1, guides arbuscular mycorrhizal fungi (AMF) to access penetration sites – i.e. unsuberized HPCs. In the M. truncatula, due to a lack of exodermis, SLs could passively enter the rhizosphere because they do not encounter the apoplastic diffusion barrier. However, we suggest that to achieve the full extent of AMF colonization, the active export of SLs, mediated by MtABCG59, is required. Its action ensures sufficiently high concentrations of SLs around the roots to attract AM fungi, which together with the characteristic root anatomical traits in M. truncatula, enables the colonization of the whole root surface.