Rhizophagus irregularis (Syn. Glomus intraradices, DAOM 197198; Krüger et al., 2012) inoculum for seedlings and root organ cultures (ROCs) mycorrhization, was produced through in vitro monoxenic cultures. These were established in bi-compartmental Petri dishes with a watertight plastic wall separating the root compartment (RC) from the hyphal compartment (HC) (Fortin et al., 2002). The RC was filled with 25 ml of solid M minimal medium and the HC with 25 ml of solid M medium lacking sugar (M-C). Cultures were started by placing an explant of Agrobacterium rhizogenes transformed-chicory (Cichorium intybus) roots colonized with the AM fungus in the RC. Once the mycelium of R. irregularis had grown over the plastic wall and completely filled the HC compartment, the medium was dissolved with sterile 10 mM citrate buffer, pH 6.0. Spores were then collected and used for plant inoculation.

To obtain the ERM, when the fungus profusely colonized the HC, its content was removed, and the HC was filled with 15 ml liquid M-C medium containing either 3.2 mM (100% N) or 0.8 mM (25% N) KNO⁻₃. The mycelium was allowed to colonize this medium over the subsequent 2 weeks. Petri dishes were examined regularly and roots were trimmed as required to prevent crossing into the HC.

For the dipeptide treatments, ERM was grown for 2 weeks in liquid M medium (100% N as KNO⁻₃). At this point, the medium of the HC was removed and replaced by fresh liquid M-C medium containing as nitrogen sources 3.2 mM nitrate, 3.2 mM nitrate and 10 mM Ala-Leu, 10 mM Ala-Leu or no N. ERM was harvested after 24 h.

In both experiments, ERM was collected with tweezers, rinsed with sterilized water, dried with sterilized filter paper, immediately frozen in liquid N and stored at −80°C until used.

To obtain seedlings colonized by R. irregularis, the Millipore sandwich method (Giovannetti et al., 1993) was used. Seeds of Medicago truncatula Gaertn cv Jemalong were first scarified using sandpaper P180–200, sterilized with 5% commercial bleach for 3 min and rinsed three times for 10 min with sterile distilled water. Germination was induced under sterile conditions in 0.6% agar/water, incubated for 5 days in the dark (25°C) and then exposed at the light for 4 days. Plants were watered with a modified Long-Ashton (LA) solution containing 3.2 μM Na₂HPO₄·12H₂O and 0.5 mM NaNO₃ nitrate as P and N sources, respectively (Hewitt, 1966) and were grown in a growth chamber under 14 h light (24°C)/10 h dark (20°C) regime. Plants were harvested 60 days post-inoculation (dpi).

For the dipeptide treatment, in a first experiment, M. truncatula mycorrhizal roots were obtained in pot cultures watered with a LA solution containing 1 mM nitrate. Two months after inoculation, mycorrhizal roots were treated for 24 h in hydroponic conditions with a LA solution containing 10 mM Ala-Leu or 1 mM nitrate or no N. In a second experiment M. truncatula mycorrhizal roots were grown in the sandwich system as described above for 60 days with a modified LA solution containing 2.5 mM Ala-Leu and 0.25 mM nitrate as N sources.

For mycorrhizal plants, only portions of the root system showing extraradical fungal structures were collected under a stereomicroscope. The colonization level was assessed according to Trouvelot et al. (1986). For the molecular analyses, roots were immediately frozen in liquid nitrogen and stored at −80°C.

Fungal protein sequences homologous to PTR2 transporters were identified by BLASTp searches within the Mycocosm database (Grigoriev et al., 2014). Prediction of trans-membrane domains was performed using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/), SOSUI (http://harrier.nagahama-i-bio.ac.jp/sosui/), HMMTOP (http://www.enzim.hu/hmmtop/) and TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) programs.

Amino acid sequences were aligned using MUSCLE (Edgar, 2004) and their phylogenetic links inferred by using the Maximum Likelihood method based on the JTT matrix-based model (Jones et al., 1992) as implemented in MEGA5 (Tamura et al., 2011).

The coding region of RiPTR2 was amplified from R. irregularis cDNAs by PCR using Phusion DNA-Polymerase (Finnzymes, Espoo, Finland) and the two following oligonucleotides containing a NotI restriction site: forward primer, 5′-TGACATTGCGGCCGCATAATGGAAGGACACATTCAA-3′; reverse primer, 5′-ACTTCGAGCGGCCGCTGTGACTATTCTTCGGATTTA-3′. The PCR product was cloned into NotI sites downstream of the S. cerevisiae constitutive PGK1 promoter in the pFL61 E. coli-yeast shuttle vector (Minet et al., 1992). Recombinant (pRiPTR2) and empty vector (EV) were used to transform W303 (MATa ura3 can1–100) yeast mutants deleted of only one or of its two known dipeptide transporter genes, PTR2 and DAL5 (Homann et al., 2005).

Yeast transformation was performed using standard protocols (Rose et al., 1990) and yeast transformants propagating either the EV or the pRiPTR2 recombinant vector were initially selected on a yeast nitrogen base minimal medium lacking uracil. Single colonies of transformants were grown over night in 5 ml of NH⁺₄-containing yeast carbon base (YCB) liquid medium. Once the OD_{600 nm} reached 0.9, serial dilutions (1:10–1:100–1:1000–1:10,000) in sterile water were prepared and 5 ml plated on YCB solid medium containing H₂0 (negative control), NH⁺₄(positive control), Ala-Tyr, Tyr-Ala or Ala-Leu (0.25 mM each). Plates were incubated at 30°C for 4 days and photographed.

Total genomic DNA was extracted from R. irregularis extraradical structures and M. truncatula and C. intybus roots using the DNeasy Plant Mini Kit (Qiagen), according to the manufacturer's instructions.

Total RNA was isolated from about 100 mg of seedling roots and 20 mg ERM using the RNeasy Plant Mini Kit (Qiagen). Samples were treated with TURBO™ DNase (Ambion) according to the manufacturer's instructions. The RNA samples were routinely checked for DNA contamination by RT-PCR analysis, using primers MtTefF 5′-AAGCTAGGAGGTATTGACAAG-3′ and MtTefR 5′-ACTGTGCAGTAGTACTTGGTG-3′ for MtTEF or RiTef-f 5′-GCTATTTTGATCATTGCCGCC-3′ and RiTef-r 5′-TCATTAAAACGTTCTTCCGACC-3′ for RiTEF (Gonzàlez-Guerrero et al., 2010) and the One-Step RT-PCR kit (Qiagen). The MtPT4 phosphate transporter gene was amplified with MtPT4F (5′-TCGCGCGCCATGTTTGTTGT-3′) and MtPT4R (5′-CGCAAGAAGAATGTTAGCCC-3′) primers (Zocco et al., 2011) and the RiPTR2 peptide transporter with RiPTR2F (5′-GGCTATATTCTTAACGATGTCG-3′) and RiPTR2R (5′-CGACCTGTTCTTCTTCCTCTT-3′) primers. Conventional PCR assays on plant genomic DNA excluded any cross-hybridization of RiPTR2 specific primers.

For single-strand cDNA synthesis about 500 ng of total RNA were denatured at 65°C for 5 min and then reverse-transcribed at 25°C for 10 min, 42°C for 50 min and 70° for 15 min in a final volume of 20 μl containing 10 μM random hexamers, 0.5 mM dNTPs, 4 μl 5× buffer, 2 μl 0.1 M DTT, and 1 μl Super-ScriptII (Invitrogen).

qRT-PCR experiments were carried out in a final volume of 20 μl containing 10 μl of iTaq™ Universal SYBR® Green Supermix (Bio-Rad), 1 μl of 3 μM specific primers, and about 20 ng of cDNA. Samples were run in the iCycler iQ apparatus (Bio-Rad) using the following program: 10 min pre-incubation at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. Each amplification was followed by melting curve analysis (60–94°C) with a heating rate of 0.5°C every 15 s. All reactions were performed with three technical replicates and only Ct values with a standard deviation that did not exceed 0.3 were considered. The comparative threshold cycle method (Rasmussen, 2001) was used to calculate relative expression levels using the plant MtTEF or the fungal RiTEF as reference genes for plant and fungal genes, respectively. The analyses were performed on three independent biological replicates. Statistical tests were carried out through one-way analysis of variance (One-Way ANOVA) and Tukey's post-hoc test, using a probability level of p < 0.05. All statistical analyses were performed using the PAST statistical package (version 2.16; Hammer et al., 2001).

M. truncatula roots colonized by R. irregularis obtained with the millipore sandwich system, as described above, were cut into 5–10 mm-long pieces, treated with ethanol and glacial acetic acid (3:1) under vacuum for 30 min and then placed at 4°C overnight. Roots were subsequently dehydrated in a graded series of ethanol (50%–70%–90% in sterilized water and 100% twice) followed by Neoclear (twice) with each step on ice for 30 min. Neoclear was gradually replaced with paraffin (Paraplast Plus; Sigma-Aldrich, St. Louis) according to Pérez-Tienda et al. (2011). A Leica AS LMD system (Leica Microsystem, Inc.) was used to collect colonized cortical cells from paraffin root sections as described by Balestrini et al. (2007).

RNA was extracted from dissected cells using the PicoPure kit protocol (Arcturus Engineering). A DNAse treatment was performed using an RNA-free DNase Set (Qiagen) in a Pico Pure column, according to the manufacturer's instructions and RNAs were eluted in 21 μl of sterile water.

All RT-PCR assays were carried out using the One Step RT-PCR kit (Qiagen). DNA contaminations were assessed using the MtTEF primers described above. Reactions with RiPTR2 or MtPT4 specific primers were carried out in a final volume of 10 μl containing 2 μl of 5× buffer, 0.4 μl of 10 mM dNTPs, 1 μl of each primer 10 mM, 0.2 μl of One Step RT-PCR enzyme mix, and 1 μl of a total RNA diluted 1:1. The samples were incubated for 30 min at 50°C, followed by 15 min incubation at 95°C. Amplification reactions were run for 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s. RT-PCR experiments were conducted on two different biological replicates of 1500–2000 microdissected cells each.

To verify whether RiPTR2 encodes a functional peptide transporter, it was expressed in S. cerevisiae W303 mutant strains deleted of either one or both of its two endogenous di/tripeptide transporter genes, PTR2 and DAL5. Both single (ptr2Δ or dal5Δ or double (ptr2Δ dal5Δ) mutant strains are unable to use many dipeptides as N sources (Homann et al., 2005).

RiPTR2-expressing yeast transformants were plated onto selective media, containing one of three different dipeptides as N sources; Ala-Tyr, Tyr-Ala or Ala-Leu. Tyr-Ala is known to be a substrate for the yeast Ptr2p but not for Dal5p, while Ala-Leu is used by Dal5p but not by Ptr2p, and Ala-Tyr is neither used by Ptr2p nor by Dal5p in the W303 background (Homann et al., 2005). In addition, H₂0 and NH⁺₄ were used as negative and positive control, respectively.

All transformants (empty or recombinant vector) were unable to grow in N-free medium (H₂O) although a background growth could be observed probably due to residual traces of N compounds present in the agar. As expected, all transformants were able to grow in the NH⁺₄-containing medium, Interestingly, ptr2Δ dal5Δ cells expressing RiPTR2 grew on Ala-Leu, Ala-Tyr and also Tyr-Ala, whereas no growth was observed for ptr2Δ dal5Δ cells transformed with the EV, indicating that RiPTR2 is able to transport these three dipeptides (Figure 2).

Ala-Leu and Ala-Tyr transport in the dal5Δ single mutant expressing RiPTR2 was also evident (Figure 2). Results obtained with ptr2Δ single mutant were difficult to interpret since this mutant, transformed with the EV, showed a high background growth on all three dipeptides. However, RiPTR2 transformants clearly diplayed an increased growth on all three dipeptides.

Taken as a whole these results demonstrated that RiPTR2 codes for a functional dipeptide transporter.

We first compared the expression profile of RiPTR2 in intraradical (IRM) and extraradical (ERM) mycelia by quantitative RT-PCR from M. truncatula plants colonized by R. irregularis grown in 0.5 mM nitrate in the sandwich system. RNA extractions were performed on ERM and on roots fragments from which the ERM was carefully removed to produce the IRM sample. Samples were normalized with the RiTEF housekeeping gene. A strong RiPTR2 expression was observed in the IRM while RiPTR2 transcripts were less abundant, and almost barely detected, in the ERM (Figure 3A). A similar result was obtained considering mycorrhizal roots of C. intybus devoided of ERM, grown in the root compartment and ERM developed in the hyphal compartment of the same ROC (Figure 3B). These results thus confirmed the strong expression in the IRM as observed in the microarray data (Tisserant et al., 2012).

We also performed a time course experiment in the sandwich system to determine RiPTR2 transcript abundance at different times (7, 14, 28, and 60 days) post-inoculation (dpi) of M. truncatula plants. Morphological analyses of roots showed almost no fungal structures at 7 or 14 dpi, while mycorrhization frequency increased from 28 to 60 dpi. Arbuscules were visible starting from 28 dpi and were slightly less abundant at 60 dpi than at 28 dpi (Figure S3). Since in the ERM RiPTR2 is expressed at negligible levels, gene expression was evaluated in whole mycorrhizal roots, without making a distinction between IRM and ERM. RiPTR2 mRNA abundance increased in parallel to the development of the intraradical phase as demonstrated by morphological data and the parallel mRNA accumulation of MtPT4, the M. truncatula phosphate transporter-encoding gene which is considered a molecular marker of arbuscule-containing cells (Harrison et al., 2002; Figures 4A,B).

The laser microdissection technique was used to specifically obtain RNA from M. truncatula arbusculated cells. The authenticity of the samples was verified using MtPT4 specific primers. RiPTR2 mRNA was detected in the two independent samples analyzed, indicating that, under these conditions, in planta RiPTR2 expression occurred in arbuscules (Figure 4C).

We then investigated whether the presence of a dipeptide (Ala-Leu), a candidate substrate of RiPTR2 as indicated by the yeast heterologous expression, could modulate RiPTR2 expression levels. This was tested in both a short- and in a long-term exposure experiments where we evaluated the RiPTR2 expression in whole mycorrhizal roots, without making a distinction between IRM and ERM.

In the first experiment (short term exposure), M. truncatula mycorrhizal plants were obtained in pot cultures watered with a Long Ashton (LA) nutrient solution containing 1 mM nitrate. After 2 months mycorrhizal roots were treated for 24 h in hydroponic conditions with a LA solution containing 10 mM Ala-Leu or 1 mM nitrate or no N. No significant difference was observed in RiPTR2 expression levels among the different treatments (Figure 5A). Plants showed rather similar mycorrhization degrees as revealed by the MtPT4 expression levels; although no N samples had higher values, they were not statistically different from those of the other two treatments (Figure 5B).

We also investigated whether the RiPTR2 expression in the ERM developed in the ROC system was responsive to 24 h exposure to 10 mM Ala-Leu. As previously observed RiPTR2 expression in the ERM was extremely low (Figure 3) and this treatment led to an apparent decrease of RiPTR2 mRNA abundance compared to the ERM kept for 24 h in a medium containing 3.2 mM nitrate or no nitrogen (Figure 6).

In the second experiment (long term exposure), M. truncatula mycorrhizal and non-inoculated plants were grown in the sandwich system for 60 days with a nutrient solution containing 2.5 mM Ala-Leu and 0.25 mM nitrate as N sources. Mycorrhizal plants grown in standard nutrient solution, that is 0.5 mM nitrate, were used as controls. Independently from the AM colonization, plants grown in the presence of Ala-Leu + nitrate have total (shoot + root) biomasses lower than plants grown in only nitrate (Figure 7A). Interestingly, when shoot to root ratio is considered, it appears that the mycorrhization has a positive effect on the plant in the presence of Ala-Leu + nitrate, in particular in resource allocation into shoots (Figure 7B). This result suggests that the AM fungal colonization contributes to the more efficient use of N, including dipeptides. This result may also be due to the higher colonization level as revealed by the MtPT4 expression (Figure 8B). No difference in RiPTR2 transcript abundance was observed in the two conditions (Figure 8A).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.