Plant growth conditions
Seeds of Medicago truncatula ecotype R108, obtained from Dr. González-Guerrero´s Laboratory (CBGP-UPM/INIA, Spain), were scarified with sulfuric acid (H2SO4), sterilized with bleach before they were germinated in 0,8% agar plates and placed in the dark at 4 °C for 48 h, following the steps described by (Tejada-Jiménez et al., 2015). After stratification, plates were moved to a growth chamber at 22 °C for 24 h with a 16h:8 h light:dark cycle. Seedlings used for phenotypic or RT-qPCR analyses were transferred to pots using perlite as a substrate and grown in the greenhouse with a long-day photoperiod (16h:8 h light:dark cycle at 18–25 °C). Greenhouse plants were irrigated every two days with Jenner’s solution or water, alternatively (57). Jenner’s solution was supplemented or not with B (to achieve control or B deficient conditions) and N (to achieve symbiotic or non-symbiotic conditions))
Nicotiana benthamiana Domin plants, obtained from Dr. González-Guerrero´s Laboratory (CBGP-UPM/INIA, Spain), were pre-germinated in peat for 10–15 days at 20–22 °C with a 16h:8 h photoperiod. They were then transplanted to peat:vermiculite 3:1 and grown for 3–4 weeks in a greenhouse at 18–25 °C and long-day photoperiod.
Arabidopsis thaliana ecotype Col-0 and nip5;1–1 mutant seeds (58), obtained from Dr. Miwa Laboratory (Hokkaido University, Japan), were sterilized first in ethanol 70% and then in bleach 50% with a droplet of Tween-20. After rinsing well the seeds with H2Od, they were kept overnight in the dark at 4 °C and then germinated in plates with half strength MS media (59). For segregation experiments, seedlings were transplanted to peat:perlite 3:1 and grown in a growth chamber at 23 °C with long-day photoperiod.
Phenotypic analysis
In order to study B-availability effects on plant growth, inoculated and non-inoculated M. truncatula plants were irrigated with different concentrations of H3BO3. Control treated plants were irrigated with 0,1 mM H3BO3, toxicity conditions were achieved irrigating plant with a final H3BO3 concentrations of 1 mM and no H3BO3 was applied for deficiency conditions. All irrigation media was treated with Amberlite® IRA743 to eliminate B traces.
Plants grown under symbiotic conditions were inoculated with Sinorhizobium meliloti FSM-MA strain (60) without receiving any external input of N (-N). Non-inoculated plants were supplemented with 20 mM NH4NO3 (+ N).
Plant tissue was collected 5 weeks after the transplant (4 weeks after inoculation) for biomass measurements, nitrogenase activity and RT-qPCR assays.
Biomass, as fresh weight, was determined immediately after plant harvesting. Biomass, as dry weight, was determined after drying the plant tissue at 60 °C for 72 h.
Nitrogenase activity was measured using the acetylene reduction assay as described by Hardy et al. (1968). Briefly, roots of nodulated plants were placed in 30 ml vials where 3 ml of air were replaced with 3 ml of acetylene. After 30 minutes, 4 replicates of 0,5 ml each were extracted and their ethylene content was measured using a gas chromatograph Shimadzu GC-2014 (Japan). A dilution of ethylene and acetylene 0,413 mg/l was used as standard. At the end, nodules were counted and weighed.
Gene candidate identification
To identify AtNIP5;1 homologous genes that putatively encode B facililator trasnporters in M. truncatula, the AtNIP5;1 (At4g10380) sequence was obtained from the TAIR database (https://www.arabidopsis.org/) and BLAST in the M. truncatula genome using the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html).
Gene expression analysis by RT-qPCR
Gene expression studies were carried out by real-time RT-PCR (Applied Biosystems®) in order to analyze transcript levels of candidate genes. ARN Extraction was carried out using the RNeasy Mini Kit (Qiagen). cDNA was obtained from 500 ng of DNA-free RNA using PrimeScript™ ( Takara Bio Inc., Japan). Primers used are indicated in Additional file 1. RNA levels were normalized by using the ubiquitin carboxy-terminal hydrolase gene as internal standard for M. truncatula expression patterns (62).
M. truncatula transformation
The Agrobacterium rhizogenes ARqua1 strain containing the vector was used to transform M. truncatula seedlings after 18 h germination in a growth chamber at 22 °C. Transformation experiments were performed following the protocols described by Boisson-Dernier et al. (2001). Transformed seedlings were later transferred to Farhaeus media plates supplemented with kanamycin (50 µg/ml) as selection marker (64). After three weeks, plants were transplanted to sterile perlite pots that were placed in the greenhouse at 18–25 °C and long-day photoperiod.
GUS staining
The MtNIP5;1 promoter:: β-glucuronidase (GUS) construct was generated using the Gateway System (Life Technologies, Carlsbad, USA). The promoter fragment of the candidate gene (2.022 kb upstream of the MtNIP5;1 start codon, P− 1174 UT848) was amplified using the primers indicated in Additional file 1, cloned in the pDONOR27 vector (Invitrogen) and transferred to the pGWB3 plasmid (65).
Gus activity assay was performed in plants 4 weeks after inoculation following the protocol described by (66) with minor modifications. Roots and nodules sections (100 µm) were incubated in a GUS buffer (0,69% PO4H2Na, 0,5 M EDTA pH 8, 0, 30% sarkosyl, 40 µl triton X-100 AND H2O) supplemented with X-Gluc (0,1 mg/ml) for 12–16 h at 28 °C in the dark, and then distained with bleach 50% and rinsed five times in H2O. Afterwards, sections were observed with a Leica DM IRB microscope.
Immunohistochemistry and confocal microscopy
MtNIP5;1 gene and its native promoter (2 kb upstream of the start codon) were amplified with the primers indicated in Additional file 1 and cloned into the plasmid pGWB13 that fuses three C-terminal hemagglutinin (HA) tags in‐frame or into the pGWB4 plasmid that fuses the GFP (green fluorescent protein) tag (65), using the Gateway system (Life Technologies, Carlsbad, USA).
Transformed plants were inoculated with S. meliloti 2011. Roots and nodules were fixed overnight in a 4% paraformaldehyde, 2.5% sucrose and PBS buffer at 4 °C. After several washes in PBS, the tissue was embedded in 6% agarose and 100 µm sections were prepared in a Vibratome 1000 plus. Sections were dehydrated using methanol series (30, 50, 70, 100% in PBS) for 5 min and then rehydrated. The immunostaining was started permeabilizing plant cell walls with 4% cellulase in PBS for 1 h at RT and with 0.1% Tween 20 in PBS for 15 min. Sections were blocked with 5% bovine serum albumin (BSA) in PBS before their incubation with anti-HA mouse monoclonal antibody (Sigma) for 2 hours at room temperature. After several washes, sections were incubated for 1 h with Alexa594-conjugated anti-mouse rabbit monoclonal antibody (Sigma). Images were acquired using a confocal laser-scanning microscope (Leica SP8) at 488 nm and 561 nm for GFP and Alexa 594, respectively.
N. benthamiana transient expression assay
The MtNIP5;1 gene was amplified using the primers indicated in Additional file 1, and then cloned into the pGWB5 plasmid, which fuses the cauliflower mosaic virus 35S RNA (CaMV 35S) promoter and the GFP tag C-terminally (65). A. tumefaciens C58C1 (67) was transformed with either p35S::MtNIP5;1-GFP or the the construct p35S::AtPIP2A- cyan fluorescent protein (CFP) (68) together with the silencing suppressor p19 of the Tomato bushy stunt virus (69). N. benthamiana 3 weeks-old leaves were then infiltrated following the protocol described by Sparkes et al. (2006). Images were taken 48 h after agroinfiltration with a confocal microscope (Leica SP8).
Bioinformatic analysis
The aminoacidic sequence of the gene candidate was obtained from the database UniProt (http://www.uniprot.org/). Protein 3D models were predicted using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I- TASSER/) and were edited with the software PyMol.
A thaliana mutant complementation assay
A. tumefaciens strains containing the construct p35S::MtNIP5;1-GFP were used to transform the nip5;1–1 A. thaliana mutant (58) using the floral dipping transformation method as described by (71). Homozygous lines containing the construct of interest were selected by kanamycin selection and PCR analysis, using the primers included in Additional file 1. Seeds of A. thaliana Col-0 (wildtype, WT), the mutant nip5;1–1, and two homozygous independent lines incorporating the construct p35S::MtNIP5;1-GFP were grown vertically in plates of ½ MS medium with two B treatments: control (100 µM B[OH]3) and deficiency (0,03 µM B[OH]3). Primary root length was then measured at 3, 5, 7 and 10 days post germination, from the root tip to the hypocotyl boundary, using the online available softwre ImageJ (http://rsbweb.nih.gov/ij/).
Statistical Analysis
One-way analysis of variance (ANOVA) followed by a Tukey HSD post-hoc test were applied to perform multiple comparisons at a probability level of 5% (p < 0.05). The SPSS Statistics 17.0 (SPSS Inc.) package was used for the statistical analyses.