The Medicago truncatula accession Jemalong A17 was grown in perlite saturated with Färhaeus medium without nitrate in a growth chamber at 21°C and 16/8-h light/darkness cycle. These plants were inoculated with Sinorhizobium meliloti strain Sm2011 (OD600 0.1, 2 mL per plant). Root nodules were collected for analysis 14 days post inoculation (dpi). Agrobacterium rhizogenes MSU440-mediated hairy root transformation was performed according to Limpens et al. (2005).

DNA sequences of M. truncatula synaptotagmins named MtSyt1, MtSyt2 and MtSyt3 were retrieved by BLAST based on homology with Arabidopsis SYT1 (Schapire et al., 2008). MtSyt1, MtSyt2 and MtSyt3 genes and their putative promoters were amplified by PCR on M. truncatula cDNA genomic DNA, respectively, using Phusion High-Fidelity DNA Polymerase (Finnzymes) and specific primers (Supplementary Table 1). PCR fragments were introduced in pENTR-D-TOPO (Invitrogen) and sequenced. Each promoter was further re-cloned into two different pENTR 4-1 vectors (Invitrogen); one is with GFP and the other without GFP (Invitrogen). To create N- or C- terminal GFP fusion these vectors together with pENTR-TOPO containing MtSyts genes and pENTR 2–3 (with or without GFP) were subsequently recombined into the modified pKGW-UBQ10::DsRED destination vector (Limpens et al., 2004) using Gateway technology (Invitrogen), pEnter clone of Exo70i was recombined to the plasmid of UBQ3-pK7WGF2-R (Limpens et al., 2009) creating N-terminal GFP fusion under Ubiquitin promoter (ProUbiq3:GFP-Exo70i).

The double RNAi constructs were created by fusing MtSyt1, MtSyt2 and MtSyt3 PCR-generated sequences using protocol adopted from Franssen et al. (2015). In a first round of PCR the short overlaps (15 bp) were introduced to PCR products by using specific primers. A mixture of two obtained PCR fragments diluted in 1:500 was used as a template in second PCR to create a single DNA fragment. The primers are listed in Supplementary Table 1.

Total RNA was extracted from roots and 14-dpi root nodules using the E.Z.N.A. The extraction was made from two transformations. Plant RNA Mini Kit (Omega Bio-Tek) and transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad). Real-time PCR was set up in a 20 μl reaction system using iQ SYBR Green Supermix (Bio-Rad). Gene-specific primers were designed with Primer-3-Plus software (Untergasser et al., 2007). Gene expression profiles were normalized against the transcription level of the reference gene UBQ10. The primers are listed in Supplementary Table 1. Results were compared with M. truncatula Gene Expression Atlas¹ and Symbimics² data. According to Symbimix database (Roux et al., 2014; INRA²). MtSyt1, MtSyt2 and MtSyt3 were expressed in the meristem, zone of infection and zone of nitrogen fixation; MtSyt1 and MtSyt3 have higher expression than MtSyt2 (Supplementary Figure 2B).

Transgenic roots and nodules were collected and washed twice in 0.1M sodium phosphate buffer, pH 7.2, incubated in β-glucuronidase (GUS) buffer under vacuum at room temperature for 30 min to allow the buffer to replace oxygen in the tissue, incubated at 37°C for 2 h. Hand-cut sections of processed nodules were analyzed using Leica DM 5500 Flu microscopes.

GFP-fused proteins were visualized on transgenic roots and hand-sectioned nodules. Imaging was done on a Zeiss LSM 5 Meta confocal laser-scanning microscope (Carl Zeiss) and Leica TCS SP8 HyD confocal microscope (Leica) with 63 oil immersion objective. Polyclonal rabbit anti-GFP antibody (Molecular Probes) at a dilution of 1:100 and secondary anti-rabbit Alexa 488 antibody (Molecular Probes), (excitation max 490, emission max 525 nm) at a dilution of 1:200 were used for signal enhancement. The mixture of Goat serum (50%) with 2% (vol/wt) BSA was used as the blocking agent. Sections were counterstained with FM4-64 (30 μg/mL) or propidium iodide (0.001%).

Tissue preparation was performed as described previously (Limpens et al., 2009). Semi-thin (0.6 μm) sections were cut using a Leica Ultracut microtome (Leica) and examined using a Leica FL light microscope. For EM immunogold analysis the tissue was fixed by high-pressure freezing method as described before by Limpens et al. (2009). The nickel grids with the sections were blocked in normal goat serum with 1% of skimmed milk or 2% BSA in PBS and incubated with the primary antibody at the dilutions given above. Goat anti-rabbit coupled with 10-nm or with 15 nm gold particles (BioCell) (1:50 dilution) were used as secondary antibody. Sections were examined using a JEOL JEM 2100 transmission electron microscope equipped with a Gatan US4000 4K × 4K camera.

The proteins were extracted from root nodules in 0.025M Tris-HCl buffer containing 1 mM EDTA, 1 mM DTT and protease inhibitors cocktail (Roche). The probes loaded to the gel: 45 μg/well of root tips extracts of transgenic roots containing GFP-tagged synaptotagmins. The proteins were separated by 12.5% SDS-PAGE and blotted to nitrocellulose (Bio-Rad). The membrane was incubated in 3% BSA as a blocking agent followed by primary anti-GFP rabbit specific antibody, 1:1000 dilution; followed by secondary antibody, anti-rabbit AP antibody produced in goat (Sigma), 1:5000 dilution. The immunosignal was revealed by NBT/BCIP staining.

Five homologs of synaptotagmin1 genes were retrieved from M. truncatula databases by BLAST analysis using Arabidopsis thaliana synaptotagmin1 sequence. Phylogenetic analysis shows that the MtSyt1 and MtSyt2 genes and A. thaliana AtSYT1, AtSYT2 belong to the same group, and annotated in NCBI as synaptotagmins7 of M. truncatula, further sub-grouping is not clearly defined (Supplementary Figure 1). MtSyt1 is a closest homolog of AtSYT1. MtSyt3 is an ortholog of AtSyt3, forming a separate group (Yamazaki et al., 2010). MtSyt4 and MtSyt5 are grouped with AtSyt4 and AtSyt5.

For this study we selected MtSyt1, MtSyt2, and MtSyt3 genes. The expression of MtSyt1, MtSyt2, and MtSyt3 in roots and nodules at 14 dpi was analyzed by qRT-PCR (Supplementary Figure 2). MtSyt1 and MtSyt2 were expressed less in nodules than in the roots, whereas MtSyt3 expression in nodules was higher. According to Symbimix database (Roux et al., 2014; INRA³) MtSyt1, MtSyt2 and MtSyt3 are expressed in the meristem, zone of infection and zone of nitrogen fixation; the expression of MtSyt1 and MtSyt3 is higher than one of MtSyt2 (Supplementary Figure 2B). To clarify the role of synaptotagmins during symbiosis development, we studied the expression, localization, and functional role of MtSyt1, MtSyt2 and MtSyt3.

For the analysis, we created constructs containing a region 2.5 kb upstream of the translational start of MtSyt1, MtSyt2 and MtSyt3 fused to GUS. Transgenic roots were obtained using Agrobacterium rhizogenes-mediated transformation. In the roots ProMtSyt1:GUS, ProMtSyt2:GUS and ProMtSyt:GUS were expressed in all cells and most strongly in root primordia, meristem, epidermis of the zone of elongation, including the root hairs (Figures 1A–B). To see whether the expression pattern may be affected by membrane tension, we performed a root bending assay. Transgenic roots were selected from the plants grown in vertically oriented agar plates, bent and fixed using the tooth sticks. Bent roots were harvested after 48 h. Bending the root shifted the GUS signal to the site of curvature (Figures 1D,E). We concluded that the expression of synaptotagmins follows the region of the root with high membrane tension caused by the bending, hence the created constructs: ProMtSyt1:GUS, ProMtSyt2:GUS, and ProMtSyt3:GUS give a functional response for mechanical stimulation. In the nodules, ProMtSyt1:GUS, ProMtSyt2:GUS, and ProMtSyt3:GUS were strongly expressed in cells undergoing rapid increase of volume: in the cells of the meristem, of distal cell layers of the infection zone (Figures 1F–H) and distal layers of the zone of fixation (Figures 1F,H) (ProMtSyt1:GUS and ProMtSyt3:GUS).

To investigate the localization of synaptotagmins in nodules and roots, constructs expressing GFP translational fusions at the C-terminal position of these genes under the control of their respective 2.5 kb native 5′ regulatory sequences were created. The size of the proteins has been analyzed by Western blot with anti-GFP antibody (Supplementary Figure 3). For confocal imaging, roots and root nodules at 14–21 dpi were hand sectioned and fixed in 1% paraformaldehyde in a phosphate buffer as previously described (Gavrin et al., 2014). The GFP signal was weak and has to be enhanced by anti-GFP antibody coupled with secondary antibody tagged by ALEXA488. Confocal microscopy of the tissue reveals that GFP-tagged proteins ProMtSyt1:MtSyt1-GFP, ProMtSyt2:MtSyt2-GFP and ProMtSyt3:MtSyt3-GFP locally accumulate in the meristem of roots, the strongest signal was found in the developing cell walls in freshly divided cells (Figure 2A). In the nodules, the immunosignal of ProMtSyt1:MtSyt1-GFP, ProMtSyt2:MtSyt2-GFP and ProMtSyt3:MtSyt3-GFP was present in apical part of the nodule: in the meristem, zone of infection and proximal cell layers of zone of fixation and in vascular bundles (Figures 2B–D). On the cellular level the signal was found in the ER-rich central areas of the cell with an enrichment of the signal over the PM of freshly divided meristematic cells and around infection threads and unwalled droplets (Figures 2E,G,H). The symbiosomes were specifically outlined by the GFP signal only in the nodules carrying ProMtSyt1:MtSyt1-GFP (Figure 2F). The nodule tissue from the negative control where the primary (anti-GFP) antibody was omitted during the labeling process does not display labeling pattern (Supplementary Figure 4A). Synaptotagmins were therefore localized in the cells with expanding membranes. The special role in symbiosome membrane expansion, according to the localization study, belongs to MtSyt1 which is localizes on symbiosome membrane. Electron microscopy (EM) immunogold analysis of the transgenic nodules carrying GFP-tagged synaptotagmin shows the signal over the PM of freshly divided cells, in ER and the symbiosome membrane in the transgenic nodules carrying ProMtSyt1:MtSyt1-GFP (Figures 3A–C). The control without primary (anti-GFP) antibody is shown on Supplementary Figure 4B.

Endoplasmic reticulum is extremely abundant in young, freshly infected cells. ER contact sites with the membranes of infection threads, unwalled droplets, and symbiosomes represented as local dilations of tubular extensions and vesicles are ubiquitously present in young infected cells. The images obtained by transmission EM show numerous fusion events with the membrane vesicles and close contacts with ER (Supplementary Figures 5A,B).

To check the functional status of GFP-tagged constructs, we compared the localization pattern of the construct with GFP at the C-terminal position against the construct containing N-terminal (transmembrane domain) GFP (ProMtSyt3-GFP: MtSyt3). The GFP positioning on the transmembrane domain resulted in the protein becoming mis-localized. According to the localization pattern, the GFP signal was not targeted to the PM and was retained in the cytoplasm forming a dot-like pattern (Supplementary Figure 6A). EM immunogold analysis has shown that the protein was mainly present over the ER and in Golgi bodies (Supplementary Figure 6B). Therefore the C-terminal constructs which were used for the localization study maintained the correct positioning in the PM of the host cell.

We believe that the null mutations in case of housekeeping genes like synaptotagmins negatively affect the development of the whole host plant and indiscriminately damage the nodules. Hereafter, to specify the role of synaptotagmins in the infected symbiotic cells and avoid the negative effects for the shoots, vascular system and non-infected nodule cells we used RNAi silencing under the control of Enod12 promoter. This promoter is active in the nodules in zone of infection (Limpens et al., 2005), this approach permits to silence genes only in the young infected cells without affecting the other cells of the nodule.

We have created the double silencing constructs of MtSyt1/MtSyt3 and MtSyt2/MtSyt3 to induce a simultaneous silencing under the Enod12 promoter. The level of silencing of MtSyt1/MtSyt3 and MtSyt2/MtSyt3 in transgenic nodules of ProENOD12:MtSyt1/MtSyt3 and ProENOD12:MtSyt2/MtSyt3 is shown in Supplementary Figure 7.

Analysis of the transgenic roots from 10 plants showed that transgenic nodules were smaller and less numerous: 2.81 ± 1.65 per plant versus 5.32 ± 1.91 in the controls. Transgenic nodules shown a distinct phenotype: a short meristem, extended zone of infection and diminished or aborted zone of fixation (Figures 4A–D versus Figures 4E,F). Zones of infection have increased numbers of cell layers (Table 1), delayed rhizobia release and symbiosome maturation. The double-silencing phenotype was observed in 65% of the nodules (n = 20). The analysis was performed on 20 nodules randomly collected from 10 plants. In the majority of affected nodules, the symbiosomes were not able to differentiate beyond stage 2/3 according to the classification of Vasse et al. (1990) and undergo premature senescence (Figure 4A). EM analysis of nodules with double silencing (ProENOD12:MtSyt1/MtSyt3 and ProENOD12:MtSyt2/MtSyt3) shown that infected cells in the extended zone of infection contain unwalled droplets without apparent bacteria release (Figures 4G,H). However, the release was not completely inhibited and the cells were finally colonized, but the maturation of the symbiosomes was restricted. The colonization of infected cells was quite slow and was taking up to 12 cell layers (cf. Figures 4A,B with Figure 4E) comparing with 3–5 in control. The number of cell layers in different nodule zones in control and RNAi nodules and the statistical analysis are presented in Table 1. The T-test was used to estimate a difference between control and RNAi nodules (P < 0.05).We have concluded that the double silencing of MtSyt1/MtSyt3 and MtSyt2/MtSyt3 negatively affects nodule meristem development, growth of infected cells, caused the reduction of bacteria release from unwalled droplets, and hampers growth and maturation of symbiosomes.

To specify the role of synaptotagmins in the formation of interface membrane in other type of symbiosis we have studied the localization of MtSyt3 in the roots inoculated by R. intraradices. The formation of arbuscules, the symbiotic intracellular extensions of hyphae is always accompanied by the rapid increase of interface membrane (Genre et al., 2009; Gutjahr and Parniske, 2013). The distribution of GFP signal of ProMtSyt1:MtSyt1-GFP and ProMtSyt3:MtSyt3-GFP was similar, it was clearly delineating the membranes of arbuscules fine branches, but not the trunks (Supplementary Figures 8A,B). The GFP signal was enhanced by anti-GFP antibody coupled with secondary antibody tagged by ALEXA488 equally as it was done for the nodule tissue.

To specify the role of synaptotagmin in tip growth we have compared the localization of synaptotagmin with the localization of Exo70i, the subunit of tethering complex exocyst in root nodule infected cells and in the cells containing arbuscules. For the study we have cloned the Exo70 subunit which is highly expressed in arbusculated roots and in the nodules. The same gene have been cloned and characterized by Zhang et al. (2015), due to this we are using the abbreviation used by Zhang et al. (2015): Exo70i, despite that this gene was cloned by us independently.

In arbusculated root cells Exo70i was strictly localized near the tip of fine branches of arbuscules in contrast to the signal of synaptotagmin which was found over the whole surface of the branches (Supplementary Figure 8C versus Supplementary Figures 8A,B). The localization pattern of this subunit over the arbuscules in our work and in the paper of Zhang et al. (2015) shows the identical pattern.

The localization of Exo70i in infected cells of the nodules was specific, it has dot-like pattern and forms a small clusters near the tips of infection threads and also was marking unwalled droplets, but was not delineating the membrane of infection thread or unwalled droplet like synaptotagmins do, neither it was labeling the symbiosome membrane (Supplementary Figure 8D).

Therefore we concluded that the distribution of synaptotagmins on the membranes of infection threads and fine branches of arbuscules was spatially overlapping on the regions of infection threads tips and the tips of arbuscular fine branches, but was not overlaying with the signal of Exo70i on the whole interface membrane.