Summary
The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron. The host plant is known to provide iron to developing bacteroids, but questions remain regarding which transporters are involved. Here, we characterize two Vacuolar Iron Transporter-Like (VTL) proteins in Medicago truncatula that are specifically expressed during nodule development. VTL4 is mostly expressed during early infection and the protein was localized to membranes and the infection thread. vtl4 mutants were delayed in nodule development. VTL8 is closely related to SEN1 in Lotus japonicus and expressed in the late stages of bacteroid differentiation. The VTL8 protein was localized to the symbiosome membrane. A mutant line lacking the tandemly-arranged VTL4 – VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a slight decrease in luminescence was observed in vtl4 mutants and a strong decrease in the 13U mutant. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. Taken together, these data indicate that VTL-type transporters are the main route for delivering iron to symbiotic rhizobia.
INTRODUCTION
Legumes and a small number of other plant species (Parasponia sp.) are able to form a symbiosis with rhizobium bacteria which enables the host plant to access N2 as a source of nitrogen. The host plant provides carbohydrates derived from photosynthesis for the energy-demanding reduction of N2 to ammonium, carried out by the bacterial nitrogenase enzyme. Successful establishment of the symbiosis in specialized structures called root nodules depends on signalling and recognition between the rhizobia and the host plant, as well as later checkpoints during nodule development (reviewed in Oldroyd et al., 2011; Suzaki et al., 2015).
Root nodules have a high requirement for iron owing to abundant iron proteins. Infected plant cells produce large amounts of haem as a cofactor in leghaemoglobins which maintain a microaerobic environment for the oxygen-sensitive nitrogenase enzyme (Downie, 2005; Ott et al., 2005; Garrocho-Villegas et al., 2007). The bacterial nitrogenase enzyme binds 12 iron in the form of iron-sulphur clusters and another 7 in the Fe-Mo cofactor (Dean et al., 1993; Howard & Rees, 2006). The nitrogen-fixing bacteria also contain numerous cytochromes and other iron proteins. When the plant is starved of iron, nodule initiation and further development is strongly impaired (O’Hara et al., 1988; Tang et al., 1990; Brear et al., 2013).
Nodule development is initiated by a signalling cascade between plant roots and the bacteria, entrapment of the bacteria in curled root hairs and formation of infection threads along which the bacteria travel into the root cortex. Division of cortex cells leads to formation of a specialized root outgrowth, the nodule. The root nodules formed by Medicago truncatula in symbiosis with Sinorhizobium meliloti or S. medicae are of the indeterminate type. The meristem persists over time and generates a gradient of cells at progressing developmental stages (Vasse et al., 1990; Roux et al., 2014), commonly divided into 4 histological zones, with the meristem as Zone I. Zone II corresponds to the infection zone where bacteria are released from the infection threads, divide and form symbiosomes – intracellular bacteria surrounded by a plant-derived membrane. In older cells of Zone II bacterial cell division stops while genome replication continues, resulting in elongated polyploid bacteroids (Mergaert et al., 2006). Synchrotron X-ray fluorescence (XRF) studies on M. truncatula indicated that iron is delivered to Zone II by the vascular bundles in the nodule (Rodríguez-Haas et al., 2013). The expression of the iron storage protein ferritin is induced in Zone II (Strozycki et al., 2007; Roux et al., 2014), corresponding to ‘iron-rich bodies’ in and near the vascular bundles detected by XRF (Rodríguez-Haas et al., 2013). In the Interzone (transition of zone II-III), infected plant cells and the bacteroids complete their differentiation with the last rounds of endoreduplication and enlargement, resulting in a striking cell morphology of tightly packed elongated rhizobia surrounding a central vacuole. Zone III comprises the major part of a functional nodule and this is where nitrogen-fixation takes place. In older nodules, a zone of senescent cells, Zone IV, is formed from which nutrients such as iron are recycled from degraded plant cells and symbionts (Van de Velde et al., 2006; Rodríguez-Haas et al., 2013).
Forward and reverse genetic studies have identified several transporters involved in iron delivery to the nodules and symbiosomes. The closely related genes L. japonicus MATE1 and M. truncatula MATE67 are highly induced during nodule development, specifically in the infection zone (Takanashi et al., 2013; Wang et al., 2017; Kryvoruchko et al., 2018). Studies in Xenopus oocytes showed that the LjMATE1 and MtMATE67 proteins can transport citrate, an organic chelator of Fe3+ in the xylem (Takanashi et al., 2013; Kryvoruchko et al., 2018). The MtMATE67 protein is localized to vascular bundles but also to the symbiosome membrane (Kryvoruchko et al., 2018). An RNAi mutant of LjMATE1 accumulated high levels of iron in the vascular bundle of roots and nodules, whereas the iron concentration in infected cells of the nodules was lower compared to wild type (Takanashi et al., 2013). These two studies indicate that the host plant delivers iron to the nodules via the xylem.
Uptake of iron into the infected cells may involve MtNRAMP1, one of 7 members of this gene family in M. truncatula with the highest relative expression in roots and nodules (Tejada-Jiménez et al., 2015). Yeast complementation studies showed that MtNRAMP1 can transport iron and manganese, similar to its closest Arabidopsis homologue which is the primary route for manganese into the cell (Cailliatte et al., 2010). MtNRAMP1 is localized to plasma membranes, including the plasma membrane of infected cells. A transposon insertion mutant of MtNRAMP1 has impaired nodule development, but still exhibited 60% nitrogenase activity compared to wild type (Tejada-Jiménez et al., 2015).
For iron to reach the bacteroid, it needs to be exported by the infected plant cell and imported across the bacteroid membrane. Five different allelic mutants in the LjSEN1 gene, encoding a Vacuolar iron Transporter-Like (VTL) protein, were identified in a screen for ineffective symbiotic (Fix-) mutants (Hakoyama et al., 2011). The VTL proteins differ from the better characterized Vacuolar Iron Transporter (VIT) proteins in that they lack a cytosolic loop thought to mediate Fe2+/H+ antiport (Labarbuta et al., 2017; Kato et al., 2019). Promoter-GUS studies showed that LjSEN1 is expressed exclusively in rhizobia-infected cells, but its subcellular localization and function in iron transport has not been documented to date.
To better understand the role of VTLs in nodule development and biological nitrogen fixation, we characterized two VTLs in M. truncatula, VTL4 and VTL8, which are exclusively expressed in nodules. Localization and mutant studies showed they play a role at early and late stages, respectively, of nodule development. VTL8 is critical for delivering iron to the bacteroids and establishment of the nitrogen-fixing bacteria.
RESULTS
M. truncatula has 8 VTL genes of which two genes are expressed in nodules
The M. truncatula genome (Mt4.0v1) was searched for homologs of A. thaliana VIT and VTL genes. We identified 2 homologs of VIT and 8 homologs of VTL as compared to one and 5 genes, respectively, in A. thaliana. Amino acid alignment of the 8 MtVTLs with the A. thaliana homologs showed only a limited phylogenetic relationship between different VTLs of the two species (Fig. 1a). Based on this phylogeny, the M. truncatula genes were assigned as VTL1-VTL8. MtVTL1, MtVTL2 and MtVTL3 are most similar to A. thaliana VTL1–VTL4. MtVTL4, MtVTL5, MtVTL6 and MtVTL7 are closely related paralogs on chromosome 4 which are more similar to A. thaliana VTL5 than the other AtVTL genes. In contrast, MtVTL8 has no direct homologue in A. thaliana, but is closely related to NODULIN-21 of soybean (Delauney et al., 1990) and SEN1 in L. japonicus (Hakoyama et al., 2011), which have strongly induced expression in nodules. Indeed, MtVTL8 is also highly expressed during the later stages of nodule development, with the highest expression in the Interzone (Zone II – III), see Fig. 1b based on data in Roux et al., (2014). Of the other MtVTL genes, only MtVTL4 transcripts are found at significant levels in nodules, with the highest levels in the early infection stage (distal zone II, ZIId).
MtVTL4 and MtVTL8 localize to membranes in different zones of nodule development
To investigate the localization of the VTL4 and VTL8 proteins in nodules, the proteins were fused via a short C-terminal linker peptide to mCherry and transiently expressed in roots under the control of their own promoters. The presence of a glycine-rich linker peptide has previously been shown to improve membrane insertion and thus stability of A. thaliana VTLs expressed in Baker’s yeast (Gollhofer et al., 2014). As a marker protein for the plasma membrane we used the aquaporin PIP2A from A. thaliana (Cutler et al., 2002) which localizes similarly to the cell membrane in M. truncatula roots (Ivanov & Harrison, 2014). The symbiosome membrane is derived from the plasma membrane, however regulatory elements that target proteins there are not well understood. The coding sequence of AtPIP2A was fused to enhanced GFP and the resulting PM-eGFP marker was expressed using the LjUBI promoter. From several membrane markers that were tested, LjUBI:AtPIP2A-eGFP was expressed in all nodule cell types and GFP was found in the plasma membrane as well as the symbiosome membrane (Fig. 2).
In nodules expressing VTL4-mCherry, red fluorescence was observed in cells of the infection zone only, corresponding to the highest level of RNA expression in this zone (Fig. 1b). VTL4-mCherry co-localized with the PM-eGFP marker to plasma membranes. VTL4-mCherry was also associated with membranes of the infection thread (Fig. 2a, arrow head in merged image). VTL8-mCherry was detected in the infected cells of the interzone only (Fig. 2b). The fusion protein was confined to the bacteroids surrounding the central vacuole (upper panel). Co-localization with the PM-eGFP marker indicates that VTL8 is localized to the symbiosome membrane.
VTL8 is required for nodule development and nitrogen fixation
To study the role of VTL4 and VTL8 in nodule development, mutants were obtained from different sources. For VTL4, two lines with a Tnt1 insertion in the coding sequence were isolated (Fig. 3a), but none were found for VTL8 in the Noble Foundation collection despite extensive screening by PCR. However, a mutant line lacking several VTL genes was isolated from a collection of Fix-mutants generated by fast neutron radiation. The rough map position of the 13U mutant was identified previously between the genetic markers h2_32m20c and DENP (Domonkos et al., 2013). Microarray hybridization using the genomic DNA of 13U identified a probe set corresponding to the gene Medtr4g094325 encoding VTL4. Further analysis by PCR amplifications identified a 30-kb deletion in the 13U genome spanning VTL4 to VTL8. Gene expression analysis confirmed the absence of VTL4 transcript in the vtl4 mutants and in the 13U line (Fig. 3b, c). While low levels of PCR product for VTL8 were detected in the 13U mutant using standard RT-PCR (Fig. 3b), no product was generated by quantitative RT-qPCR (Fig. 3c). Possibly, aspecific priming to VTL paralogues occurred in the RT-PCR reaction, but not with more stringent qPCR conditions.
13U plants grew less vigorously than the parental wild type (Fig. 4a) and nodules were arrested early in development, lacking the typical pink colour of leghaemoglobin (Fig. 4b). Previous studies showed bacterial colonization of the infection zone and interzone of the 13U mutant nodules and only sporadic infection within the nitrogen fixation zone (Domonkos et al., 2013; Fig. 4c,d). To investigate the morphology and arrangement of rhizobia, nodule sections were stained with the nucleic acid binding dye SYTO13. No significant difference was found in the morphology of bacteria, but in the differentiation zone of 13U mutant nodules the elongated rhizobia were disorganized rather than orientated toward the vacuoles of infected cells found in wild type (Fig. S1).
In order to demonstrate which gene or genes caused the symbiotic phenotype of 13U, genetic complementation experiments were carried out. Transformation with constructs expressing VTL4 and VTL8 individually or together showed that expression of VTL8 alone in the 13U mutant reverted the phenotypes to wild type. Specifically, nodules developed fully to nitrogen-fixing competency including the expression of leghaemoglobin and restoring the wild-type nodule zonation (Fig. 4b and e). Expression of VTL4 alone in the 13U mutant did not have any effect, although co-expression with VTL8 increased nodule fresh weight to above wild-type values (Fig. S2). Thus, the function of VTL8 is essential for nodule maturation, whereas VTL4, VTL5, VTL6 and VTL7 only play a minor role if any.
MtVTL4 is required for early bacteroid development
The two vtl4 mutant lines were indistinguishable from wild type with respect to shoot and root development (Fig. 5a). However, vtl4 mutants had a relatively larger proportion of immature nodules compared to wild type (Fig. 5b). At 35 days post inoculation, wild-type plants had 20% immature nodules, compared to 34% and 57%, respectively, in vtl4-1 and vtl4-2 mutant plants. The number of immature nodules is more significant in the vtl4-2 allele, in which the Tnt1 insertion is closer to the start of the open reading frame. While no macroscopic differences were observed from the outside of the nodules (Fig. 5c), cross sections stained with SYTO13 showed that infected cells in zone II have a larger central vacuole and less elongated bacteroids (Fig. 5d). To quantify the difference, the diameter of the vacuoles of infected cells was measured in images of the differentiation zone of wild type and the vtl4-1 mutant using ImageJ software. On average, the vacuoles in vtl4-1 are 22.3 ± 5.7 μm (n = 284 cell images) and those in the R108 wild type 19.1 ± 4.0 µm (n = 88 cell images). This subtle cell morphological phenotype occurs in the zone where VTL4 expression reaches its highest levels in nodules (Fig. 1b).
Host plant VTLs are required for iron delivery to the bacteroids
It has previously been suggested that LjSEN1 and its homologs transport iron out of the infected plant cell into the peribacteroid space, based on sequence homology between VTLs and VIT proteins (Hakoyama et al., 2011; Brear et al., 2013; González-Guerrero et al., 2016). In addition, Arabidopsis VTLs can complement the Δccc1 yeast mutant defective in vacuolar iron transport, although growth complementation was only partial (Gollhofer et al., 2014), and VIT proteins can transport manganese as well as iron (Lapinskas et al., 1996).
To provide evidence for iron transport in vivo, we developed a transcriptional reporter in the bacteria using an iron-inducible promoter driving the lux operon. With the prerequisite that the bacterial promoter is active during nodule development, luminescence would be correlated with the iron status of the bacteria (deficient or sufficient). Most well-studied iron homeostasis genes, for example those for rhizobactin biosynthesis, are not expressed during nodule development and bacteroid differentiation (see Table S2 with data from Roux et al., 2014). However, the expression of mbfA (membrane-bound ferritin A) is strongly induced in the proximal zone II and interzone, while its transcript levels are low in non-differentiated bacteria of Zone IId (Fig. 6a). The mbfA promoter is negatively regulated by the Iron-regulated repressor (Irr) which binds to the Iron Control Element (ICE) in the promoter (Rudolph et al., 2006) (Fig. 6b). In addition to the wild-type promoter sequence of mbfA, we also made a reporter construct with a mutated ICE sequence, to prevent binding of Irr and achieve constitutive expression of mbfA-lux. Both reporter constructs were first tested in free-living S. meliloti 1021. Luminescence readings showed that the wild-type mbfA promoter was fully repressed under low iron but induced in the presence of iron (Fig. 6c). In contrast, the ICE mutant promoter construct (PmbfAICE:lux) was de-repressed in the absence of iron, although some Fe-dependent regulation was still observed.
Next, wild-type and mutant M. truncatula seedlings were inoculated with bacteria expressing the mbfA-lux reporter, and luminescence was analysed at 21 dpi using a NightOwl imaging system. S. meliloti 1021 was used to inoculate all plant genotypes. While less effective in nodulating M. truncatula Jemalong, the parent line of the 13U mutant, the number of nodules were sufficient for quantitative analysis. Luminescence was only observed in nodules, where bacteria are concentrated and provided with iron (Fig. 7a). Quantification of the signal showed approximately 50% luminescence in nodules of vtl4 mutants, and only 10% in the 13U mutant, compared to their respective wild types (Fig. 7b). Using the ICE mutant reporter, the luminescence signal was similar in the vtl4 mutants and parental line. In the 13U line, luminescence was slightly increased compared to the control line (Fig. 7c). Thus, the number of bacteria in Zone II and the Interzone in wild-type and mutant nodules are comparable, regardless of their difference in nodule development. Taken together, the lower expression of the bacterial iron reporter in the M. truncatula vtl4 and 13U mutants indicates that the nodule-specific VTL proteins are required for iron delivery to the bacteroids.
VTL4 and VTL8 mediate iron transport into yeast vacuoles
To confirm that VTL4 and VTL8 are able to transport iron, the genes were expressed in yeast for functional complementation assays. The VTL genes were cloned into the pYES2 plasmid under the control of a galactose-inducible promoter. The plasmids were transformed into a Δccc1 yeast mutant which lacks the vacuolar iron transporter Ccc1 (Li et al., 2001). Its inability to store iron in the vacuole leads to severely impaired growth in the presence of excess iron in the medium (Fig. 8). Growth can be restored by expressing another vacuolar iron transporter such as Arabidopsis VIT1 (Kim et al., 2006), used here as a positive control alongside the wild-type strain (Fig. 8). VTL4 as well as VTL8 were able to rescue growth of the Δccc1 strain on medium with 5 mM Fe sulphate. Functional complementation was only seen using Δccc1 in the DY150 (W303 derivative) genetic background, but not in the BY4741 strain. This may be due to differences in salt tolerance between the two strains causing an indirect effect on Fe homeostasis (Petrezxelyova et al., 2010).
DISCUSSION
Because of their high demand for metals, nodules provide an interesting system to study the function of transporters and other metal homeostasis genes in a highly compartmentalized plant cell. After iron is imported across the plasma membrane into the infected cell, it is distributed between the mitochondria, plastids containing the iron storage protein ferritin, and the differentiating bacteroids. Because of toxicity of ‘free’ iron (not bound to chelators or proteins), the timing of distribution and thus gene expression is crucial. In M. truncatula, 2 of its 8 VTL genes appear to be involved in iron delivery to the bacteroids, but at different times of the infection process. This may reflect the findings of older studies in peanut and lupin, that iron is required for both nodule initiation and further development (O’Hara et al., 1988; Tang et al., 1990). VTL4 is providing iron to the bacteria, before any differentiation, when they are dividing in the infection thread. The minor effects of knocking out VTL4 on nodule and bacteroid development suggest that VTL4 is not the only source of iron. At this stage, the bacteria may still have some iron stored from before infection, or simply need little iron. In contrast, VTL8 is critical for full differentiation of the bacteroids into nitrogen-fixing symbionts. In line with this, VTL8 expression peaks just before the induction of the bacterial nif genes for nitrogenase when large amounts of iron need to reach the bacteroids for incorporation in the iron-sulphur cofactors of the abundant nitrogenase protein.
Phylogenetic analysis shows that VTL8, LjSEN1 and GmNOD21 form a separate clade of VTL genes. It is therefore likely that the gene was recruited specifically for symbiosis in the ancestral legume species. Why was a VIT-like gene, but not VIT, co-opted for this function? VIT is a Fe2+/H+ antiporter, but the iron species transported by VTLs is not known. Because the iron-binding loop is missing in VTLs, it is possible VTLs transport both Fe2+ and Fe3+, or Fe3+ exclusively. Interestingly, VTLs are highly homologous to the transmembrane domain of MbfA, which also lacks the Fe-binding loop but has an additional N-terminal ferritin-like domain (Bhubhanil et al., 2014; Sankari & O’Brian, 2014). It has been suggested that this domain oxidizes Fe2+ to Fe3+, which is then transported by the membrane domain. The ferritin-like domain may also help drive transport in one direction, namely to the outside of the cell. It would be interesting to see if plant ferritin, which accumulates transiently in Zone II, is able to associate with VTL8 to deliver iron to the peribacteroid space. Normally the 24-mer iron-storage protein ferritin is localized on the periphery of plastids in plants (Roschzttardtz et al., 2013; Moore et al., 2018). So, either plastids interact with the symbiosome membrane, or a cytosolic form of ferritin exists in infected cells.
It is still not clear how iron exported from the plant cell into the peribacteroid space is taken up by the bacteroids. The peribacteroid space undergoes acidification towards the onset of nitrogen fixation as shown by fluorescent probes (Pierre et al., 2013). The pH is a critical factor for the solubility of iron, with a low pH enhancing solubility. Most bacteria species have separate transport systems for Fe2+ and Fe3+-chelate complexes. Iron uptake genes that are active in free-living rhizobium (Johnston et al., 2001) are generally not induced in the symbiont stage (Table S2, Roux et al., 2014, and reviewed in (Abreu et al., 2019). The Fe2+ transporter FeoAB plays a role in iron transport in Bradyrhizobium and feoA or feoB deletion strains produced ineffective nodules on soybean (Sankari & O’Brian, 2016). However, there are no feoAB genes in the S. meliloti genome. Alternatively, iron may be taken up as Fe3+-citrate of Fe3+-malate via tricarboxylic acid transporters, which are highly induced. The Fe-responsive lux reporter developed for this study should help to fully characterize the bacterial iron transporters that are active in the nodules. It is important to note that the strongly induced MbfA expression indicates bacteroids are saturated with iron, and need to export it back to the peribacteroid space. Thus, low-affinity uptake systems may be sufficient for the bacteroids’ Fe requirements.
In summary, this study provides confirmation that VTL8 / SEN1 is indeed the main iron transporter across the plant symbiosome membrane, but it also opens up new questions regarding the iron homeostasis in nodules. In addition to identifying the Fe-species that is exported, keys questions are how Fe is delivered to VTL8 / SEN1 and how Fe is partitioned between haem biosynthesis for leghaemoglobin and export to the bacteroids.
MATERIALS AND METHODS
Plant growth and bacterial strains
Medicago truncatula Jemalong and M. truncatula subsp. tricycla (R108) genotypes were used as wild-type controls for the 13U (Domonkos et al., 2013) and vtl4 mutant lines, respectively. The Tnt1-insertion mutants vtl4-1 (NF17463) and vtl4-2 (NF21016) were purchased from the Samuel Roberts Noble Foundation (Ardmore, Oklahoma). M. truncatula seeds were scarified with sandpaper before being surface sterilised with 10% (w/v) sodium hypochlorite for 4 min. Seeds were then washed 5 times with distilled sterile water and imbibed in the dark at room temperature for 4 h. Seeds were plated on sterile water agar, and stratified by placing the plates upside down at 4 °C for 3-7 days. The plates were then moved to 20 °C for 16 h to allow the seeds to germinate. Seedlings were transferred either to sterile medium or planted out on a 50:50 mixture of Terragreen and sand for inoculation with rhizobium. Plants were grown in long day conditions (16 h light, 8 h dark) at 22 °C, light intensity 180-200 μmol photons m−2 s−1.
Two different bacterial strains were used, as indicated in the figure legends. Sinorhizobium (Ensifer) medicae WSM419 (pXLGD4 PhemA:lacZ, tetR) was used for nodulating M. truncatula Jemalong and the 13U mutant; Sinorhizobium meliloti 1021 (PnifA:lacZ, tetR) was used for M. truncatula subsp. tricycla (R108) and the vtl4 mutants. S. meliloti 1021 can also establish nitrogen-fixing symbiotic interaction with Jemalong albeit less effectively, and was used in the lux reporter assays for all plant lines. Bacteria were grown in TY medium (1% (w/v) tryptone, 0.3% (w/v) yeast extract, 6 mM CaCl2), either in liquid or solid medium with 1% (w/v) agar.
Identification of the 13U mutation and genotyping of vtl4 lines
A combined approach of genetic mapping and microarray-based Affymetrix GeneChip hybridization was applied to identify the deletion in the 13U mutant line which was isolated from a collection of fast neutron radiation mutants. Initial analysis had placed 13U in linkage group 4 of M. truncatula (Domonkos et al., 2013). The 13U mutant was crossed with the A20 genotype and more than 250 F2 individuals were scored for symbiotic phenotypes in relation to the genetic markers amplified with the primer pairs listed in Table S1. To identify deletions in the genome of 13U, genomic DNA from the 13U mutant was labeled and hybridized to an Affymetrix GeneChip, as described previously (Murray et al., 2011). To determine the boundaries of the deletion on chromosome 4, the region was scanned by PCR amplification using primers designed for the predicted genes in the region (Table S1). For genotyping vtl4 mutants, genomic DNA was used as template for PCR reactions with one primer set designed to amplify the wild-type VTL4 gene and a second primer set with one primer in the VTL4 coding sequence and the other in the Tnt1 sequence to detect the insertion.
Gene expression analysis
To assess VTL4 and VTL8 transcript levels, total RNA was extracted from wild-type and mutant nodules using the RNeasy Mini kit (QIAGEN, Germany) and treated with DNase (Turbo DNase kit, Agilent). cDNA was produced using Thermo SuperScript II Reverse Transcriptase and an anchored oligo-dT primer, and used as template for either standard RT-PCR with products separated by agarose gel electrophoresis, or for quantitative RT-qPCR. RT-qPCR reactions were made using SensiFAST master-mix (Bioline), each with 20 ng of cDNA. Reactions were measured in a Bio-Rad CFX-96 real-time PCR system and cycled as per the Bioline protocol. The expression values were normalized to that of the UPL7 gene (UBIQUITIN PROTEIN LIGASE 7, Medtr7g103210.1)
Agrobacterium rhizogenes-mediated complementation of M. truncatula mutants
Fragments containing the genes VTL4 and VTL8 including the 1.6 and 1.5 kb native promoter sequences and 1.2 and 0.9 kb 3’untranslated regions, respectively, were amplified with the oligonucleotides listed in Table S1 and using BAC (Bacterial Artifical Chromosome) clone mth2-28d20 as a template. The destination vector pKGW-R (gateway.psb.ugent.be/vector/show/pKGW_RedRoot/) was linearized by digestion with restriction enzymes AatII and SpeI. The PCR-generated gene fragments and the linearized vector were fused using the In-Fusion HD Cloning Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Constructs were introduced into ARqua1 strain of A. rhizogenes and used for hairy root transformation as described in the Medicago truncatula Handbook (www.noble.org/MedicagoHandbook).
Fluorescence microscopic analysis of nodules
Transformed roots were identified by fluorescence of the DsRed marker protein. To analyze nodule occupancy by rhizobia, nodule sections were stained with the DNA-binding fluorescent dye SYTO13 (Invitrogen, Eugene, Oregon). The preparation of nodule sections was carried out as described earlier (Domonkos et al., 2013). Sections were stained in 1×PBS (pH 7.4) containing 5 μM SYTO13 for 20 min and rinsed with 1×PBS before analysis. Images were acquired with a Leica TCS SP8 confocal laser scanning microscope with the following configuration: objective lens: HCX PL FLUOTAR 10x/0.30 (dry, NA:0.3), PL FLUOTAR 40x/1.00 OIL and HC PL APO CS2 63x/1.40 OIL, scanning mode: sequential unidirectional; excitation: 488 nm; main dichroic beamsplitter: DM488/552; detection range for the SYTO13 channel was 507-564 nm.
Protein localization
The VTL4 promoter (2566 nt), VTL8 promoter (2096 nt) and coding sequences were domesticated for Golden Gate assembly. A glycine-rich linker sequence (Table S1) was added to the C-terminus followed by the coding sequence of mCherry. As a marker for the plasma membrane, the A. thaliana PIP2A (AT3G53420) sequence was fused to eGFP and placed behind the promoter of L. japonicus UBIQUITIN (GenBank DQ249171.1) The sequences were assembled into backbone vector pL1-R1 and transformed into Agrobacterium rhizogenes strain ARqua1. M. truncatula seeds were sterilized and germinated on water-agar containing 3 mg/ml Nystatin. Approximately a quarter of the root of germinated seedlings was removed before dipping in a suspension of A. rhizogenes bearing the plasmid of interest. Seedlings were then plated out on modified Fahraeus medium (0.7 mM KPO4, 0.8 mM Na2PO4, 0.50 mM MgSO4, 0.7 mM CaCl2, 20 µM Fe-citrate, pH 6.0, and micronutrients (5 µM H3BO3, 9 µM MnSO4, 0.8 µM ZnSO4, 0.3 µM CuSO4 and 0.5 µM H2MoO4) with Nystatin. Plants were grown at a 45° angle in the dark for a week before transferring to fresh plates lined with filter paper in the light. After 3 weeks plants were transferred to fresh modified Fahraeus plates containing Nystatin and 100 nM aminoethoxyvinylglycine (AVG) and inoculated with 0.5 mL S. meliloti 1021 at an OD600 of 0.03 and grown for 21 days. Nodules were harvested, hand-bisected and mounted on microscope slides on double-sided tape, with water as the mounting medium and hi-vac silicone grease as a spacer. Fluorescence was imaged using a Leica TCS SP5 confocal microscope. For eGFP detection the excitation wavelength was 488 nm, emission was measured at 493 – 550 nm with a Hybrid (HyD) detector; for mCherry detection the excitation wavelength was 561 nm, emission was measured at 575 – 650 nm; and for DAPI (DAPI, or 4′,6-diamidino-2-phenylindole) the excitation wavelength was 405 nm, emission was measured at 435 – 477 nm.
PmbfAp:lux reporter and expression analysis in S. meliloti 1021
Nucleotides −487 to +104, containing the promoter sequence and part of the coding sequence of the mbfA gene (SMc00359) were PCR-amplified from S. meliloti 1021 (see Table S1 for primer sequences) and cloned using the BamHI and KpnI restriction sites into pIJ11268 upstream of the lux operon (Frederix et al., 2014). A de-repressed version of the reporter was made by mutating the Iron Control Element (ICE) from TTCTAA to AGCTTC (−19 to −14) by site-directed mutagenesis. The plasmids were transferred from Escherichia coli to S. meliloti by conjugation using a helper strain carrying plasmid pRK2013. For luminescence assays, overnight bacterial cultures were diluted in UMS medium (Wheatley et al., 2017) containing 20 mM 3-morpholinopropane-1-sulfonic acid-KOH pH 7, 10 mM glucose, 10 mM NH4Cl, 8.5 mM NaCl, 2 mM MgSO4, 0.5 mM K2HPO4, 0.51 mM CaCl2, 1x Trace Elements (1 µM Na2-EDTA, 0.6 µM ZnSO4, 0.8 µM Na2MoO4, 4 µM H3BO3, 0.9 µM MnSO4, 0.08 µM CuSO4, 4 µM CoCl2, 3 µM thiamine, 4.2 µM D-pantothenic acid, 0.4 µM biotin, without or with 80 µM FeSO4 as indicated. Luminescence and OD600 of triplicate cultures for each strain and condition were measured in a CLARIOstar microplate reader (BMG LABTECH).
Luminescence imaging and quantification
Plants were inoculated with S. meliloti 1021 carrying the PmbfA:lux reporter plasmid or the mutant form PmbfAICE:lux at 7 days postgermination, and grown for a further 21 days. Plants were dug up, roots rinsed and blotted dry and imaged using the NightOWL II LB 983 in-vivo imaging system with IndiGO software (Berthold Technologies, Bad Wildbad, Germany). Luminescent nodules were identified using the automated peak picking tool and the luminescence in photons per mm2 calculated from the output. Five plants per line were analysed with each inoculum.
Yeast complementation
The yeast strain DY150, which is derived from W303, was used as wild type. The Δccc1 strain in this background carries a genomic deletion of CCC1, initially identified as Cross-Complements Ca2+1, but later shown to mediate vacuolar iron transport (Li et al., 2001). Plant genes were cloned into shuttle vector pYES2 under the control of the GAL1 promoter for galactose-inducible expression. The coding sequence of Arabidopsis VIT1 (AT2G01770) was used as a positive control for functional complementation. Yeast were transformed using the lithium-acetate method and positive transformants were selected on synthetic dropout medium lacking uracil (DSCK102, Formedium, Hunstanton UK) with glucose as carbon source (SD). Overnight cultures of selected colonies were grown in SD-Ura, then spotted onto 2% (w/v) agar plates of SGal-Ura with or without 5 mM FeSO4. Plates were photographed after 4 days (control) or 5 days (with iron).
Author Contributions
J.H.W., R.T.G., G.K.K., A.D., B.H., E.M.B. and M.F. performed the research and helped with designing experiments and data analysis; P.K. and J.B. analysed and interpreted the data and wrote the manuscript.
Acknowledgements
We thank Jeremy Murray, Andy Breakspear and Giles Oldroyd for their generous advice to J.H.W.’s PhD research and for providing gene sequences for Golden Gate cloning. We also like to thank Jeremy Murray for help with identification of the deleted genome region in the 13U line; Allan Downie (John Innes Centre) for the lux plasmid; Penelope Smith (La Trobe University) for sending AtVIT1 and MtVTL4 in pYES2; Thomas Buckhout (Humboldt University Berlin) for the Δccc1 and wild-type yeast strains; Eva Wegel (John Innes Centre) and Zoltán Tóth (Agricultural Biotechnology Institute) for confocal microscopy; Hannah Justice and Heather Bland for phenotype analysis. We thank Kristina Miró (ABC, Gödöllő) for skillful technical assistance. This study was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Grants BB/J004553/1 and BB/P012574/1 (R.G.T., M.F. and J.B.), the Gatsby Charitable Foundation (J.H.W.), and the Hungarian Scientific Research Fund grants NKFIH OTKA 67576 and 119652 (P.K.).