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•Legume–rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals – flavonoids – before infection. Host plants sense counterpart rhizobial signalling molecules – Nod factors – through transient changes in intracellular free-calcium. Here we investigate the potential involvement of Ca2+ in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae.
•By using aequorin-expressing rhizobial strains, we monitored intracellular Ca2+ dynamics and the Ca2+ dependence of nod gene transcriptional activation.
•Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca2+ that were essential for the induction of nod genes. Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca2+ variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca2+ signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca2+ signalling event.
•These findings demonstrate a key role played by Ca2+ in sensing and transducing plant-specific flavonoid signals in rhizobia and open up a new perspective in the flavonoid–NodD paradigm of nod gene regulation.
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The symbiotic association between leguminous plants and Gram-negative soil bacteria, collectively called rhizobia, yields benefits for the plant through nitrogen acquisition and provides a unique, environmentally favourable, alternative to nitrogen fertilizers. The integration of the two symbionts requires a multistep, strictly regulated developmental programme, resulting in the differentiation of root nodules. Within these specialized structures, bacteria, in the form of bacteroids, fix nitrogen by reducing atmospheric N2 to ammonia (Oldroyd & Downie, 2008).
A number of rhizobial species nodulate legumes in a host-specific manner. The two interacting organisms communicate through a mutual exchange of diffusible signal molecules at multiple levels with distinct degrees of specificity (Den Herder & Parniske, 2009). The first step of this molecular cross-talk occurs in the soil interface surrounding the plant root – the rhizosphere – which represents a highly dynamic forefront for interactions between plants and soil microorganisms.
Plant roots continuously produce and secrete into the rhizosphere complex mixtures of chemicals, through which plants modify their environmental proximity and shape soil microbial communities (Badri & Vivanco, 2009). The chemical composition of root exudates is genetically controlled (Michallef et al., 2009) and highly variable, and includes an array of primary and secondary metabolites (Bais et al., 2006). Some components of root exudates, such as sugars, organic acids and amino acids, induce in soil bacteria a chemotactic response towards the root that represents the first step in plant colonization. Among the chemical information delivered by legumes to rhizobia through root exudation, flavonoids are considered crucial for initiating the symbiotic programme in the bacterial partner. Flavonoids are compounds produced by plant secondary metabolism that have multiple roles during legume nodulation (Cooper, 2007; Reddy et al., 2007). The distinct pattern of secreted flavonoids confers specificity to root exudates, enabling the selection of the compatible plant host–microsymbiont pair. Flavonoids initially act as bacterial growth promoters and chemotactic signals, favouring rapid proliferation of rhizobia in the potential infection zone of the plant root. Furthermore, flavonoids are primarily responsible in compatible rhizobia for the expression, among a wide range of genes involved in the symbiosis (Perret et al., 1999), of nodulation (nod) genes encoding enzymes required for the synthesis of the rhizobial signalling molecules known as Nod factors (Dénariéet al., 1996). Some flavonoids act as antagonists (anti-inducers) of nod gene transcriptional activation that is triggered by inducing flavonoids. Thus, the ensuing level of nod gene induction is the result of both stimulatory and inhibitory effects (Cooper, 2004).
According to the currently accepted model, expression of the inducible nod genes in rhizobia is mediated by the transcriptional activator NodD, which has been hypothesized to be involved in flavonoid perception (Schlaman et al., 1992). Several studies have suggested that activation of NodD is mediated by its direct interaction with flavonoids (Peck et al., 2006; Li et al., 2008). In Rhizobium leguminosarum bv. viciae an accumulation of the flavonoid inducer naringenin has been shown to occur in the cytoplasmic membrane (Recourt et al., 1989), where NodD is also localized (Schlaman et al., 1989). Nevertheless, whether such an interaction actually occurs still remains to be ascertained. As a result of nod gene induction, Nod factor is produced, released and perceived by the host plant, which in turn activates its own morphogenetic programme. Early events in the plant-signalling pathway leading to nodulation include root-hair membrane depolarization and ion fluxes. Upon detection of Nod factor, an influx of Ca2+ occurs very quickly, followed by Ca2+ oscillations within and around the nucleus (Oldroyd & Downie, 2006; Sieberer et al., 2009). The information encoded in the Ca2+ spiking is deciphered by a specific Ca2+/calmodulin-dependent protein kinase (Lévy et al., 2004; Mitra et al., 2004), which regulates the expression of plant-nodulation genes through specific transcriptional regulators (Kalóet al., 2005; Smit et al., 2005).
Although much is known about the signalling cascade activated in legumes by Nod factors, information on signal transduction in the bacterial partner is still lacking. In a previous work we found that Mesorhizobium loti, the specific symbiont of Lotus japonicus, detects host-plant root exudates through transient elevations in the level of intracellular Ca2+ (Moscatiello et al., 2009). As flavonoids that specifically activate nod gene expression in M. loti are still unknown, it was not possible to ascertain their putative role in the root exudate-induced Ca2+ signal. For this reason we decided to study flavonoids in R. leguminosarum bv. viciae, in view of the in-depth knowledge of nod gene regulation by specific flavonoids in this bacterium, as well as its well-established genetics and the availability of symbiotically defective mutants. Here we demonstrate that flavonoid inducers trigger transient increases of intracellular Ca2+ in R. leguminosarum bv. viciae, which are essential for the activation of nod gene expression. This novel, early event in the symbiotic route, represented by a Ca2+ signal, was found to be positioned upstream of NodD activity, suggesting that the symbiotic signalling pathway induced by flavonoids in the bacterial microsymbiont may be more complex than has hitherto been thought.
Materials and Methods
Flavonoids (naringenin, luteolin, hesperetin, genistein and daidzein) were obtained from Sigma-Aldrich (St Louis, MO, USA). Coelenterazine was purchased from Molecular Probes (Leiden, the Netherlands). The strigolactone analogue GR24 was provided by B. Zwanenburg (Nijmegen, the Netherlands).
Bacterial strains and growth conditions
Rhizobium leguminosarum bv. viciae strain 300 (Johnston & Beringer, 1975) and R. leguminosarum strain 8401 (which lacks the Sym plasmid and carries either the pIJ1477 plasmid (nodC–lacZ fusion) or the pIJ1478 plasmid (nodD–lacZ fusion) (kindly provided by A. Downie, Norwich, UK)) were grown at 28°C in Tryptone-Yeast (TY) medium containing antibiotics (30 μg ml−1 of kanamycin and 2 μg ml−1 of tetracycline), where appropriate. Escherichia coli JM109 (Yanish-Perron et al., 1985) was grown at 37°C in Luria–Bertani (LB) medium containing 30 μg ml−1 of kanamycin, where appropriate.
Preparation of plant root exudates and arbuscular mycorrhizal fungal germination medium
Germination of sterilized seeds of Vicia sativa subsp. nigra (Vergerio Mangimi S.R.L., Padova, Italy) and spores of Glomus intraradices (Premier Tech Biotechnologies, Riviere-du-Loup, Quebec, Canada), lyophilization and resuspension of plant root exudates and fungal germination medium, respectively, were carried out as previously described (Navazio et al., 2007; Moscatiello et al., 2009).
Transformation of R. leguminosarum
The apoaequorin expression vector pAEQ80 (Moscatiello et al., 2009), and pIJ1518 containing cloned nodD (kindly provided by A. Downie) were introduced into the Rhizobium strains used in this study by the freeze–thaw method, as described by Vincze & Bowra (2006).
Ca2+ measurements with recombinant aequorin
Induction of apoaequorin expression was carried out by inoculating a loopful of pAEQ80-containing Rhizobium strains into 30 ml of TY medium supplemented with 30 μg ml−1 of kanamycin and 1 mM isopropyl thio-β-d-galactoside (IPTG) and culturing the bacteria at 28°C overnight until an absorbance of 0.25 at 600 nm was reached. After extensive washing with buffer A (25 mM Hepes, 125 mM NaCl, 1 mM MgCl2, pH 7.5), apoaequorin was reconstituted to aequorin by incubating the cells for 90 min with 5 μM coelenterazine in buffer A (Campbell et al., 2007). Measurements of Ca2+ were carried out in the presence of 6 mM CaCl2 (the same concentration as in TY medium), which was added to the cells 10 min before the experiments. Briefly, aequorin-expressing bacteria (50 μl) were placed in the luminometer chamber in close proximity to a low-noise photomultiplier, with a built-in amplifier discriminator, whose output was captured using a Thorn EMI photon-counting board (Thorn Electrical & Music Industries (EMI), London, UK). The experiments were terminated by lysing the cells with 15% ethanol in a Ca2+-rich solution (0.5 M CaCl2 in H2O) to discharge the remaining aequorin pool. Luminescence was calibrated offline into cytosolic Ca2+ concentration ([Ca2+]cyt) values by using a computer algorithm based on the Ca2+ response curve of aequorin (Brini et al., 1995).
When needed, cells were pretreated for 10 min with 3 mM LaCl3 or 1 mM EGTA in buffer A.
Extraction of RNA, and reverse transcription–polymerase chain reaction (RT-PCR) analysis of gene expression, were carried out on cells prepared in parallel to those used for measurements of Ca2+, as previously described (Moscatiello et al., 2009). The oligonucleotide primers used in this study (Supporting Information Table S1) were designed against nodA, B, C and D and glutamine synthetase II (GSII) gene sequences from R. leguminosarum (Young et al., 2006), and the aequorin gene from Aequorea victoria (Inouye et al., 1985). To amplify the 16S ribosomal RNA (rRNA) gene, Y1 and Y2 primers were used (Young et al., 1991).
R. leguminosarum 8401 pIJ1518 pIJ1477, containing both a cloned nodD and a nodC–lacZ gene fusion, and R. leguminosarum 8401 pIJ1478, containing a nodD–lacZ gene fusion (Rossen et al., 1985), were grown for 5 h with the specified compounds. The β-galactosidase activity assay was performed as described by Miller (1972).
The statistical significance of differences between means was evaluated using the Student’s t-test: *P <0.05, **P <0.01, ***P <0.005.
Flavonoids that induce nod gene expression activate transient intracellular elevations of Ca2+ in R. leguminosarum bv. viciae
To analyse the participation of flavonoids in the generation of the transient increase in [Ca2+]cyt, we constructed an apoaequorin-expressing strain of R. leguminosarum bv. viciae. Fig. 1(a) shows that root exudates of V. sativa subsp. nigra do indeed elicit a transient elevation of [Ca2+]cyt. The effects of the major flavonoids that induce nod genes (Zaat et al., 1987; Begum et al., 2001) were then tested. Naringenin (10 μM) triggered a rapid elevation in the level of intracellular Ca2+ that was absent in buffer-only controls (Fig. 1b). The naringenin-specific [Ca2+]cyt transient peaked at 1.40 ± 0.22 μM after about 100 s and slowly decreased to basal levels within 15 min. The RT-PCR analyses of gene expression confirmed the well-known activation by naringenin of the common nodABC genes in these apoaequorin-expressing cells (Fig. 1b). Luteolin and hesperetin (10 μM), which are also active inducers of nod gene expression in R. leguminosarum, generated transient elevations in [Ca2+]cyt with kinetics similar to those induced by naringenin (Fig. 1c). By contrast, in apoaequorin-expressing E. coli, none of the flavonoids tested elicited any change in Ca2+, although the same cells were found to be responsive to other stimuli, such as oxidative stress (10 mM H2O2), hypo-osmotic shock (three volumes of distilled water) and 10 mM external Ca2+ (Fig. S1).
The isoflavones daidzein and genistein (10 μM) were unable to promote induction of the nod gene in this species of Rhizobium and were found to be equally unable to induce any detectable transient increase in Ca2+ (Fig. 2a). No effect on either Ca2+ homeostasis or nod gene activation was observed with increasing concentrations (up to 50 μM) of the above isoflavones (data not shown).
As noninducing flavonoids have been demonstrated, in Sinorhizobium meliloti, to act as competitive inhibitors of inducers, preventing the transcriptional activator NodD from promoting nod gene induction (Peck et al., 2006), we determined whether inducers and noninducers compete also in the generation of the Ca2+ signal. Competition experiments were carried out by examining the effect of increasing concentrations of daidzein on the naringenin-elicited elevation of [Ca2+]cyt. While confirming the competitive inhibition of noninducers at the level of nod gene transcription, no effect was observed on the magnitude and overall kinetics of the naringenin-activated Ca2+ transient (Fig. 2b,c).
Naringenin-induced expression of nod genes is Ca2+ dependent
To evaluate the role of Ca2+ in the bacterial symbiotic signalling pathway activated by flavonoids, we evaluated the effect of blocking the naringenin-induced Ca2+ change on the downstream activation of nod genes. Fig. 3(a) shows a dramatic attenuation of the naringenin-induced Ca2+ transient, regardless of whether Ca2+ activity is lowered in the extracellular medium with the chelator EGTA or if Ca2+ entry into cells is putatively inhibited by the Ca2+-channel blocker LaCl3. Induction of nodABC genes by naringenin was effectively prevented by the abolition of the intracellular Ca2+ change (Fig. 3b), demonstrating that expression of common nodABC genes requires an upstream Ca2+ signal.
In control experiments, it was verified by RT-PCR analysis that neither LaCl3 nor EGTA significantly affected the expression of constitutive genes such as GSII (Fig. 4a) or nodD (Fig. 4b), ruling out potential general effects of these pharmacological agents on gene activation. Beta-galactosidase assays, which were carried out in a R. leguminosarum pSym− (cured) strain containing either the pIJ1477 plasmid (nodC–lacZ fusion) and pIJ1518 (cloned nodD), or the pIJ1478 plasmid (nodD–lacZ fusion) (Rossen et al., 1985), confirmed the inhibitory effect played by EGTA specifically on the expression of common nodulation genes (Fig. 4c,d).
Other common components of plant root exudates do not trigger intracellular Ca2+ changes in rhizobia
To determine whether rhizobia sense, through Ca2+, other chemical signals in addition to specific flavonoids of V. sativa root exudates, we tested the chemoattractants homoserine (Armitage et al., 1988), mannitol, galactose and pyruvate (Miller et al., 2007). The basal level of Ca2+ was not increased by any of the compounds tested (Fig. 5a), suggesting that Ca2+ is not involved in the perception of these chemoeffectors.
In view of the ability of legumes to enter a dual symbiosis with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi, GR24, a synthetic analogue of strigolactones – recognition signals for AM fungi released by plant roots (Akiyama et al., 2005) – was applied to R. leguminosarum cells. No transient elevation in the intracellular concentration of Ca2+ was detected, suggesting that rhizobia do not perceive, at least through Ca2+ signalling, diffusible molecules specifically addressed to the other legume microsymbiont (Fig. 5b).
Rhizobia in the rhizosphere can experience the presence of the AM fungal co-partner through the signalling molecules (Myc factor) released by the AM fungus and recognized by the plant host. The germination medium of G. intraradices did not induce, in R. leguminosarum, any detectable change in the concentration of Ca2+ (Fig. 5b), supporting the lack of Ca2+-mediated dialogue between the two symbionts of leguminous plants. None of the tested compounds triggered the activation of nod gene expression (Fig. 5c).
These data suggest that the association between the Ca2+ transient and nod gene induction by flavonoids is not only required, but is also specific.
The transcriptional activator NodD is not involved in the generation of the Ca2+ signal
To investigate the position of the Ca2+ signal with respect to the activity of the transcriptional activator, NodD, the R. leguminosarum 8401 Sym− strain, which lacks its symbiotic plasmid, was tested in the Ca2+ measurement assays upon treatment with inducing flavonoids. This Sym− mutant, which does not possess nodD or the other nod genes, retained its ability to respond to naringenin with an unchanged Ca2+ response compared with the wild-type strain. The same occurred in the Sym− strain carrying the pIJ1477 plasmid, which has a nodC–lacZ fusion (Fig. 6a). The noninducing flavonoid, daidzein, failed to trigger any transient elevation in [Ca2+]cyt (Fig. 6a), as observed in the wild-type strain. No nodC transcriptional activation was detected in response to naringenin, which was restored by reintroducing the cloned nodD gene into the bacterial strain (Fig. 6b). The above results indicate that NodD per se is not involved in generating the Ca2+ increase, and plays its role downstream of the Ca2+ signal.
The present results demonstrate that flavonoids, known to be specific nod gene inducers in R. leguminosarum bv. viciae, trigger Ca2+-mediated signalling in the bacterial symbiont. The transient intracellular Ca2+ changes elicited by inducing flavonoids in aequorin-expressing rhizobial cell cultures appear to be central in encoding biological information that is subsequently decoded into a downstream event (activation of nod gene expression) related to symbiosis signal transduction. The pivotal involvement of Ca2+ transients in processing plant symbiotic signals by rhizobia is evident from the following: the specificity of the plant-signalling molecules that activate the Ca2+ influx (intracellular Ca2+ increases are triggered by the inducing flavonoids naringenin, luteolin and hesperetin, but not by the noninducing flavonoids daidzein and genistein or other molecules contained in root exudates, such as chemoattractants) and the specificity of the downstream event, which is blocked in the absence of the Ca2+ signal (nodABC genes, which are involved in the synthesis of Nod factors, are no longer transcriptionally induced when the naringenin-dependent Ca2+ elevation is inhibited). These findings indicate the Ca2+ dependence of the expression of common nod genes and highlight the link between Ca2+ signals and transcriptional regulation.
We have recently demonstrated that plant cells perceive diffusible molecules (Myc factor) released by endomycorrhizal fungi through variations in intracellular Ca2+ levels (Navazio et al., 2007) and it is plausible that AM fungi also use Ca2+ to sense plant signals during the early stages of AM symbiosis (Requena et al., 2007). In view of the lack of Ca2+ response by rhizobia to either plant-to-fungus or fungus-to-plant signals, it seems that the Ca2+-mediated initial symbiotic conversation is limited to the specific matching pair (rhizobium and its host legume).
The conventional model of nod gene transcriptional activation implies an interaction between flavonoids and the transcriptional activator, NodD, which promotes the expression of nod genes (Schlaman et al., 1992). The exact mechanism by which NodD responds to inducing flavonoids to initiate nod gene transcription has not been fully unravelled. It has been demonstrated in R. leguminosarum that modulation of DNA bending by NodD in response to naringenin allows the formation of an active transcriptional complex that induces nod genes (Fisher & Long, 1993; Chen et al., 2005). Peck et al. (2006) showed in S. meliloti that noninducing flavonoids act as competitive inhibitors of inducers by antagonizing nod gene transcriptional activation. Our competition experiments with naringenin/daidzein in R. leguminosarum showed that daidzein affects neither the magnitude nor the overall kinetics of the naringenin-activated Ca2+ transient, while confirming the inhibitory effect of noninducing flavonoids at the level of nod gene induction. Thus, inducing and noninducing flavonoids do not compete for the generation of the Ca2+ signal, suggesting that competition for the same ligand-binding site is unlikely.
The presence of an unchanged Ca2+ response upon stimulation with inducing flavonoids in the Sym− strain of R. leguminosarum indicates that NodD is not involved in originating the transient elevation of [Ca2+]cyt, which is therefore located, timewise, upstream of NodD activity. The conserved ability of the cured derivative to respond to naringenin with a Ca2+ transient also suggests that NodD may not be directly responsible for the earliest events of flavonoid perception. The occurrence of a transmembrane signalling system able to couple flavonoid perception to nod gene induction via the involvement of a secondary signal has been previously hypothesized (Djordjevic et al., 1987). The lack of nod gene activation in the Sym− strain, and the rescue of nodABC gene expression upon the re-introduction of the nodD gene, confirm that NodD is nevertheless essential for the transcriptional activation of common nod genes (Mulligan & Long, 1985), being a central regulator in the flavonoid-induced Ca2+ signalling.
Based on these results, an early crucial step seems to precede and prime NodD-induced nod gene transcription along the symbiotic signalling pathway activated in R. leguminosarum by inducing flavonoids. After detection of the specific inducers, a transient elevation of intracellular Ca2+ is generated and the message subsequently transduced to NodD. Thus, Ca2+ signalling links flavonoid sensing to NodD-mediated gene activation. Noninducing flavonoids, in view of their ability to antagonize nod gene activation without triggering a Ca2+ signal, may operate through a Ca2+-independent pathway, which converges at the level of NodD by interfering with the inducer-activated signalling pathway.
NodD is not likely to be per se the Ca2+ sensor, because its amino acid sequence lacks evident sites of Ca2+ binding. Other possibilities may be envisaged, such as modulation of NodD activity by a calmodulin-like protein or the direct Ca2+-dependent phosphorylation of NodD (the amino acid sequence of the protein shows several potential phosphorylation sites). Alternatively, additional Ca2+-regulated factors may play a role as activators or repressors of nod gene transcriptional regulation. Our results are not in conflict with the data of Burn et al. (1987, 1989) who isolated R. leguminosarum bv. viciae mutants (class IV) with the ability to activate the transcription of nod genes in the absence of inducers. The authors hypothesized that single amino acid substitutions in NodD might stabilize the protein in an active conformational state with the ability to promote nod gene transcription in a flavonoid-independent manner. This conformational change of NodD would allow the requirement of Ca2+-dependent signal transduction activated by flavonoids to be bypassed. The same applies for the nodD flavonoid-independent transcriptional activation (FITA) mutations described by Spaink et al. (1989).
The Ca2+-mediated perception of plant-to-microbe symbiotic signalling molecules discloses an additional level of complexity in the communication network between legumes and their nitrogen-fixing endosymbionts. Based on the well-known wide effect of flavonoids on gene expression that goes beyond the activation of nod genes (Perret et al., 1999), future work can be aimed at elucidating additional signalling pathways possibly mediated by Ca2+, such as those leading to the secretion of nodulation signalling proteins via the export pathways (Krehenbrink & Downie, 2008; Downie, 2010).
It remains to be established whether the implication of Ca2+ in transducing the symbiotic message is restricted to rhizobia, such as M. loti (Moscatiello et al., 2009) and R. leguminosarum bv. viciae (this paper), which utilize the Nod factor strategy for nodulation, or is a universal signalling mode used by all nitrogen-fixing symbiotic bacteria. This is a crucial issue for consideration in future studies, in view of the increasing evidence for the rhizobial phylogenetic diversity underlying the variety of infection-process mechanisms used by rhizobia to enter into symbiosis with legumes (Masson-Boivin et al., 2009) and owing to the recent demonstration of the possibility of converting a plant pathogen into a legume symbiont (Marchetti et al., 2010).
Our findings concerning how rhizobia sense plant flavonoids support the notion that both legumes and rhizobia use the same intracellular transducer – Ca2+– to process the molecular information that is reciprocally exchanged during the early steps of their mutualistic encounter. This confirms the universality of Ca2+ signalling (Dominguez, 2004; Clapham, 2007; Dodd et al., 2010) and its crucial role in the molecular communications underpinning plant–microorganism symbiotic associations.
Deciphering the Ca2+-based bacterial perception of symbiosis-related plant signals contributes to the acquisition of a unified perspective into rhizobial and plant reprogramming for endosymbiosis.
We are grateful to A. Downie (Norwich, UK) for kindly providing R. leguminosarum strain 8401, 8401 pIJ1477 (nodC–lacZ), 8401 pIJ1478 (nodD–lacZ) and E. coli containing the pIJ1518 (nodD) plasmid. We thank R. Rizzuto (Padova, Italy) for helpful discussions, and D. Sanders (York, UK) for critical reading of the manuscript. This work was supported by Progetti di Ricerca di Ateneo from the University of Padova (CPDA063434) and Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (2008WKPAWW) to L.N.