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- Materials and Methods
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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.
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- Materials and Methods
- Supporting Information
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.