Legumes acquired the ability to engage in a symbiotic interaction with soil-borne bacteria and establish a nitrogen-fixing symbiosis in a novel root organ, the nodule. Most legume crops and the model legumes Medicago truncatula and Lotus japonicus are infected intracellularly in root hairs via infection threads that lead the bacteria towards a nodule primordium in the root cortex. This infection process, however, does not reflect the great diversity of infection strategies that are used by leguminous plants. An alternative, intercellular invasion occurs in the semiaquatic legume Sesbania rostrata. Bacteria colonize epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium. This infection mode evolved as an adaptation to waterlogged conditions that inhibit intracellular invasion. In this review, we discuss the molecular basis for this adaptation and how insights into this process contribute to general knowledge of the rhizobial infection process.
Plants have evolved symbiotic interactions with soil-borne micro-organisms to enhance nutrient uptake. An ancient symbiosis of plants and Glomeromycota fungi, called arbuscular mycorrhiza, involves fungal hyphae that penetrate the root and form ‘arbuscules’ inside cortical cells (Parniske, 2008). These arbuscules are the interface for the uptake of phosphorus (Pi) and inorganic nitrogen (N) as well as micronutrients that benefit the plant. The evolutionarily more recent bacterial endosymbioses for biological N fixation are restricted to a limited number of subgroups of the Eurosid I clade of flowering plants; they include actinorhizeal interactions with Gram-positive Frankia bacteria and legume interactions with Gram-negative rhizobia (Doyle, 1998; Zhu et al., 2007; Wang et al., 2009). Legume roots exude numerous compounds, among which flavonoids serve as signalling molecules for compatible rhizobia; these rhizobia produce a return signal, designated the Nod factor (NF), which consists of a chitin oligomer with an acyl chain at the nonreducing end and various decorations at both the reducing and nonreducing ends (D’Haeze & Holsters, 2002). Recognition of NFs by Lysin motif (LysM)-type plant receptors triggers the formation of novel organs on the roots, called nodules. Within these nodules, differentiated bacteria reduce atmospheric N and provide fixed N to the host.
Nodule organogenesis is a complex interplay of two distinct programmes: bacterial infection and organ initiation. Best studied is the infection process in model legumes, such as Medicago truncatula, in which responsive root hair cells form a curl that is occupied by bacteria. Within the curl, local invagination of the plasma membrane and inverted tip growth result in the formation of a tubular infection thread (IT) that guides the bacteria towards the inner cortical cells where organogenesis is initiated (Oldroyd & Downie, 2004). Upon reaching the dividing cells of the nodule primordium, bacteria are released from the ITs and are internalized into the plant cells. During this process, they become surrounded by a plant membrane and differentiate into N-fixing bacteroids, resulting in a new organelle, called the symbiosome (Oldroyd & Downie, 2004). However, this infection mechanism is not the only existing one and other colonization modes have been observed in different legumes (Guinel & Geil, 2002).
Forward genetic screens in model legumes have identified numerous genes involved in the establishment of both rhizobia and mycorrhiza symbioses. Interestingly, several genes are needed for the two symbioses, which have been designated ‘common Sym genes’; to date, genes encoding a leucine-rich repeat (LRR) receptor-like kinase (SymRK), cation channels, nuclear pore complex proteins, a calcium- and calmodulin-dependent protein kinase (CCaMK), and a nuclear coiled coil protein have been characterized (Parniske, 2008). This ‘common Sym’ pathway functions in a specific cellular response to NFs: the generation and interpretation of calcium oscillations that occur in and around the nucleus of responsive root hair cells (Oldroyd & Downie, 2006). Oscillations start within minutes of NF application and have a distinctive asymmetric shape with a rapid increase in cytosolic calcium concentrations and a slower decrease back to baseline. In M. truncatula, spiking frequencies differ from cell to cell, but the average period between spikes is c. 100 s. It has been hypothesized that information is encoded in this frequency and spike shape, activating specific gene expression cascades that result in nodule initiation (Oldroyd & Downie, 2006). CCaMK is probably involved in decoding the spiking signal and instigating downstream signalling and specificity. Gain-of-function mutations in the gene encoding this protein result in the spontaneous formation of nodules in the absence of bacteria, revealing the essential role of calcium signalling in the establishment of the symbiosis (Gleason et al., 2006; Tirichine et al., 2006).
Our understanding of nodulation signalling has greatly improved, propelled forwards by research on model legumes. However, the legume family is huge, with diverse nodulation features that cannot be reflected in the few species studied to date. In this review, we discuss research carried out over the past decade in the tropical legume Sesbania rostrata, which has versatile nodulation features and a dual rhizobial infection pathway. Thanks to these studies, key questions in nodulation research could be addressed from a new angle and the results obtained broadened our knowledge of rhizobial infection.
Water-tolerant nodulation in Sesbania rostrata
Sesbania rostrata (Brem.) is a semi-aquatic legume indigenous to the Sahel region of Africa. As an adaptation to growth in temporarily flooded habitats, it carries rows of dormant adventitious root primordia that develop into roots when they become submerged. Remarkably, these adventitious root primordia can develop into ‘stem nodules’ (Duhoux, 1984; Ndoye et al., 1994). Moreover, on waterlogged and mainly hairless roots, nodules are formed at lateral root bases (Ndoye et al., 1994). In both cases, bacterial infection does not happen via the root hair curling mechanism, but occurs intercellularly in the outer cortex (Ndoye et al., 1994; Goormachtig et al., 1998). Bacteria enter via epidermal fissures at sites of side-root emergence and thus gain direct access to the cortex (Ndoye et al., 1994; Goormachtig et al., 1998). Interestingly, and in direct contrast to the situation in M. truncatula, nodulation on stems and submerged roots of S. rostrata is not inhibited by the gaseous plant hormone ethylene, but actually requires it (Oldroyd et al., 2001; D’Haeze et al., 2003), which makes sense because ethylene does not diffuse easily in water and accumulates; hence, S. rostrata acquired an ethylene-dependent method of nodulation to access biologically fixed N under conditions that inhibit the default nodulation mode (Goormachtig et al., 2004b). This cortical infection pathway has been studied in great detail.
Rhizobia present in an epidermal fissure induce localized cortical cell death in an NF-dependent manner. This process involves the production of hydrogen peroxide, ethylene and gibberellins and allows the bacteria to proliferate in cortical infection pockets (IPs) (D’Haeze et al., 1998, 2003; Lievens et al., 2005). Intercellular and, eventually, intracellular ITs are launched from IPs towards the incipient nodule primordium and a fully functional nodule is formed. Presumably, the IP is a signalling centre that provides a source of concentrated NF signal and a positional cue that allows the establishment of suitable signalling gradients, resulting in proper organogenesis and infection (Goormachtig et al., 2004a) (Fig. 1). While hydroponically grown S. rostrata roots are bald, some root hair initials occur at the lateral root bases which, when challenged with purified NFs, respond with outgrowth and deformation, but are not invaded by bacteria (Mergaert et al., 1993; D’Haeze et al., 2000; Lievens et al., 2005).
The canonical root hair infection pathway exists in S. rostrata
In contrast to growth under submergence, S. rostrata roots grown under nonflooded conditions have susceptible root hairs that are invaded via root hair curling and intracellular ITs (Goormachtig et al., 2004b). As in M. truncatula, this root hair infection is inhibited by ethylene (Goormachtig et al., 2004b). Interestingly, the requirements of NF structure for signalling in root hair invasion were more stringent than those for intercellular, cortical invasion under waterlogged conditions (D’Haeze et al., 2000; Goormachtig et al., 2004b). This observation was reminiscent of work in M. truncatula in which NF structure stringencies differed for infection initiation and cortical cell division (Ardourel et al., 1994), leading to a model with a nonstringent signalling receptor complex for root hair deformation and gene expression and a stringent entry receptor complex for bacterial entry into the root hair cells (Ardourel et al., 1994). Thus, because in S. rostrata the NF structural requirements associated with root hair invasion are more stringent than those with intercellular cortical invasion, this stringent entry response only occurs in root hair infection and is not necessary for intercellular infection, where rhizobia enter the cortex directly. The root hair initials at the lateral root bases of hydroponically grown roots show a nonstringent response to NFs and do not become infected, suggesting that the stringent response for invasion signalling is missing (Figs 1 and 2). These observations inspired experiments to investigate the mechanism behind the loss of the stringent epidermal response in hydroponic Sesbania roots.
A role for common Sym genes in hydroponic nodulation?
It was a challenging idea that maybe intercellular infection of S. rostrata did not require the common Sym genes that had been isolated in other legumes (Oldroyd & Downie, 2004). The common Sym gene SymRK is needed for bacterial infection and NF-induced calcium spiking in the epidermis in M. truncatula (Oldroyd & Downie, 2004). Hydroponically grown transgenic roots of S. rostrata with strongly reduced SrSymRK transcript levels still formed nodules and IPs, but closer inspection showed that these nodules were severely impaired. Swollen and deformed ITs in an otherwise differentiated nodule were no longer able to release bacteria, resulting in nodules unable to fix N (Capoen et al., 2005). This surprising result revealed two things. First, rhizobia skip the epidermal stages of nodulation and utilize a different infection approach via intercellular IPs, the formation of which seems to be independent of SymRK activity. The second observation has more widespread implications: at least one early nodulation gene has an additional, previously unknown, role at later stages of nodule development, namely bacterial release into the primordium cells. The intermediate stages, such as cell division initiation and IT elongation, were not affected, although the ITs were deformed (Fig. 1). It is probable that more common Sym genes will have functions at later stages of nodulation, because many of them are expressed in mature nodules in the zone of bacterial release and differentiation (Limpens et al., 2005).
Calcium oscillations differ in the two infection events in S. rostrata
In S. rostrata, ethylene is required for IP formation (D’Haeze et al., 2003), but inhibits root hair invasion (Goormachtig et al., 2004b). In M. truncatula, NF-induced calcium spiking is extremely sensitive to ethylene because addition of the ethylene biosynthesis precursor 1-aminocyclopropane-3-carboxylic acid (ACC) abolishes spiking, whereas the ethylene biosynthesis inhibitor l-α-(2-aminoethoxyvinyl)-glycine (AVG) enhances spiking (Oldroyd et al., 2001). Given the differences between the molecular mechanisms at the early stages of intercellular and intracellular infections, and especially the opposite requirements for the plant hormone ethylene, the elucidation of the role of calcium spiking in both processes became central.
Root hair initials close to the fissures at lateral root bases respond to NFs with outgrowth and deformation, but not with infection. Visualization of NF-induced calcium changes in these cells indeed revealed calcium oscillations in these hydroponically grown cells (Capoen et al., 2009) and the spiking patterns differed distinctly from those described for root hair invasion in M. truncatula. Hydroponic, NF-induced spiking in the root hair initials of submerged roots was faster than that in M. truncatula, and the spikes were almost symmetrical, whereas in M. truncatula they had a rapid upward slope and a gradual downward slope (Ehrhardt et al., 1996). Hydroponic roots of S. rostrata grown in the presence of the ethylene biosynthesis inhibitor AVG develop root hairs that can be invaded for nodulation (Goormachtig et al., 2004b). These root hairs present a calcium spiking signature similar to that seen in M. truncatula, that is, slow and asymmetric (Capoen et al., 2009). These data show that legumes with a particular calcium signature can undergo intracellular root hair infection.
In M. truncatula, the spiking frequency can be modulated with jasmonic acid and this effect is exacerbated in the ethylene-insensitive mutant sickle, an indirect indication that the ethylene status influences calcium oscillations (Sun et al., 2006). Addition of either jasmonic acid or AVG to hydroponic cells of S. rostrata significantly altered the spiking pattern; in other words, the frequency decreased and the spikes became asymmetric (Capoen et al., 2009). When hydroponically grown roots were pretreated with either of these compounds, thus slowing down the intrinsic spiking frequencies of root hair cells, after the addition of rhizobia, intracellular infection was observed in lateral root base-associated root hair initials (Capoen et al., 2009). Hence, a change in spiking frequency alters the ability of cells to become infected by rhizobia.
In order to ascertain the role of calcium in deeper cell layers, CCaMK, the kinase that presumably interprets the calcium signal in root hair cells, was knocked down in S. rostrata (Capoen et al., 2009). Transgenic roots with strongly reduced CCaMK transcript levels no longer produced N-fixing nodules. However, IPs were found in these lines, suggesting that IP formation does not depend on calcium spiking, as opposed to IT formation and nodule organogenesis (Capoen et al., 2009) (Fig. 1).
Intercellular invasion is widespread throughout the legume phylogeny
Intercellular invasion by rhizobia is a widespread adaptation to situations that do not allow root hair infection. Infection through epidermal fissures occurs in distantly related legumes in distinct subfamilies (Guinel & Geil, 2002). This type of infection is more primitive and transcriptionally more simple than the more complex IT symbiosis that seems to have evolved later. However, some legumes retained intercellular infection strategies to cope with adverse conditions (Capoen et al., 2007; Sprent, 2007). Indeed, the basal Mimosoid legume Neptunia plena, like S. rostrata a water-tolerant legume capable of nodulation via intercellular infection, can form also intracellular ITs when roots grow under aerated conditions (Goormachtig et al., 2004b). Hence, these legumes have the ability to switch to intercellular invasion under the high-ethylene, root hair invasion-inhibiting conditions under which their roots usually thrive upon submergence.
Nevertheless, recent findings indicate that a predisposition for intercellular invasion might not be exclusive to water-tolerant legumes. In Lotus japonicus, several mutants have been identified with altered root hair morphology, of which root hairless 1 (Ljrhl1) showed intercellular infection reminiscent of IP formation in S. rostrata, which led to infected nodules (Karas et al., 2005). It would be interesting to see whether another Lotus species, Lotus creticus, a water stress-tolerant legume, has a similar or even enhanced propensity for intercellular infection.
Perhaps water-tolerant legumes have recruited and elaborated on an inefficient crack entry infection system that is present in many legumes. As intercellular infection is transcriptionally less complex (Capoen et al., 2007), it might be more easily induced in nonlegumes than the highly regulated root hair invasion. This aspect of legume biology might become important in the future, as transferring the nodulation process to nonlegumes is a dream of many legume biologists. Sesbania rostrata has proved to be a useful model system with which to study intercellular infection and great progress has been made in the understanding of the molecular underpinnings of this process.
Certain environmental conditions have a strong effect on the presence of hairs on roots or their physiological state, leading to situations where nodulation is repressed and plants must adapt. In flooded habitats, ethylene accumulates (Bailey-Serres & Voesenek, 2008) and may inhibit root hair infection, by interfering with NF-induced calcium spiking (Oldroyd et al., 2001). Hence, an alternative invasion strategy has been developed that is independent of the stringent uptake mechanism in the epidermis. In contrast to root hair infection, IP formation requires ethylene as well as production reactive oxygen species and gibberellins (D’Haeze et al., 2003; Lievens et al., 2005). Bacteria proliferate massively in these IPs and must generate sufficient amounts of NFs. Indeed, when applied exogenously, millimolar quantities of NFs can induce the formation of nodule-like structures on hydroponically grown roots of S. rostrata (Mergaert et al., 1993). Strong knockdown of CCaMK expression does not interfere with IP formation, but calcium spiking interpretation clearly is required for nodule organogenesis (Capoen et al., 2009).
Despite the occurrence of some NF signalling in the axillary root hairs at lateral root bases of submerged S. rostrata roots, this response is incapable of generating the appropriate signature necessary to accommodate intracellular infection (Capoen et al., 2009). Only when calcium spiking frequencies are altered pharmacologically can these root hairs be infected by rhizobia (Capoen et al., 2009). In M. truncatula, it has been suggested that appropriate signalling by the signalling receptor complex affects the function of the entry receptor complex (Geurts et al., 2005) (Fig. 2). Indeed, some gene sets that are still expressed in the hair curling (hcl) mutant, which is affected in the proposed entry receptor, are not expressed in an NF perception mutant (nfp) or in NF signalling mutants, such as doesn't make infections 1 (dmi1), dmi2, dmi3, nodulation signaling pathway 1 (nsp1) and nsp2 (mutated in genes encoding a nuclear cation channel, an LRR-RLK, CCaMK, and two GRAS proteins, respectively) (Geurts et al., 2005;Mitra et al., 2004). This observation suggests that, to obtain a fully functional signalling-to-entry pathway, an appropriate calcium spiking frequency needs to be established. The current paradigm dictates that calcium spiking is involved in transcriptional reprogramming of cells to allow accommodation (Oldroyd & Downie, 2006). It seems reasonable to assume that certain components required for bacterial entry are only expressed upon recognition of NFs and only then can entry occur (Fig. 2).
The common Sym gene SymRK is not involved in signalling for cortical invasion and nodule initiation at lateral root bases, because infected nodules develop on SymRK knockdown lines; however, SymRK seems to be necessary for proper IT maintenance and, most importantly, for bacterial release into plant cells and the establishment of functional symbiosomes (Capoen et al., 2005).
Intracellular infection of root hairs proceeds identically in M. truncatula and S. rostrata and requires the same common Sym pathway (Capoen et al., 2005, 2009). This observation reveals that intercellular infection is different from the default infection pathway at both the morphological and the molecular levels.
Sesbania rostrata has become a new model organism that complements model legume research in many ways. The fact that it does not necessarily require epidermal infection stages to nodulate allows it to be used as an elegant system with which to infer late functions for genes that are essential for epidermal infection. Sesbania rostrata also excels as a model for intercellular invasion and water-tolerant nodulation, two traits that might become very important in future attempts to transfer nodulation to crops, such as rice (Oryza sativa).
The authors thank Martine De Cock for help with preparing the manuscript and Karel Spruyt for the illustrations. This work was supported by the European Union Grain Legume Integrated Project (Food-CT-2004-506223). GO is funded by a David Philips Fellowship and a Grant in aid of the BBSRC, a Wolfson Research Merit award of the Royal Society, and the European Molecular Biology Organization (EMBO) Young Investigator Program. WC is indebted to the EMBO for a long-term fellowship.