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Keywords:

  • symbiosis;
  • quorum sensing;
  • biofilm;
  • Nod factor

Abstract

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

Rhizobia adopt many different lifestyles including survival in soil, growth in the rhizosphere, attachment to root hairs and infection and growth within legume roots, both in infection threads and in nodules where they fix nitrogen. They are actively involved in extracellular signalling to their host legumes to initiate infection and nodule morphogenesis. Rhizobia also use quorum-sensing gene regulation via N-acyl-homoserine lactone signals and this can enhance their interaction with legumes as well as their survival under stress and their ability to induce conjugation of plasmids and symbiotic islands, thereby spreading their symbiotic capacity. They produce several surface polysaccharides that are critical for attachment and biofilm formation; some of these polysaccharides are specific for their growth on root hairs and can considerably enhance their ability to infect their host legumes. Different rhizobia use several different types of protein secretion mechanisms (Types I, III, IV, V and VI), and many of the secreted proteins play an important role in their interaction with plants. This review summarizes many of the aspects of the extracellular biology of rhizobia, in particular in relation to their symbiotic interaction with legumes.


Foreword

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

This article will focus mostly on the aspects of extracellular biology associated with Rhizobium–legume interactions, ranging from initial attraction and attachment through to root colonization and infection. It will start with a summary overview of the essential events leading to nodule formation on legumes by almost all rhizobia. These events include exchange of signals between the two prospective symbiotic partners and the ability of the bacteria to initiate developmental changes in legumes leading to the formation of infected nitrogen-fixing nodules. The review will then focus on how the extracellular biology of rhizobia modifies and enhances these different stages of interactions with legumes, particularly those that are infected via root-hair infections. To optimize legume infection and form effective nitrogen-fixing nodules, rhizobia export different surface polysaccharides and proteins, deliver proteins into plant cells and use quorum-sensing regulation of gene expression to coordinate their behaviour in ways that enhance and spread their symbiotic capacity. Rhizobia are also highly attuned to recognize and utilize various plant-made components, some of which act as signals and others as nutrients during different stages of the interaction.

Overview of Rhizobium–legume interactions

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

Root attachment

Rhizobia survive in soil over periods of several years even in the absence of their legume hosts. This survival is probably attributable to their ability to grow and survive in the rhizosphere of various different plant types. Most rhizobia are in the Alphaproteobacteria and are closely related to nonsymbiotic soil bacteria (Sawada et al., 2003); however, rhizobia are phylogenetically widely distributed, even extending into the Betaproteobacteria (Chen et al., 2003b), and the evolution of new diverse strains of rhizobia is attributed to the horizontal transfer of symbiosis genes into different types of bacteria (Young & Haukka, 1996; Chen et al., 2003b). The distinctive ability of rhizobia to enter into a symbiosis with legume plants, leading to the formation of nodules containing many nitrogen-fixing bacteria, develops as a consequence of several different stages of interaction, each with increasing specificity for a given host–legume pairing.

Among other compounds, some root-derived phenolics can act as chemotactic attractants (Armitage et al., 1988; Dharmatilake & Bauer, 1992). Mutations affecting flagellum formation can delay nodulation, although it is possible that this could be due to effects on biofilm formation on roots (Fujishige et al., 2006). The attachment of rhizobia to roots and root hairs is a very important step in the initiation of the symbiosis and, as described below, involves both secreted proteins and surface polysaccharides. At least three aspects of root attachment are of crucial importance. Firstly, close proximity to roots and root hairs ensures a supply of nutrients that enable the bacteria to grow on and around the root so that a biofilm can rapidly build up on root hairs anchored via the bacteria that are directly attached to the root hairs. In addition, those bacteria that are attached to the roots hairs are best placed to be in a position to win the lottery that can determine whether they will be the ones that can successfully initiate infection, which is a clonal event. This is important because the winner, derived from a single attached cell or microcolony, gets a chance to multiply within the plant to very high numbers. Thirdly, although root-hair deformation can occur in response to Nod-factor signals, root-hair curling (which is when the root-hair curls tightly back on itself, entrapping bacteria) is only possible when induced by attached rhizobia; this root-hair curling is essential for most infections in many legumes. Therefore, being in the right place at the right time to initiate nodulation signalling can be critical.

Signalling between rhizobia and their host plants

Signalling between rhizobia and legumes is one of the best examples of signal exchange identified in interactions between bacteria and their eukaryotic hosts. The fundamentals of the signalling between the prospective symbiotic partners were established some years ago and in essence are relatively simple (Downie, 1998; Perret et al., 2000; Spaink, 2000). The legumes secrete signals, usually phenolics (often flavanoids or isoflavanoids), which can passively diffuse across the bacterial membrane (Recourt et al., 1989); the bacteria recognize these using a positively acting transcription factor, usually encoded by nodD. Different legumes secrete different types of signals and rhizobia have different NodD proteins (frequently more than one) that are attuned to recognizing these root-exudate signals. The activated NodD proteins bind to highly conserved bacterial promoters (at so-called nod boxes) and induce the expression of several genes. Many of these genes are involved in the biosynthesis and secretion of signalling molecules called Nod factors, which are oligomers of usually four or five 1,4-linked N-acetyl-glucosamine residues that carry an N-linked acyl group (Fig. 1). These Nod factors are primary determinants of which legumes the bacteria will be able to nodulate: the different biovars and species of rhizobia generate a diverse range of Nod factors (Downie, 1998; Perret et al., 2000). Host–legume specificity is determined by the precise substitutions on the Nod factors (Fig. 1). For example, the N-linked acyl chain can vary, and there are acetyl, carbamoyl, methyl and suphuryl substituents that can be attached to the N-acetyl-glucosamine backbone by a variety of nod gene products. Additional sugars such as fucose and arabinose can also be added to different locations, and in turn, these sugars can carry various substituents that can further contribute to signalling specificity (Downie, 1998; Perret et al., 2000).

image

Figure 1.  Diversity of Nod factors. Nod factors usually contain four (as shown) or five β-1-4-linked N-acetyl-glucosamine residues. Different decorations are attached to this Nod factor backbone and the major sites of decorations are indicated as I–VI. The different types of decorations generated by a variety of rhizobia are indicated in the box and the nod gene products responsible are indicated in parentheses. References describing the biosynthesis of Nod factors and the functions of the nod gene products can be found in reviews (Downie, 1998; Perret et al., 2000; Spaink, 2000).

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Nod factors can induce plant responses at astonishingly low concentrations; root-hair deformation and calcium oscillations (called calcium spiking) can be induced in root hairs by as little as 10−13 M Nod factor (Oldroyd et al., 2001). The plant signalling pathway induced by Nod factors is currently being worked out using legume mutants defective for nodulation. There appear to be multiple different Nod-factor receptor proteins on root-hair plasma membranes and two of these receptors are required for plant responses (Radutoiu et al., 2003). A signalling pathway has been identified in legumes as being essential for nodulation (Oldroyd & Downie, 2008), and because several of the components in the signalling pathway are also required for the much more ancient symbiosis with mycorrhizal fungi that is found in most land plants, it is likely that legumes learned to adapt and modify this ancient signalling pathway so that they could establish a symbiosis with nitrogen-fixing bacteria (Parniske, 2008). During their coevolution, different rhizobia and legumes have developed the ability to use various different Nod factor structures as a highly specific means of communication. The nitrogen-fixing symbiosis that exists between actinorrhizal bacteria belonging to the Frankia genus and several plant genera clearly shares an evolutionary link with the legume symbiosis because the two groups of plant symbionts are phylogenetically closely linked within the Eurosid II clade of plants (Parniske, 2008). Understanding the specific requirements for many of these interactions can be difficult to define in the laboratory, given the various different environmental conditions that may have to be understood to analyse any given system.

Until recently, it had been thought that Nod factor-based signalling was essential for legume nodulation, but recent reports on nodulation by bacteria lacking nod genes have led to the realization that at least for a few unusual rhizobia and legumes, it is possible to establish nodules without using Nod factors. Little is known about the bacterial signals and how the plants respond. There is circumstantial evidence that signals based on purines may be involved in this pathway (Giraud et al., 2007). In normal nodulation, activation of a cytokinin pathway can allow legumes to induce nodules in the absence of rhizobia (Gonzalez-Rizzo et al., 2006; Tirichine et al., 2007). Because cytokinins are related to purines, it has been speculated that some rhizobia may have acquired the ability to short circuit the normal Nod-factor signalling pathway, activating nodule morphogenesis at the level of cytokinins. It had already been recognized that a purine precursor aminoimidazole carboxamide ribonucleotide played a role in helping rhizobia infect legumes (Newman et al., 1994).

Legume infection

Initiation of infection

The formation of infected legume nodules capable of fixing nitrogen requires the bacteria to activate two plant programmes: one leading to nodule morphogenesis and the other leading to nodule infection. In some legumes, Nod factors alone can induce nodule morphogenesis (Truchet et al., 1991; Relic et al., 1994), and this appears to occur as a consequence of modifying existing plant hormonal signalling systems, including the cytokinin pathway (Relic et al., 1994; Hirsch et al., 1997). In parallel with, and closely linked to, nodule morphogenesis is the initiation of infection. From a plant perspective, this is a critical stage because allowing bacterial entry gives rise to the potential for nonsymbiotic bacteria to try to enter and take advantage of the plant. It is not surprising that several different aspects of signalling come into play to enable bacterial infection.

There are two broad modes of bacterial infection and these are primarily determined by the legume species. Both types require Nod-factor signalling (but see the exceptions cited above) and require the appropriate bacterial surface polysaccharides. In one type, the bacteria enter the roots at sites where the epidermal layer is broken, for example at sites of lateral root emergence. This intercellular initiation of infection is thought to be the more ancient mechanism of root infection (Sprent, 2007), and initial colonization occurs by the bacteria growing between cortical cells of the root. This type of infection has probably been best characterized in the legume Sesbania rostrata. In this legume, the bacteria appear to grow in the intercellular spaces, forming infection foci, where they may form a signalling centre that can induce nodulation and infection (Goormachtig et al., 2004a, b). This type of infection has been reviewed recently (Den Herder et al., 2006; Capoen et al., 2010). The second type of root infection initially occurs by intracellular infection, starting in root hair cells, and this will be the system focussed on in this review.

In many legumes such as peas, clovers, vetch, alfalfa and Medicago spp., Lotus spp., beans and soybean, infection is usually initiated by bacteria, attached near root hair tips, producing Nod factors, which promote the deformation of root hairs such that the root hair growth bends back on itself, entrapping bacteria in a so-called ‘shepherds crook’ structure. The trapped bacteria continue to grow, forming an infection focus. Continued production of Nod factors stimulates the root hair to set up an intracellular infection structure, which develops by an ingrowth of the membrane and cell wall. The bacteria grow within this narrow tube in a column usually only a couple of cells wide, keeping up with the continued growth at the tip of the elongating infection thread (Gage, 2004; Oldroyd & Downie, 2004). The rapid growth of bacteria at the tip tends to select out a single bacterial strain even if more than one strain becomes entrapped initially. This selection even works with two near-identical rhizobial strains in the infection (Gage, 2002) and can select against potential ‘cheaters’, which will attempt to coinfect the nodule niche without conferring any benefit to the plant (Kiers & Denison, 2008). The successful infecting strains continue to produce Nod factors during growth in the infection thread, based on analysis of reporter gene expression (Schlaman et al., 1991) and on the complementation of glucosamine auxotrophy (in a glucosamine synthase mutant) by induction of the nodulation gene nodM, which encodes a glucosamine synthase (Fig. 1) (Marie et al., 1992).

It seems that for the establishment of infection threads, it is critical that the bacteria produce the correct type of Nod factor, because bacterial mutants lacking specific nod genes (determining specific additions) can form arrested infections, even though they can induce many other plant responses such as root-hair deformation, plant gene induction, calcium spiking, etc. (Ardourel et al., 1994; Walker & Downie, 2000; Oldroyd & Downie, 2004). In addition to promoting active growth of infection threads at their tips, they also promote the establishment of preinfection thread structures in cells that the infection thread is approaching, but has not reached (van Brussel et al., 1992). The growing infection thread must find this preinfection thread structure to allow the infection thread to bridge between cells or to change from being in the intercellular space, and this can promote changes in direction of its growth. In addition to Nod-factor specificity, surface polysaccharides (both exopolysaccharide and lipopolysaccharide) and secreted proteins can also play important roles in infection.

Reactive oxygen species play an important role in the initiation of infection in legumes (D'Haeze et al., 2003; Jamet et al., 2007; Cardenas et al., 2008; Chang et al., 2009), and rhizobia have several enzymes such as catalases and superoxide dismutases that help them survive the oxidative stress (Crockford et al., 1996; Herouart et al., 1996; Santos et al., 2000; Jamet et al., 2003; Vargas et al., 2003; Hanyu et al., 2009). Nod factors can induce the production of reactive oxygen species in root hairs (Cardenas et al., 2008), and it has been proposed that plant-produced reactive oxygen species are involved in cross-linking of glycoproteins in the matrix of the infection threads (Brewin, 2004). Therefore, during the symbiosis, the ability of bacteria to deal with extracellular oxidative stress is clearly important.

Release from infection threads and differentiation of nitrogen-fixing bacteroids

Infection threads extend between cells, forming a continuous system of cellular tubes and intercellular spaces within which rhiozobia grow. As the infection thread gets close to the growing meristem, it branches and ramifies into most of the cells in the growing nodule and delivers the bacteria into these nodule cells. The bacteria are budded off the end of the infection threads before the synthesis of the plant cell wall surrounding the infection thread. The endocytic budding process results in the bacteria being released into the plant cell surrounded by a plant-made membrane (Gage, 2004; Monahan-Giovanelli et al., 2006). The bacteria then differentiate, and in parallel, express many genes required for nitrogen fixation (Fischer, 1994; Mesa et al., 2008). The environment within the nodule is very unusual and forms a rather special case of an interaction that involves quite specific nutrient uptake systems, a specialized electron transport chain that enables them to respire (and generate ATP) efficiently at low oxygen concentrations (Preisig et al., 1996), and the bacteria have quite specific modifications to their lipopolysaccharide surface (Kannenberg & Carlson, 2001). Thus, the symbiotic environment forms a very special type of extracellular biology interaction within the plant cells.

Rhizobial extracellular components associated with root attachment

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

It has been estimated that up to 20% of plant photosynthate can be released from roots in the form of mucilage and metabolites (Estabrook & Yoder, 1998). Therefore, attachment to roots and root hairs provides an excellent niche for microbial growth. To this end, it appears that rhizobia have multiple mechanisms that enable them to attach to roots and these include surface polysaccharides and secreted/surface proteins (Matthysse & Kijne, 1998; Rodriguez-Navarro et al., 2007). The relative contribution of different components can be affected by factors such as pH, concentrations of Ca2+ and Mg2+, specific growth conditions and pretreatment of roots, etc (see the review by Rodriguez-Navarro et al., 2007).

Roles of polysaccharides

An early proposal had been that binding of plant lectins to rhizobial surface polysaccharides was the mechanism determining specificity between rhizobia and their specific legume hosts (Dazzo & Hubbell, 1975; Dazzo et al., 1976). It is now accepted that this ‘lectin hypothesis’ was incorrect and that the major determinant of host specificity is in fact determined by Nod factors rather than surface polysaccharides (Oldroyd & Downie, 2008). However, it is evident that binding of plant lectins to bacterial polysaccharide can influence legume nodulation and this occurs in pea: as illustrated in Fig. 2, a root-hair-expressed lectin binds to a polarly localized glucomannan surface polysaccharide produced by Rhizobium leguminosarum, promoting bacterial binding to root hairs (Laus et al., 2006). A mutation specifically blocking the production of this glucomannan considerably reduced both root-hair attachment by R. leguminosarum and the ability of the mutant strain to compete with the wild type during nodule infection (Williams et al., 2008). It is likely that the accumulation of R. leguminosarum on the root hairs enhances delivery of Nod factors to the root hairs and as proposed previously (van Rhijn et al., 1996), the accumulation of rhizobia on root hairs might reduce (but not abolish) the level of Nod-factor specificity as a consequence of increased levels of Nod factors delivered to root hairs. This could explain the observation (Diaz et al., 1989) that transgenic clover roots expressing a pea lectin could be nodulated by R. leguminosarum bv. Viciae, which normally nodulates pea, but not clover. This also fits with the observation that even with lectin transgenic roots, appropriate types of Nod factors were still required for nodulation and infection (van Rhijn et al., 2001). It is likely that plant lectin–bacterial surface polysaccharide interactions (such as that with glucomannan) are found in other legumes, because there appears to be a soybean lectin that promotes attachment of Bradyrhizobium japonicum to root hairs (Lodeiro & Favelukes, 1999; Lodeiro et al., 2000).

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Figure 2.  Biofilms formed by Rhizobium leguminosarum in vitro and in vivo. The image on the left shows a structured biofilm with water-filled channels formed in vitro during static culture of R. leguminosarum [labelled with green fluorescent protein (GFP)]. Such biofilms are not formed by exopolysaccharide -defective mutants and are altered by blocking export of proteins via the prsDE-encoded Type I secretion system (Russo et al., 2006). The image was captured using confocal microscopy. A model is shown for R. leguminosarum attachment and subsequent biofilm formation on legume root hairs as described previously (Laus et al., 2006). Under acidic conditions, a plant lectin is localized on the root hair tips and binds to the glucomannan polysaccharide expressed at the pole of R. leguminosarum. Under basic conditions, the root lectin is solubilized from the root hair tip and an alternative mechanism of attachment occurs. This involves the extracellular rhizobial protein rhicadhesin, which attaches to the rhizobial cell surface and the root hair in a calcium-dependent manner. At acidic pH, the rhicadhesin is released from the bacterial surface and so does not function in attachment. The bacteria then aggregate on the root hair, forming a biofilm or a cap and this biofilm structure requires cellulose (which is not required for the in vitro biofilm) (Smit et al., 1987; Laus et al., 2005a; Williams et al., 2008). The confocal image on the right shows a root-hair biofilm formed by GFP-labelled R. leguminosarum. The confocal images were captured as described previously (Russo et al., 2006; Williams et al., 2008) and were kindly provided by Alan Williams and Fang Xie.

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In addition to the glucomannan, the acidic exopolysaccharide is required both for attachment of R. leguminosarum to root hairs (Williams et al., 2008) and for infection (see below). After the initial attachment or R. leguminosarum to root hairs, bacteria accumulate around the root hairs in a cap or a biofilm, and this requires cellulose fibrils (Smit et al., 1987; Laus et al., 2005a; Williams et al., 2008). However, the cellulose fibrils are not required for infection (Smit et al., 1987; Ausmees et al., 1999; Laus et al., 2005a, b) or competitiveness in nodule infection (Williams et al., 2008), although it appears that a cellulose-defective mutant was less able to infect long root hairs; furthermore, the production of cellulose in mutants lacking the acidic exopolysaccharide caused agglutination in infection threads, contributing to decreased infection (Laus et al., 2005a, b). It seems likely (Fig. 2) that cellulose allows a biofilm to develop on root hairs (Smit et al., 1987; Williams et al., 2008) and that this may enhance the growth of those rhizobia that do not succeed in infecting the roots; this role of cellulose in biofilm development on root hairs seems to be plant specific because the normal structured in vitro biofilm formed by R. leguminosarum bv. Viciae (Fig. 2) was unaffected in mutants defective for cellulose formation (Russo et al., 2006; Williams et al., 2008).

Although mutations affecting lipopolysaccharide formation significantly perturb biofilm formation in vitro (D. M. Russo et al., unpublished data), lipopolysaccharide mutants of R. leguminosarum were relatively unaffected in terms of root-hair attachment (Smit et al., 1989) as was a mutant defective for a gel-forming polysaccharide (Williams et al., 2008). However, there is some evidence for some plant lectins (e.g. peanut lectin) binding specifically to lipopolysaccharide (Jayaraman & Das, 1998), and so this could be a mechanism of specific binding of some rhizobia.

Secreted proteins and attachment

Secreted proteins are important for attachment. An R. leguminosarum protein factor called rhicadhesin has been shown to play an important role in attachment to root hairs (Smit et al., 1992), particularly at alkaline pH (Laus et al., 2006), but the gene encoding this protein has not yet been identified. The rhicadhesin functions widely to promote attachment of rhizobia and agrobacteria and a similar protein has been identified in a Bradyrhizobium spp. (Dardanelli et al., 2003). An attempt to isolate rhicadhesin from R. leguminosarum using a phage display library to identify proteins adhering to R. leguminosarum (Ausmees et al., 2001) identified several Rhizobium-adhering proteins (RAPs). These proteins are secreted across the inner and outer membranes via a Type I secretion system, which is encoded by the prsD and prsE genes (Russo et al., 2006). Several other proteins including predicted cadherins (calcium-binding adherence proteins) were secreted via this PrsDE system (Krehenbrink & Downie, 2008), and this may in part explain the abnormal in vitro biofilm seen with the prsD mutant (Russo et al., 2006). However, the prsD and prsE mutants appeared to infect legumes normally (Finnie et al., 1998; Krehenbrink & Downie, 2008), and although the RapA1 protein played a role in attachment to roots, it was not required for nodulation (Mongiardini et al., 2008).

A B. japonicum polarly localized lectin expressed on the bacterial surface is involved in binding to soybean root hairs (Loh et al., 1993, 1994). Binding occurs predominantly on young root hairs and bacterial mutants lacking antigenically detectable lectin were significantly reduced for binding to root hairs and for nodulation of soybeans (Ho et al., 1994).

Nod factors

The production of Nod factors by Sinorhizobium meliloti enhanced the production of biofilms in vitro. Surprisingly, an enhanced biofilm was observed even in the absence of added inducers of the nodulation genes. A nod gene inducer enhanced biofilm levels and the enhanced biofilm was not formed by a nodC mutant. However, mutation of genes required for host-specific modifications to the Nod factor did not decrease biofilm formation. This indicates that the core Nod factor (the acylated N-acetyl-glucosamine oligosaccharide) enhances attachment, possibly by changing surface hydrophobicity; the enhanced attachment was also seen on root hairs (Fujishige et al., 2008).

Extracellular signalling in symbiotic interactions

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

In addition to Nod factor-mediated signalling required to initiate legume infection and initiate nodule morphogenesis in most legumes, rhizobia can also use many other methods of signalling including extracellular polysaccharides, secreted proteins, quorum-sensing signalling and possibly the production of metabolites that play a role in plant cytokinin signalling. The establishment of the symbiosis may also benefit from suppression of plant defence responses and recognition of the types of bacteria infecting the plant.

Nod-factor signalling

Most legume-nodulating rhizobia produce Nod factors in response to exudates from legume roots; the synthesis, secretion, diversity and regulation of production of these Nod factors have been reviewed in great detail (Downie, 1998; Perret et al., 2000; Spaink, 2000). It appears that there are multiple levels of Nod-factor recognition by legumes. Nod-factor perception requires a legume receptor (or receptor complex) that contains extracellular LysM domains that are likely to bind the chitin backbone of the Nod factor; activated intracellular kinase domains induce the signalling pathway. Binding and recognition of the Nod factors by these receptors can result in at least three separate outputs (Miwa et al., 2006). At very low concentrations, the Nod factors induce cytoskeletal changes that accompany root-hair deformation; the continuous polar growth towards the source of the Nod factor (Esseling et al., 2003) can ultimately entrap surface-attached bacteria in the crook of a curled root hair. In parallel with this, there is the activation of a calcium-signalling pathway that induces calcium oscillations around the root-hair nucleus (Downie & Walker, 1999). Both of these responses can be observed even at 10−12–10−13 M Nod factor, but calcium oscillations are not required for root-hair deformations because plant nodulation mutants that have lost calcium oscillations retain root-hair deformation (Catoira et al., 2000; Miwa et al., 2006). There are some Nod-factor structural requirements for these responses, based on analysis of various Nod factors lacking surface decorations; for example loss of the sulphate group reduced sensitivity to the S. meliloti Nod factor by over six orders of magnitude, whereas loss of an acetyl group had very little effect (Oldroyd et al., 2001).

Relatively high concentrations of the Nod factor (around 10−8–10−9 M) induce a calcium influx across the root-hair plasma membrane (Ehrhardt et al., 1992; Shaw & Long, 2003; Oldroyd & Downie, 2004; Miwa et al., 2006). It has been proposed (Miwa et al., 2006) that the root-hair deformation leads to an entrapment of bacteria, resulting in the accumulation of a Nod factor within curled root hairs: the accumulated Nod factor is then thought to induce a calcium influx and membrane depolarization, which are involved in establishing the initiation of infection thread growth. It appears that the Nod-factor structural requirements for initiation and maintenance of infection thread growth are relatively strict, because mutations in the nodL and nodFE genes (which affect addition of an acetyl group and the type of acyl group, respectively; Fig. 1) block most infections on alfalfa and vetch (Ardourel et al., 1994; Walker & Downie, 2000), while other substitutions such as glycosylations and carbamoyl groups are important for infection of S. rostrata plants by Azorhizobium caulinodans (D'Haeze et al., 2000; Goormachtig et al., 2004a, b). In addition to these structural requirements, the amounts of Nod factors generated by rhizobial strains can be very important. This was first observed during the nodulation of a specific variety of pea called cultivar Afghanistan that needs a specific Nod factor modification (acetylation at site V; Fig. 1) for infection to proceed (Firmin et al., 1986; Geurts et al., 1997); surprisingly, it was observed that high levels of cognate Nod factors can inhibit nodulation (Hogg et al., 2002). A similar situation may occur with certain varieties of soybean that are sensitive to high levels of Nod-factor production (Jitacksorn & Sadowsky, 2008).

Rhizobial Nod factors affect the expression of many (over 750) plant genes (El Yahyaoui et al., 2004; Mitra et al., 2004; Lohar et al., 2006). Many of the induced genes are called early nodulin (ENOD) genes and these include genes associated with cytoskeletal remodelling, cell wall deposition, cell growth and division. In addition, there is upregulation of plant genes associated with the signalling and transcriptional responses induced by the Nod factors. It also appears that there is a decrease in the expression of genes associated with plant defence responses.

Surface polysaccharides

The initiation of infection thread growth by legumes to allow access of potential symbionts is a critical checkpoint because infection by nonsymbionts could allow the development of an unproductive infection and possibly even an initiation of pathogenesis. In addition to requirements for Nod-factor structural specificity, infection requires surface polysaccharides, and it seems that these have biological activity rather than simply acting as an inert protective coat that envelops the bacteria. Most rhizobia have surface polysaccharides (examples shown in Fig. 3), which are essential for legume infection (reviewed by Leigh & Coplin, 1992; Fraysse et al., 2003; Becker et al., 2005; Jones et al., 2007; Gibson et al., 2008), but the most comprehensive work on the synthesis and role of the surface polysaccharides has been carried out with S. meliloti. For nodulation of alfalfa (Medicago sativa), either of two surface polysaccharides (known as succinoglycan or EPSI and galactoglucan or EPSII; Fig. 3) is sufficient to allow infection, although the succinoglycan-mediated infection (Glazebrook & Walker, 1989; Pellock et al., 2000) is more efficient. There appears to be plant specificity in the recognition, because in the absence of succinoglycan, galactoglucan cannot sustain infection of Medicago truncatula (Pellock et al., 2000). The synthesis of succinoglycan results in the production of a high-molecular-weight polymer of repeating octasaccharide subunits (containing one galactose and seven glucose residues decorated with succinyl, pyruvyl and acetyl groups; Fig. 3), but in addition, low-molecular-weight oligomers (one to three octasaccharide repeats) are also produced. It is these low-molecular-weight oligomers that are most critical for the initiation of infection (Battisti et al., 1992; Urzainqui & Walker, 1992; Wang et al., 1999). This was established using a mutant (exoH) that was defective for succinylation and produced normal amounts of the high-molecular-weight exopolysaccharide, but little of the low-molecular-weight exopolysaccharide. This mutant was defective for infection, and so the surface exopolysaccharide is unlikely to act as a protective sheath (Leigh et al., 1987), although it appears that surface exopolysaccharide can protect some rhizobia from reactive oxygen species formed during infection (D'Haeze et al., 2004).

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Figure 3.  Structures of exopolysaccharides produced by some rhizobia. The chemical structures of the exopolysaccharide repeat units of different rhizobia are shown along with that of the closely related strain Agrobacterium tumefaciens. Sinorhizobium meliloti EPSI from is also known as succinoglycan because of the presence of a succinyl group, whereas EPSII is also known as galactoglucan. This figure and the appropriate citations of the structural determinations were published previously (Laus et al., 2005b) and the figure is reproduced with permission from the American Phytopathological Society.

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It seems likely that the exopolysaccharide can suppress plant defence responses; there is increased expression of predicted defence-related genes in M. truncatula inoculated with a succinoglycan-deficient mutant compared with the control strain producing the succinoglycan (Jones et al., 2008). The ability of various different forms of exopolysaccharide to promote the symbiosis may point to a general property of the polysaccharides such as ion binding, gel formation or some other activity.

Purified rhizobial lipopolysaccharide components can also suppress defence reactions such as production of reactive oxygen species in plants (Albus et al., 2001; Fraysse et al., 2003; Scheidle et al., 2005), and lipopolysaccharide mutants are often defective for establishment of the symbiosis (Lerouge & Vanderleyden, 2002; Campbell et al., 2003; D'Haeze et al., 2004; Beck et al., 2008). Often the effects are seen later in the symbiosis than those seen with exopolysaccharide mutants (Perotto et al., 1994; GarciadelosSantos & Brom, 1997; Gao et al., 2001; Vedam et al., 2004) and the arrest of infection appears to be similar to that observed in a plant defence response (Perotto et al., 1994). Sometimes, mutations affecting lipopolysaccharide can abolish the symbiosis in some legumes, but not in others (Niehaus et al., 1998). It is clear that the plant can influence the lipopolysaccharide by inducing modifications: in Rhizobium sp. NGR234, flavonoids induce changes in the rhamnan O-antigen in parallel with induction of expression of nodulation genes (Theunis et al., 2004; Broughton et al., 2006), and during differentiation of bacteroids, there are changes in the structure of the R. leguminosarum lipopolysaccharide that make it more hydrophobic (Kannenberg & Carlson, 2001).

Sinorhizobium meliloti produces capsular polysaccharides (KPS) analogous to the group II K antigens of Escherichia coli (Reuhs et al., 1993); they are made up of small repeating units of hexoses and 3-deoxy-d-manno-2-octulosonic acid (Kdo, normally a component of lipopolysaccharide) or other 1-carboxy-2-keto-3-deoxy sugars (Reuhs et al., 1998). KPS can play a role in nodulation (Petrovics et al., 1993). In the absence of succinoglycan (EPSI) and galactoglucan (EPSII), the KPS can allow infection of alfalfa, but the infection threads are often stunted and abnormal and terminate more frequently (Pellock et al., 2000). However, in the related species, Sinorhizobium fredii, which nodulates soybean and pigeon pea, mutations in rkpH or rkpG blocking the production of the KPS were very significantly reduced for nodulation, whereas a succinoglycan (exoA) mutant was not significantly affected (Parada et al., 2006). This indicates that the type of surface polysaccharide recognized by different plant species can vary significantly and reinforces the idea that the surface polysaccharides are not simply providing a protective sheath.

Secreted proteins that influence legume interactions

Different rhizobia have the potential to secrete proteins into the periplasm via the general export pathway and the TAT secretion system and some of these proteins appear to leak into the extracellular growth medium (Krehenbrink & Downie, 2008). In addition, different rhizobia have Type I, Type III, Type IV and Type VI secretion systems that can influence the symbiosis; these will be dealt with under the different types of protein secretion systems. Protein secretion systems in rhizobia have recently been reviewed (Fauvart & Michiels, 2008; Krehenbrink & Downie, 2008; Deakin & Broughton, 2009).

Symbiosis-specific proteins secreted via the general export pathway

There are many proteins that are secreted via the general export pathway using a typical N-terminal transit peptide and it is likely that many of these will be expressed during infection (and many other stages of the symbiosis). For example, there are several periplasmic substrate-binding proteins, many of which could be involved in nutrient acquisition (Mauchline et al., 2006), but these will not be dealt with here. One protein of particular symbiotic significance is a cellulase (CelC2) that is predicted to be exported via the general export pathway with a typical transit peptide. This cellulase can erode the noncrystalline cellulose in the root-hair cell wall and is thought to allow rhizobial penetration during the initial phase of infection in root-hair curls. Mutation of the gene in Rhizobium leguminosarum bv. trifolii resulted in an inability of the bacteria to initiate infections in root hairs and those nodules that did form were uninfected (Robledo et al., 2008). It is not clear how the cellulase crosses the rhizobial outer membrane; it is evident that several periplasmic enzymes can leak into the growth medium (Krehenbrink & Downie, 2008), but it is also possible that there is an alternative secretion mechanism, although it is unlikely to be a Type II system because rhizobia that have a similar cellulase gene lack a Type II secretion system (Krehenbrink & Downie, 2008; Robledo et al., 2008).

Symbiosis-specific proteins secreted via the TAT pathway

Proteins secreted via the TAT (twin arginine translocation) pathway are secreted in their folded form, some containing cofactors, and the proteins have a signal peptide usually containing a distinctive pair of consecutive twin arginine residues in the motif RRXΦΦ, where X is any residue and Φ a hydrophobic residue. Mutation of the TAT transporter (encoded by the tatABC genes) in R. leguminosarum resulted in loss of the ability to fix nitrogen (Meloni et al., 2003; Krehenbrink & Downie, 2008), probably because the mutants are defective for an essential exported iron–sulphur protein required for bacteroid respiration. However, the mutants also had abnormalities in infection (Meloni et al., 2003), possibly because of a lack of export of proteins required for outer-membrane biogenesis, as has been observed in E. coli (Ize et al., 2003).

Type I secretion pathway influences attachment and infections

Proteins secreted via this pathway are translocated across the inner and outer membranes without a periplasmic intermediate stage (Koronakis et al., 2004). NodO, which is present in some rhizobia and can extend the ability to nodulate different legumes, is one such protein (Economou et al., 1994; van Rhijn et al., 1996; Vlassak et al., 1998). NodO is a calcium-binding protein that forms cation-selective channels in membranes (Economou et al., 1990; Sutton et al., 1994), and it has been proposed that NodO may complement Nod factor function by promoting the movement of cations across the root-hair membrane (Miwa et al., 2006) because in the absence of host-specific Nod factors nodO is required to initiate infection in root hairs (Walker & Downie, 2000). NodO is secreted via the prsDE-encoded secretion system, which can also secrete glycanases (Finnie et al., 1997, 1998; York & Walker, 1997). These glycanases can cleave nascent exopolysaccharides produced by rhizobia (York & Walker, 1998; Zorreguieta et al., 2000) and are required for the formation of a normal biofilm (Russo et al., 2006).

Among the several other proteins that are secreted via the PrsDE Type I secretion system are several predicted adhesins (including the Rhizobium-adhering proteins ‘Rap’ described above), at least some of which primarily play a role in attachment and biofilm formation rather than infection (Russo et al., 2006; Krehenbrink & Downie, 2008; Mongiardini et al., 2008). The PrsDE system is one of three predicted Type I secretion systems in R. leguminosarum bv. Viciae strain 3841, but even a triple mutant infected peas normally (Krehenbrink & Downie, 2008). A delay in nodulation was observed if prsD was mutated in a strain lacking also mutated in nodE and this was due to lack of export of NodO (Finnie et al., 1997). Mutation of prsD abolished symbiotic nitrogen fixation in R. leguminosarum bv. Viciae (strain A34 but not 3841) and bv. Trifolii strain TA1; the block occurred late because fully infected nodules with normal-looking bacteroids were formed (Finnie et al., 1997; Mazur et al., 1998), but the cause of the defect was not established.

Type III secretion system for the delivery of proteins into plant cells

The Type III secretion pathway is derived from the bacterial flagellar secretion system and has evolved into a mechanism that can deliver proteins from the bacterial cytoplasm into the cytoplasm of eukaryotic cells (Saier, 2004). Many bacterial pathogens of animals and plants use this secretion system as a crucial part of their pathogenicity by delivering various proteins (often called effectors) into their hosts (Hueck, 1998). The secretion complex involves about 20 different proteins, some of which act in the cytoplasm, some constitute a barrel-like structure that crosses the bacterial inner and outer membranes and others form a needle-like structure that conveys proteins into the eukaryotic cells. Effector proteins, which have an amino-terminal signal, move in their unfolded form down a narrow channel within the centre of the complex. Some, but not all, rhizobia have such secretion systems and it is very clear that, when present, they can play an important role in symbiosis (Deakin & Broughton, 2009). Type III secretion systems characterized in B. japonicum USDA110, Mesorhizobium loti MAFF303099, Rhizobium etli CNPAF512, Rhizobium sp. NGR234 and S. fredii strains HH103 and USDA257, have recently been reviewed in considerable detail (Deakin & Broughton, 2009), and so only the key points will be dealt with here.

The synthesis of the Type III secretion system and the effectors it expresses is regulated by flavonoids and NodD, which, in addition to inducing nod gene expression, induces the expression of ttsI, a gene encoding a regulator that binds to highly conserved promoter elements (tts boxes) upstream of operons encoding the secretion machinery and effectors (Wassem et al., 2008). The number of such promoter elements can vary significantly, from seven tts boxes in M. loti MAFF303099 (Deakin & Broughton, 2009) to about 30 in B. japonicum USDA110 (Zehner et al., 2008). However, not all genes downstream of tts boxes are involved in Type III secretion, and so identifying genes encoding effectors is not always simple. Because some of the rhizobial effector proteins are similar to those from bacterial pathogens, some likely functions can be deduced. The secreted proteins have been called Nops (nodulation outer protein, Nop) and this term includes secreted components of the secretion machinery as well as the effector proteins delivered into the plant cell (Viprey et al., 1998; Marie et al., 2003). Some of the effectors promote symbiosis on certain legumes, whereas on other legumes they may either have no effect or can even significantly reduce symbiotic proficiency (Deakin & Broughton, 2009). By analogy with plant defence responses against bacteria (Bent & Mackey, 2007), the impairment of symbiotic proficiency could result from the plant recognizing the effectors and mounting a defence response.

In Rhizobium sp. NGR234 five effectors have been identified (NopL, NopP, NopT, NopJ and NopM). NopL and NopP are required for optimal nodulation of the legumes Flemingia congesta and Tephrosia vogelii (Marie et al., 2003). Both NopP and NopL are phosphorylated by plant kinases and expression of nopL in tobacco cells blocked the induction of some pathogenesis-related defence proteins such as chitinases (Bartsev et al., 2003, 2004; Ausmees et al., 2004; Skorpil et al., 2005). NopT, which is structurally similar to pathogenic effector proteases, enhances nodulation on T. vogelii, but it considerably decreases symbiotic proficiency on Crotalaria junceaea. This phenotype presumably requires the protease activity, because mutation of the protease-active site significantly restored symbiotic proficiency (Kambara et al., 2009). NopJ and NopM can also have negative effects on the nodulation of some legumes, but no effects of mutations were seen in those legumes in which the Type III secretion system reduced symbiotic proficiency; such simple tests, however, can be misleading if other Nops have a deleterious effect (Marie et al., 2003). NopJ is similar to the YopJ family of proteins that act as acetyl transferases that inactivate MAP kinases. NopM contains leucine-rich repeats that are involved in protein–protein interactions, but its precise role is yet to be established (Kambara et al., 2009).

A B. japonicum Type III system secretes a homologue of NopP, other proteins of unknown function including GunA2, NopE1 and NopE2 and probably secretes NopL, NopM and NopT homologues (Krause et al., 2002; Suss et al., 2006; Zehner et al., 2008; Hempel et al., 2009). NopE1 and NopE2 are translocated into plant cells, where they appear to undergo self-cleavage in the presence of calcium. This cleavage is important for their activity, which represses nodulation in mung bean and stimulates nodulation in soybean (Wenzel et al., 2010). Sinorhizobium fredii HH103 secretes NopL, NopM and NopP (Rodrigues et al., 2007; Lopez-Baena et al., 2008) via a Type III system and M. loti MAFF303099 and Bradyrhizobium elkanii also use Type III systems to secrete several flavonoid-inducible effector proteins, some of which are similar to pathogen effectors and probably interfere with plant metabolism (Okazaki et al., 2009; Sanchez et al., 2009).

Type IV pathway

Mesorhizobium loti strain R7A secretes at least two effectors Msi059 and Msi061 into plant cells and it is predicted that Msi061 may be involved in the ubiquitinylation of plant proteins, thereby targeting them for degradation via proteasomes, whereas Msi059 may be a protease in its own right (Hubber et al., 2004, 2007). Loss of the Type IV secretion system decreased symbiotic competence on Lotus Japonicus, but enabled M. loti to form effective nodules on Leucaena leucocephala, which M. loti R7A was otherwise unable to infect.

Type V and Type VI pathways

Autotransporter proteins (Type V) are secreted via the general export pathway into the periplasm using an N-terminal transit peptide and then a C-terminal domain inserts into the outer membrane, where it catalyses the translocation of the N-terminal region into the extracellular medium (Henderson et al., 2004). Such proteins have a distinctive autotransporter domain near the C-terminus and three such proteins were identified in R. leguminosarum bv. Viciae strain 3841. However, mutation of all three autotransporter genes had no observed effect on the symbiotic phenotype (Krehenbrink & Downie, 2008).

Type VI secretion systems, which have only recently been recognized, mediate the translocation of proteins across the bacterial envelope. Around 20 components appear to be involved and the genes are fairly widely conserved (Bingle et al., 2008). In fact, the imp locus (Type VI) secretion system was identified in R. leguminosarum at an early stage because mutation of this locus enabled a strain of R. leguminosarum bv. trifolii to fix nitrogen on pea (Roest et al., 1997), which is normally a nonhost plant. Proteins translocated by this system were identified and showed similarity to ribose-binding proteins from other bacteria. It was not tested whether mutations in the genes encoding the identified secreted proteins allowed nitrogen fixation on pea (Bladergroen et al., 2003).

Quorum-sensing signals

Many strains of rhizobia use N-acyl-homoserine lactone (AHL)-mediated regulation of gene expression to induce gene expression in relation to their population density. This quorum-sensing type of regulation usually depends on the AHLs accumulating during growth and as the signals reach a threshold concentration, they can induce gene expression, with one of the induced genes usually encoding the AHL synthase (by convention given the gene letter I as in luxI, rhiI, etc.), resulting in a positive feedback loop. This type of regulation, which is common among rhizobia, has been reviewed in considerable detail (Wisniewski-Dye & Downie, 2002; Sanchez-Contreras et al., 2007; Downie & Gonzalez, 2008), and affects many aspects of rhizobial physiology including induction of transfer of plasmids and integrated symbiosis islands, regulation of surface polysaccharide production and growth-inhibitory effects. It is evident that mutations in closely related genes in different rhizobial species can have markedly different effects on the interaction with legumes, and so what holds for one strain need not be valid with another. It is also clear that within a single rhizobial species, different isolates may have a different range of AHL-dependent quorum-sensing regulatory systems. This section will deal primarily with those quorum-sensing regulatory systems that have a direct effect on the symbiosis; for details of the role of quorum-sensing regulation on phenotypes such as conjugation, swarming, motility and adaptation to abiotic stresses (Table 1), and the AHLs produced by these systems, readers are referred to other reviews (Wisniewski-Dye & Downie, 2002; Gonzalez & Marketon, 2003; He et al., 2003; Hirsch et al., 2003; Daniels et al., 2004; Somers et al., 2004; Barnett & Fisher, 2006; Gao et al., 2006; Gonzalez & Keshavan, 2006; Downie & Gonzalez, 2008; Gurich & Gonzalez, 2009).

Table 1.   Phenotypes regulated by quorum sensing in rhizobia
Rhizobial strainGenesPhenotypes regulatedReferences
Sinorhizobium meliloti
 strain Rm1021csinR/sinIEPSII production, swarmingMarketon & Gonzalez (2002), Marketon et al. (2002), Gonzalez & Marketon (2003), Teplitski et al. (2003), Gao et al. (2005), Glenn et al. (2007), Bahlawane et al. (2008), McIntosh et al. (2008)
expREPSI and EPSII production, swarming, motility; regulates visNPellock et al. (2002), Marketon et al. (2003), Hoang et al. (2004), Gao et al. (2005), Glenn et al. (2007), Bahlawane et al. (2008), Hoang et al. (2008), McIntosh et al. (2008)
 strain Rm41traR/traIPlasmid transferMarketon & Gonzalez (2002)
 strains RU10/406 and Rm1021visN/visRMotility (flagellar regulon: fli, mot, fla and che genes)Sourjik et al. (2000), Hoang et al. (2004), Hoang et al. (2008)
R. leguminosarum
 bv. ViciaecinR/cinI/cinSGrowth inhibition; regulation of EPS cleavage (PlyB)Lithgow et al. (2000), Edwards et al. (2009)
rhiR/rhiINodulation efficiencyCubo et al. (1992), Rodelas et al. (1999)
traR/traIPlasmid transferWilkinson et al. (2002), Danino et al. (2003)
expRBiofilm formationEdwards et al. (2009)
 bv. PhaseoliraiR/raiIUnknownWisniewski-Dye & Downie (2002), Wisniewski-Dye et al. (2002), Edwards et al. (2009)
R. etli
 strain CNPAF512cinR/cinINitrogen fixation, symbiosome development, growth inhibition, swarmingDaniels et al. (2002), Daniels et al. (2004), Daniels et al. (2006)
raiR/raiINitrogen fixation, growth inhibitionRosemeyer et al. (1998), Daniels et al. (2002)
 strain CFN42traR/traI (p42a)Plasmid transferTun-Garrido et al. (2003)
Mesorhizobium loti R7A and NZP 2213traR/traI2, traI2Symbiosis island transfer Legume nodulationRamsay et al. (2006), Yang et al. (2009)
Mesorhizobium. tianshanensemrtR/mrtIGrowth rate, nodulationZheng et al. (2006), Cao et al. (2009)
Mesorhizobium huakii Biofilms and legume nodulationWang et al. (2004), Gao et al. (2006)
B. japonicum
 USDA110Unknownnod gene control via bradyoxetinLoh et al. (2002), Loh & Stacey (2003), Jitacksorn & Sadowsky (2008)
 USA 10/290 and B. elkaniiUnknownUnknownBrelles-Marino & Bedmar (2001), Pongsilp et al. (2005)

The first identified rhizobial quorum-sensing regulatory genes were the rhiR and rhiI genes found in R. leguminosarum bv. Viciae (Table 1), in which they influence infection as a result of the production of C6-HSL (an AHL carrying a C6 acyl chain), which induces the rhiABC genes of unknown function (Cubo et al., 1992). The infection-defective phenotype caused by rhi mutations was only seen in a mutant strain already compromised for nodulation because of the deletion of some nod genes. In fact, it appears that nodulation is normal in a strain lacking AHL production because mutations in all four genes encoding AHL synthesis (cinI, rhiI, raiI and traI) did not block nodulation as long as all the nod genes are present (Rodelas et al., 1999; Wilkinson et al., 2002; Wisniewski-Dye & Downie, 2002). The cinI–cinR regulatory system does control the expression of the other quorum-sensing regulatory systems in R. leguminosarum (Lithgow et al., 2000; Wisniewski-Dye & Downie, 2002). This is at least in part due to a small gene (cinS) coexpressed with cinI. It appears that both CinS and ExpR are required to regulate the expression of the glycanase (PlyB) that cleaves the surface polysaccharide. Although this exopolysaccharide is required for infection and mutations affecting its cleavage affect biofilm formation, mutations in cinS, cinI or expR did not significantly affect nodulation (Edwards et al., 2009).

Whereas mutation of cinI or cinR had little observed effect on the symbiosis with pea, mutation of the R. etli cinI gene (very similar to cinI in R. leguminosarum) significantly impaired growth, and decreased symbiotic nitrogen fixation and development of nitrogen-fixing symbiosomes in nodules (Daniels et al., 2002). CinI in R. leguminosarum produces 3-OH-C14:1-HSL (Lithgow et al., 2000) and probably the same AHL is made in R. etli (Daniels et al., 2002), but it is not known why the lack of cinI has such different effects in R. etli and R. leguminosarum. Similarly, Mesorhizobium huakuii contains genes (mrtR and mrtI) that are almost identical to those in R. leguminosarum, but in M. huakuii, these play a critical role in infection and symbiotic nitrogen fixation (Gao et al., 2006; Zheng et al., 2006; Cao et al., 2009), although the reason for their requirement is not known.

Both the raiI and raiR genes are also both found in some strains of R. leguminosarum and R. etli, in which RaiI produces 3OH-C8- and C8-HSLs and RaiR responds to these AHLs (Rosemeyer et al., 1998; Wisniewski-Dye & Downie, 2002). Genes other than raiI that are regulated by this system have not been identified. Whereas mutation of raiI caused increased nodule number and nitrogenase activity on Phaseolus beans, no such effect was noted for mutation of raiI in R. leguminosarum during pea nodulation (Rosemeyer et al., 1998; Wisniewski-Dye et al., 2002).

In S. meliloti isolates, the sinI and sinR genes appear to be widely conserved. SinI produces several AHLs in the range C12-HSL to C18-HSL (Marketon et al., 2002; Teplitski et al., 2003; Gao et al., 2005). Mutation of sinI or sinR causes delayed and reduced nodulation and it is evident that they function together with another LuxR regulator called ExpR. Among other roles, ExpR acting in concert with SinI and SinR is important for the production of EPSII (Pellock et al., 2002; Hoang et al., 2004), and for full expression of exo genes required for EPSI (Glenn et al., 2007), both of which can be important for the symbiosis. In addition, ExpR regulates genes involved in the control of expression of components required for motility (Hoang et al., 2008).

There is an expR-like gene in a similar location in R. leguminosarum and its product acts together with CinS to influence exopolysaccharide cleavage, although unlike in S. meliloti, expR is not essential for exopolysaccharide production (Edwards et al., 2009). The role of ExpR seems to be quite complex because it seems to operate together with SinI-made AHLs for expression of some genes (Gurich & Gonzalez, 2009), while it appears to induce other genes without a need for AHLs (Edwards et al., 2009; Gurich & Gonzalez, 2009). In addition, ExpR can act as a repressor when bound to AHLs (generated at a high population density) with other genes such as those involved in motility and chemotaxis (Bahlawane et al., 2008). This repression function appears to be important because a flagellar biosynthesis mutation can suppress the negative effect of the sinI mutation during infection. It appears that expressing flagellar proteins during the symbiosis may hinder infection and ExpR plays a role in coordinating both induction and repression of gene expression: this occurs by ExpR-mediated repression of VisN expression that is normally needed for induction of rem, which encodes a global regulator of motility and swarming (Gurich & Gonzalez, 2009). In addition to affecting motility, chemotaxis and flagellar gene expression, ExpR stimulates the expression of genes involved in the production of galactoglucan and depolymerization of succinoglycan (Glenn et al., 2007; Hoang et al., 2008; Gurich & Gonzalez, 2009), placing ExpR in a key position with regard to controlling several changes important for symbiosis. Paradoxically, ExpR is not required for the symbiosis and many analyses of the symbiosis have been performed with what was thought to be a wild-type strain, Rm1021, but that actually carried an insertion element within expR (Pellock et al., 2002).

Some Bradyrhizobium strains can generate AHLs (Brelles-Marino & Bedmar, 2001; Pongsilp et al., 2005), but not much is known about what they regulate. A different type of quorum-sensing regulation has been reported in B. japonicum (Loh et al., 2001, 2002; Jitacksorn & Sadowsky, 2008) in which a compound called bradyoxetin accumulates at high population densities, particularly during iron limitation. Bradyoxetin induces the nodulation gene nolA, which then leads to repression of nod gene expression (Loh & Stacey, 2003) probably via NodD2, and this is important for competitive nodulation on some types of soybean (Jitacksorn & Sadowsky, 2008). However, this appears to be rather specific to nodulation signalling and may be considered rather differently from other quorum-sensing regulatory systems.

Taken as a whole, the work with quorum-sensing regulatory mutants is somewhat enigmatic. It is clear that the quorum-sensing control systems regulate genes important for the symbiosis and yet may not be required. It is evident from work in S. meliloti that after the bacteria enter the plant, the ExpR/Sin quorum-sensing system is repressed and the rhi system in R. leguminosarum is switched off (Dibb et al., 1984; Gurich & Gonzalez, 2009; Karunakaran et al., 2009). Conversely, there is good evidence in the R. etli–bean symbiosis that the cin and rai systems are expressed in planta (Rosemeyer et al., 1998; Daniels et al., 2002), and it seems likely that Mesorhizobium strains are similar based on symbiotic phenotypes (Gao et al., 2006; Zheng et al., 2006; Cao et al., 2009). It is possible that expression of rhizobial quorum-sensing regulated genes could be influenced by the host plant, because in the galegoid legumes (such as alfalfa, pea and clover), the bacteroids are induced to differentiate into a terminal form (Mergaert et al., 2006), whereas in other legumes terminal bacteroid differentiation does not occur and so there may be a role for quorum-sensing regulation in the nodule.

It may be that different legumes themselves play a key role influencing bacterial quorum-sensing regulation, because they appear to have the potential to detect AHLs (Chen et al., 2003a, b; Mathesius et al., 2003; Gao et al., 2007) and to generate metabolites that can block AHL detection by quorum-sensing regulators, or to produce compounds that can activate bacterial quorum-sensing regulators (Gao et al., 2003). The possible role of using quorum-sensing signals and/or mimics and antagonists in communication between legumes and rhizobia has been discussed in some depth (Gao et al., 2003; Hirsch et al., 2003; Mathesius et al., 2003; Sanchez-Contreras et al., 2007).

Concluding remarks

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

One of the features of rhizobia is their relatively large genomes, ranging from about 7 to 9 Mb and it is probable that this is correlated with the environmental complexity in which they live. They must be able to survive various stresses in the soil environment and take advantage of growth niches afforded by the roots of many different plant types. Although many soil saprophytes are well adapted to such environments, rhizobia also have to be able to compete efficiently with other bacteria to grow on the root hairs of legumes and this alone will require significant additional specialization. Added to this are the requirements to (1) enter into a multilayered signalling dialogue with legumes, (2) infect roots without inducing defence reactions and the associated ability to suppress plant defences, (3) deal with the stresses such as reactive oxygen species imposed by plants, (4) grow at close to their maximal growth rate in infection threads and (5) survive and fix nitrogen in a very low oxygen environment in nodules. A significant part of the genome is dedicated to the production of the various signals, protein secretion systems, surface characteristics and the regulation of the interaction with their complex environment. Good progress has been made, but there are still niches that are only beginning to be investigated. One is the growth and survival within infection threads and transcriptomic studies are beginning to provide an insight into relevant environmental changes that occur in that environment. Another relatively unexplored area is how some rhizobia survive nodule senescence and the various stresses this imposes, and this too is an area where transcriptomics approaches will facilitate progress. One of the problems with defining the importance of any system in such environments is the likelihood that the rhizobia probably have alternative mechanisms for dealing with the stresses and so simple mutations may not reveal an effect. Furthermore, a phenotype may only be observed on some legume that is not particularly amenable to growth in the laboratory and/or seeds are not readily available. The identification of the roles of surface polysaccharides, quorum-sensing signalling, protein secretion and even some of the modifications in Nod-factor signalling have all been beset by such experiences. However, transcriptomics and analysis of strains carrying multiple mutations will help in future research.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References

I would like to thank Jan Kijne for his thoughts, insightful suggestions and corrections, Michael Göttfert, Juan Gonzalez and my student Marij Frederix for commenting on this manuscript, and Mark Laus for providing Fig. 3, which was reproduced from (Laus et al., 2005a, b) with the permission of the American Phytopathological Society. Alan Williams and Fang Xie kindly gave me unpublished images of the R. leguminosarum biofilms shown in Fig. 2. I would like to thank them and all the other members of my research group past and present whose work stimulated my interest in the background of much of the research described here. The relevant research in my laboratory is funded by the BBSRC by a grant in aid and by grant BB/E017045.

References

  1. Top of page
  2. Abstract
  3. Foreword
  4. Overview of Rhizobium–legume interactions
  5. Rhizobial extracellular components associated with root attachment
  6. Extracellular signalling in symbiotic interactions
  7. Concluding remarks
  8. Acknowledgements
  9. References