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Critical Reviews in Plant Sciences, 23(4):293–316 (2004) Copyright C Taylor and Francis Inc. ISSN: 0735-2689 print / 1549-7836 online DOI: 10.1080/07352680490480734 Plant Cell Wall Remodelling in the Rhizobium–Legume Symbiosis Nicholas J. Brewin∗ John Innes Centre, Norwich, NR4 7UH, Great Britain Referee: Professor Ann Hirsch, Department of Molecular Cell & Developmental Biology, UCLA, 405 Hilgard Avenue, Los Angeles, CA 90095-1606, USA Table of Contents I. INTRODUCTION ............................................................................................................................................ 294 II. EVOLUTIONARY CONSIDERATIONS .......................................................................................................... 295 III. PREINFECTION SIGNALLING AT THE ROOT SURFACE ........................................................................... 296 IV. CORTICAL CELL ACTIVATION BY NOD-FACTOR ..................................................................................... 298 V. PLANT CELL WALL GLYCOPROTEINS ....................................................................................................... 300 VI. INITIATION OF AN INFECTION THREAD .................................................................................................. 304 VII. TRANSCELLULAR INFECTION THREADS ................................................................................................. 305 VIII. ENDOCYTOSIS AND THE DEVELOPMENT OF SYMBIOSOMES ............................................................... 309 IX. DIFFERENTIATION OF THE SYMBIOSOME COMPARTMENT ................................................................ 309 X. CONCLUSIONS AND HISTORICAL PERSPECTIVE .................................................................................... 310 ACKNOWLEDGMENTS ........................................................................................................................................... 311 REFERENCES .......................................................................................................................................................... 311 ∗ Corresponding author. E-mail: [email protected] Abbreviations: AGP, Arabinogalactan protein; GPI-anchor, Glycosylphosphatidylinositol lipid anchor; HRGP, Hydroxyproline-rich glycoprotein; IT, Infection thread; NF, Nod-factor (Lipochitin oligosaccharide); LPS, lipolysaccharide; PRP, proline-rich protein; UDP, uridine diphosphate Colonization of host cells by rhizobium bacteria involves the progressive remodelling of the plant–microbial interface. Following induction of nodulation genes by legume-derived flavonoid signals, rhizobium secretes Nod-factors (lipochitin oligosaccharides) that cause root hair deformations by perturbing the growth of the plant cell wall. The infection thread arises as a tubular ingrowth bounded by plant cell wall. This serves as a conduit for colonizing bacterial cells that grow and divide in its lumen. The transcellular orientation of thread growth is controlled by the cytoskeleton and is coupled to cell cycle reactivation and cell division processes. In response to rhizobium infection, host cells 293 294 N. J. BREWIN synthesize several new components (early nodulins) that modify the properties of the cell wall and extracellular matrix. Root nodule extensins are a legume-specific family of hydroxyproline-rich glycoproteins targeted into the lumen of the infection thread. They have alternating extensin and arabinogalactan (AGP) glycosylation motifs. The structural characteristics of these glycoproteins suggest that they may serve to regulate fluid-to-solid transitions in the extracellular matrix. Extensibility of the infection thread is apparently controlled by peroxide-driven protein cross-linking and perhaps also by modification of the pectic matrix. Endocytosis of rhizobia from unwalled infection droplets into the host cell cytoplasm depends on physical contact between glycocalyx components of the plant and bacterial membrane surfaces. As endosymbionts, bacteroids remain enclosed within a plant-derived membrane that is topologically equivalent to the plasma membrane. This membrane acquires specialist functions that regulate metabolite exchanges between bacterial cells and the host cytoplasm. Ultimately, however, the fate of the symbiosome is to become a lysosome, causing the eventual senescence of the symbiotic interaction. Keywords I. cellulose synthesis, extensin, infection threads, nitrogen fixation, root nodules, proline rich glycoproteins INTRODUCTION Because plant cells, unlike animal cells, are nonmotile and bounded by rigid cell walls, it is often found that the properties of the plant extracellular matrix play a critical role in the process of tissue and cell morphogenesis. This principle is vividly illustrated by the example of the rhizobium–legume symbiosis. Remodelling of the plant cell wall forms the particular focus of the present review as a means to address the fundamental questions of symbiosis. What is special about legumes that allows them to establish a nitrogen-fixing endosymbiosis with rhizobium? What is an infection thread and how is it formed? How is the process of cell cycle activation coupled to the invasion process? How are host defense responses suppressed during symbiosis? The legumes (Fabaceae) are a large and diverse family of higher plants with over 18,000 species (Sprent, 2001). A distinctive characteristic of legumes is their ability to engage in a nitrogen-fixing symbiosis with diverse groups of gramnegative root nodule bacteria (Spaink et al., 1998). Examples include rhizobium, allorhizobium, azorhizobium, bradyrhizobium, mesorhizobium, sinorhizobium, and some species of burkholderia, ralstonia and methylobacterium. These bacteria are collectively called rhizobia (Perret et al., 2000; Brewin, 2002; Broughton, 2003). It is estimated that over 90% of all legume species are nodulated by rhizobium strains. Nodulation is predominant in the two major subfamilies, the papilionoids and mimosoids, but it is much less frequent among members of the more primitive caesalpinioid group, where only 21% of species have been reported to be nodulated (Sprent, 2001). During legume nodule development, host cells and their symbiotic partners undergo a progressive metamorphosis that leads to the development of new forms of molecular interaction at the plant–microbial cell interface (Brewin, 1991). Tissue and cell colonization by rhizobium is generally associated with some form of infection thread structure. Infection threads are defined as columns of invading bacteria embedded in a plant extracellular matrix and bounded by plant cell wall material (Brewin, 1998). They may be intercellular, intracellular, or transcellular, but in all cases it is clear that their development involves a remodelling of plant cell walls. In cases where there is no evidence of intracellular infection threads, e.g., peanut (Uheda et al., 2001) and lupin (Lotocka et al., 2000), there is definite evidence of an interaction between rhizobia and the plant cell wall, in particular with the middle lamella. In many (but not all) examples of legume root nodule symbiosis, there is a second class of intracellular structure termed the symbiosome (Roth and Stacey, 1989). Symbiosomes represent the discrete intracytoplasmic stage in which nitrogen-fixing bacteroids are enclosed, individually or collectively, within a plant-derived membrane envelope termed the peribacteroid membrane. This membrane is topologically related to the plasma membrane but it is not associated with plant cell wall components, e.g., cellulose and pectin (Rae et al., 1992). How are the observed changes in morphology related to underlying molecular mechanisms? Because root nodules are inessential structures (required only for growth under conditions of N-deprivation), it has been possible to identify mutations in “symbiotic genes” that are impaired in nodule development or function. Mutational analysis in genetically amenable model legumes such as Medicago truncatula, Lotus japonicum, and Pisum sativum has helped to identify key components in the developmental pathways associated with nodule morphogenesis (Stougaard et al., 2001; Tsyganov et al., 2002; Ben Amor et al., 2003). In some cases, the relevant genes have recently been cloned and sequenced, giving an indication of underlying mechanism (Downie and Parniske, 2002; Mitra et al., 2004). However, mutational analysis can only identify unique gene functions that are characteristic of signal-transduction cascades. It is less successful at dissecting complex genetic networks or systems involving closely related gene families such as the structural proteins involved in plant cell walls. In addition to direct mutational approaches, functional genomics and proteomics have helped to identify large numbers of plant (Gyorgyey et al., 2000; Mathesius et al., 2001; Lamblin et al., 2003) and bacterial (Ampe et al., 2003) genes that are upregulated (or sometimes downregulated) at different times during the infection process (Colebatch et al., 2002). Learning how, when, and where these genes function requires that molecular genetics should be complemented by cytological and biochemical approaches. For example, the use of reporter gene fusions helps to monitor transcriptional control, and the use of epitopetagged transgenic variants yields information on the ultimate location of gene products. Another analytical approach has involved the use of monoclonal antibodies and polyclonal antisera that react with cell surface components, including plant and bacterial cell wall polysaccharides, glycoproteins, and glycolipids (Perotto et al., 1991). Because of the structural and genetic complexity of these glycoconjugates at the plant–microbial interface, their nature and PLANT CELL WALL REMODELLING IN SYMBIOSIS function have often been difficult to analyze by forward or reverse genetic approaches. Yet there are many different forms of glycoconjugate, and they clearly play an important role providing positional information during the infection process. In the article that follows, I shall describe the key morphological changes observed during the development of root nodule symbiosis, particularly in P. sativum, M. truncatula, and L. japonicum. I shall approach this problem from the perspective of the interacting cell surfaces and their progressive differentiation, analyzing cellular development within the context of the development of the nodule as a whole. II. EVOLUTIONARY CONSIDERATIONS Let us begin by setting the rhizobium–legume symbiosis in an evolutionary context. Comparative genomics, comparative anatomy, and comparative physiology can point to some intriguing correlations that may help to frame questions for future experimental analysis. How did the root nodule symbiosis arise some 60 million years ago (Soltis et al., 1995), and what were the most probable antecedents? Legumes belong to the Eurosid Clade I. This taxonomic group includes virtually all known examples of root nodule symbiosis that occur in higher plants. Furthermore, the occurrence of root nodule symbiosis is restricted to a subgroup termed the FaFaCuRo, comprising the orders Fabales, Fagales, Cucurbitales, and Rosales (Kistner and Parniske, 2002). Judging from the distribution of nodulation in relation to the phylogenetic tree for the evolution of this clade, it seems probable that root nodule symbiosis has arisen independently on several occasions (Doyle et al., 1997; Gualtieri and Bisseling, 2000). In four of the five nonlegume taxonomic groups that establish root nodule symbiosis, the endosymbiont is the actinomycete, frankia (Berg, 1999; Pawlowski et al., 2003). Examples of actinorhizal plants include alnus, ceonothus, casuarina, and datisca spp. (Santi et al., 2003) frankia is related to the gram-positive group of filamentous pathogenic streptomycetes such as Streptomyces acidiscabes (Loria et al., 2003). By contrast, in the nodulated legumes and in parasponia (a nonlegume belonging to the Ulmaceae) the colonizing bacteria belong to the cluster of gram-negative bacteria collectively described as rhizobia (Perret et al., 2000). In both rhizobial and actinorhizal symbiosis, tissue colonization by bacterial cells is often via columnar infection thread structures similar to those found in legumes (Pawlowski and Bisseling, 1996). However, not all actinorhizal plants have root hair infection threads: in ceonothus, elaeagnus, cercocarpus, and shepherdia, infection involves the so-called crack-entry mode, with infection threads being formed at a later stage of development (Berg, 1999). This seems to be similar to the infection process in the legume sesbania (D’Haeze et al., 2003). The common features of the actinorhizal and rhizobial symbioses and their phylogenetic restriction to the FaFaCuRo taxonomic group may indicate that members of this group have a common characteristic that leads to a “predisposition” to establish a root nodule symbiosis (Doyle, 1998). 295 On the bacterial side, it may be worth reflecting on the fact that Agrobacterium spp. and Sinorhizobium spp. are taxonomically related (Spaink et al., 1998). The invasion strategies of Agrobacterium tumefaciens and Agrobacterium rhizogenes each depend on the exploitation of a point of plant cell damage that becomes, in effect, an incipient wound meristem. Bacterial virulence (vir) genes are transcriptionally activated by acetosyringone (a wound hormone). Following transfer of T-DNA to host cells, they are stimulated to divide, creating a gall tissue as a consequence of a local perturbation in auxin/cytokinin ratios. In the case of sinorhizobium (and other rhizobia), nod gene activation occurs in response to host plant flavonoids, another product of wounding and tissue damage (Peters and Verma, 1990). In many respects, the process of nodule initiation resembles the induction of a wound meristem (Baron and Zambryski, 1995). The consequence of Nod-factor (NF) production by rhizobia is the activation of cortical cell divisions (Long, 1996), and this is also associated with a local change in auxin/cytokinin ratios (Mathesius et al., 1998a, 1998b). It is these cortical cells with activated cell cycle processes that become the target for colonization and the site for infection thread development. These correlations point towards a possible evolutionary relationship between rhizobium colonization processes and wound- or stressinduced cell cycle activation. Other hypotheses about the evolutionary origin of nodulation have been discussed by Hirsch and LaRue (1997). Another interesting discovery has been the common genetic pathway controlling infection by zygomycete mycorrhizal fungi (Order Glomales) and Rhizobium spp. (Duc et al., 1989; Kosuta et al., 2003). These microsymbionts establish different forms of root symbiosis. Whereas the nitrogen-fixing rhizobium symbiosis is host specific and confined to the Leguminosae (Fabaceae), the mineral-scavenging arbuscular mycorrhizal (AM) symbiosis is very promiscuous and widespread among all groups of land plants (Harrison, 1999). A number of mutations have been identified in genes that apparently have a pleiotropic effect on the initiation of both symbioses (Albrecht et al., 1998; Kistner and Parniske, 2002). In L. japonicus, at least six genes constitute the common pathway. Among them, the SYMRK gene was the first to be characterized by positional cloning. It encodes a leucine-rich repeat (LRR) receptor-like kinase that is involved in the perception of both mycorrhizal and rhizobial signals, probably at the junction of the common pathway (Endre et al., 2002; Stracke et al., 2002). Some genes of the common pathway may be concerned with the suppression of host defence responses (Albus et al., 2001; Novero et al., 2002). During mycorrhiza formation, fungal hyphae penetrate the epidermis of the root and grow towards the inner cortex, sometimes by intercellular and sometimes by transcellular penetration (Kistner and Parniske, 2002; Pawlowski et al., 2003). Then, in the phase of intense biotrophic interaction, the hyphae form highly branched intracellular arbuscules bounded by a specialized form of host plasma membrane (Harrison, 1999). Thus, the course of infection is quite similar to that of rhizobium-induced infection threads. This raises 296 N. J. BREWIN an interesting question. Does the existence of genes controlling both pathways indicate that rhizobium has effectively become a mimic of the AM infection process in legumes (Parniske, 2000)? Using the inexorable logic of evolutionary biology, it seems obvious that the plant cell wall constitutes a major obstacle to the establishment of endosymbiosis. Each plant cell is enclosed within a “cardboard box,” a network of cellulose fibrils comprising the cell wall that would be completely impenetrable to microorganisms. Thus, in the context of evolution there might be two strategies to circumvent this obstacle. The first strategy involves the dissolution of the cell wall in a mature plant cell: this strategy has been adopted by many pathogens, e.g., erwinia, but not apparently by endosymbionts. The second strategy involves remodelling the process of cell wall growth at the point of inception, i.e., at the stage when the cell is still expanding or at the even earlier stage of cell division. As a result of local inhibition of cellulose synthesis and local modification of the extracellular matrix, an endosymbiont such as rhizobium, frankia, or glomus could, in effect, create a subcellular point of entry through the cellulose network with minimum damage to the host cell and with minimum elicitation of stress and defense responses (Parniske, 2000). III. PREINFECTION SIGNALLING AT THE ROOT SURFACE As a prelude to infection, rhizobial cells accumulate on the legume root surface as a biofilm, which is normally described as a close-knit microbial community that colonizes the surface of a solid substratum. The bacteria within this rhizosphere community interact with each other through a quorum-sensing system based on the exchange of homoserine lactone signalling molecules (Wisniewski-Dye and Downie, 2002). The contact zone immediately adjacent to the root is known as the rhizoplane. Its composition is influenced by cells and substances released from the root cap and by the surface properties of root hairs and epidermal cells (Loria et al., 2003). Bacterial adhesins (Mathysse and Kijne, 1998) and plant cell surface lectins may serve to tether the attachment of particular classes of bacterial cells to the root surface (van Rhijn et al., 2001). An augmented population of bacterial cells in the biofilm could potentiate an otherwise weak bacterial signal, thereby enhancing their capacity to infect the host plant. Presumably it is through this mechanism that transgenic plants expressing foreign lectin genes (van Rijn et al., 1998, 2001) are capable of being nodulated by heterologous rhizobia and establish infection threads in response to heterologous strains. Apart from promoting bacterial growth in the rhizoplane, plant lectins may have other more specific roles in cellular morphogenesis (Diaz et al., 2000), but the basis for specificity in this interaction is still unclear (Brewin and Kardailsky, 1997; Kijne et al., 1997). In the pea symbiosis, a nodule-specific lectin-like glycoprotein has been shown to be attached to the surface of exnodule bacteroid cells (Bolanos et al., 2004), while in M. truncatula a symbiotic role has been investigated for a class of apyrase lectins that may bind to bacterial surfaces or bacterial signal molecules (Navarro-Gochicoa et al., 2003a). Furthermore, a new family of receptor-like kinases has recently been described (Navarro-Gochicoa et al., 2003b), indicating that lectins are likely to have a variety of roles in cell surface recognition and signal transduction. Flavonoids secreted by host legumes act as inducers of nod gene activity in rhizobial cells (Shimada et al., 2003). This in turn results in the secretion of lipochitin oligosaccharide signal molecules by rhizobia at the root surface (Schultze and Kondorosi, 1998; D’Haeze and Holsters, 2002). These Nodfactors (NFs) act as mitogens and morphogens within the host legume root tissues, but the molecular mechanisms involved are still not understood (Limpens and Bisseling, 2003). The core structure of all NFs is that of a beta-(1,4)-linked N -acetyl glucosamine backbone, with a fatty acyl substitution on the N acetyl group of the terminal nonreducing sugar. Various decorations, particularly on the reducing and nonreducing ends of the oligosaccharide backbone, confer host specificity, e.g., the sulphate group is normally essential for nodulation in Medicago spp. (Figure 1a), although apparently Nod-factor sulphation is not required for Sinorhizobium fredii to nodulate alfalfa (Noreen et al., 2003). Using fluorescent derivatives of NF, it was shown (Goedhart et al., 2000) that most or all of the NF applied to roots of Vicia sativa became rapidly and irreversibly associated with the plant cell wall, presumably by hydrogen bonding to cellulose or hemicellulose fibers. What is not yet clear is how this apparently nonspecific binding of NF to mature plant cell walls is coupled to host-specific recognition and a cellular response to NF that might be occurring at sites where cell wall construction is still in progress. Is the primary action of NF to perturb the process of cell wall biosynthesis in root hairs and epidermal cells, or does the primary effect involve cell surface recognition followed by intracellular propagation of a signal transduction chain (Pingret et al., 1998; Carvalho-Niebel et al., 2002; Ane et al., 2004)? There is increasing indication from genetic analysis and positional cloning that several classes of membrane-spanning proteins and receptor kinases are involved in the early stages of the signal-response pathway leading to infection either by rhizobium, or by arbuscular mycorrhizal symbionts (Krussell et al., 2002; Limpens et al., 2003; Levy et al., 2004; Mitra et al., 2004). In L. japonicus, two LysM-type serine/threonine receptor kinases encoded by NFR1 and NFR5 enable the host legume to recognize its bacterial symbiont Mesorhizobium loti (Madsen et al., 2003; Radutoiu et al., 2003). However, the primary molecular response to NF binding is still unclear at the present time, as is the precise role of specific NF-binding proteins (Etzler et al., 1999), such as the receptor kinase MtLECRK1 (NavarroGochicoa et al., 2003a, 2003b). Early cytological responses to NF in root hair cells have been described in several legumes (Cardenas et al., 1998; Esseling et al., 2003). The first event, detectable only a few seconds after Nod factor application, is a Ca2+ influx at the root hair tip, causing an increase in cytosolic Ca2+ (Felle et al., 1999). This is associated with a transient depolarization of the plasma membrane PLANT CELL WALL REMODELLING IN SYMBIOSIS 297 FIG. 1. Bacterial compounds affecting cell wall biosynthesis. (a) Nod Factor from S. meliloti, a lipochitin oligosaccharide that induces cell wall deformations in the root hairs of legumes (Lerouge et al., 1990). Nod-factors generally consist of an oligosaccharide backbone comprising four or five residues of N -acetyl-Dglucosamine (GlcNAC), beta-(1,4)-linked, of which the nonreducing terminal residue is substituted at the C2 position with an acyl chain. Host specificity depends on the length and degree of unsaturation of the acyl chain, and on specific substitutions at the reducing and nonreducing terminal glucosamine residues. In the case of S. meliloti, sulphate decoration of NF is essential for the induction of most symbiotic responses in medicago. Synthesis of Nod-factors is encoded by nod-genes. During the course of evolution, these genes have spread to many groups of gram-negative soil bacteria by horizontal gene transfer (Broughton, 2003), thus conferring the ability to nodulate legumes. ( b) Thaxtomin, a cellulose synthesis inhibitor secreted by the filamentous bacterium S. acidiscabes. Thaxtomin modifies the process of cell wall biosynthesis and thereby facilitates tissue and cell invasion by this root pathogen (Scheible et al., 2003). This di-peptide derivative is a member of a family of 4-nitroindol-3-yl-containing 2,5 dioxypiperazines produced by S. scabies and S. acidiscabies. Synthesis is encoded by two peptide synthase genes, txtA and txtB, associated with a pathogenicity island. During evolution, this pathogenicity region has apparently spread by horizontal gene transfer to other root pathogenic actinomycetes (Loria etal., 2003). (Lhuissier et al., 2001; Shaw and Long, 2003a). Within 20–30 min following application of NF, there is suppression of peroxide release from roots (Shaw and Long, 2003b) and a reorganization of the actin and microtubule networks in root hair cells (Timmers et al., 1999; Sieberer and Emons, 2000, Sieberer et al., 2002). There is also a second calcium event, the induction of periodic oscillations of cytoplasmic calcium concentration (Ca2+ spiking), which is observed in the perinuclear area (Ben Amor et al., 2003; Oldroyd and Long, 2003; Shaw and Long, 2003a). Following application of NFs, the earliest observed morphological effect is the cessation of tip-growth within 1 h (Lhuissier et al., 2001). The arrest of apical growth is associated with the swelling of root hair tips. NF-induced swelling is even observed with the Nod− mutants, representing genes of the common path- way for rhizobial and mycorrhizal infection of lotus and medicago. Exceptions are NFR1 and NFR5 from L. japonicus and the NFP mutant from M. trucatula (Ben Amoret al., 2003). These mutants are apparently completely insensitive to NF, and they are presumed to identify some of the earliest components of the signal-transduction pathway. Some hours after application of NF, cell wall deformation occurs in root hairs when NF induces a reinitiation of tip growth in a different direction (Figure 2). The original growth point ceases to be active and a new apex is initiated, often leading to a branched root hair cell. Further changes in the stability and dominance of the apical growth point lead to intense root hair curling, which is apparently a precondition for the initiation of an infection thread (IT). Using a spot-inoculation system, it has been shown that local application of NF is able to 298 N. J. BREWIN FIG. 2. Induction of root hair deformation by rhizobium-derived Nod-factor. Model for root hair curling by NF secreted at a point source by a bacterial cell or microcolony (adapted from Esseling et al., 2003). (a) Normal apical growth of a root hair cell, showing uniform cell wall growth around the apical dome (arrows). (b) Localized application of Nod-factor (from an attached bacterial cell) results in a disproportionate rate of cell wall growth on the side of the apical dome closest to the site of NF application. (c) Increased cell wall growth and progressive amplification of the bacterial microcolony producing NF leads to root hair deformation and eventually to root hair curling (d). induce root hair curling towards the point of application on root hair cells of M. truncatula (Esseling et al., 2003). This suggests a model for root hair curling in which a host-specific bacterium attaches to the root hair apical dome and locally excretes NF, which is then immobilized within the cell wall ( Goedhart et al., 2000). This provides the positional cue for root hair morphogenesis. The presence of NF results in a local perturbation of cell wall biosynthesis, and the continual supply of NF from the bacterial source-cell causes progressive reorientation of tip growth towards the NF-producing bacterial cells. Thus the bacteria gradually become enclosed and entrapped within the so-called shepherd’s crook (Figure 2). These studies indicate a possible role for the machinery of cellulose synthesis in the remodelling of root hair cell walls (Mulder and Emons, 2001). It should be remembered that NFs are beta(1,4)-linked glucan oligomers (D’Haeze and Holsters, 2002). Because of their structural resemblance to cellulose and chitin oligomers, it is possible that they might interfere directly or indirectly with the process of cellulose or hemicellulose biosynthesis. Root hair swelling is also observed following application of cellulose synthesis inhibitors (D. J. Sherrier and N. J. Brewin, unpublished observations) and other stress-inducing factors (Esseling et al., 2004). Conceivably, therefore, NF may act as an inhibitor of some specific component of the machinery for cell wall synthesis in tip-growing root hair cells (Carol and Dolan, 2002; Doblin et al., 2002). This local inhibition could be the trigger for early host cell responses and ultimately for host cell invasion by rhizobium. In a similar way, it has been suggested that thaxtomin (Figure 1b), a cellulose synthesis inhibitor secreted by the root pathogen Streptomyces acidiscabes, facilitates tissue and cell invasion by this filamentous bacterium by modifying the process of cell wall biosynthesis (Loria et al., 2003). On a rather speculative note, it would be interesting to consider whether any components or processes involved in the regulation of cell wall biosynthesis and polar growth (Foreman et al., 2003) might also be involved in the NF signal response pathway (Kimura and Kondo, 2002). However, the situation is still confused because of the general lack of understanding regarding the mechanism of cellulose synthesis and its integration into the process of cell growth and development (Doblin et al., 2002). IV. CORTICAL CELL ACTIVATION BY NOD-FACTOR A distinctive feature of the rhizobium-legume symbiosis is that the rhizobial cells colonize host tissues which are the product of cell-cycle reactivation in the root cortex. In addition to the direct effects of NF on root hair and epidermal cell walls, cell cycle reactivation is observed in the cortex and pericycle following application of purified NF to host legume roots (Figure 3). The relationship between cortical cell activation and pericycle cell activation can vary between different legumes, depending on the predisposition of morphogenetic fields in the root cortex (van Spronsen et al., 2001). In Medicago sativa, the earliest morphological event observed is the activation of cells of the inner cortex, 18–24 h after inoculation with rhizobia (Timmers et al., 1999). These vacuolated cells first undergo anticlinal divisions. Subsequently there are periclinal divisions as the primordium becomes established. The same is true in pea (Scheres et al., 1990) and clover (Lotocka et al., 1997), although the process takes a day or two longer. All of these legumes are characteristic of the class that develop cylindrical (or branched) nodules with apical indeterminate meristems in which the mitotic cells are uninfected by rhizobia (Brewin, 1991). By contrast, in Phaseolus vulgaris (Tate et al., 1994) and in soybean (Calvert et al., 1984) the first cells to divide are in the hypodermis, i.e., immediately subjacent to the epidermis. These legumes give rise to spherical nodules with determinate meristems in which the mitotic cortical cells are themselves invaded by rhizobia. A similar process occurs in L. japonicus (Hayashi et al., 2000), although in this legume cell divisions are initiated in the middle regions of the cortex (Szczyglowski et al., 1998). Apparently nodule development in pea, but not in lotus, is susceptible to salicylic acid, an inhibitor of oxylipin signalling (van Spronsen et al., 2003). This inhibition may be correlated with the presence of a polyunsaturated acyl component for NF in Rhizobium leguminosarum and other rhizobia that infect members of the Galegae tribe. PLANT CELL WALL REMODELLING IN SYMBIOSIS 299 FIG. 3. Morphological effects associated with cell cycle activation by Nod-factor. (a) Root hair deformation. (b) Cell cycle activation in the outer cortex leads to centralization of nuclei and the formation of transcellular cytoplasmic strands (van Brussel et al., 1992), sometimes referred to as preinfection threads (PITs) (Timmers et al., 1999). PITs mark the subsequent course for IT development. (c) Cell division in the inner cortex (double arrowheads) leads to development of the nodule primordium which becomes the target for host cell invasion by rhizobium. (d) and (e) Cell division in the endodermis and pericycle leads to the development of the nodule vasculature and the outer uninfected cortex of the mature nodule. The diagram represents a longitudinal section for a legume root that will develop nodules with indeterminate meristems, e.g., pea and alfalfa. In many legumes that give rise to spherical nodules, e.g., soybean and Phaseolus vulgaris, the first cortical cell divisions are in the outer cortex, while in L. japonicus the first cortical divisions are in the meso-cortex (Hayashi et al., 2000). Vascularization of the developing nodule tissue apparently arises by reactivation of pericycle and endodermal cells that are adjacent to the cortical cell primordium (Bond, 1948). As a result of morphogenetic gradients arising from the vascular tissue, nodule primordia are preferentially initiated on the axis adjacent to the xylem poles (Yang et al., 1994). The pericyclederived tissues develop as a sheath surrounding the host-infected cells that originate from the cortical component of the nodule primordium. The nodule endodermis, with its specialized cell wall (Rae et al., 1991) apparently represents the boundary between the tissue types derived from pericycle and cortical cells, respectively. Vascularization of the nodule primordium and the associated development of transfer cells (Dahiya and Brewin, 2000) indicates that the nodule primordium becomes an intense sink for photosynthate. This seems to correlate with the observed activation of the early nodulin gene ENOD40 in pericycle cells of legumes (Imaizumi-Anraku et al., 2000; Santi et al., 2003). In some respects, cortical cell reactivation by rhizobium resembles what is observed during the development of a wound meristem in the root cortex (Brewin, 1991; Mathesius et al., 1998a, 1998b). Consistent with this observation is the fact that a number of wound- and stress-inducible genes have been shown to be expressed as an early response to rhizobium-derived NFs (Gyorgyey et al., 2000; Mathesius et al., 2001; Lamblin et al., 2003), including a rhizobium-induced peroxidase (Ramu et al., 2002). During the development of the rhizobium–legume symbisosis many of the cellular responses resemble those described for wound induction or the impairment of cellulose synthesis in nonlegumes. For example, a mutation in the cellulose synthase gene CeSA3 of arabidopsis causes the induction of a set of stress responses mediated by jasmonate and ethylene (Ellis et al., 2002), ectopic synthesis of lignin and other defense responses (Cano-Delgado et al., 2003). A similar cascade of stress-induced gene activation may be involved in mediating the downstream effects of NF application, because genes for lipoxygenases (the source of jasmonate) are known to be transcribed in the apical region of developing nodule tissue (Wisniewski et al., 1999), and ethylene has many effects on nodule initiation (Heidstra et al., 1997; Guinel and Giel, 2002). In the ethylene hypersensitive pea mutant R50 (sym16), rhizobium infection results in abnormal development of the nodule primordium in the root cortex with a reduced number of anticlinal cell divisions and a complete absence of periclinal divisions (Guinel and Sloetjes, 2000). Consequently, there is severe disorientation of infection thread development. As with a wound meristem, perturbing the cytokinin-to-auxin ratio in host tissues can simulate many of the effects of NF on root cortical cells (Cooper and Long, 1994; Hirsch et al., 1997). 300 V. N. J. BREWIN PLANT CELL WALL GLYCOPROTEINS It is noteworthy that many of the genes that are upregulated during the infection process encode proteins with putative signal peptides (Miklashevichs et al., 2001). Many of these “early nodulins” are (hydroxy)proline rich (glyco)proteins (HRGPs) that are targeted to the cell wall and extracellular matrix (Cassab, 1998). Presumably these proteins are candidates for the remodelling of plant cell walls during nodule initiation and for the regulation of infection thread development. Examples of the various classes are given in Table 1. The HRGPs found in the cell walls of developing nodules are representative of all the major groups that are found in higher plants (Cassab, 1998). The superfamily of HRGPs ranges from the lightly arabinosylated, proline-rich proteins (often with POxxx repeating pentapeptide motifs), through the cross-linked extensins, which are periodic and highly arabinosylated, to the arabinogalactan proteins (AGPs), which are the most highly glycosylated and the least periodic. In the case of legumes, there appears to be a fourth group typified by the glycoprotein component of gum arabic (derived from acacia trees) and root nodule extensin, a component of IT (Rathbun et al., 2002). These complex glycoproteins carry alternating glycomotifs for extensin and AGP-type glycosylation patterns (Goodrum et al., 2000) and, because of their high carbohydrate content, they are more soluble than normal extensins. On the basis of accumulating information relating primary structure to glycosylation patterns, it is becoming possible to predict the form and function of cell wall glycoproteins (Kieliszewski, 2001). The key variables are the periodicity and repetitiveness of their peptide sequence motifs and the extent of two consecutive posttranslational modifications—proline hydroxylation and glycosylation (Kielisewski and Schpak, 2001). Thus, the peptide sequence of cell wall polypeptides also determines their pattern of glycosylation (Showalter, 2001; Zhao et al., 2002). Although the rules for prolyl hydroxylation have not been completely established (Gaspar et al., 2001), it is clear that some dipeptide combinations are always hydroxylated (e.g., Ala-Pro, Pro-Ala, Pro-Pro, Pro-Val), while others are not (e.g., Lys-Pro, Tyr-Pro, Phe-Pro). According to the recently proposed “hydroxyproline contiguity hypothesis,” Hyp arabinosylation increases with Hyp contiguity, while clustered noncontiguous Hyp residues are sites for the addition of large arabinogalactan polysaccharides (Tan et al., 2004; Kieliszewski, 2001). Thus, motifs with contiguous hydroxyproline (Hyp or O) residues, e.g., SPPPP, are targets for the addition of short arabinoside chains, while galactosylation occurs on clustered noncontiguous blocks of Hyp. Galactosylation is then followed by the addition of large arabinogalactan (AG) blocks, which are built around a 1-3, betalinked galactose backbone. In relation to Table 1, there are four general classes of HRGP. Extensin-type proteins with SPPPP motifs and a high Tyr content have been reported to be downregulated at the onset of root hair deformation in Vigna unguilata (Arsenijevic-Maksimovic et al., 1997), but this class of glycoproteins is still abundant in cell wall extracts from nodule tissue (Frueauf et al., 2000). A second class of glycoprotein consists of proteins with predominantly AGP motifs, e.g., ENOD5 (Fruhling et al., 2000) and the putative arabinogalactan proteins ENOD16 and ENOD20 (Vernoud et al., 1999), each of which appears to have a metalbinding plastocyanin motif near the N-terminus and a hydrophobic Cterminus that could be part of a GPI-anchor domain (Showalter, 2001). Thirdly, there is the legume-specific class of root nodule extensins (Rathbun et al., 2002) which comprises glycoproteins with alternating extensins and AGP motifs: MtN12 is an example of this class (Gamas et al., 1996). Finally, there is the group of proline-rich glycoproteins, including ENOD11 (Journet et al., 2001), which appears to be closely associated with IT development and ENOD12 (Pichon et al., 1992), which is expressed not only in cells with ITs but also in meristematic cells. Another family member, MsENOD10 (Löbler and Hirsch, 1993), was identified in one cultivar of alfalfa, suggesting a functional redundancy of proline-rich proteins in legume nodule development. AGPs are a diverse family of proteoglycans implicated in many aspects of plant growth and development, including embryogenesis and cell proliferation (Schultz et al., 2000). While many of these proteins appear to be soluble components of the extracellular matrix, others are membrane associated through a C-terminal lipid anchor, comprising glycosylphosphatidyl inositol (Showalter, 2001; Johnson et al., 2003). GPI-anchored AGPs appear to be major component of the glycocalyx associated with the external face of the plasma membrane of all plant cells. Monoclonal antibody markers indicate that AGPs are also abundant on the IT membrane and also on the inward face of the peribacteroid membrane of the symbiosome compartment (Perotto et al., 1991; Sherrier et al., 1999). The large side chains of arabinogalactans project from the membrane surface into the extracellular matrix where they are presumed to interact with other plant cell wall component (or potentially with bacterial cell wall components if they are in close proximity). However a precise role for AGPs in cell surface attachment or signalling has yet to be established. In pea nodule membranes, monoclonal antibody MAC207 recognises a high molecular weight GPI-anchored AGP (Sherrier et al., 1999), while MAC206 recognises what is probably a low molecular weight GPI-anchored AGP that is apparently expressed very strongly in the peribacteroid membrane (Bradley et al., 1988; Perotto et al., 1991). Interestingly, it has recently been shown that bacteroid cells isolated by sucrose gradient centrifugation from pea nodule homogenates still carry the plant-derived MAC206 antigen but not the MAC207 antigen, suggesting that a low molecular weight glycosylphosphatidylinositol-(GPI)-anchored AGP may become physically associated with the surface of bacterial cells (Bolanos et al., 2004). Recently in arabidopsis, some fasciclinlike AGPs were identified as putative cell adhesion molecules, and there may be counterparts operating in the rhizobium– legume symbiosis (Johnson et al., 2003). The root nodule extensins are a class of glycoproteins that are apparently abundant in the infection thread lumen. They 301 EMBL code Sequence YGQPWNNYPK PYVYKSPPPP SPPPPYVYKS PPSPSPPPPY KSPPPPSPSP PYYYKSPPPP SPPPPYYYKS PPSPSPPPPY QTPPYYYNAP SPSPPPPYYY PPPPSPSPPP VYKSPPPPSP PPPYYYKSPP SPSPPPPYVY PPPPSPSPPP YYKSPPPPSP PYYYKSPPPP KSPPPPSPSP PYYYKSPPPP SPPPPYYYKS PPSPSPPPPY KSPPPPSPSP PYYYKSPPPP SPPPPYYPYL 60 Extensin motifs 120 Tyr-rich 180 Downregulated by 240 rhizobium 300 360 420 480 489 ArsenijevicMaksimovic et al., 1997 PLGLADKSPS PLLLKPPLPK PPLPKPPVNK QGFAEYYLYP VHKPPRKESP Proline-rich proteins associated with IT development MtENOD11 AJ297721 MASFFLYSLG LVFLSALTLV PPTYKPPIKK QPINKSPNKK PPQKKPPSRK RPINTPPNKK MtENOD12 X68032 MASFSLSILV FFFSALVLVP VHKPPHKDPP VNKPPQKESP HNMPPNPIYT PPHKEPPSRK PPHKKPSNKR AYRPPQTKPP THRHPPAEDN TPIHKNPTNT RPINTPPDKK PPPYGNQPPP VNKPSHKEPP IHF DSENSWKFPL PTRHALTRWA DHYDGNTMVV LKKTGIHHFI IPHPPRRSLP SPPSPSPSPS PSLAPSPSDS VASLAPSSSP PVYNPPIYKP PPLLKPPFPK SIHF VNKPPHKEPP 60 120 174 60 103 Pichon et al., 1992 Journet et al., 2001 (Continued on next page) PPXXX Basic amino acids Low Tyr PPXXX Basic amino acids SNYQFIVGDT 60 Plastocyanin SGKKRHCRLG 120 Extensin PSPSPSPSPR 180 AG-glycosylation SDESPSPAPS 240 ? GPI anchor site 268 Greene et al., 1998 YSESTDYLVG DRCGIRGEHV SPPTPRSSTP LSKSPSPSES AMMMFLIF MSSSSPILLM ITFQYNNKTE LKLAVVVMVA STPIPHPRKR PSSSGSKGGG MtENOD20 X99467 FIFSIWMLIS SVHEVEEEDY PVLSSPPPPP SPASPSPSPS AGHGFLEVSI Greene et al., 1998 MASSSPILLM IIFSMWLLIS HSESTDYLIG DSHNSWKVPL PSRRAFARWA SAHEFTVGDT 60 Plastocyanin ILFEYDNETE SVHEVNEHDY IMCHTNGEHV EHHDGNTKVV LDKIGVYHFI SGTKRHCKMG 120 AG-glycosylation LKLAVVVQNK HDLVLPPLIT MPMPPSPSPS PNSSGNKGGA AGLGFIMWLG VSLVMMMFLI 180 GPI anchor site Fruhling et al., 2000 TVAADDYKPY PPPPSPSPPP YYKSPPPPSP PPPYYYKSPP SPSPPPPYYY PPPPSPSPPP YYKSPPPPSP PPPYYYKSPP AJ250498 MASSSTSPIL LMIIFSMCLL FSYSESTEYI AGDTESSWKV NFPSRDALID WATRHQFTYS 60 Low Tyr DTVVNEDEDE DDCNTKIHSK LGDMVVTKRP LFLPPLITLP LSPSPAPAPN SSGAAAGRGF 120 AG-glycosylation IVLLEVSLAM LMFLIWL 137 GPI anchor site LAFAICLMAI HKYPPYYYKS PPSPSPPPPY KSPPPPSPSP PYYYKSPPPP SPPPPYYYKS PPSPSPPPPY KSPPPPSPSP Scheres et al., 1990 MGTRQWPRLI SPSPPPPPYV PPPYYYKSPP SPSPPPPYYY PPPPSPSPPP YYKSPPPPSP PPPYYYKSPP SPSPPPPYVY YNSPPPPAY VYHSPPPPVH HTYPKPVYHS PPPPVHTYPH PKPVYHSPPP VPHPKPVYHS PPPPVHTYPP HVPHPVYHSP PPPVHSPPPP MASSSSPIFL MIIFSMWLLF SYSESTEYLV RDSENSWKVN FPSRDALNRW VTRHQLTIHD 60 Low Tyr TIDVVDEDDC NTEIRSKLGG DFVVTKRPLV LPPLITLPLS PSPAPAPSLS GAAAGHGFIV 120 AG-glycosylation WLGASLPMLM FLIWL 135 GPI anchor site S45139 X91836 Y15369 Reference SPPPPKDPHH YSSPPPPPPP 60 Tyr- and His- rich Rathbun et al., 2002 YKYPSPPPPP VHTYPHPHPV 120 Basic amino acids PPPAYSPPPP AYYYKSPPPP 180 Extensin motifs 183 AG glycosylation Conserved 3 -UTR PVHTYPKPVY HSPPPPVHTY 60 Tyr- and His- rich Gamas et al., 1996 HYYYKSPPPP YHN 113 Basic amino acids Extensin motifs AG glycosylation Conserved 3 -UTR Comments/ predominant motifs MtENOD16 X99466 VfENOD5 AGPs PsENOD5 Extensins Root hair Extensin MtN12 (fragment) RNEs (with alternating AGP and extensin domains) PsRNE1 AF397026 MRSLMASAAL ILALAMLFLS FPSEISANQY SYSSPPPPVH VHTYPHPHPV YHSPPPPVHT YPHPHPVYHS PPPPTPHKKP YHSPPPPPTP HKKPYKYPSP PPPPAHTYPP HVPTPVYHSP YHH Name TABLE 1 Examples of proline-rich proteins involved in the rhizobium–legume infection process 302 EMBL code MtPRP4 LLLLGVVMLT PYEKPPPVYP PPEYQPPHEK PSYEKPPPYE YKPPHEKPPP EKPPGYNPPP KPLVEKPPTH KPPVEKPPVH KPLVEKPPVH KPPVEKPPVH KPPVEKPPVE KLPVYKPPVE KLPVYKPPVE KPQVHKPPVE KPPVHKPPFE PPPTYEKPPP VYKPPIFPPP YQPPHEKPPP EYQPPHENPP EKPPPEYQPP QEKPPPVYPP KPPYDKPPYE KPPHEKPPHE PPEYKPPHEK PPPPEYPPYV KPPVENPQFY KPHIEKPPVH KPSVEKPPVH KPPVEKPPVH KPSVEKPPVY KPPVEKPPLH KPHVEKPPVN KPPVEKPPVH KPPVEKTPMH KPPVEKPPVH KPPVHKPPVE KPPVHKPPVE KPPVHKPPVE KPPVHKPPVE KPTEYKPPIE KFPVYKPPVE NPPVHKPLVE KPPVYKPPVE TPVLANYYEP PPIEKPPTYE PPYEKPPPVY PPPYEKPPPE PPPEYQPPHE KPPPEYQPPH KPPHEKPPYE KPPHEKPPHE EYKPPHEKPP PYEKPPHEKP YGHYPPSKKN Comments/ Sequence L23504 MASISFLVLL LFALYIIPQG LANYEKPPEY HPPIEKPPIY KPPVEKPPAY KPPVEHHPVY KPPVEKPPVH KPPVEKPPVH KPPVEKLPVY KPPVEKPPVH KPPVEKLPVY KPPVEKPPVY KPPVEKLPVY KPHVEKPPVY KPLVEKPPLH KLPVYKPPVE KPPVYKPHVE KPPLHKPPVE KPPVYKPHVE KPPLHKPPVE KPPVHKPPVE KPPVYKPPVE KPPVHKPPVE KPPLHKPQVE KPPVHKSPVK KLPVYKPPAE KPPVYKPPVE KPPIYTPPL Other Proline-rich proteins SrENOD2 X63339 MSSLHYSLVT YEKPPPVYSP PEYQPPHEKP PYEKPPHEKP KPPHEKPPPE KPPPEYKPPH Name Reference 60 PPXXX 120 Basic amino acids 180 240 300 360 420 480 540 549 Wilson et al., 1994 60 PPXXX Chen et al., 1998 120 Basic amino acids 180 3 -UTR controls expression 240 300 330 predominant motifs TABLE 1 Examples of proline-rich proteins involved in the rhizobium–legume infection process. (Continued) PLANT CELL WALL REMODELLING IN SYMBIOSIS 303 FIG. 4. Glycosylation motifs of root nodule extension. Deduced amino acid sequence of RNE-1 (EMBL# AF397026; Rathbun et al., 2002), following removal of the leader peptide. The major sites of glycosylation are eleven extensin type SPPPP motifs (underlined) and eleven potential sites for addition of arabinogalactantype motifs (bold). Molecular modelling indicates that the entire structure adopts the conformation of an extended rod (S. Jenkyns, M. C. Durrant, E. A. Rathbun and N. J. Brewin, unpublished observations). Motifs at the N–terminus (YSY) and at the C-terminus (YYY) are candidates for the formation of intermolecular cross-links (Brady and Fry, 1997), while elsewhere in the molecule adjacent Tyr residues may be subject to intramolecular cross-linking. Comparison of the C-terminal sequence of RNE indicates strong conservation relative to counterparts from other legumes. were originally identified in a high-throughput screen involving monoclonal antibodies (VandenBosch et al., 1989). The DNA sequence of the corresponding cDNAs (Rathbun et al., 2002) revealed that they encode a family of sequence-repetitive structural glycoproteins. They have been described as root nodule extensins (RNE) because of their close involvement with the rhizobium infection process and because all examples of this gene family are apparently restricted to the legumes. Figure 4 shows the deduced 150 amino acid sequence of the secreted polypeptide of RNE-1, which is typical of this family of (glyco)proteins. It indicates the following interesting features that may have implications for protein complex formation in symbiotic development. 1. Ten basic residues (mainly Lys) offer scope for attachment to negatively charged surfaces, e.g., bacterial polysaccharides (Campbell et al., 2003) and plant pectins (Macdougall et al., 2001). 2. Nineteen Tyr-residues offer scope for peroxide-based protein cross-linking, either through intramolecular isodityrosine residues or by intermolecular cross-bridges (Brady and Fry, 1997). 3. Twenty two His-residues, often clustered, seem to confer the ability to complex with copper ions in the extracellular matrix. 4. Eleven extensin motifs (Ser-Pro4 ) are predicted to carry relatively small arabinose glycosylations. 5. Eleven non-contiguous (hydroxy)-proline residues are predicted to be targets for large glycan substitutions, each comprising a 1-3, beta-linked galactose backbone with one or two oligosaccharide side chains (Kieliszewski, 2001). The promoters of several proline-rich protein (PRP) genes have been used extensively in promoter-GUS reporter systems to monitor the downstream effects of NF activation (Kosuta et al., 2003). However, the functions of the gene products have not been established, although the cytological expression of ENOD10, ENOD11, ENOD12, and ENOD20 correlates closely with the process of IT formation. ENOD12 has been shown to be inessential for nodule development in M. sativa (Csanadi et al., 1994), perhaps indicating a certain redundancy of function between different members of this family of glycoproteins. Many of the early nodulin genes that encode cell wall glycoproteins, e.g., ENOD12 (Albrecht et al., 1998) and ENOD11 (Chabaud et al., 2002), are also induced during mycorrhizal infections. These proteins may serve to modulate the dynamics of the physical interaction between plant cell wall components and the invading microsymbiont. Some early nodulin genes encode proline-rich cell wall glycoproteins that are not directly associated with the infection process (Chen et al., 1998). For example, the ENOD2 gene is expressed in the outer (uninfected) cortex of the nodule, a region that is thought to serve as a gas diffusion barrier regulating the physiology of the central infected tissue (Rae et al., 1991). However, there is no direct evidence that the expression of ENOD2 transcript is affected by the prevailing concentration of oxygen surrounding the nodule (Wykoff et al., 1998). Other PRPs are expressed in the meristematic zone of the nodule rather than being directly associated with the infection process. Examples include PRP4, a member of the repetitive proline-rich gene family (Wilson et al., 1994). Root nodule extensins and proline-rich early nodulins have a biased amino acid composition and hence a high net positive charge. It is still unclear to what extent these (glyco)proteins interact with the pectin network during nodule development (MacDougall et al., 2001). Of particular interest might be the status of the pectin species rhamnogalacturanan II (RGII), which contains apiose residues that bind to borate ion (Brown et al., 2002; Rodriguez-Carvajal et al., 2003). Nodule morphogenesis has been shown to be particularly sensitive to borate deprivation (Brenchley and Thornton, 1925), and in the absence of borate there is impairment of IT development (Bolanos et al., 1996). Thus it seems likely that RGII-borate complexes interact with components involved in the infection process. Proline and hydroxyproline-rich glycoproteins have also been implicated in other forms of cross-linking in the extracellular matrix. These glycoproteins may act as priming sites for p-hydroxycinnamyl alcohols, which are the precursors for lignin biosynthesis (Cassab, 1998). Furthermore, three of the amino acid residues that are most abundant in PRPs and HRGPs, namely Val, Pro, and Lys, are targets for peroxidation and subsequent cross-linking. Glycine-rich proteins (GRPs) are also expressed at several stages of symbiotic nodule development in Medicago spp. (Kevei et al., 2002). They are thought to be unglycosylated components of the extracellular matrix, but their function in cell surface interactions is still unknown. Several examples of GRP genes are expressed exclusively in nodule tissues and are apparently induced by bacterial infection at different points in the 304 N. J. BREWIN invasion process. Differences in kinetics and localization of gene activation, as well as the primary structure of the proteins, suggest that they have nonredundant roles in nodule organogenesis. A number of other nonstructural proteins that have been described may play a catalytic role in the remodelling of plant cell walls during rhizobium-induced infection. These include: PG3 from M. truncatula, encoding a root hair polygalacturonase that is upregulated in response to infection (Munoz et al., 1998; Rodriguez-Llorente, 2003); extensin peroxidase from white lupin (Jackson et al., 1999; Price et al., 2003); coppercontaining diamine oxidase, which may provide a local source of peroxide in the extracellular matrix of pea (Laurenzi et al., 2001) and in the lumen of infection threads (Wisniewski et al., 2000); and a nodule-induced putative polyamine transporter that may excrete polyamines into the extracellular matrix of pea nodules (E. Butelli and N. J. Brewin, unpublished observations). In view of the apparent preponderance of proline-rich proteins in nodule development, it is perhaps significant that a gene encoding cyclophilin (a proline isomerase) is strongly expressed in the meristematic zone of lupin nodules (Nuc et al., 2001). Expansins are also an important class of cell wall proteins that are upregulated in response to rhizobial inoculation (Giordano and Hirsch, 2004). Several classes of protein in the extracellular matrix appear to be involved in binding ions that could in turn regulate the activity of metallo-enzymes such as cysteine proteases (Vincent and Brewin, 2000) or oxidases in the extracellular matrix pea (Laurenzi et al., 2001). For example, in pea and related legumes, an extensive class of proteins with conserved cysteine-rich motifs has been identified (Kato et al., 2002), and in M. trucatula ENOD16 and ENOD20 appear to have a plastocyanin-related domain near the N-terminus. These proteins bear His, Cys, and Met residues that may be involved in complexing with metal ions in the extracellular matrix (Greene et al., 1998). Both RNE1 and MtN12 (Table 1) have a high histidine content and may be concerned with the binding of copper using a Type 2 binding site similar to that in diamine oxidase (Laurenzi et al., 2001). When RNE1 protein was expressed ectopically in insect cells, it was found that the protein product (25–30 kDa) was capable of binding to nickel-affinity resin, suggesting a role for the histidine residues in RNE. When the same gene product (80–100 kDa) was isolated from tobacco leaves or pea nodules, there was similar evidence of binding to these resins (E. A. Rathbun, S. Gucciardo, and N. J. Brewin, unpublished observations). However, attachment of AG blocks to proline residues (Figure 4) may influence the activity of divalent cations, e.g., copper, to adjacent His residues on the polypeptide of RNE in the plant extracellular matrix. VI. INITIATION OF AN INFECTION THREAD Infection of legume host cells begins when rhizobia become entrapped between adjacent root hair cell walls (Figure 5). Similarly, in the case of the Gunnera/Nostoc endosymbiosis, physical entrapment of the prokaryote between two papillae in the infection chamber is an essential precondition for host cell invasion FIG. 5. Initiation of an infection thread in a root hair cell following cell wall deformation. (a) Bacterial cells are enclosed in the cleft that arises when growth of the root hair cell wall is deformed by the local effects of Nod factor. Peroxidebased cross-linking of the extracellular matrix may result in an outer skin that effectively isolates the enclosed bacterial cells (Wisniewski et al., 2000). (b) Continued secretion of plant glycoproteins into the enclosed space and continued growth and division of entrapped bacteria generates an inward pressure on the plant cell wall. (c) Localized rupture and partial degradation of the cell wall in the infection pocket (van Spronsen et al., 1992) generates a point of intrusion. (d) Reorganization of cytoskeletal structures generates an inward apical growth point for IT initiation. The onset of this stage has a highly specific structural requirement for Nod Factor (Catoira et al., 2001). The high concentrations of NF produced by the bacterial microcolony in the infection pocket may also be important for the initiation of the IT (Walker and Downie, 1999). (Uheda and Silvester, 2001). In the angiosperm gunnera, infection occurs only in the enclosed space between adjacent papillae, and dividing gunnera cells surrounding this cavity become the sites of cyanobacterial infection. This situation has interesting parallels with the rhizobium–legume symbiosis, indicating that physical constraint and cell cycle activation may be preconditions for penetration of the cellulose sheath and consequent intracellular invasion of plant cells by prokaryotic symbionts. In the case of legume root hairs, entrapment normally follows NF-induced root hair deformation when the root hair cell wall undergoes 360-degree curling (Esseling et al., 2003). Live bacterial cells are apparently a requirement for the initiation of an inwardly growing IT from the birefringent infection pocket, or “hyaline spot,” at the center of a curled root hair cell (Gage and Margolin, 2000). It is still not clear how an IT is initiated, but several elements in the process can be identified as a result of genetic and cytological analysis, and these are illustrated in Figure 5. A major bacterial signal for IT initiation is NF. At the point of IT initiation, there appears to be a highly stringent requirement for NF structure (Catoira et al., 2001). Furthermore, the entrapped microcolony of rhizobial cells apparently generates a high local concentration of NF, which may be required in order to reach a threshold concentration for IT initiation. Disabled strains of rhizobium producing NF with suboptimal structures PLANT CELL WALL REMODELLING IN SYMBIOSIS still induce normal root hair deformations but create numerous enlarged infection pockets without any progression towards IT initiation (Walker and Downie, 2000). Thus, the localized secretion of NF within the infection pocket apparently serves as a morphogenic organising center, providing positional information for cell wall remodelling through reorientation of the underlying plant cytoskeleton. In the case of S. meliloti, bacterial extracellular polysaccharide (succinoglycan) is also important for the initiation and propagation of ITs (Cheng and Walker, 1998; Wang et al., 1999). An exoY mutant (unable to make succinoglycan) cannot initiate ITs, resulting in the development of an empty nodule comprised exclusively of uninfected tissue. On the other hand, overproduction of extracellular polysaccharide (EPS) reduces the ability to colonize curled root hair cells, although it does not affect the subsequent rate of IT propagation. An exoZ mutant, impaired in acetyl modification, shows some slight impairment in IT initation and propagation, while an exoH mutant, impaired in succinylation, cannot form extended ITs. On the plant side, an important component of IT initiation probably depends on targeted secretion and cell wall biosynthesis. This “pull” mechanism is controlled by the cytoskeleton in such a way as to create an inwardly growing cellulose tunnel. The development of cell wall ingrowths is a common plant cell response to regions of intense metabolic flux, as for example in the development of labyrinthine wall ingrowths in transfer cells (Dahiya and Brewin, 2000), and in the development of cell wall thickenings and callose plugs adjacent to the point of attachment of bacterial and fungal pathogens (Panstruga and SchulzeLefert, 2002). Using a monoclonal antibody (MAC 265), it has been demonstrated that bacteria in the hyaline spot of legume root hair cells and in the lumen of ITs are embedded in a plant extracellular matrix (Rae et al., 1992) containing root nodule extensins (Rathbun et al., 2002). Hence, the targeted secretion of root nodule extensin and other components of the extracellular matrix could be a key factor governing the initiation of IT development. Another aspect of plant cell wall growth that leads to IT initiation is the process of entrapment. Continued growth and division of bacterial cells within a physically confined space will provide a “push” mechanism for entry, counteracting the turgour pressure of the host plant cell. Root nodule extensins are secreted from curled root hair cells (Rae et al., 1992), and under oxidative conditions these glycoproteins apparently become cross-linked and insolubilized (Wisniewski et al., 2000). Cross-linking of the extracellular matrix might serve to isolate the bacteria enclosed within the kink of the curled root hair cell. Thus, if a solid crust formed on the outside of the hyaline spot, the continued division of enclosed bacteria in the infection pocket could provide an intrusive force focussed on a point of weakness in the cell wall of the infection pocket (van Spronsen et al., 1994). In other words, peroxide-driven protein cross-linking could be the biochemical mechanism that occludes the infection pocket by enforcing a fluid-to-solid transition in the outer skin surrounding the en- 305 closed bacteria (Figure 5). As newly synthesized components of the extracellular matrix are targeted into the occluded space, this would further contribute to the intrusive growth of the IT. It is worth remembering that not all legumes are colonized by rhizobia through deformed root hair cells. Even in white clover, colonization by crack entry at the point of lateral root emergence is a well-recognized alternative (Mathesius et al., 2000). Similarly, in arachis (Uheda et al., 2001) infections are not initiated directly from within root hair cells but by intercellular penetration from wound tissue associated with the point of lateral root emergence. In the stem-nodulating legume Sesbania rostrata, rhizobial cells gain access to host tissues at the point of lateral root emergence and appear to colonize a pre-existing wound-activated meristematic region of root tissue (D’Haeze et al., 2003). Thus, in all cases of cell colonization involving rhizobia, a common feature is that the host cells have been involved in cell cycle activation, although this does not always involve root hairs. In lupin, rhizobia induce root hair deformations and penetrate the root hair cell wall, but instead of forming ITs bacteria colonize the space between the cell wall and plasma membrane. There are no intracellular or transcellular ITs, and bacteria are apparently released directly from infection droplets into the cytoplasm of cells in the root cortex, which then become meristematic (Lotocka et al., 2000). VII. TRANSCELLULAR INFECTION THREADS The use of fluorescent-tagged (GFP) derivatives of S. meliloti has indicated that bacterial cell division is restricted to the growing apex of the IT (Gage et al., 1996). Within the IT of alfalfa, the bacteria appear to be aligned longitudinally in one or a few columns of cells, with their longitudinal axis more or less parallel with the long axis of the IT. On the basis of extensive observations using mixed populations of bacteria carrying different fluorophores, it has been proposed that the growth zone of the IT extends approximately 60 µm behind the apex in alfalfa host cells (Gage, 2002). Given the dimensions of rhizobial cells (approximately 1 µm in length, and with two or three columns of cells arranged in parallel in the lumen of the IT), this implies that only 100–150 bacterial cells are actively involved in the propagation of the IT. The mean growth rate of an infection thread was observed to be approximately 10 µm per hour, and the maximum rate of cell doubling for S. meliloti was found to be 4 h, although this rate decreased with increasing distance from the growing tip of the IT. These parameters were deduced from the observed sectoring of nonclonal subpopulations within the IT lumen following infection with a mixed inoculant population comprising two distinguishable cell types. The model predicts that only the subpopulation of bacterial cells that is established at the apex of the IT will multiply indefinitely. The transcellular orientation of IT growth is apparently determined by the cytoskeleton of the host cell and there appear to be cytoskeletal connections between the growing apex of the IT and the host cell nucleus (Timmers et al., 1999). During the initiation of indeterminate nodules, root cells of the inner cortex 306 N. J. BREWIN respond to NF by re-entering the cell cycle and dividing anticlinally, thereby giving rise to the nodule primordium. As part of the process of cell cycle activation, the nucleus moves from the periphery to a central position, supported by radial cytoplasmic strands (Figure 2). This also occurs in cells of the outer cortex, but these cells usually progress no further and apparently arrest in the G2 phase of the cell cycle (Yang et al., 1994). The cells in this region give rise to columns with radially aligned transcellular cytoplasmic bridges called preinfection threads (PITs). These aligned PITs chart the course that the nascent IT will follow as its growth is propagated across successive layers of cortical cells (van Brussel et al., 1992). The composition of the IT, as deduced by electron microscopy, indicates that it is a tubular ingrowth of the primary plant cell wall (Figure 6). The IT wall of Pea and Vicia spp. is composed of cellulose and pectin, and the lumen contains plant extracellular matrix glycoproteins that are antigenically similar to those found in intercellular spaces in root tissues and at the root cap (Rae et al., 1992). Electron microscopy also reveals that each bacterial cell is sheathed by a capsule of bacterial exopolysaccharide (Rathbun et al., 2002). Extension growth of the IT probably depends on secretion of matrix glycoprotein into the lumen FIG. 6. Topology of infection threads at three stages of development. (a) Root hair deformation and IT initiation. (b) Transcellular IT in the root cortex, e.g., pea, and also found in the invasion zone of a maturing nodule with indeterminate meristem. (c) An unwalled infection droplet from which there is endocytosis, i.e., uptake of bacteroids into membrane-enclosed sacs (symbiosomes) in the host cell cytoplasm (Rae et al., 1992). Transverse sections (insets (d) and (e)) are shown as enlargements on the right-hand side. This illustrates the relationship between the topology of an IT (as a transcellular tunnel in stages (a) and (b), relative to an incipient cell plate that is laid down after mitosis. In each case, growth and orientation of cell wall (cw) and plasma membrane (pm) is controlled by the cytoskeleton. The plant extracellular matrix (Ecm) is topologically equivalent to the lumen of the IT (VandenBosch et al., 1989). Note that stages (a) and (b) are not seen in lupinus (Lotocka et al., 2000) and arachis; stage (b) is much reduced in phaseolus and glycine, in which rhizobial cells are released from the apical point of an IT ingrowth, sometimes called an infection peg (Brewin, 1998); stage (c) is not observed in andira, in which rhizobial cells develop the capacity for nitrogen fixation within tubular fixation threads that are still bounded by plant cell wall (de Faria et al., 1987). (Rathbun et al., 2002) and on multiplication of rhizobia at the growing tip (Gage, 2002). The linear arrangement of bacterial cells within the thread could reflect the viscometric properties of RNE macromolecules, which adopt an extended rodlike conformation and may be concatenated end-to-end by intermolecular tyrosine cross-linking (according to unpublished computer modelling by S. Jenkyns and M. C. Durrant). Furthermore, the EPS capsule surrounding each rhizobial cell suggests that the bacteria are, in effect, held as an emulsion within the RNE matrix. Mutational analysis confirms the importance of EPS synthesis for the sustained growth of ITs (Pellock et al., 2000). Alfalfa nodule invasion by S. meliloti can be mediated by any one of three polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). There are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both initiation and extension of ITs. However, EPS II is significantly less efficient at mediating both invasion steps, and K antigen is less efficient at mediating IT extension. In the case of EPS-II–mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen–producing strains. Additionally, EPS-I– and K-antigen–mediated ITs are eight to ten times more likely to have aberrant morphologies than those mediated by succinoglycan (Pellock et al., 2000). Thus the relationships between S. meliloti polysaccharides and plant cell surfaces seem likely to be subtle, complex, and subject to functional redundancy. Therefore the role of individual components will be difficult to dissect by mutational analysis. Perhaps the preliminary data are pointing towards two general mechanisms. On the one hand it has been proposed that EPS components serve as signal molecules to suppress host defense responses, and on the other hand the bacterial capsule could itself serve as a physical barrier that is impermeable to antimetabolites such as phytoalexins and reactive oxygen species. The presence of reactive oxygen species, including hydrogen peroxide, has been detected in the IT lumen (Santos et al., 2000; Herouart et al., 2002). Peroxide-induced cross-linking (Wisniewski et al., 2000) may explain why ITs appear to undergo a progressive hardening so that they become resistant to digestion by cell-wall–degrading enzymes such as driselase (Higashi et al., 1987). It can therefore be presumed that regulation of the concentration of reactive oxygen species (ROS) is likely to play an important role in the rhizobium–legume symbiosis. For example, bacterial superoxide dismutase (SOD) apparently plays a protective role in the symbiotic process (Santos et al., 2000). In Sinorhizobium meliloti, the sodA gene encodes the sole cytoplasmic SOD. Following deletion of this gene, the resulting SOD-deficient mutant nodulated poorly and displayed abnormal infection. It has also been observed that suppression of peroxide release by host plant root cells is an early response to the application of NF (Shaw and Long, 2003a). Presumably growth of the IT and the enclosed bacterial cells depends on the IT lumen being maintained in a fluid state. PLANT CELL WALL REMODELLING IN SYMBIOSIS By appropriate regulation of the rate of peroxide-driven protein cross-linking in the extracellular matrix, a dynamic equilibrium would be established between the apical growth processes confined to the region 60 µm behind the growing tip (Gage, 2002), relative to the progressive solidification of the infection thread lumen (Herouart et al., 2002). Another component of a fluid-to-solid transition might involve the enzymic removal of the arabinogalactan (AG) side chains from root nodule extensin (RNE) in the luminal matrix. In gum arabic glycoprotein, the AG side chains are composed of a backbone of galactose residues, linked 1-3, beta (Goodrum et al., 2000) and a similar backbone is probably present in RNE. Thus, under stress-induced conditions, the cleavage of the AG oligosaccharide backbone with a 1-3, beta endogalactosidase would convert RNE into a more conventional extensin that would probably be more susceptible to intermolecular protein cross-linking by peroxide. On the basis of the predicted physical and biochemical properties of RNE, a model for the propagation of IT growth is proposed (Figure 7). In the case of incompatible infections, e.g., with lipopolysaccharide-defective mutants of rhizobium, the growth of ITs is abnormal and they frequently abort (Perotto et al., 1994). In 307 these circumstances, the balance between a fluid and a solid IT matrix may be tilted towards a defense reaction. It is not yet clear which enzymes and signal molecules are concerned with regulation of ROS in the extracellular matrix or with the general suppression of host defense responses during symbiotic development. Lipolysaccharide (LPS) components of S. meliloti have been implicated in suppression of an infection-induced oxidative burst (Albus et al., 2001). In alfalfa cell cultures, the addition of purified LPS of S. meliloti suppressed the alkalinization and oxidative burst reactions that are normally induced by elicitors. However, cell cultures of the nonhost tobacco reacted differently: in these cell cultures, the S. meliloti LPS caused an alkalinization of the culture medium and an oxidative burst reaction. Therefore, it is concluded that S. meliloti LPS released from the bacterial surface might function as a specific signal molecule, promoting the symbiotic interaction and suppressing a pathogenic response in the host plant, alfalfa (Campbell et al., 2003). It is interesting to note that LPS of S. meliloti is sulphated (Keating et al., 2002), apparently by the same genetic system responsible for the sulphation of NF (Figure 1a). Furthermore a undine diphosphate (UDP)-glucuronic acid epimerase mutant FIG. 7. Model for the growth of an IT. The IT is bounded by a cylindrical primary plant cell wall that grows at its apex as an intrusive tube within the plant cytoplasm. The plant cytoskeleton controls the orientation of apical growth by targeting pectin-containing cytoplasmic vesicles to this growth point and by focusing the synthesis of cellulose microfibrils at the apex (Rae et al., 1992). Rhizobial cells within the lumen of an IT are embedded in a plant extracellular matrix comprising RNE and other plant glycoproteins that are secreted into the lumen (Rathbun et al., 2002). Initially, the matrix is a fluid phase, and the bacterial cells are able to grow and divide (Gage, 2002). Approximately 60 µm behind the growing point, rhizobial cells cease to divide, and the luminal matrix becomes solidified as a result of protein cross-linking. Hydrogen peroxide is the agent of cross-linking (Herouart et al., 2002) and the properties of RNE suggest that it may be the main target for oxidative cross-linking. Cleavage of the arabinogalactan side chains (Figure 4) would convert RNE from a soluble glycoprotein into a more conventional extensin that would probably be more susceptible to protein cross-linking by peroxide, thus promoting the fluid-to-solid transition in the IT matrix. Feedback control systems, that regulate the rate of protein cross-linking by peroxide, could modulate IT development (Campbell et al., 2003; Shaw and Long, 2003b). In an effective symbiosis, the rate of apical growth would be balanced by the rate of matrix solidification, but in an abortive infection, e.g., from a lipopolysaccharide-defective rhizobial strain (Perotto et al., 1994), enhanced levels of peroxide would cause arrest of the IT. Bacterial cells encapsulated by extracellular polysaccharide (Fraysse et al., 2003) are held as an emulsion in the matrix of RNE. Superoxide dismutase and other bacterial enzyme systems may help rhizobial cells to survive in the presence of peroxide (Santos et al., 2000). Sustained growth of an IT may depend on suppression of cellulose synthesis at the apex. In order to maintain growth of the IT, bacteria within the lumen probably continue to secrete Nod-factor or, alternatively, there may be another local signal required for autoregulation of IT propagation. EPS of low molecular weight may also act as a signal molecule governing IT development (Cheng and Walker, 1998). 308 N. J. BREWIN that resulted in LPS structures with reduced sulphation showed impaired nodule development and abnormal infection structures that aborted prematurely. Symbiotically defective plant mutants that show defects in infection thread development have been described in many legumes (Morzhina et al., 2000; Voroshilova et al., 2001; Tsyganov et al., 2002; Tansengco et al., 2003). Analysis of the mutant phenotypes has indicated how the genetic program for IT formation is closely interlinked with the program for development of the nodule meristem and with the morphological and metabolic differentiation of host cells and endophytes. When the corresponding genes have been identified by positional cloning, it will be much easier to develop a framework for understanding the genetic and biochemical factors that control IT growth. Similarly, a comprehensive analysis of bacterial expression profiles during IT development will identify many new bacterial components involved in the infection process (Ampe et al., 2003). This study will be greatly helped by the use of fluorescent probes, both to visualize bacteria in plant tissues and to monitor the expression of individual bacterial genes during the process of infection (Stuurman et al., 2000). The role of early nodulin gene MtN6 will be interesting to study because it appears to be a novel marker for the events preceding IT development (Mathis et al., 1999). Following infection of M. truncatula roots by S. meliloti, MtN6 transcripts accumulate in the outer cortical cells containing preinfection structures. At later stages MtN6 expression is observed ahead of growing ITs in the infection zone of mature nodules. In soybean, GmN6L is similar in sequence to MtN6 (Trevaskis et al., 2002). However, GmN6L is a late nodulin that is apparently not involved in the infection process. The GmN6L gene was strongly expressed in the infected zone of nodules, and GmN6L protein was found in symbiosomes isolated from mature soybean nodules. Homology between GmN6L and FluG, a protein involved in signalling in Aspergillus nidulans, suggests that GmN6L may play a role in communication between the host and microsymbionts during symbiotic nitrogen fixation. One key question remains: how does an IT exit from the host cell, and what controls the localized cell wall degradation that must accompany this process (van Spronsen et al., 1994)? As indicated in Figure 6, the structure and development of an IT is a tubular analogue of a postmitotic cell plate (Brewin, 1991; Rae et al., 1992). This model integrates cell cycle reactivation by rhizobium with the process of IT development. The transcellular orientation of IT growth is controlled by the cytoskeleton and is coupled to the process of cell cycle activation in the root cortex and in the nodule primordium. It is well known that rhizobiumderived NF induces cortical cell reactivation in legume roots and simultaneously initiates a cell colonization process through the formation of ITs as transcellular tunnels. As part of the cellcycle–activation process, there is centralization of cortical cell nuclei and the development of aligned transcellular strands (preinfection structures) that chart the future course of the IT. The proposal that an IT is a tubular analogue of a postmitotic cell plate has recently become testable by using an extensin gene product associated with cell division in arabidopsis. The Root-ShootHypocotyl gene (Hall and Cannon, 2002) encodes an extensintype protein that is targeted to the mother cell wall at the point of fusion of the postmitotic cell plate. Thus, following mitosis and cell division, the localization of RSH-Extensin corresponds to the previous position of the preprophase band. In legumes, it is not yet known whether a preprophase band is associated with the transcellular strands known as preinfection structures that are induced by NF as part of the process of cortical cell activation. However, the introduction into medicago of a GFPmarked RSH-extensin gene might make it possible to answer this question. The prediction would be that the advancing IT fuses with the mother cell wall at a point that is associated with RSH-extensin because the orientation of IT growth is based on the same cytoskeletal structures that are involved in cell plate formation (Figures 2 and 6). Another interesting question relating to the process of IT growth is the mechanism whereby Golgi vesicles containing RNE are targeted to the polar growth point at the apex of the IT. It has been observed (Rathbun et al., 2002) that the 3 -UTR (untranslated region) of some RNE cDNA clones shows very strong conservation of sequence (even between Pea and medicago transcripts). Furthermore, it has been shown by molecular modelling (M. Crespi, E. A. Rathbun and N. J. Brewin, unpublished observations) that the 3 -UTR of RNE cDNAs can adopt a stable hairpin-duplex structure. Thus it is possible that cytoplasmic targeting of RNE could be achieved at the mRNA level by specific coupling between RNE-mRNA and microtubule-associated proteins that serve to concentrate the transcript at the growing point of the IT. There are precedents for the cellular targeting of mRNAs in animal cells. For example, in the drosophila oocyte, microtubule-dependent processes govern the asymmetric positioning of specific mRNAs that function as cytoplasmic determinants (Januschke et al., 2002) Although NF is clearly involved in the initiation of the first IT and in the initiation of the nodule primordium, it is still unclear whether NF is required for the cell-to-cell reinitiation of ITs as they propagate into host cells derived from the nodule primordium. In Vigna unguiculata there is some indication that growth of an IT can be sustained even when the bacteria within the thread are not themselves producing NF, provided that NF is supplied from an exogenous source (Relic et al., 1994). Detailed cytological investigations of V. unguiculata showed that, in the presence of appropriate Nod factors, a mutant of sinorhizobium NGR234 deleted for nodulation genes nodABC was able to enter roots through ITs in the same way as the wild-type bacterium. This leads to the interesting possibility that there may be a second local (plant-derived) signal molecule involved in propagation of IT development (or alternatively in feedback control). In view of the intimate involvement of RNE in the regulation of IT development and its complex pattern of glycosylation, it is possible that an oligosaccharide derived from this glycoprotein could be the source of signal molecule(s) that activate PLANT CELL WALL REMODELLING IN SYMBIOSIS transcription of nodulin genes and other components of the NF signal transduction pathway. Such a plant-derived oligosaccharide signal might be expected to be a ligand for one of the plant receptor kinases recently identified by mutation as a nod-gene product (Limpens and Biselling, 2003). VIII. ENDOCYTOSIS AND THE DEVELOPMENT OF SYMBIOSOMES It is not known what drives the transition of rhizobial cells from the extracellular environment (the apoplast) to the intracytoplasmic phase of symbiosis, but clearly this involves a further remodelling of the plant cell wall. Infection droplets are intracellular structures that represent unwalled outgrowths from ITs. They permit direct contact between rhizobial cells and the plant cell membrane, allowing uptake (endocytosis) into the cytoplasmic compartment (Figure 8). The first condition for endocytosis is that rhizobial cells should come into direct contact with host cell plasma membrane without the intervention of plant cell wall as a barrier (Rae et al., 1992). Sometimes, e.g., in phaseolus (Cermola et al., 2000), endocytosis occurs at the tips of FIG. 8. Model for endocytosis and the development of symbiosomes. Uptake of rhizobial cells depends on surface contact with plasma membrane without an intervening barrier of cell wall material. This situation arises either (as illustrated for pea) following rupture of a transcellular IT as a result of continued expansion of the host cell, or (as in P. vulgaris) as a result of extrusion through the apex of the IT. At this point, the cellulose sheath apparently collapses and the pectic matrix degenerates (Rae et al., 1992). There may be a stabilizing role for rhamnogalactan II and plant cell wall glycoproteins at this transitional phase (L. Bolanos, personal communication). Close contacts at the plant–microbial interface (Bradley et al., 1986; Bolanos et al., 2004) may depend on plant arabinogalactan proteins and glycolipids (Perotto et al., 1991). The structure of bacterial lipopolysaccharide is important for the process of endocytosis (Kannenberg and Carlson, 2001), and a number of bacterial functions have been shown to be important for the endosymbiotic state, e.g., bacA in S. meliloti (Ferguson et al., 2002). Following endocytosis of bacteria (now called bacteroids), the organelle-like structure is called a symbiosome. The symbiosome membrane and the enclosed bacteroids become metabolically adapted for the process of symbiotic nitrogen fixation (Lodwig and Poole, 2003). 309 short intracellular ITs, termed infection pegs (Brewin, 1998). In other legumes, e.g., pea, endocytosis occurs when matrix material apparently extrudes beneath a point of rupture in the IT wall, creating an infection droplet. Using monoclonal antibodies as markers for in vitro studies, there is evidence for physical association between the bacterial cell surface and the glycoproteins and glycolipids that constitute the glycocalyx on the external face of the plasma membrane (Bradley et al., 1988; Perotto et al., 1991). When sucrose-purified bacteroids were isolated by differential centrifugation from pea nodules, they were shown to be associated with a lectin-like glycoprotein (recognized by monoclonal antibody MAC254) and also with a small AGP-like glycolipid recognized by MAC206 (Bolanos et al., 2004). PsNLEC1 is a lectin-like glycoprotein associated with the peribacteroid fluid, while the small AGP-like glycolipid recognized by MAC206 is associated with the symbiosomal membrane. Both of these antigens are strongly expressed in symbiosomes, suggesting that they may play a direct role in surface interactions with nodule bacteria. In Astragalus sinicus (Fujie et al., 1998) a novel nodulin gene has been identified that localizes to the plant cell wall of bacteria-infected cells but not to uninfected cells. On the bacterial side, the BacA gene (required for intracellular survival in Brucella abortis as well as in S. meliloti) appears to control modification of the cell envelope, including the development of lipid-A derivatives with long-chain fatty acids (Ferguson et al., 2002). Lipid A and O-chain modifications cause rhizobium lipopolysaccharides to become hydrophobic during bacteroid development (Kannenberg and Carlson, 2001), and this is associated with distinct changes in immunological characteristics. This hydrophobic property may be a driver for plantmicrobial cell surface interactions, interacting with membrane AGPs associated with the glycocalyx of the plasma membrane and the symbiosomal membrane (Perotto et al., 1991). However, Sharypova et al. (2003) isolated a mutant of S. meliloti that was unable to synthesize the C-28 hydroxylated fatty acid but still produced effective nodules. This suggests that a combination of several factors may be involved in adaptation to the endosymbiotic environment, including host-strain specificity (Niehaus et al., 1998), the nature of bacterial surface polysaccharides (Campbell et al., 2003), the presence of specific adhesins (Oke and Long, 1999), and the extent of nutritional and hypo-osmotic flexibility (Ferraioli et al., 2002). IX. DIFFERENTIATION OF THE SYMBIOSOME COMPARTMENT Symbiosomes represent the discrete cytoplasmic stage in which nitrogen-fixing bacteroids are enclosed, individually or collectively, within a plant-derived membrane envelope termed the peribacteroid membrane. In many legume hosts (e.g., pea), bacteroids divide within the symbiosome compartment and the peribacteroid membrane divides concomitantly. In other legumes, e.g., phaseolus and glycine, bacteroids accumulate in groups of 8–12 within symbiosomes as a result of symbiosome 310 N. J. BREWIN fusion (Fedorova et al., 1999; Cermola et al., 2000) and/or of continuing bacteroid division. Mutational analysis is perhaps most advanced in the pea system, and a number of developmental blocks have been identified (Imaizumi-Anraku et al., 2000; Morzhina et al., 2000; Voroshilova et al., 2001). For example, mutation of sym 31 causes a developmental block in symbiosome differentiation that is apparently associated with an altered pathway for vesicle targeting of cytoplasmic vehicles (Dahiya et al., 1998). In this mutant, the symbiosome is apparently indistinguishable from a prelysosomal vesicle enclosed by plasma membrane. The supporting cytoplasmic infrastructure for symbiosomes apparently includes a modified array of actin micofilaments (Davidson and Newcomb, 2001). In host cells containing bacteroids, there is a network of cytoplasmic microfilaments that is equally abundant in all regions of the cytoplasm and may interact with the bacteroids and organelles. There is also evidence for nodule-specific regulation of phosphatidyl inositol transfer proteins in L. japonicus (Kapranov et al., 2001) and of a special role for small GTP-binding proteins in membrane targeting in Mesembryanthemum crystallinum (Bolte et al., 2000). As a result of membrane differentiation, the peribacteroid membrane becomes specialized for osmoregulation by acquisition of Nodulin 26, an aquaporin (Dean et al., 1999). It also assumes a specialized metabolic function in the regulation of metabolic exchanges between bacteroids and the host cytoplasm. This has been examined by proteomics (Saalbach et al., 2002) and by bacterial mutant and gene expression studies (Lodwig and Poole, 2003). Bacteroids are normally sustained within symbiosomes as a prelytic compartment of the endomembrane system. Ultimately, however, symbiosomes enter a senescent phase, along with the host cells that harbor them. In some plant mutants, symbiosome senescence is induced prematurely, e.g., in peas carrying mutations in sym 13, sym 26, and sym 27 (Voroshilova et al., 2001). Although the molecular mechanisms for these mutations are not understood, it is presumed that they affect the nature of vesicle targeting pathways to the symbiosome compartment (Andreeva et al., 1998; Vincent and Brewin, 2000). Defining what is essential for the metabolic control of biological nitrogen fixation must be tempered by an awareness of the diversity that has developed in the rhizobium–legume symbiosis during the course of evolution. There are over 18,000 species in the legume family the Fabaceae (Sprent, 2001), but only a few dozen have been studied morphologically and only two or three model species have been subject to genetic and molecular analysis. Evolutionary diversity is illustrated by the fact that, in some primitive species, e.g., gleditsia, there are apparently infection threads in the root cortex without the development of a nodule (de Faria, 1999). In other primitive legumes, e.g., andira (de Faria et al., 1987), there are no symbiosomes and hence no intracellular bacteroids that are specialized for nitrogen fixation. Instead, bacterial nitrogen fixation takes place within tubular infection threads (termed fixation threads) that are still bounded by plant cell walls. In the light of this evolutionary diversity (Szczyglowski and Amyot, 2003), it is clear that there is a wide gulf between nodule morphogenesis (on the one hand) and the fundamental molecular biology of symbiosis (on the other). X. CONCLUSIONS AND HISTORICAL PERSPECTIVE One of the fascinations of biology is the relationship between structure and function. Pliny the Elder (75 AD) was one of the first to record the agronomic benefits of legumes: “It is universally agreed that no manure is more beneficial than a crop of lupins turned in by the plough or with forks before the plants form pods.” This observation was put on a scientific footing by Hellriegel and Willfarth in their classic physiological study (1888) entitled Where Do Legumes Get Their Nitrogen From?, as reviewed by Quispel a century later (1988). The fundamental anatomy of IT development was described by Brenchley and Thornton (1925), and the essential features of nodule morphogenesis were described by Bond (1948). What is exciting in the era of molecular genetics is the opportunity to relate morphogenesis to molecular biology and hence to describe what is unique about the rhizobium–legume symbiosis. The specificity of the symbiotic interaction is based on a molecular dialogue in which legume-derived flavonoids induce transcription of rhizobial nod-genes, and rhizobial NFs induce cortical cell division, root hair deformation, and the initiation of IT development (Limpens and Bisseling, 2003). During the process of nodule development that follows this initial molecular dialogue, rhizobial cells are progressively internalized by plant cells. Successive changes in cell morphology are matched by successive changes in molecular architecture in the cell wall and associated surface structures. As the host–symbiont interaction develops, the rhizobial cells move progressively from the outside of the cell inwards towards the vacuolar compartment of the Golgi-derived endomembrane system. The first effects of rhizobium-derived NFs are seen as deformations of the primary cell wall of root hairs and may involve perturbations in cellulose biosynthesis. Next, the extracellular matrix, comprising cell wall glycoproteins and pectic components, is modified to accommodate IT development. This is apparently associated with the extensive and targeted secretion of a legume-specific family of plant glycoproteins, RNE. Transcellular ITs apparently develop in the context of a developing meristem that resembles a wound meristem. The subsequent process of endocytosis depends on the surface properties of the plasma membrane glycocalyx, comprising membrane AGPs and glycolipids. During the differentiation of symbiosomes and the development of the capacity for biological nitrogen fixation, vesicle-targeting pathways are modified to control the metabolism of bacteroids. Finally, an autolytic process is induced and symbiosomes go into senescence. “Topobiology” has been defined as the study of the placedependent regulation of cell development that results from the interactions of molecules at cell surfaces (Cassab, 1998). Not surprisingly, in the case of the rhizobium–legume symbiosis, PLANT CELL WALL REMODELLING IN SYMBIOSIS we have learned that these interactions are complex and multifaceted. A large number of plant and bacterial genes and gene products cooperate during the course of nodule development. But to what extent are these genes and functions unique to the process of symbiotic interaction? Many of the legume genes that are critical for symbiosis will soon be identified by genomic approaches in model legumes, but their functions will only be established in the context of plant cell biology. Preliminary indications suggest that tissue and cell colonization by rhizobium involves many gene functions that are also involved in plant responses to other forms of biotic and abiotic stress. In general, the colonization process appears to depend on suppression of host defense responses that would otherwise arrest bacterial development—in the extracellular matrix, by causing abortion of ITs, or, in the cytoplasmic phase, by inducing early senescence of the symbiosome organelle. Although it is still not yet possible to identify the unique components of the rhizobium–legume interaction, it is certainly becoming possible to make some more informed guesses. First, cell wall remodelling is a direct or indirect host cell response to rhizobium-derived NF. Perhaps this involves some form of inhibition of cell wall biosynthesis (as with a root pathogen such as S. acidiscabes, which produces thaxtomin). Second, tissue and cell invasion by rhizobium involves targeted secretion of RNE and other proline-rich (glyco) proteins into the extracellular matrix. Third, peroxide-driven protein cross-linking apparently leads to fluid-to-solid phase transitions in the extracellular matrix, which affects the progress of IT growth. Fourth, IT development is closely coupled to the process of cell cycle activation in the root cortex. The nodule meristem seems to be a composite between a cortical wound meristem and a modified lateral root meristem. Fifth, the transcellular orientation of IT growth probably involves close coupling between cell wall remodelling and the underlying architecture of the cytoskeleton. Future genetic and genomic analysis will help to identify the components of these processes that are unique to symbiosis and the components that play a more general role in the stress biology of legumes. ACKNOWLEDGMENTS I thank Allan Downie and Keith Chater for their critical reading of the manuscript. The John Innes Centre is supported by a grant-in-aid from the UK Biotechnology and Biological Research Council. 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