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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
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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
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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
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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
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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).
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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
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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.
REFERENCES
Albrecht, C., Geurts, R., Lapeyrie, F., and Bisseling, T. 1998. Endomycorrhizae
and rhizobial Nod factors both require SYM8 to induce the expression of the
early nodulin genes PsENOD5 and PsENOD12A. Plant J. 15: 605–614.
Albus, U., Baier, R., Holst, O., Puhler, A., and Niehaus, K. 2001. Suppression of
an elicitor-induced oxidative burst reaction in Medicago sativa cell cultures by
Sinorhizobium meliloti lipopolysaccharides. New Phytologist 151: 597–606.
Ampe, F., Kiss, E., Sabourdy, F., and Batut, J. 2003. Transcriptome analysis of
Sinorhizobium meliloti during symbiosis. Genome Biol. 4: art–R15.
311
Andreeva, I. N., Kozharinova, G. M., and Izmailov, S. F. 1998. Senescence of
legume nodules. Russ. J. Plant Physiol. 45: 101–112.
Ane, H. M., Kiss, G. B., Denarie, J., and Cook, D. R. 2004. Medicago truncatula
DMI1 required for bacterial and fungal symbioses in legumes. Science 303:
1364–1367.
Arsenijevic-Maksimovic, I., Broughton, W. J., and Krause, A. 1997. Rhizobia
modulate root-hair-specific expression of extensin genes. Mol. Plant Microbe
Interact. 10: 95–101.
Baron, C. and Zambryski, P. C. 1995. Notes from the underground—Highlights
from plant-microbe interactions. Trends Biotechnol. 13: 356–362.
Ben Amor, B., Shaw, S. L., Oldroyd, G. E. D., Maillet, F., Penmetsa, R. V., Cook,
D., Long, S. R., Denarie, J., and Gough, C. 2003. The NFP locus of Medicago
truncatula controls an early step of Nod factor signal transduction upstream
of a rapid calcium flux and root hair deformation. Plant J. 34: 495–506.
Berg, R. H. 1999. Frankia forms infection threads. Can. J. Botany-Revue Canadienne de Botanique 77: 1327–1333.
Bolanos, L., Brewin, N. J., and Bonilla, I. 1996. Effects of boron on Rhizobiumlegume cell-surface interactions and nodule development. Plant Physiol. 110:
1249–1256.
Bolanos, L., Redondo-Nieto, M., Rivilla, R., Brewin, N. J., and Bonilla, I. 2004.
Cell surface interactions of Rhizobium and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules. Mol.
Plant Microbe Interact. 17: 216–223.
Bolte, S., Schiene, K., and Dietz, K. J. 2000. Characterization of a small GTPbinding protein of the rab 5 family in Mesembryanthemum crystallinum with
increased level of expression during early salt stress. Plant Mol. Biol. 42:
923–936.
Bond, L. 1948. Origin and developmental morphology of root nodules of Pisum
sativum. Bot. Gaz. 109: 411–434.
Bradley, D. J., Butcher, G. W., Galfre, G., Wood, E. A., and Brewin, N. J. 1986.
Physical association between the peribacteroid membrane and lipopolysaccharide from the bacteroid outer-membrane in Rhizobium-infected pea root
nodule cells. J. Cell Sci. 85: 47–61.
Bradley, D. J., Wood, E. A., Larkins, A. P., Galfre, G., Butcher, G. W., and
Brewin, N. J. 1988. Isolation of monoclonal antibodies reacting with peribacteroid membranes and other components of pea root nodules containg Rhizobium leguminosarum. Planta 173: 149–160.
Brady, J. D. and Fry, S. C. 1997. Formation of Di-isodityrosine and loss of
isodityrosine in the cell walls of tomato cell-suspension cultures treated with
fungal elicitors or H2O2. Plant Physiol. 115: 87–92.
Brenchley, W. E. and Thornton, H. G. 1925. The relation between the development, structure and functioning of nodules on Vicia faba, as influenced by the
presence or absence of boron in the nutrient medium. Proc. Roy. Soc. London
Ser. B 98: 373–398.
Brewin, N. J. 1991. Development of the legume root nodule. Ann. Rev. Cell Biol.
7: 191–226.
Brewin, N. J. 1998. Tissue and cell invasion by Rhizobium: the structure and
development of infection threads and symbiosomes. In: The Rhizoibaceae, pp.
417–429, Spaink, H. P., Kondorosi, A., and Hooykaas, P. J. J., Eds., Kluwer,
Dordrecht.
Brewin, N. J. 2002. Pods and nods: a new look at symbiotic nitrogen fixing
bacteria. Biologist 49: 113–117.
Brewin, N. J. and Kardailsky, I. V. 1997. Legume lectins and nodulation by
Rhizobium. Trends Plant Sci. 2: 92–98.
Broughton, W. J. 2003. Roses by other names: taxonomy of the Rhizobiaceae.
J. Bacteriol. 185: 2975–2979.
Brown, P. H., Bellaloui, N., Wimmer, M. A., Bassil, E. S., Ruiz, J., Hu, H.,
Pfeffer, H., Dannel, F., and Romheld, V. 2002. Boron in plant biology. Plant
Biology 4: 205–223.
Calvert, H. E., Pence, M. K., Pierce, M., Malik, N. S. A., and Bauer, W. D.
1984. Anatomical analysis of the development and distribution of Rhizobium
infections in soybean roots. Can. J. Botany-Revue Canadienne de Botanique
62: 2375–2384.
Campbell, G. R. O., Sharypova, L. A., Scheidle, H., Jones, K. M., Niehaus, K.,
Becker, A., and Walker, G. C. 2003. Striking complexity of lipopolysaccharide
312
N. J. BREWIN
defects in a collection of Sinorhizobium meliloti mutants. J. Bacteriol. 185:
3853–3862.
Cano-Delgado, A., Penfield, S., Smith, C., Catley, M., and Bevan, M. 2003.
Reduced cellulose synthesis invokes lignification and defense responses in
Arabidopsis thaliana. Plant J. 34: 351–362.
Cardenas, L., Vidali, L., Dominguez, J., Perez, H., Sanchez, F., Hepler, P. K.,
and Quinto, C. 1998. Rearrangement of actin microfilaments in plant root
hairs responding to Rhizobium etli nodulation signals. Plant Physiol. 116:
871–877.
Carol, R. J. and Dolan, L. 2002. Building a hair: tip growth in Arabidopsis
thaliana root hairs. Philos. Transac. Roy. Soc. London Series B-Biol. Sci.
357: 815–821.
Carvalho-Niebel, F., Timmers, A. C. J., Chabaud, M., Defaux-Petras, A., and
Barker, D. G. 2002. The Nod factor-elicited annexin MtAnn1 is preferentially
localised at the nuclear periphery in symbiotically activated root tissues of
Medicago truncatula. Plant J. 32: 343–352.
Cassab, G. I. 1998. Plant cell wall proteins. Ann. Rev. Plant Physiol. Plant Mol.
Biol. 49: 281–309.
Catoira, R., Timmers, A. C. J., Maillet, F., Galera, C., Penmetsa, R. V., Cook,
D., Denarie, J., and Gough, C. 2001. The HCL gene of Medicago truncatula
controls Rhizobium-induced root hair curling. Development 128: 1507–1518.
Cermola, M., Fedorova, E., Tate, R., Riccio, A., Favre, R., and Patriarca, E. J.
2000. Nodule invasion and symbiosome differentiation during Rhizobium etli
Phaseolus vulgaris symbiosis. Mol. Plant Microbe Interact. 13: 733–741.
Chabaud, M., Venard, C., Defaux-Petras, A., Becard, G., and Barker, D. G.
2002. Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular
mycorrhizal fungi. New Phytol. 156: 265–273.
Chen, R., Silver, D. L., and de Bruijn, F. J. 1998. Nodule parenchyma-specific
expression of the Sesbania rostrata early nodulin gene SrEnod2 is mediated
by its 3 untranslated region. Plant Cell 10: 1585–1602.
Cheng, H. P. and Walker, G. C. 1998. Succinoglycan is required for initiation
and elongation of infection threads during nodulation of alfalfa by Rhizobium
meliloti. J. Bacteriol. 180: 5183–5191.
Colebatch, G., Trevaskis, B., and Udvardi, M. 2002. Symbiotic nitrogen fixation
research in the postgenomics era. New Phytolo. 153: 37–42.
Cooper, J. B. and Long, S. R. 1994. Morphogenetic rescue of Rhizobium meliloti
nodulation mutants by trans-zeatin secretion. Plant Cell 6: 215–225.
Csanadi, G., Szecsi, J., Kalo, P., Kiss, P., Endre, G., Kondorosi, A., Kondorosi,
E., and Kiss, G. B. 1994. ENOD12, an early nodulin gene, is not required
for nodule formation and efficient nitrogen-fixation in alfalfa. Plant Cell 6:
201–213.
D’Haeze, W., De Rycke, R., Mathis, R., Goormachtig, S., Pagnotta, S.,
Verplancke, C., Capoen, W., and Holsters, M. 2003. Reactive oxygen species
and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume. Proc. Natl. Acad. Sci. USA 100: 11789–11794.
D’Haeze, W. and Holsters, M. 2002. Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology 12: 79R–105R.
Dahiya, P. and Brewin, N. J. 2000. Immunogold localization of callose and other
cell wall components in pea nodule transfer cells. Protoplasma 214: 210–218.
Dahiya, P., Sherrier, D. J., Kardailsky, I. V., Borisov, A. Y., and Brewin, N. J.
1998. Symbiotic gene sym31 controls the presence of a lectinlike glycoprotein
in the symbiosome compartment of nitrogen-fixing pea nodules. Mol. Plant
Microbe Interact. 11: 915–923.
Davidson, A. L. and Newcomb, W. 2001. Changes in actin microfilament arrays
in developing pea root nodule cells. Can. J. Botany-Revue Canadienne de
Botanique 79: 767–776.
de Faria, S. M., McInroy, S. G., and Sprent, J. I. 1987. The occurrence of infected
cells with persistent infection threads in legume root nodules. Can. J. Bot. 65:
553–558.
de Faria, S. M. 1999. Nodulation of forest legumes. In: Soil Fertility, Soil Biology
and Plant Nutrition, pp. 667–686, Sequira, J. O., Ed., Federal University of
Brazil, Buenos Aires.
Dean, R. M., Rivers, R. L., Zeidel, M. L., and Roberts, D. M. 1999. Purification
and functional reconstitution of soybean nodulin 26. An aquaporin with water
and glycerol transport properties. Biochemistry 38: 347–353.
Diaz, C. L., Spaink, H. P., and Kijne, J. W. 2000. Heterologous rhizobial
lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions
in red clover roots, transformed with the pea lectin gene. Mol. Plant Microbe
Interact. 13: 268–276.
Doblin, M. S., Kurek, I., Jacob-Wilk, D., and Delmer, D. P. 2002. Cellulose
biosynthesis in plants: from genes to rosettes. Plant Cell Physiol 43: 1407–
1420.
Downie, J. A. and Parniske, M. 2002. Plant biology—fixation with regulation.
Nature 420: 369–370.
Doyle, J. J. 1998. Phylogenetic perspectives on nodulation: evolving views of
plants and symbiotic bacteria. Trends Plant Sci. 3: 473–478.
Doyle, J. J., Doyle, J. L., Ballenger, J. A., Dickson, E. E., Kajita, T., and Ohashi,
H. 1997. A phylogeny of the chloroplast gene rbcL in theLeguminosae: taxonomic correlations and insights into the evolution of nodulation. Am. J. Botany
84: 541–554.
Duc, G., Trouvelot, A., and Sagan, M. 1989. First report of non-mycorrhizal
plant mutants obtained in pea and fababean. Plant Sci. 60: 215–222.
Ellis, C., Karafyllidis, I., Wasternack, C., and Turner, J. G. 2002. The Arabidopsis
mutant cev1 links cell wall signaling to jasmonate and ethylene responses.
Plant Cell 14: 1557–1566.
Endre, G., Kereszt, A., Kevei, Z., Mihacea, S., Kalo, P., and Kiss, G. B. 2002. A
receptor kinase gene regulating symbiotic nodule development. Nature 417:
962–966.
Esseling, J. J., Lhuissier, F. G. P., and Emons, A. M. C. 2003. Nod factorinduced root hair curling: continuous polar growth towards the point of nod
factor application. Plant Physiol. 132: 1982–1988.
Esseling, J. J., Lhuissier, F. G. P., and Emons, A. M. C. 2004. A non-symbiotic
root hair growth tip phenotype in NORK-mutated legumes: implications for
Nod factor-induced signalling and formation of multi-faceted root hair pockets
for bacteria. Plant Cell 16: 933–966.
Etzler, M. E., Kalsi, G., Ewing, N. N., Roberts, N. J., Day, R. B.,
and Murphy, J. B. 1999. A nod factor binding lectin with apyrase
activity from legume roots. Proc. Natl. Acad. Sci. USA 96: 5856–
5861.
Fedorova, E., Thomson, R., Whitehead, L. F., Maudoux, O., Udvardi, M. K.,
and Day, D. A. 1999. Localization of H+ -ATPase in soybean root nodules.
Planta 209: 25–32.
Felle, H. H., Kondorosi, E., Kondorosi, A., and Schultze, M. 1999. Elevation
of the cytosolic free [Ca2+] is indispensable for the transduction of the nod
factor signal in alfalfa. Plant Physiol. 121: 273–279.
Ferguson, G. P., Roop, R. M., and Walker, G. C. 2002. Deficiency of a Sinorhizobium meliloti bacA mutant in alfalfa symbiosis correlates with alteration of
the cell envelope. J. Bacteriol. 184: 5625–5632.
Ferraioli, S., Tate, R., Cermola, M., Favre, R., Iaccarino, M., and Patriarca, E. J. 2002. Auxotrophic mutant strains of Rhizobium etli reveal new
nodule development phenotypes. Mol. Plant Microbe Interact. 15: 501–
510.
Foreman, J., Demidchik, V., Bothwell, J. H. F., Mylona, P., Miedema, H., Torres,
M. A., Linstead, P., Costa, S., Brownlee, C., Jones, J. D. G., Davies, J. M.,
and Dolan, L. 2003. Reactive oxygen species produced by NADPH oxidase
regulate plant cell growth. Nature 422: 442–446.
Fraysse, N., Couderc, F., and Poinsot, V. 2003. Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis. Eur. J. Biochem. 270:
1365–1380.
Frueauf, J. B., Dolata, M., Leykam, J. F., Lloyd, E. A., Gonzales, M.,
Vandenbosch, K., and Kieliszewski, M. J. 2000. Peptides isolated from cell
walls of Medicago truncatula nodules and uninfected root. Phytochem. 55:
429–438.
Fruhling, M., Hohnjec, N., Schroder, G., Kuster, H., Puhler, A., and Perlick, A. M. 2000. Genomic organization and expression properties of the
VfENOD5 gene from broad bean (Vicia faba L.). Plant Sci. 155: 169–
178.
Fujie, M., Nakanishi, Y., Murooka, Y., and Yamada, T. 1998. AsNODc22, a
novel nodulin gene of Astragalus sinicus, encodes a protein that localizes
along the cell wall of bacteria-infected cells in a nodule. Plant Cell Physiol.
39: 846–852.
PLANT CELL WALL REMODELLING IN SYMBIOSIS
Gage, D. J. 2002. Analysis of infection thread development using Gfpand DsRed-expressing Sinorhizobium meliloti. J. Bacteriol. 184: 7042–
7046.
Gage, D. J., Bobo, T., and Long, S. R. 1996. Use of green fluorescent protein
to visualize the early events of symbiosis between Rhizobium meliloti and
alfalfa (Medicago sativa). J. Bacteriol. 178: 7159–7166.
Gage, D. J. and Margolin, W. 2000. Hanging by a thread: invasion of legume
plants by rhizobia. Curr. Opin. Microbiol. 3: 613–617.
Gamas P., Niebel, F. D. C., and Lescure, N. 1996. Use of subtractive hybridisation to identify new Medicago truncatula genes induced during root nodule
development. Mol. Plant Microbe Interact. 9: 233–242.
Gaspar, Y., Johnson, K. L., McKenna, J. A., Bacic, A., and Schultz, C. J. 2001.
The complex structures of arabinogalactan-proteins and the journey towards
understanding function. Plant Mol. Biol. 47: 161–176.
Giordano, W. and Hirsch, A. M. 2004. The expression of MaEXP1, a Melilotus
alba expansin gene is up-regulated during the sweetclover-Sinorhizobium
meliloti interaction. Mol. Plant Microbe Interact. 17: 613–622.
Goedhart, J., Hink, M. A., Visser, A. J. W. G., Bisseling, T., and Gadella, T. W. J.
2000. In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls. Plant J. 21:
109–119.
Goodrum, L. J., Patel, A., Leykam, J. F., and Kieliszewski, M. J. 2000. Gum arabic glycoprotein contains glycomodules of both extensin and arabinogalactanglycoproteins. Phytochem. 54: 99–106.
Greene, E. A., Erard, M., Dedieu, A., and Barker, D. G. 1998. MtENOD16 and
20 are members of a family of phytocyanin-related early nodulins. Plant Mol.
Biol. 36: 775–783.
Gualtieri, G. and Bisseling, T. 2000. The evolution of nodulation. Plant Mol.
Biol. 42: 181–194.
Guinel, F. C. and Geil, R. D. 2002. A model for the development of the rhizobial
and arbuscular mycorrhizal symbioses in legumes and its use to understand
the roles of ethylene in the establishment of these two symbioses. Can. J.
Botany-Revue Canadienne de Botanique 80: 695–720.
Guinel, F. C. and Sloetjes, L. L. 2000. Ethylene is involved in the nodulation
phenotype of Pisum sativum R50 (sym 16), a pleiotropic mutant that nodulates
poorly and has pale green leaves. J. Exper. Botany 51: 885–894.
Gyorgyey, J., Vaubert, D., Jimenez-Zurdo, J. I., Charon, C., Troussard, L.,
Kondorosi, A., and Kondorosi, E. 2000. Analysis of Medicago truncatula nodule expressed sequence tags. Mol. Plant Microbe Interact. 13: 62–
71.
Hall, Q. and Cannon, M. C. 2002. The cell wall hydroxyproline-rich glycoprotein
RSH is essential for normal embryo development in Arabidopsis. Plant Cell
14: 1161–1172.
Harrison, M. J. 1999. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Ann. Rev. Plant Physiol. Plant Mol. Bio. 50: 361–389.
Hayashi, M., Imaizumi-Anraku, H., Akao, S., and Kawaguchi, M. 2000. Nodule
organogenesis in Lotus japonicus. J. Plant Res. 113: 489–495.
Heidstra, R., Yang, W. C., Yalcin, Y., Peck, S., Emons, A. M., vanKammen, A.,
and Bisseling, T. 1997. Ethylene provides positional information on cortical
cell division but is not involved in Nod factor-induced root hair tip growth in
Rhizobium-legume interaction. Development 124: 1781–1787.
Herouart, D., Baudouin, E., Frendo, P., Harrison, J., Santos, R., Jamet, A., Van
de Sype, G., Touati, D., and Puppo, A. 2002. Reactive oxygen species, nitric
oxide and glutathione: a key role in the establishment of the legume-Rhizobium
symbiosis? Plant Physiol. Biochem. 40: 619–624.
Higashi, S., Kushiyama, K., and Allan, D. 1987. Electron-microscopic observations of infection threads in driselase-treated nodules of Astragalus sinicus.
Can. J. Microbiol. 32: 947–952.
Hirsch, A. M., Fang, Y., Asad, S., and Kapulnik, Y. 1997. The role of phytohormones in plant-microbe symbioses. Plant and Soil 194: 171–184.
Hirsch, A. M. and LaRue, T. A. 1997. Is the legume nodule a modified root or
stem or an Organ sui generis? Crit. Rev. Plant Sci. 16: 361–392.
Imaizumi-Anraku, H., Kouchi, H., Syono, K., Akao, S., and Kawaguchi, M.
2000. Analysis of ENOD40 expression in alb1, a symbiotic mutant of
Lotus japonicus that forms empty nodules with incompletely developed nodule vascular bundles. Mol. Gen. Genet. 264: 402–410.
313
Jackson, P., Paulo, S., Brownleader, M., Freire, P. O., and Ricardo, C. P. P.
1999. An extensin peroxidase is associated with white-light inhibition of
lupin (Lupinus albus) hypocotyl growth. Austr. J. Plant Physiol. 26: 29–36.
Januschke, J., Gervais, L., Dass, S., Kaltschmidt, J. A., Lopez-Schier, H.,
St Johnston, D., Brand, A. H., Roth, S., and Guichet, A. 2002. Polar transport
in the Drosophilia oocyte requires Dynein and Kinesin I cooperation. Curr.
Biol. 12: 1971–1981.
Johnson, K. L., Jones, B. J., Bacic, A., and Schultz, C. J. 2003. The fasciclin-like
arabinogalactan proteins of arabidopsis. A multigene family of putative cell
adhesion molecules. Plant Physiol. 133: 1911–1925.
Journet, E. P., El Gachtouli, N., Vernoud, V., de Billy, F., Pichon, M., Dedieu, A.,
Arnould, C., Morandi, D., Barker, D. G., and Gianinazzi-Pearson, V. 2001.
Medicago truncatula ENOD11: A novel RPRP-encoding early nodulin gene
expressed during mycorrhization in arbuscule-containing cells. Mol. Plant
Microbe Interact. 14: 737–748.
Kannenberg, E. L. and Carlson, R. W. 2001. Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during
bacteroid development. Molecular Microbiol. 39: 379–391.
Kapranov, P., Routt, S. M., Bankaitis, V. A., de Bruijn, F. J., and Szczyglowski,
K. 2001. Nodule-specific regulation of phosphatidylinositol transfer protein
expression in Lotus japonicus. Plant Cell 13: 1369–1382.
Kato, T., Kawashima, K., Miwa, M., Mimura, Y., Tamaoki, M., Kouchi, H., and
Suganuma, N. 2002. Expression of genes encoding late nodulins characterized
by a putative signal peptide and conserved cysteine residues is reduced in
ineffective pea nodules. Mol. Plant Microbe Interact. 15: 129–137.
Keating, D. H., Willits, M. G., and Long, S. R. 2002. A Sinorhizobium meliloti
lipopolysaccharide mutant altered in cell surface sulfation. J. Bacteriol. 184:
6681–6689.
Kevei, Z., Vinardell, J. M., Kiss, G. B., Kondorosi, A., and Kondorosi, E. 2002.
Glycine-rich proteins encoded by a nodule-specific gene family are implicated
in different stages of symbiotic nodule development in Medicago spp. Mol.
Plant Microbe Interact. 15: 922–931.
Kieliszewski, M. J. 2001. The latest hype on Hyp-O-glycosylation codes. Phytochem. 57: 319–323.
Kieliszewski, M. J. and Shpak, E. 2001. Synthetic genes for the elucidation of
glycosylation codes for arabinogalactan-proteins and other hydroxyprolinerich glycoproteins. Cell. Mol. Life Sci. 58: 1386–1398.
Kijne, J. W., Bauchrowitz, M. A., and Diaz, C. L. 1997. Root lectins and rhizobia.
Plant Physiol. 115: 869–873.
Kimura, S. and Kondo, T. 2002. Recent progress in cellulose biosynthesis. J.
Plant Res. 115: 297–302.
Kistner, C. and Parniske, M. 2002. Evolution of signal transduction in intracellular symbiosis. Trends Plant Sci. 7: 511–518.
Kosuta, S., Chabaud, M., Lougnon, G., Gough, C., Denarie, J., Barker, D. G.,
and Becard, G. 2003. A diffusible factor from arbuscular mycorrhizal fungi
induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula. Plant Physiol. 131: 952–962.
Krusell, L., Madsen, L. H., Sato, S., Aubert, G., Genua, A., Szczyglowski,
K., Duc, G., Kaneko, T., Tabata, S., de Bruijn, F., Pajuelo, E., Sandal, N.,
and Stougaard, J. 2002. Shoot control of root development and nodulation is
mediated by a receptor-like kinase. Nature 420: 422–426.
Lamblin, A. F. J., Crow, J. A., Johnson, J. E., Silverstein, K. A. T., Kunau, T. M.,
Kilian, A., Benz, D., Stromvik, M., Endre, G., Vandenbosch, K. A., Cook,
D. R., Young, N. D., and Retzel, E. F. 2003. MtDB: a database for personalized
data mining of the model legume Medicago truncatula transcriptome. Nucleic
Acids Res. 31: 196–201.
Laurenzi, M., Tipping, A. J., Marcus, S. E., Knox, J. P., Federico, R., Angelini,
R., and McPherson, M. J. 2001. Analysis of the distribution of copper amine
oxidase in cell walls of legume seedlings. Planta 214: 37–45.
Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G., Prome, C., and
Denarie, J. 1990. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature
344: 781–784.
Levy , J., Denarie, J., and Debelle, F. 2004. A putative Ca- and calmodulindependent protein kinase required for bacterial and fungal symbioses. Science
303: 1361–1364.
314
N. J. BREWIN
Lhuissier, F. G. P., De Ruijter, N. C. A., Sieberer, B. J., Esseling, J. J., and
Emons, A. M. C. 2001. Time course of cell biological events evoked in legume
root hairs by Rhizobium Nod factors: state of the art. Ann. Botany 87: 289–
302.
Limpens, E. and Bisseling, T. 2003. Signaling in symbiosis. Curr. Opin. Plant
Biol. 6: 343–350.
Limpens, E. and Franken, C., Smit, P., Willemse, J., Bisseling, T., and Geurts, R.
2003. LysM domain receptor kinases regulating rhizobial Nod factor- induced
infection. Science 302: 630–633.
Löbler, M. and Hirsch, A. M. 1993. A gene that encodes a proline-rich nodulin
with limited homology to PsENOD12 is expressed in the invasion zone of
Rhizobium-meliloti-induced alfalfa root nodules. Plant Physiol. 103: 21–30.
Lodwig, E. and Poole, P. 2003. Metabolism of Rhizobium bacteroids. Crit. Rev.
Plant Sci. 22: 37–78.
Long, S. R. 1996. Rhizobium symbiosis: Nod factors in perspective. Plant Cell
8: 1885–1898.
Loria, R., Coombs, J., Yoshida, M., Kers, J., and Bukhalid, R. 2003. A paucity
of bacterial root diseases: Streptomyces succeeds where others fail. Physiol.
Mole. Plant Pathol. 62: 65–72.
Lotocka, B., Kopcinska, J., and Golinowski, W. 1997. Morphogenesis of root
nodules in white clover. I. Effective root nodules induced by the wild type Rhizobium leguminosarum biovar. trifolii. Acta Societatis Botanicorum Poloniae
66: 273–292.
Lotocka, B., Kopcinska, J., Gorecka, M., and Golinowski, W. 2000. Formation
and abortion of root nodule primordia in Lupinus luteus L. Acta Biologica
Cracoviensia Series Botanica 42: 87–102.
MacDougall, A. J., Brett, G. M., Morris, V. J., Rigby, N. M., Ridout, M. J., and
Ring, S. G. 2001. The effect of peptide behaviour–pectin interactions on the
gelation of a plant cell wall pectin. Carbohyd. Res. 335: 115–126.
Madsen, E. B., Madsen, L. H., Radutoiu, S., Olbryt, M., Rakwalska, M.,
Szczyglowski, K., Sato, S., Kaneko, T., Tabata, S., Sandal, N., and Stougaard,
J. 2003. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425: 637–640.
Mathesius, U., Bayliss, C., Weinman, J. J., Schlaman, H. R. M., Spaink, H.
P., Rolfe, B. G., McCully, M. E., and Djordjevic, M. A. 1998a. Flavonoids
synthesized in cortical cells during nodule initiation are early developmental
markers in white clover. Mol. Plant Microbe Interact. 11: 1223–1232.
Mathesius, U., Keijzers, G., Natera, S. H. A., Weinman, J. J., Djordjevic, M.
A., and Rolfe, B. G. 2001. Establishment of a root proteome reference map
for the model legume Medicago truncatula using the expressed sequence tag
database for peptide mass fingerprinting. Proteomics 1: 1424–1440.
Mathesius, U., Schlaman, H. R. M., Spaink, H. P., Sautter, C., Rolfe, B. G.,
and Djordjevic, M. A. 1998b. Auxin transport inhibition precedes root nodule
formation in white clover roots and is regulated by flavonoids and derivatives
of chitin oligosaccharides. Plant J. 14: 23–34.
Mathesius, U., Weinman, J. J., Rolfe, B. G., and Djordjevic, M. A. 2000. Rhizobia can induce nodules in white clover by “hijacking” mature cortical cells
activated during lateral root development. Mol. Plant Microbe Interact. 13:
170–182.
Mathis, R., Grosjean, C., de Billy, F., Huguet, T., and Gamas, P. 1999. The
early nodulin gene MtN6 is a novel marker for events preceding infection
of Medicago truncatula roots by Sinorhizobium meliloti. Mol. Plant Microbe
Interact. 12: 544–555.
Matthysse, A. G. and Kijne, J. W. 1998. Attachment of Rhizobiaceae to plant
cells. In: The Rhizobiaceae, pp. 235–249. Spaink, H. P., Kondorosi, A., and
Hooykaas, P. J. J., Eds., Kluwer, Dordrecht.
Miklashevichs, E., Rohrig, H., Schell, J., and Schmidt, J. 2001. Perception and
signal transduction of rhizobial NOD factors. Crit. Rev. Plant Sci. 20: 373–
394.
Mitra, R. M., Gleason, C. A., Edwards, A., Hadfield, J., Downie, J. A., Oldroyd,
G. E. D., and Long, S. R. 2004. A Ca/Calmodulin-dependent protein kinase
required for symbiotic nodule development: gene identification by transcriptbased cloning. Proc. Nat. Acad. Sci. USA 101: 4701–4705.
Morzhina, E. V., Tsyganov, V. E., Borisov, A. Y., Lebsky, V. K., and Tikhonovich,
I. A. 2000. Four developmental stages identified by genetic dissection of pea
(Pisum sativum L.) root nodule morphogenesis. Plant Sci. 155: 75–83.
Mulder, B. M. and Emons, A. M. C. 2001. A dynamical model for plant cell
wall architecture formation. J. Math. Biol. 42: 261–289.
Munoz, J. A., Coronado, C., Perez-Hormaeche, J., Kondorosi, A., Ratet, P., and
Palomares, A. J. 1998. MsPG3, a Medicago sativa polygalacturonase gene
expressed during the alfalfa Rhizobium meliloti interaction. Proc. Natl. Acad.
Sci. USA 95: 9687–9692.
Navarro-Gochicoa, M. T., Camut, S., Niebel, A., and Cullimore, J. V. 2003a.
Expression of the apyrase-like APY1 genes in roots of Medicago truncatula
is induced rapidly and transiently by stress and not by Sinorhizobium meliloti
or Nod factors. Plant Physiol. 131: 1124–1136.
Navarro-Gochicoa, M. T., Camut, S., Timmers, A. C. J., Niebel, A., Herve, C.,
Boutet, E., Bono, J. J., Imberty, A., and Cullimore, J. V. 2003b. Characterization of four lectin-like receptor kinases expressed in roots of Medicago
truncatula. Structure, location, regulation of expression, and potential role
in the symbiosis with Sinorhizobium meliloti. Plant Physiol. 133: 1893–
1910.
Niehaus, K., Lagares, A., and Puhler, A. 1998. A Sinorhizobium meliloti
lipopolysaccharide mutant induces effective nodules on the host plant Medicago sativa (Alfalfa) but fails to establish a symbiosis with Medicago truncatula. Mol. Plant Microbe Interact. 11: 906–914.
Noreen, S., Schlaman, H. R. M., Bellogin, R. A., Buendia-Claveria, A. M.,
Espuny, M. R., Harteveld, M., Medina, C., Ollero, F. J., Olsthoorn, M. M. A.,
Soria-Diaz, M. E., Spaink, H. P., Temprano, F., Thomas-Oates, J., Vinardell,
J. M., Yang, S. S., Zhang, H. Y., and Ruiz-Sainz, J. E. 2003. Alfalfa nodulation
by Sinorhizobium fredii does not require sulfated Nod-factors. Func. Plant
Biol. 30: 1219–1232.
Novero, M., Faccio, A., Genre, A., Stougaard, J., Webb, K. J., Mulder, L.,
Parniske, M., and Bonfante, P. 2002. Dual requirement of the LjSym4gene for
mycorrhizal development in epidermal and cortical cells of Lotus japonicus
roots. New Phytol. 154: 741–749.
Nuc, K., Nuc, P., and Slomski, R. 2001. Yellow lupine cyclophilin transcripts
are highly accumulated in the nodule meristem zone. Mol. Plant Microbe
Interact. 14: 1384–1394.
Oke, V. and Long, S. R. 1999. Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis. Mol. Microbiol. 32: 837–
849.
Oldroyd, G. E. D. and Long, S. R. 2003. Identification and characterization of
nodulation-signaling pathway 2, a gene of Medicago truncatula involved in
Nod factor signaling. Plant Physiol. 131: 1027–1032.
Panstruga, R. and Schulze-Lefert, P. 2002. Live and let live: insights into powdery
mildew disease and resistance. Mole. Plant Pathol. 3: 495–502.
Parniske, M. 2000. Intracellular accommodation of microbes by plants: a common developmental program for symbiosis and disease? Curr. Opin. Plant
Biol. 3: 320–328.
Pawlowski, K. and Bisseling, T. 1996. Rhizobial and actinorhizal symbioses:
what are the shared features. Plant Cell 8: 1899–1913.
Pawlowski, K., Swensen, S., Guan, C. H., Hadri, A. E., Berry, A. M., and
Bisseling, T. 2003. Distinct patterns of symbiosis-related gene expression
in actinorhizal nodules from different plant families. Mol. Plant Microbe
Interact. 16: 796–807.
Pellock, B. J., Cheng, H. P., and Walker, G. C. 2000. Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides. J.
Bacteriol. 182: 4310–4318.
Perotto, S., Brewin, N. J., and Kannenberg, E. L. 1994. Cytological evidence for
a host-defense response that reduces cell and tissue invasion in pea nodules
by lipopolysaccharide-defective mutants of Rhizobium leguminosarum strain
3841. Mol. Plant Microbe Interact. 7: 99–112.
Perotto, S., Vandenbosch, K. A., Butcher, G. W., and Brewin, N. J. 1991. Molecular composition and development of the plant glycocalyx associated with
the peribacteroid membrane of pea root-nodules. Development 112: 763–
773.
Perret, X., Staehelin, C., and Broughton, W. J. 2000. Molecular basis of symbiotic promiscuity. Microbiol. and Mol. Biol. Rev. 64: 180–195.
Peters, N. K. and Verma, D. P. S. 1990. Phenolic compounds as regulators of
gene expression in plant-microbe relations. Mol. Plant Microbe Interact. 3:
4–8.
PLANT CELL WALL REMODELLING IN SYMBIOSIS
Pichon, M., Journet, E. P., and Dedieu, A. 1992. Rhizobium meliloti induces
transient expression of the early nodulin gene ENOD12 in the differentiating
root epidermis of transgenic alfalfa. Plant Cell 10: 1199–1211.
Pingret, J. L., Journet, E. P., and Barker, D. G. 1998. Rhizobium nod factor
signaling: evidence for a G protein-mediated transduction mechanism. Plant
Cell 10: 659–671.
Price, N. J., Pinheiro, C., Soares, C. M., Ashford, D. A., Ricardo, C. P., and
Jackson, P. A. 2003. A biochemical and molecular characterization of LEP1,
an extensin peroxidase from lupin. J. Biol. Chem. 278: 41389–41399.
Quispel, A. 1988. Hellriegel and Wilfarth’s discovery of symbiotic nitrogen
fixation one hundred years ago, In: Nitrogen Fixation: Hundred Years After,
pp. 3–12. Bothe, H., de Bruijn, F., and Newton, W., Eds., Gustav Fischer,
Stuttgart.
Radutoiu, S., Madsen, L. H., Madsen, E. B., Felle, H. H., Umehara, Y., Gronlund,
M., Sato, S., Nakamura, Y., Tabata, S., Sandal, N., and Stougaard, J. 2003.
Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425: 585–592.
Rae, A. L., Bonfantefasolo, P., and Brewin, N. J. 1992. Structure and growth of
infection threads in the legume symbiosis with Rhizobium-leguminosarum.
Plant J. 2: 385–395.
Rae, A. L., Perotto, S., Knox, J. P., Kannenberg, E. L., and Brewin, N. J. 1991.
Expression of extracellular glycoproteins in the uninfected cells of developing
pea nodule tissue. Mol. Plant Microbe Interact. 4: 563–570.
Ramu, S. K., Peng, H. M., and Cook, D. R. 2002. Nod factor induction of reactive
oxygen species production is correlated with expression of the early nodulin
gene rip1 in Medicago truncatula. Mol. Plant Microbe Interact. 15: 522–528.
Rathbun, E. A., Naldrett, M. J., and Brewin, N. J. 2002. Identification of a family
of extensin-like glycoproteins in the lumen of Rhizobium-induced infection
threads in pea root nodules. Mol. Plant Microbe Interact. 15: 350–359.
Relic, B., Perret, X., Estradagarcia, M. T., Kopcinska, J., Golinowski, W.,
Krishnan, H. B., Pueppke, S. G., and Broughton, W. J. 1994. Nod factors
of Rhizobium are a key to the legume door. Mol. Microb. 13: 171–178.
Rodriguez-Carvajal, M. A., du Penhoat, C. H., Mazeau, K., Doco, T., and
Perez, S. 2003. The three-dimensional structure of the mega-oligosaccharide
rhamnogalacturonan II monomer: a combined molecular modeling and NMR
investigation. Carbohyd. Res. 338: 651–671.
Rodriguez-Llorente, I. D., Perez–Hormaeche, J., Dary, M., Caviedes, M. A.,
Kondorosi, A., Ratet, P., and Palomares, A. J. 2003. Expression of MsPG3GFP fusions in Medicago truncatula ‘hairy roots’ reveals preferential tip
localization of the protein in root hairs. Eur. J. Biochem. 270: 261–269.
Roth, L. E. and Stacey, G. 1989. Bacterium release into host cells of nitrogenfixing soybean nodules: the symbiosome membrane comes from three sources.
Eur. J. Cell Biol. 49: 13–23.
Saalbach, G., Erik, P., and Wienkoop, S. 2002. Characterisation by proteomics
of peribacteroid space and peribacteroid membrane preparations from pea
(Pisum sativum) symbiosomes. Proteomics 2: 325–337.
Santi, C., von Groll, U., Ribeiro, A., Chiurazzi, M., Auguy, F., Bogusz, D.,
Franche, C., and Pawlowski, K. 2003. Comparison of nodule induction in
legume and actinorhizal symbioses: the induction of actinorhizal nodules
does not involve ENOD40. Mol. Plant Microbe Interact. 16: 808–816.
Santos, R., Herouart, D., Puppo, A., and Touati, D. 2000. Critical protective
role of bacterial superoxide dismutase in Rhizobium-legume symbiosis. Mol.
Microbiol. 38: 750–759.
Scheible, W. R., Fry, B., Kochevenko, A., Schindelasch, D., Zimmerli, L.,
Somerville, S., Loria, R., and Somerville, C. R. 2003. An Arabidopsis mutant
resistant to thaxtomin A, a cellulose synthesis inhibitor from Streptomyces
species. Plant Cell 15: 1781–1794.
Scheres, B., van Engelen, F., van der Knap, E., van de Wiel, C., van Kammen,
A., and Bisseling, T. 1990. Sequential induction of nodulin gene expression
in the developing pea nodule. Plant Cell 2: 687–700.
Schultz, C. J., Johnson, K. L., Currie, G., and Bacic, A. 2000. The classical
arabinogalactan protein gene family of arabidopsis. Plant Cell 12: 1751–
1767.
Schultze, M. and Kondorosi, A. 1998. Regulation of symbiotic root nodule
development. Annu. Rev. Genet. 32: 33–57.
315
Sharypova, L. A., Niehaus, K., Scheidle, H., Holst, O., and Becker, A. 2003.
Sinorhizobium meliloti acpXL mutant lacks the C28 hydroxylated fatty acid
moiety of lipid A and does not express a slow migrating form of lipopolysaccharide. J. Biol. Chem. 278: 12946–12954.
Shaw, S. L. and Long, S. R. 2003a. Nod factor elicits two separable calcium
responses in Medicago truncatula root hair cells. Plant Physiol. 131: 976–
984.
Shaw, S. L. and Long, S. R. 2003b. Nod factor inhibition of reactive oxygen
efflux in a host legume. Plant Physiol. 132: 2196–2204.
Sherrier, D. J., Prime, T. A., and Dupree, P. 1999. Glycosylphosphatidylinositolanchored cell-surface proteins from Arabidopsis. Electrophoresis 20: 2027–
2035.
Shimada, N., Aoki, T., Sato, S., Nakamura, Y., Tabata, S., and Ayabe, S.
2003. A cluster of genes encodes the two types of chalcone isomerase
involved in the biosynthesis of general flavonoids and legume-specific 5deoxy(iso)flavonoids in Lotus japonicus. Plant Physiol. 131: 941–951.
Showalter, A. M. 2001. Arabinogalactan-proteins: structure, expression and
function. Cell. Mol. Life Sci. 58: 1399–1417.
Sieberer, B. and Emons, A. M. C. 2000. Cytoarchitecture and pattern of cytoplasmic streaming in root hairs of Medicago truncatula during development
and deformation by nodulation factors. Protoplasma 214: 118–127.
Sieberer, B. J., Timmers, A. C. J., Lhuissier, F. G. P., and Emons, A. M. C.
2002. Endoplasmic microtubules configure the subapical cytoplasm and are
required for fast growth of Medicago truncatula root hairs. Plant Physiol.
130: 977–988.
Soltis, D. E., Soltis, P. S., Morgan, D. R., Swensen, S. M., Mullin, B. C., Dowd,
J. M., and Martin, P. G. 1995. Chloroplast gene sequence data suggest a single
origin of the predisposition for symbiotic nitrogen-fixation in angiosperms.
Proc. Natl. Acad. Sci. USA 92: 2647–2651.
Spaink, H. P., Kondorosi, A., and Hooykaas, P. J. J. 1998. The Rhizobiaceae.
Kluwer, Dordrecht.
Sprent, J. I. 2001. Nodulation in Legumes. Royal Botanical Gardens, Kew,
London.
Stougaard, J. 2001. Genetics and genomics of root symbiosis. Curr. Opin. Plant
Biol. 4: 328–335.
Stracke, S., Kistner, C., Yoshida, S., Mulder, L., Sato, S., Kaneko, T., Tabata, S.,
Sandal, N., Stougaard, J., Szczyglowski, K., and Parniske, M. 2002. A plant
receptor-like kinase required for both bacterial and fungal symbiosis. Nature
417: 959–962.
Stuurman, N., Bras, C. P., Schlaman, H. R. M., Wijfjes, A. H. M., Bloemberg,
G., and Spaink, H. P. 2000. Use of green fluorescent protein color variants
expressed on stable broad-host-range vectors to visualize rhizobia interacting
with plants. Mol. Plant Microbe Interact. 13: 1163–1169.
Szczyglowski, K. and Amyot, L. 2003. Symbiosis, inventiveness by recruitment?
Plant Physiol. 131: 935–940.
Szczyglowski, K., Shaw, R. S., Wopereis, J., Copeland, S., Hamburger, D.,
Kasiborski, B., Dazzo, F. B., and de Bruijn, F. J. 1998. Nodule organogenesis
and symbiotic mutants of the model legume Lotus japonicus. Mol. Plant
Microbe Interact. 11: 684–697.
Tan, L., Qiu, F., Lamport, D. T. A., and Kieliszewski, M. J. 2004. Structure
of a hydroxyproline (Hyp)-arabinogalactan polysaccharide from repetitive
Ala-Hyp expressed in transgenic Nicotiana tabacum. J. Biol. Chem. 279:
13156–13165.
Tansengco, M. L., Hayashi, M., Kawaguchi, M., Imaizumi-Anraku, H., and
Murooka, Y. 2003. Crinkle, a novel symbiotic mutant that affects the infection
thread growth and alters the root hair, trichome, and seed development in Lotus
japonicus. Plant Physiol. 131: 1054–1063.
Tate, R., Patriarca, E. J., Riccio, A., Defez, R., and Iaccarino, M. 1994. Development of Phaseolus vulgaris root nodules. Mol. Plant Microbe Interact. 7:
582–589.
Timmers, A. C. J., Auriac, M. C., and Truchet, G. 1999. Refined analysis of
early symbiotic steps of the Rhizobium-Medicago interaction in relationship
with microtubular cytoskeleton rearrangements. Devel. 126: 3617–3628.
Trevaskis, B., Wandrey, M., Colebatch, G., and Udvardi, M. K. 2002. The
soybean GmN6L gene encodes a late nodulin expressed in the infected
316
N. J. BREWIN
zone of nitrogen-fixing nodules. Mol. Plant Microbe Interact. 15: 630–
636.
Tsyganov, V. E., Voroshilova, V. A., Priefer, U. B., Borisov, A. Y., and
Tikhonovich, I. A. 2002. Genetic dissection of the initiation of the infection
process and nodule tissue development in the Rhizobium-pea (Pisum sativum
L.) symbiosis. Ann. Botany 89: 357–366.
Uheda, E., Daimon, H., and Yoshizako, F. 2001. Colonization and invasion of
peanut (Arachis hypogaea L.) roots by gusA-marked Bradyrhizobium sp. Can.
J. Botany-Revue Canadienne de Botanique 79: 733–738.
Uheda, E. and Silvester, W. B. 2001. The role of papillae during the infection
process in the Gunnera-Nostoc symbiosis. Plant and Cell Physiol. 42: 780–
783.
van Brussel, A. A. N., Bakhuizen, R., van Spronsen, P. C., Spaink, H. P.,
Tak, T. K., Lugtenberg, B. J. J., and Kijne, J. W. 1992. Induction of preinfection thread structures in the leguminous host plant by mitogenic lipooligosaccharides of Rhizobium. Science 257: 70–72.
van Rhijn, P., Fujishige, N. A., Lim, P. O., and Hirsch, A. M. 2001. Sugarbinding activity of pea lectin enhances heterologous infection of transgenic
alfalfa plants by Rhizobium leguminosarum biovar viciae. Plant Physiol. 126:
133–144.
van Rhijn, P., Goldberg, R. B., and Hirsch, A. M. 1998. Lotus corniculatus
nodulation specificity is changed by the presence of a soybean lectin gene.
Plant Cell 10: 1233–1249.
van Spronsen, P. C., Bakhuizen, R., van Brussel, A. A. N., and Kijne, J. W. 1994.
Cell-wall degradation during infection thread formation by the root- nodule
bacterium Rhizobium leguminosarum is a 2-step process. Eur. J. Cell Biol.
64: 88–94.
van Spronsen, P. C., Gronlund, M., Bras, C. P., Spaink, H. P., and Kijne, J. W.
2001. Cell biological changes of outer cortical root cells in early determinate
nodulation. Mol. Plant Microbe Interact. 14: 839–847.
van Spronsen, P. C., Tak, T., Rood, A. M. M., van Brussel, A. A. N., Kijne, J. W.,
and Boot, K. J. M. 2003. Salicylic acid inhibits indeterminate-type nodulation
but not determinate-type nodulation. Mol. Plant Microbe Interact. 16: 83–91.
VandenBosch, K. A., Bradley, D. J., Knox, J. P., Perotto, S., Butcher, G. W.,
and Brewin, N. J. 1989. Common components of the infection thread matrix
and the intercellular space identified by immunocytochemical analysis of pea
nodules and uninfected roots. EMBO J. 8: 335–341.
Vernoud, V., Journet, E. P., and Barker, D. G. 1999. MtENOD20, a Nod factorinducible molecular marker for root cortical cell activation. Mol. Plant Microbe Interact. 12: 604–614.
Vincent, J. L. and Brewin, N. J. 2000. Immunolocalization of a cysteine protease
in vacuoles, vesicles, and symbiosomes of pea nodule cells. Plant Physiol.
123: 521–530.
Vincent, J. L., Dahiya, P., and Brewin, N. J. 2000. Localized expression of
cathepsin B-like sequences from root nodules of pea (Pisum sativum). Mol.
Plant Microbe Interact. 13: 778–780.
Voroshilova, V. A., Boesten, B., Tsyganov, V. E., Borisov, A. Y., Tikhonovich,
I. A., and Priefer, U. B. 2001. Effect of mutations in Pisum sativum L. genes
blocking different stages of nodule development on the expression of late
symbiotic genes in Rhizobium leguminosarum bv. viciae. Mol. Plant Microbe
Interact. 14: 471–476.
Walker, S. A. and Downie, J. A. 2000. Entry of Rhizobium leguminosarum bv.
viciae into root hairs requires minimal nod factor specificity, but subsequent
infection thread growth requires nodO or nodE. Mol. Plant Microbe Interact.
13: 754–762.
Wang, L. X., Wang, Y., Pellock, B., and Walker, G. C. 1999. Structural characterization of the symbiotically important low-molecular-weight succinoglycan
of Sinorhizobium meliloti. J. Bacteriology 181: 6788–6796.
Wilson, R. C., Long, S. R., Maruoka, E. M., and Cooper, J. B. 1994. A new
proline-rich early nodulin from Medicago truncatula is highly expressed in
nodule meristematic cells. Plant Cell 6: 1265–1275.
Wisniewski-Dye, F. and Downie, J. A. 2002. Quorum-sensing in Rhizobium.
Antonie Van Leeuwenhoek Int. J. General Mole. Microbiol. 81: 397–407.
Wisniewski, J. P., Gardner, C. D., and Brewin, N. J. 1999. Isolation of lipoxygenase cDNA clones from pea nodule mRNA. Plant Mol. Biol. 39: 775–
783.
Wisniewski, J. P., Rathbun, E. A., Knox, J. P., and Brewin, N. J. 2000. Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular
matrix: implications for pea nodule initiation by Rhizobium leguminosarum.
Mol. Plant Microbe Interact. 13: 413–420.
Wycoff, K. L., Hunt, S., Gonzales, M. B., Vandenbosch, K. A., Layzell, D.
B., and Hirsch, A. M. 1998. Effects of oxygen on nodule physiology and
expression of nodulins in alfalfa. Plant Physiol. 117: 385–395.
Yang, W. C., de Blank, C., Franssen, H., and Bisseling, T. 1994. Rhizobium
nod factors reactivate the cell cycle during infection and nodule primordium
formation, but the cell cycle is only completed in primordium formation. Plant
Cell 6: 1415–1426.
Zhao, Z. D., Tan, L., Showalter, A. M., Lamport, D. T. A., and Kieliszewski, M.
J. 2002. Tomato LeAGP-1 arabinogalactan-protein purified from transgenic
tobacco corroborates the Hyp contiguity hypothesis. Plant J. 31: 431–444.