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Annu. Rev. Genet. 2011.45:119-144. Downloaded from www.annualreviews.org
Giles E.D. Oldroyd, Jeremy D. Murray,

Philip S. Poole, and J. Allan Downie
by George Mason University on 05/14/13. For personal use only.
John Innes Center, Norwich Research Park, Norwich NR4 7UH, United Kingdom;
email: giles.oldroyd@bbsrc.ac.uk
Annu. Rev. Genet. 2011. 45:11944 Keywords

First published online as a Review in Advance on nodulation, nitrogen xation, symbiosis, bacterial infection, legumes
August 11, 2011
The Annual Review of Genetics is online at Abstract

genet.annualreviews.org
Rhizobial bacteria enter a symbiotic association with leguminous plants,
This articles doi:
resulting in differentiated bacteria enclosed in intracellular compart-
10.1146/annurev-genet-110410-132549
ments called symbiosomes within nodules on the root. The nodules
Copyright c 2011 by Annual Reviews.
and associated symbiosomes are structured for efcient nitrogen xa-
All rights reserved
tion. Although the interaction is benecial to both partners, it comes
0066-4197/11/1201-0119$20.00
with rigid rules that are strictly enforced by the plant. Entry into root
cells requires appropriate recognition of the rhizobial Nod factor signal-
ing molecule, and this recognition activates a series of events, including
polarized root-hair tip growth, invagination associated with bacterial
infection, and the promotion of cell division in the cortex leading to the
nodule meristem. The plants command of the infection process has
been highlighted by its enforcement of terminal differentiation upon
the bacteria within nodules of some legumes, and this can result in a
loss of bacterial viability while permitting effective nitrogen xation.
Here, we review the mechanisms by which the plant allows bacterial in-
fection and promotes the formation of the nodule, as well as the details
of how this intimate association plays out inside the cells of the nodule
where a complex interchange of metabolites and regulatory peptides
force the bacteria into a nitrogen-xing organelle-like state.
119
INTRODUCTION collectively referred to as rhizobia. Thus,

legumes are important crop plants that can en-
Rhizobia: bacteria Unlike animals, plants must capture nitrogen,
with the capability to rich soil nitrogen and that have protein-rich
generally in the form of nitrates and ammo-
interact with legumes seeds important in human and animal nutrition.
nia, from the soil. Although nitrogen gas (N2 )
to form nitrogen- The legume-rhizobial symbiosis results in
is widespread, the biologically active forms of
xing nodules the formation of root nodules that provide an
nitrogen are often so limiting that they can re-
Symbiosis: an environment suitable for N2 -xation by rhizo-
strict plant growth. To overcome this problem
intimate interaction bia. In broad terms, the formation of symbiotic
between organisms several plants enter into benecial symbioses
N2 -xing nodules requires two developmental
that often benets with bacteria that can convert N2 into ammo-
both partners processes: nodule organogenesis and bacterial
nia. Chief among these symbioses are legumes
infection (Figure 1). Although these can be sep-
that associate with a wide range of bacteria,
arated genetically, they must be coordinated in
NF
both a spatial and temporal fashion (Figure 2)
to ensure nodule organogenesis at the site of
bacterial infection (116). Both nodule organo-

NFR1/NFR5 Epidermis genesis and bacterial infection require plant
Bacterial infection recognition of the rhizobial signaling molecule

(NAP1, PIR1, Cerberus, RPG, FLOT2/4, Nod factor (NF). This is a decorated lipochi-
SymRem1, Vapyrin, MMPL1, nsRING, PUB1)
tooligosaccharide that is symbiosis specic, is
sufcient to activate nodule organogenesis, and
can induce cellular changes associated with the
SYMRK, NUP85, initiation of bacterial infection (115).
Calcium CCaMK, Cyclops, Gene induction,
NUP133, NENA, oscillations NSP1, NSP2 e.g. ERN1, NIN
Castor, Pollux NF recognition involves receptor-like
kinases with extracellular LysM domains
that bind chitooligosaccharides (83, 89, 132)
Inner/mid cortex
(Figure 1). This recognition activates calcium
Cytokinin oscillations in the nuclear region (32) and ac-
signaling
RR1 Inhibition of tivation of this signaling pathway also requires
LHK1/CRE1 NSP1, NSP2, NIN polar auxin
RR4 transport symbiosis receptor-like kinase (SYMRK) (33,
149), components of the nuclear pore (50,
64, 140), and two cation channels located on
Nodule organogenesis the nuclear envelope (4, 22, 60). Perception
(HAP2.1, ERF1, etc.) of the calcium oscillations involves a calcium
and calmodulin dependent protein kinase
Figure 1 (CCaMK) (80, 101, 156) that interacts with
A schematic representing the genetic pathways involved in the activation of and phosphorylates a protein of unknown func-
nodule organogenesis and bacterial infection. Epidermal cells are capable of tion (CYCLOPS) (99, 179). Downstream of
perceiving Nod factor (NF) through receptor-like kinases (NFR1/NFR5) that CCaMK and CYCLOPS there are several tran-
activate calcium oscillations via a suite of proteins. Perception of the calcium scription factors (nodulation signalling pathway
oscillations involves the calcium-activated kinase CCaMK, which functions
[NSP]1, NSP2, ERF required for nodulation
with transcription factors (such as NIN and ERN1) to activate gene expression.
This signaling pathway has two outcomes: the initiation of bacterial infection at [ERN]1, nodule inception [NIN]) that regulate
the epidermis and the promotion of cell division in the cortex via an unknown NF-induced gene expression (3, 55, 63, 94, 142,
diffusible signal (dotted line). In the inner or mid-cortex, NF-induced cytokinin 147). Once CCaMK is activated, upstream sig-
signaling involves the cytokinin receptor LHK1/CRE1, response regulators, naling components of the nodule organogenesis
and the transcription factors NSP1, NSP2, and NIN. A target for cytokinin
pathway are redundant (54, 90), implying that
signaling is the suppression of polar auxin transport that is sufcient to promote
nodule organogenesis. Although the transcription factors have been placed the primary role of the signaling pathway is the
upstream of the changes to polar auxin transport, they could equally lie activation of CCaMK, which then promotes
downstream. nodule morphogenesis (Figure 1).
120 Oldroyd et al.

Rhizobial
bacteria
Infection
thread
Epidermis Pre-infection
thread
Outer cortex
Inner cortex
Endodermis
Pericycle
Figure 2
Nodulation involves the coordinated development of bacterial infection and nodule organogenesis. Cell
division (indicated with dotted lines) in the inner or mid-cortex and pericycle is initiated early in the interaction
between the root and rhizobial bacteria and precedes the initiation of infection events. Bacteria are
entrapped in a curled root hair, and from this site infection threads (ITs) are initiated. The route of the IT is
predicted by pre-infection threads that are densely cytoplasmic subdomains with aligned cytoskeleton. ITs
progress into the inner cortex where the nodule primordium has formed through a series of cell divisions.
From these divided cells, the nodule meristem forms (154).
Nodulation in legumes evolved from the NODULE ORGANOGENESIS

much more ancient symbiotic association
that occurs between plants and arbuscular Section Introduction
mycorrhizal fungi (120). This conclusion is We focus here on determinate and indeter-
based on the fact that components of the minate nodule development, which occur in Nodules: structures
NF signaling pathway are also required for different species of legumes. Indeterminate formed on the roots of
mycorrhizal signaling (115), suggesting that nodules have a tip-growing meristem, whereas some plant species,
including most
the signaling pathway initially functioned in determinate nodules have a transient meristem, legumes, that provide
mycorrhizal signaling and was later recruited and these two forms of nodules develop from a suitable environment
in an ancestor of the legumes for recognition different positions in the root: Indeterminate for N2 xation
of rhizobia. The mechanisms that drove this nodules originate from inner cortical cells Nod factor (NF):
evolutionary step have become clearer with the abutting the endodermis, whereas determinate a rhizobial signaling
recent discovery that arbuscular mycorrhizal nodules arise from the central cortex. In both molecule that
fungi produce NF-like molecules (92). cases, multiple cell layers separate the site of promotes the
nodulation of legume
The mechanisms of symbiosis signaling have initial bacterial contact at the root surface from roots
been extensively reviewed (115, 116, 120), and the site of cortical cell division that leads to
Arbuscular
we have chosen not to review this signaling the nodule primordium. Because nodulation is mycorrhizal fungi:
pathway again, but rather to focus on the de- activated by NF application (158) and by gain- fungi that colonize
velopmental events that underpin the formation of-function mutations of CCaMK (45, 156), we plant roots, forming
of this symbiosis. This includes nodule organo- conclude that NF recognition at the root sur- highly branched
genesis, bacterial infection, and the processes face is likely to be sufcient to activate nodule hyphal structures in
root cortical cells to
that facilitate the accommodation of rhizobia organogenesis in the root cortex, and this must promote metabolite
inside the plant cell and ultimately support N2 involve a diffusible signal. Nodule organo- exchange
xation. genesis requires the integration of NF and
www.annualreviews.org Legume-Rhizobial Symbiosis 121

hormonal signaling [particularly auxin and cy- at the root surface is not the primary source of
tokinin (26)], and in this section we review how increased cytokinin production.
these hormones control nodule development A direct result of cytokinin signaling is a
Auxin: a plant
hormone that controls and how differences in the relative functions of CRE1-dependent change in polar auxin trans-
many aspects of plant these hormones appear to discriminate between port (Figure 3), which is suppressed by both
development and is a nodule meristem and a root meristem. rhizobia and by NF (51, 128, 161). The auxin-
generally associated inducible promoter GH3 is downregulated at
with meristematic
the earliest stages of rhizobial interactions with
tissues
clover roots and this suppression of the auxin
Cytokinin: a plant Plant Hormones That response occurs at localized sites of rhizobial
hormone that often Coordinate Nodulation
functions in or NF treatment (95). Such induced changes
combination with Roles for cytokinin and auxin were initially to polar auxin transport are highly signi-
auxin to dene the based on observations of nodule initiation fol- cant because auxin transport inhibitors can
transition from cell lowing modications to cytokinin levels (25) induce nodule organogenesis (56). Thus, we
division to
and application of auxin transport inhibitors can conclude that localized cytokinin signal-
differentiation
(56). Subsequently, a gain-of-function muta- ing in the root cortex and pericycle, activated
tion in the cytokinin receptor gene LHK1 of upon recognition of rhizobia, leads to the lo-
Lotus japonicus was shown to activate spon- calized suppression of polar auxin transport
taneous nodule formation (157) and loss-of- (Figure 3), which induces nodule morphogen-
function mutations of LHK1, and its ortholog esis. The suppression of polar auxin transport
CRE1 in Medicago truncatula blocked nodule and the downregulation of GH3 imply that low
formation, but allowed bacterial infection (111, levels of auxin are associated with the initiation
128). From these studies, we can conclude of the nodule, but once initiated, the nodule
that activation of cytokinin signaling is neces- meristem shows strong activation of auxin re-
sary and sufcient for the induction of nodule sponsive promoters (51).
morphogenesis. To understand better the relevance of these
Response regulators are the primary targets changes in auxin and cytokinin for meristem
of cytokinin signaling and are among the earli- formation, it is useful to consider how auxin and
est genes induced by cytokinin. These markers cytokinin inuence the formation of root meris-
have revealed that cytokinin signaling is acti- tems. The root apical meristem of Arabidopsis is
vated at a very early stage in the initiation of a highly zoned structure with the quiescent cen-
the nodule meristem (86, 128). The earliest inter and stem cell niche at the tip of the meristem.
duction of response regulators in M. truncatula There is a region of cell division behind this re-
is in the inner cortical cells and pericycle cells ferred to as the proximal meristem, followed
that mark the very rst responses to rhizobia by a region of cell expansion called the elonga-
in the inner root tissues (128). The response tion zone, and nally a zone of cell differenti-
regulators are observed prior to extensive cell ation, marked by the emergence of root hairs
division and continue to be expressed in the di- (Figure 3). During the initiation and mainte-
viding cortical cells and nodule meristem. From nance of the root apical meristem, cytokinin and
this, we can conclude that cytokinin signaling auxin function in an antagonistic manner and
during nodulation is restricted to the pericy- appear to act in different zones of the meris-
cle and cortical cells where cell divisions occur tem (107, 139). Auxin accumulates at the tip of
during nodule initiation (154). Thus, a mecha- the root apical meristem, where the stem cell
nism must exist that allows localized cytokinin niche resides, and this is crucial to maintain cell
signaling in the inner root tissues. The fact that division. In contrast, cytokinin appears to act
response regulators are not activated in the root at the boundary between the proximal meris-
epidermis or outermost cortical cell layer (128) tem and the elongation zone, and is associated
suggests that the initial site of rhizobial contact with the transition from cell division to cell
122 Oldroyd et al.

Vascular
Cortex strand
Epidermis Pericycle Root hair
Lateral root initiation
Differentiation zone
Auxin PINs
Nodule initiation
CRE1
Rhizobia Localized
Cytokinin
NF high auxin
Cytokinin
NIN signaling
Aux1
PINs
Localized
low auxin
Elongation zone
High cytokinin RR1

Low auxin
SHY2 PINs
Proximal meristem
Stem cell
niche
RR7 Cytokinin
High auxin RR15 signaling
Low cytokinin
Root cap
Figure 3
The roles of auxin and cytokinin during root meristem development. The root apical meristem is divided into the stem cell niche where
the quiescent center resides, the proximal meristem where cell divisions occur, the elongation zone where cells expand, and the
differentiation zone where the specialized structures of cells emerge. As a result of auxin transporters (PINs), auxin traverses down the
root and accumulates at the tip in the region of the stem cell niche. A cycle of auxin ow is very apparent in the proximal meristem
region but reduced in the elongation zone, likely through cytokinin suppression of PINs. A gradient of relative importance of cytokinin
and auxin is established with high auxin at the stem cell niche and high cytokinin signaling at the transition zone between the proximal
meristem and the elongation zone. This gradient is maintained at least in part through antagonisms between auxin and cytokinin that
are facilitated by auxin induction of RR7 and RR15 (that suppress cytokinin signaling) and cytokinin induction of SHY2 (that inhibits
PIN expression). In lateral roots, localized accumulations of auxin in the pericycle mark the site where the lateral root emerges.
Cytokinin can suppress lateral root emergence by blocking the localized accumulations of auxin, probably through the suppression of
PINs. During nodule development, localized cytokinin signaling in the root cortex is necessary and sufcient for the initiation of the
nodule primordia. These increases in cytokinin signaling block auxin transport via the suppression of PINs, and it appears that low
auxin levels may be associated with the initiation of the nodule primordium. Activation of nodule organogenesis requires Nod factor
(NF) recognition at the root surface, and this induces the transcription factor NIN, which may be sufcient to induce cytokinin
signaling in the cortex through the upregulation of CRE1.

differentiation (Figure 3) (107, 139). Regions positive regulator of nodule initiation and nod-
of high auxin and low cytokinin signaling at ule meristem maintenance (38). Indeed, cy-
the root tip versus high cytokinin signaling tokinin appears to be the primary activator of
and low auxin at the transition between the the nodule meristem, responsible for the reg-
proximal meristem and the elongation zone are ulation of polar auxin transport, which leads
maintained by antagonism between auxin- and to nodule organogenesis (38). In both primary
cytokinin-mediated regulation. At the root tip, and lateral root apical meristems, cytokinin sig-
auxin activates the response regulators RR7 and naling is suppressed in the cells that will be-
RR15 and together these suppress cytokinin sig- come the meristematic cells (75, 108), whereas
naling (Figure 3) (108). Conversely at the prox- in nodule development it is the activation of cy-
imal meristem-elongation zone transition, cytokinin signaling that is associated with the nod-
tokinin activates the transcription factor SHY2, ule meristem (87, 128). In both primary and lat-
which suppresses expression of the PIN auxin eral root meristems, cytokinin suppresses PINs,
transporters (29), and the resulting decreased leading to localized suppression of auxin levels,
auxin ow reduces intracellular auxin levels in which ultimately suppresses meristem forma-
this region (125) (Figure 3). tion or maintenance (124, 139). In a comparable
Auxin and cytokinin are also important for fashion, cytokinin also suppresses PINs and po-
initiation of lateral root meristems (12, 124). lar auxin transport during nodule initiation, and
Lateral roots initiate from pericycle cells, and markers of auxin signaling are downregulated in
auxin appears to be the central player in dening the inner root tissue at the site of rhizobial in-
the point in the pericycle from which the lateral oculation (95). Thus, nodule initiation appears
root meristem is initiated (Figure 3). Localized to be associated with high cytokinin and low
cellular accumulations of auxin are the earliest auxin levels; this implies that these hormones
markers for the site of the lateral root meris- play opposite roles in the activation of the dif-
tem (12, 124), and these auxin accumulations ferent lateral organs of the legume root.
occur as a result of either physical distortions Proposing that auxin represses nodule de-
to the root (76) or an intrinsic oscillation at the velopment and cytokinin induces nodule de-
root apical meristem (105). Once initiated, the velopment would be a radical shift away from
auxin levels are enhanced through activation the accepted dogma that places auxin as the key
of the AUX1 auxin importer (Figure 3) and promoter of root development. However, some
through the suppression of a negative regulator observations support this. Suppression of cy-
of auxin signaling (124). In a manner compa- tokinin signaling through regulation of CRE1
rable to its function in the root apical meris- leads to reduced nodule numbers and increased
tem, cytokinin inhibits lateral root formation, numbers of lateral roots (47), whereas the con-
probably through its suppression of PINs (75), verse occurs with suppression of auxin signal-
which abolishes the ability to accumulate auxin ing (73). In addition, ABA promotes lateral
(Figure 3). Lateral root primordia are highly root emergence and suppresses nodulation in
sensitive to cytokinin levels at the stage of initi- M. truncatula, and this appears at least in part
ation, but become insensitive to cytokinin once to occur through its antagonism of cytokinin
initiated (75). Consistent with this is the local- responses (31). Thus, it would appear that the
ized suppression of cytokinin signaling at the hormone balance achieved in the root follow-
earliest stages of lateral root formation, but ing recognition of rhizobial bacteria is one that
once initiated, the lateral root tip becomes a can promote nodule initiation but suppress lat-
site for cytokinin production, and markers of eral root initiation. This antagonism between
cytokinin signaling are strongly induced in the lateral roots and nodules supports the hypoth-
mature lateral root primordium (12, 124). esis that the roles for cytokinin and auxin are
In contrast to the root apical meristem and reversed in nodule initiation as compared to
the initiation of a lateral root, cytokinin is a lateral root initiation. However, based on the
124 Oldroyd et al.

observations that auxin-inducible markers are type B response regulators (6). RR4 is induced
induced close to the site of nodule initiation (51) during nodulation, and at least one mechanism
and that addition of NFs can promote the emer- for the induction of RR4 is through the tran-
gence of lateral roots (114), alternative views on scription factor EFD (ethylene response factor
the roles of auxin in nodule initiation can and required for nodule differentiation) (166). This
have been proposed. The difference in inter- transcription factor is a negative regulator
pretations may lie in the subtle differences be- of nodule number and appears to achieve
tween the initiation of anticlinal cell division to this through the induction of RR4. Thus,
form what has been referred to as the nodule EFD induction of RR4 suppresses cytokinin
primordia versus the later divisions that lead to signaling and inhibits nodule initiation (166).
the nodule meristem (155). Clearly, further re- Additional transcription factors present in
search is necessary and in particular a detailed the nodule meristem and necessary for nodule
analysis of the relative importance of these development are HAP2-1 of M. truncatula
hormones at these different stages of nodule and ERF1 of L. japonicus (8, 23). The genes
development. regulated by these transcription factors are not

known, but silencing of HAP2-1 and ERF1
revealed functions in the maintenance and

Transcription Factors Associated possibly initiation of the nodule meristem (8,
With the Nodule Meristem 23). HAP2-1 expression is tightly regulated by
Several transcription factors required for nod- microRNA169 (23) and, in addition, a second
ule development are induced during nodulation microRNA, 166, is also induced in the nodule
(Figure 1). The B-type response regulator RR1 meristem (17). The targets of microRNA166
is induced during nodulation and is likely to are class III homeodomain-leucine zipper
be associated with signal transduction down- transcription factors, and overexpressing
stream of the cytokinin receptor LHK1/CRE1 microRNA166 reduced the nodule number,
(128). The transcription factors NSP2 and NIN implying a role for these transcription factors
that are essential for nodulation (63, 94) are during nodule development (17). A likely target
strongly induced during nodulation and by cy- for these transcription factors is the activation
tokinin addition (47). Both NIN and NSP2 are of the cell cycle to allow de novo meristem
required for the spontaneous nodule organo- development and the continued cell divisions
genesis activated by the gain-of-function mu- that are necessary for meristematic activity. A
tation in LHK1 (90, 157). Therefore, it can surprising recent discovery is the necessity for
be concluded that NIN and NSP2 act down- a putative nitrate transporter for maintenance
stream of cytokinin perception by CRE1/LHK1. of the nodule meristem (180). The function of
NIN and NSP2 also appear to play an essential this and the relative roles for the different tran-
role prior to cytokinin signaling, allowing NF scription factors during nodule organogenesis
recognition to activate gene expression in the will no doubt emerge from future research.
epidermis and linking the epidermal NF sig-
naling response to nodule organogenesis in the
cortex (94, 117) (Figure 1). Section Conclusions
Although the type B response regulator Recognition of rhizobia at the root surface ini-
RR1 is a positive regulator of cytokinin sig- tiates nodule organogenesis, and this involves
naling, the type A response regulator RR4, the activation of cytokinin signaling in the root
which is also induced during nodulation (47), cortex, leading to suppression of polar auxin
is likely to function as a negative regulator. transport. The activation of cytokinin and the
These type A response regulators appear to suppression of polar auxin transport are both
be primary targets of cytokinin signaling and necessary and sufcient for nodule organogen-
lack the DNA-binding domains present on the esis. Thus, in a fashion comparable to primary

and lateral root development, the relative may be associated with the infection focus and
balance of cytokinin and auxin dictates nodule probably plays a role in the reorientation of po-
organogenesis. However, the roles for these lar growth from an outward growing root-hair
Infection threads
(ITs): plant-made hormones appear to be reversed during nodule cell to an inward growing invagination (102).
tube-like structures development compared with root develop- The nucleus migrates close to the infection fo-
that promote ment. Several transcription factors facilitate cus, and the cytoskeleton within the root hair
colonization of roots the activation of cytokinin signaling following aligns along the length of the root hair form-
and nodules by
NF recognition and transduce the signal from ing a pre-IT structure. This serves to guide the
rhizobia
cytokinin signaling to the promotion of nodule inward growth of the IT initiated from the in-
organogenesis. fection focus (154, 162). Cell enlargement has
ceased at this point, but the plasma membrane
INFECTION BY RHIZOBIA invaginates to form a thin tubular IT that is
lined by a new growing cell wall (Figure 4).
Section Introduction This can be considered as an ingrowth of the
root-hair tip, and it continues to extend through

Rhizobia must traverse the legume root epider-
the root hair and through the root-hair cell
mis and root cortex to gain access to developing
body until it joins the cell wall at the interface

nodule primordia, where the bacteria are then
with the underlying cortical cell. At this loca-
released into the nodule cells in which they x
tion, the cell wall has been degraded (162), and a
N2 . Rhizobial infection can occur via root hairs,
new IT is initiated in the apposed cortical cell at
via cracks in the epidermis and via interstitial
the point where the epidermal IT terminated.
infections between epidermal cells (reviewed in
The process of IT development then continues
39). However, the most common mode of infec-
through multiple cortical cells into the cells of
tion is the formation of infection threads (ITs)
the developing nodule.
in growing root hairs, and this involves redif-
ferentiation of the root-hair cell to enable it to
make the IT. In this section, we review recent Cytoskeletal Changes
developments in our understanding of root-hair
NFs induce rearrangements of the cytoskele-
infection and then consider the requirements
ton, and changes in microtubule organization
for alternative modes of infection.
occur 3 to 10 minutes after NF exposure (173).
In growing root hairs, there is a temporary
Infection via Root Hairs shortening of the endoplasmic microtubule ar-
When root hairs perceive NF, there is an ini- ray in response to NFs (163), and both NFs and
tial transient interruption of symmetrical polar rhizobia seem to transiently decrease micro-
growth, and this is sometimes accompanied by tubule dynamics (145). These responses may
swelling at the root-hair tip, after which growth be part of the redirection of root-hair growth
resumes at the tip or along the root hair to form that occurs during the initial interaction. This
a branch (34). The new growth can result in is supported by the observation that destabi-
the root hair bending back upon itself or, in the lization of microtubules leads to the loss of di-
case of a branch, against the main part of the rectionality of plant root-hair growth (13), and
root hair; both result in NF-producing bacte- similarly, NF-induced changes in microtubule
ria becoming entrapped between appressed cell reorganization are associated with the reori-
walls, forming a so-called infection pocket (40). entation of root-hair growth (145). Root-hair
The rhizobia entrapped in these infection pock- growth can be redirected by application of Ca2+
ets continue to divide, forming colonies that or NFs on the side of root hairs (14, 34); micro-
are referred to as infection foci, and it is from tubule reorganization will be required to estab-
these foci that root-hair ITs start to develop. An lish the redirection of growth in response to NF
NF-induced calcium inux at the root-hair tip signaling, possibly through the action of Ca2+ .
126 Oldroyd et al.

PM
IT cell wall
IT cell matrix
Cortical cytoplasm
NF signaling may be localized to
ER-rich cytoplasmic bridge (37)
microdomains with flotillins and
remorins (53, 79). NF-producing rhizobia
The IT is surrounded by an array of microtubules

(158).
A Ca2+ gradient is needed for vesicle fusion

and cell wall flexibility at the growing tip. Golgi-derived vesicles may be required to deliver
cell wall and matrix materials and signaling

components to the growing IT apex.
Small G proteins localized at the apex may

determine where growth takes place. Dynamic F-actin (as well as microtubules) in the
subapex may participate in turnover of PM
proteins, and F-actin may be further needed for
vesicle delivery.
Nucleus
A nearby nucleus is important for early
signaling events and tip growth.
Axial actin cables. Actin is probably required for

nuclear movement during IT development.
The vacuole controls cell turgor.
Figure 4
A model for the growth of rhizobial infection threads (ITs) in root hairs. Nod factors (NFs) produced by rhizobia within root hairs are
perceived by an NF receptor complex that may reside in signaling microdomains at the infection thread apex. Although the details of
IT growth are not yet understood, root hair and pollen tube growth provide useful models. One such commonality may be the
production of reactive oxygen species (ROS), which can be induced by NFs (134). Although the role of NF-induced ROS is not clear,
NADPH oxidases were shown to be required for both pollen tube and root-hair growth (129), so they may play an analogous role in IT
growth. One of the main roles of ROS is the activation of calcium channels (reviewed in 150), which is also required for polar growth.
During polar growth, this effect can be local; for example, in pollen tubes Ca2+ inux is restricted to a small area of the plasma
membrane (PM) at the apex of growing pollen tubes (127). Calcium, another important player, is known to be important in tip growth
processes, such as cell wall exibility at the growing apex (57) and vesicle fusion into the target membranes (143). Phospholipids are also
important for establishing the Ca2+ gradient that is found at the tips of growing cells (reviewed in 183). The cytoskeleton is also
essential for IT growth. Microtubules help to determine the direction of growth at the site of Ca2+ inux (13), and actin provides the
infrastructure for vesicle delivery at the site of growth in both root hairs and pollen tubes (reviewed in 141). For example, F-actin,
which is found in the subapex of growing root hairs (15), has been shown, along with microtubules, to participate in the recycling and
endocytosis of PM subdomains, suggesting a key role for the cytoskeleton in signaling (9). Small G proteins also play crucial roles in
support of tip growth. For example, F-actin dynamics during pollen tube growth are dependent on small G proteins (reviewed in 183).
The proximity of the nucleus to the site of root-hair growth is signicant, and its movement is mediated by actin (69).

Microtubules and actin coordinately act in of clover by Rhizobium leguminosarum biovar

plants to set up the framework for directional trifolii because deletion of the gene encoding
cell expansion (126). Within ve minutes of this cellulase blocked infection (137). However,
NF addition, actin rearrangements can be seen it has yet to be determined whether this effect
in legume root hairs (1, 20, 28). There is a is because of the resulting increased levels of
transient dissolution of thick actin bundles and bacterial cellulose, which can suppress infec-
an accumulation of diffuse actin (20, 28, 172, tion (77), or whether the bacterial cellulase is
181). Actin is required for root-hair growth necessary for degrading the plant cell wall (137).
(136) and is formed and reorganized by the
polymerization of G-actin monomers and the
nucleation of new laments via the action of Infection Thread Progression
the ARP2/3 complex (148). The activa- The invaginating plant cell wall, along with the
tion of the ARP2/3 complex requires the extended plasma membrane, grows as a hollow
SCAR/WAVE complex, and in legumes, mu- tube within the root-hair cell and the rhizobia
tations in NAP1 and PIR1, components of the grow within the glycoprotein matrix inside the
SCAR/WAVE complex, alter root hair and tri- hollow of the tube, in essence topologically still
chome growth and block infection by rhizobia outside of the cell. Bacterial surface polysaccha-
(103, 181); this is correlated with a loss of nor- rides are required at this stage and may act as
mal NF-induced rearrangement of actin la- additional signalling molecules to the host plant
ments in nap1 and pir1 mutants (181). Actin (41). The sustained growth of the IT requires
is required for vesicle delivery to tip-growing the active delivery of membrane vesicles to its
cells (171), and so a role for actin in NF-induced growing tip and the direction of growth appears
root-hair growth, IT initiation, and IT progres- to be governed by the nucleus, which precedes
sion is expected (Figure 4). its growth and is linked to the growing tip by
the cytoskeleton (39, 113). The establishment
of the growing IT tube may have parallels with
Initiation of Infection Threads the establishment of the cell plate during cell
The entrapment of rhizobia in a sealed infec- division. Normally, after mitosis in plant cells,
tion pocket (40) is probably required to allow a scaffold structure made up of microtubules,
the localized plant cell wall degradation that microlaments, and endoplasmic reticulum is
is necessary for IT initiation without causing formed between the divided nuclei. This struc-
cell rupture (135, 159). The source of enzymes ture, called a phragmoplast, is the site of forma-
for degradation of the plant cell wall could tion of the new plant cell wall. It was suggested
be bacterial, plant, or possibly both. Plant that, if a single nucleus can initiate formation of
cell walldegrading enzymes that are induced a new cell wall without requiring nuclear divi-
during infection by rhizobia include pectin- sion, and then the nucleus starts to move, the re-
methylesterase (82) and polygalacturonase sulting cell wall that formed would be like a tube
(110), and these enzymes have homologs that rather than a at structure and that this could
are expressed during pollen-tube growth, sug- explain how the tubular IT is established (18).
gesting a role in polarized growth (110, 138). The growing IT reaches the base of the root-
Plant cell walldegrading enzymes would have hair cell, and as it approaches the nucleus in
to be secreted to a localized site on the mem- the adjacent cortical cell starts to reposition it-
brane to locally loosen or degrade the cell wall. self and a centrally located cytoplasmic bridge
For instance, during the initiation of root-hair forms to establish a pre-IT in this cell (154).
growth, there is an increased xyloglucanase ac- This aligns with the growing IT tip, and there
tivity associated with the localized weakening of is localized degradation of the plant cell walls,
the cell wall (168). There is some evidence for allowing the IT to continue its growth into the
a role for a bacterial cellulase in the nodulation next cell layer. This process is repeated at each
128 Oldroyd et al.

Table 1 Genes involved in rhizobial infection

Stage required or
Locus phenotype Protein Process
NAP1/RIT1 (103, 181) Curling, IT progression, Nck-associated protein 1 Actin rearrangements
cortical infection
PIR1 (181) Curling, IT progression 121F-specic p53 inducible Actin rearrangements
RNA
NIN (142) Microcolony formation RWP-RK TF Transcription
NSP1 (55, 147) Microcolony formation GRAS TF Transcription
NSP2 (55, 63, 117) Microcolony formation GRAS TF Transcription
CYCLOPS/IPD3b (99, 178, 179) IT initiation Nuclear coiled-coil Unknown
API (153) IT initiation, cortical
infection
BRUSH (91) IT initiation
ERN1 (100) IT initiation AP2/ERF TF Transcription

FLOT2 (53) IT initiation Flotillin Unknown
FLOT4 (53) IT initiation Flotillin Unknown

Itd1 (88) IT initiation
Itd4 (159) IT initiation
LOT1 (118) IT initiation Ethylene regulation
CRINKLE (151, 152) IT progression Actin rearrangements
LIN/CERBERUS (71, 177) IT progression Putative E3 ligase Protein turnover/targeting
MMPL1 (24) IT progressiona Putative matrix Protein degradation/stabilization
metalloendoproteinase
MtCRE1 (128) IT progression Cytokinin receptor Cytokinin sensing
nsRING (144) IT progression Putative E3 ligase Protein turnover/targeting
PUB1 (96) IT progressiona Putative E3 ligase Protein turnover/targeting
RPG (7) IT progression Coiled-coil Unknown
SYMREM1 (79) IT progression Remorin Unknown
VAPYRIN b (112, 131) IT progression Major sperm protein and Unknown
ankyrin domains
ALB1 (59, 178) Undened
LHK1 (111) Hyperinfected Cytokinin receptor Cytokinin sensing
SICKLE (122, 123) Hyperinfected EIN2 (Nramp-like protein) Ethylene response mediator
NIP (164, 180) Thick ITs NRT1 transporter family
a
phenotype seen with overexpression.
b
also has an arbuscular mycorrhizal phenotype.
cell junction, thereby allowing the infection to foci but very few ITs (cyclops, ern1), and others
be propagated into the cortex. There are many in which a few ITs are formed. The growth
legume mutants that are defective for infection of these ITs is usually abnormal or arrested
(Table 1 and Figure 1). Usually, such mu- (lin/cerberus, vapyrin, rpg, nap1, pir1, symrem1).
tants can form nodules but most of the nodules
are uninfected, at least in the early stages of
the symbiosis. These infection mutants include Alternative Modes of Infection
some that lack the ability to initiate infection Although infection via root hairs is common in
foci (nin, nsp1, nsp2), some which form infection many legumes, it is absent from legumes such

as peanut and lupin, and infections can occur also occur with a Bradyrhizobium sp., which
in some legume mutants that lack root hairs lacks the ability to produce NFs (43). Thus, it
(16, 48). Legumes, such as Sesbania rostrata, appears that a basic mode of NF-independent
that grow under conditions of waterlogging rhizobial infection exists, and in L. japonicus
(which suppresses root-hair growth) have the this involves rhizobia growing between root
capability of infection via root hairs under cells and directly infecting nodule cells from
dry conditions and infection through cracks the intercellular space, without the need for
in the root epidermis under waterlogged ITs. Rhizobia can also colonize L. japonicus
conditions. During crack invasion foci of via cracks in the epidermis, but this pathway
bacterial colonization form in intercellular requires NFs and permitted more efcient in-
pockets produced by localized cell death at the fection of the cortex than the NF-independent
site of bacterial entry (27, 30). These infection mode. On a root hairless mutant of L. japonicus
pockets appear to fulll analogous roles to the most common route of infection was the
infection foci in root-hair curls (49), and it is NF-dependent infection of root hairlike
from these infection pockets that ITs develop. structures that originated from the root cortex
Although rhizobial colonization of rather than the epidermis, but crack entry was
L. japonicus occurs through root hairs, it also observed (65). Thus, it appears that L.
has become apparent from studies of a mutant japonicus has maintained the capability to be in-
that lacks root hairs and the use of gain-of- fected by rhizobia via multiple different routes,
function mutations in CCaMK and LHK1 that which are also reected in modes of infection
there are alternative modes of infection (65, observed in a variety of legume species.
90). Surprisingly, a CCaMK gain-of-function
mutant could be infected and form some
N2 -xing nodules even when inoculated with Signaling During Rhizobial Infection
a mutant of Mezorhizobium loti unable to make NF signaling is normally essential during
NFs. This mode of infection requires SYMRK bacterial infection and specic NF receptors
(90), implying that SYMRK does not play control NF recognition in this process. In
a role in NF recognition. NF-independent M. truncatula, a so-called entry receptor is en-
colonization of the roots of Acacia species can coded by LysM receptor-like kinase 3 (LYK3)
and this LysM receptor-like kinase is involved
in recognition of specic NF structures made
Nod factor
by Sinorhizobium meliloti NF (83, 146), and in
LYK3 NFP LYK3 NFP pea the SYM37 locus appears to play a similar
role (81). The regulation of these receptors
and possibly their specic location within the
plasma membrane is important during the
p
infection process. An E3 ubiquitin ligase, Plant
PUB1 PUB1
U-box protein 1 (PUB1), interacts with and is
Ub PUB1 target phosphorylated by LYK3 (96). RNAi knock-
PUB1 target down of PUB1 in a lyk3 mutant (which normally
forms aborted ITs) resulted in partial restora-
tion of infection, demonstrating that PUB1
Degradation Inactive PUB1
(infection blocked) (infection proceeds) negatively regulates bacterial infection and that
Figure 5 it may be regulated by LYK3 (Figure 5). In ad-
A model for PUB1s role in rhizobial infection (96). In uninfected cells, PUB1 dition, two other putative E3 ubiquitin ligases,
ubiquitinates a protein required for infection so that it is degraded by the LUMPY INFECTIONS (LIN)/CERBERUS
proteasome. In cells that develop successful infections, LYK3 perceives Nod and nodule specic RING nger (nsRING),
factors and phosphorylates PUB1 to deactivate it. are required for infection (71, 144, 177). The
130 Oldroyd et al.

ubiquitin proteasome in plants has been shown tion and degrees of downstream signaling.
to regulate receptor-like kinases including Analogous complexes may be required for
CERK1 (chitin-elicitor receptor kinase 1), NF recognition, and the role of otillins and
Bacteroids:
which is closely homologous to LYK3 (42), remorins may be to incorporate NF recep- differentiated rhizobia
and FLS2 (Flagellin-insensitive 2), a leucine tors into membrane microdomains, allowing that x N2 inside the
rich repeat (LRR) receptor-like kinase sim- effective recognition and signaling. cells of nodules of
ilar to SYMRK (46). Thus, we expect that some leguminous
species
ubiquitinylation can regulate the amounts Section Conclusions
and cellular location of nodulation receptor
The infection of legume roots by rhizobia
kinases important for bacterial colonization. In
usually occurs via plant-made ITs that initiate
addition, other proteins may be targeted by E3
from infection foci inside a curled root hair or
ligases during infection.
at infection pockets during crack entry. Inva-
Lipid rafts are important during rhizobial
sion through root hairs appears to be the more
infection, and these specialized compartments
advanced state, and L. japonicus that normally

of the plasma membrane may be associated with
shows root-hair entry has still maintained
receptor functions necessary for NF recogni-
the capability to be infected via crack entry

tion and signaling. The otillins FLOT2 and
or via intercellular invasion. Surprisingly, a
FLOT4 are required for IT initiation and local-
constitutively active form of CCaMK can allow
ize to microdomains in root hairs, with FLOT4
some colonization of nodules by rhizobia not
relocalizing to growing root hair tips after
producing NFs. However, in the normal case
rhizobial infection (53). Flotillins are used as
specic NF receptors are required for bacterial
markers for lipid rafts and may serve to recruit
entry. Associated with NF perception is the
multiprotein signaling complexes that regulate
regulation of ubiquitin-mediated proteolysis,
cytoskeletal remodeling to lipid raft domains
which is emerging as an important feature
(70, 74). Symbiotic Remorin 1 (SYMREM1)
of infection. Flotillin and remorin proteins,
is important during rhizobial colonization
which promote trafcking and aggregation of
(79), and the remorin protein family, to which
membrane proteins, are required for infection
SYMREM1 belongs, is associated with for-
by rhizobia, possibly by acting as scaffolds for
mation of lipid rafts. Although SYMREM1
recruitment of membrane proteins involved in
localizes to the PM and is enriched in
nodulation signaling.
detergent-insoluble lipid fractions, it has not
yet been shown to localize in membrane
microdomains (79). The precise function of INTRACELLULAR
remorin proteins is unknown, but they are ACCOMODATION OF RHIZOBIA
required for viral cell-to-cell movement in
Section Introduction
plants (133). Loss of SYMREM1 was asso-
ciated with aborted ITs and reduced nodule In considering intracellular accommodation of
number. Specic interactions occur between rhizobia with the ultimate development of
SYMREM1 and the NF receptors NFP (Nod N2 xing bacteroids, it is important to con-
factor perception) and LYK3, and the NF- sider this as a spatial and temporal develop-
signaling component SYMRK; this suggests mental process. Rhizobia begin as free-living
that this remorin may recruit these receptors bacteria growing down the IT, with bacte-
to lipid rafts and this function may be critical rial cell division restricted to a zone behind
during rhizobial infection (79). In animals the growing tip (37), leading eventually to mi-
and microorganisms, receptors function in crosymbionts surrounded by the plant-derived
large complexes associated with membrane symbiosome membrane. There are signicant
microdomains, and such complexes are metabolic challenges during growth down ITs:
important to dene specicity of recogni- redox stress (61), the need to export plant

defense metabolites such as phytoalexins, the would with its normal hosts, vetch and pea (98).
need to metabolize compounds such as GABA Thus, it can be concluded that some legumes
and malonate (V. Ramachandran, A.K. East, K. take control of the bacterial cell cycle and deter-
Endoreduplication:
progression through Ramakrishnan, A. Downie, P.S. Poole, unpub- mine the endoreduplication state of bacteroids.
the cell cycle without lished results), and the possible accumulation Transcriptome analysis of M. truncatula
associated cell division, of polyhydroxybutyrate for use in a burst of nodules revealed the induction of more than
leading to cells with carbon metabolism during bacteroid formation 400 nodule specic cysteine-rich peptides,
many copies of the
(85) (Figure 6). Growth in the IT is a key stage which are also induced in the nodules of
genome
for selection of competitive rhizobia, and this other closely related indeterminate nodu-
stage provides a checkpoint for the plant be- lating species but are absent in determinate
cause bacteria mutated in cell surface compo- nodulators such as L. japonicus (2, 68, 97).
nents such as LPS and EPS, as well as BacA, These defensin-like peptides contain 60 to 90
either fail to be released from ITs or fail to amino acids with conserved cysteine motifs
form mature symbiosomes (reviewed in 41). In but otherwise show extensive sequence di-
this section, we consider what factors govern vergence. These peptides induce membrane
bacterial development and persistence and how permeabilization, endoreduplication, and loss
the plant takes control of bacterial differentia- of viability in free-living S. meliloti, all features
tion during the development of the symbiosome characteristic of their bacteroid form (160).
within the cells of the nodule. Furthermore, when one of these cysteine-rich
peptides from M. truncatula was expressed
Bacteroid Development in L. japonicus, the bacteroids became greatly
In plants such as L. japonicus, symbiosomes usu- enlarged and were permeable to propidium
ally contain two or more bacteroids of simi- iodide, indicating that the cysteine-rich peptide
lar size to free-living bacteria, whereas symbio- could control bacterial development. These
somes from indeterminate nodules, as produced peptides colocalize with bacteroids (160) and
by M. truncatula, contain a single swollen bac- targeted secretion across the peribacteroid
teroid that can be pleomorphic in shape (85, membrane appears to be the function of a signal
119), although there are exceptions to this cor- peptidase encoded by DNF1 (170). Mutation
relation (119). Bacteroids of S. meliloti from of dnf1 blocked bacteroid differentiation (170),
M. truncatula nodules undergo endoreduplica- and the cysteine peptides became trapped in
tion to yield a chromosome count of 24 com- the endoplasmic reticulum rather than in the
pared to one to two for free-living bacteria (98). bacteroids (160). Thus, DNF1 is required to
In this respect, bacteroids resemble their host target these cysteine-rich peptides to sym-
cortical cells, which also undergo endoredupli- biosomes where they are incorporated into
cation (167). M. truncatula bacteroids, but not bacteroids and drive bacteroid development.
free-living bacteria, are permeable to propid- The BacA protein from S. meliloti is es-
ium iodide, indicating membrane permeability sential for bacteroid development (44) and
(98), and these bacteroids are terminally differ- may promote uptake of the cysteine-rich
entiated and so cannot be cultured from nodules peptides. BacA belongs to the ATP-binding
(although undifferentiated bacteria can be cul- cassette superfamily (ABC) of transporters,
tured from M. truncatula nodules). In stark con- has homology to very long chain fatty acid
trast, bacteroids from determinate L. japonicus (VLCFA) exporters, and indeed is required for
or Phaseolus vulgaris retain a chromosome num- modication of S. meliloti lipid A with VLCFAs
ber of one to two, are impermeable to propid- (35). Mutation of bacA alters sensitivity to
ium iodide, and can be regrown from nodules. antimicrobial peptides, including increas-
When R. leguminosarum bv. viciae was modied ing resistance to bleomycin and Bac7 and
so it could colonize L. japonicus nodules, it did increasing sensitivity to detergent and van-
not undergo endoreduplication as it normally comycin (66, 93). The altered sensitivity to
132 Oldroyd et al.

peptides is not due to the absence of VLCFAs rates (84). Although the bacteroids formed
alone because biosynthetic mutants of VLCFAs by these strains appeared relatively normal
can form a successful symbiosis with alfalfa in electron micrographs, they accumulated
(36) and do not show altered resistance to polyhydroxybutyrate, which is unusual in
bleomycin or Bac7. A bacA mutant of R. legumi- mature pea bacteroids and suggests impaired
nosarum bv. viciae on its indeterminate host pea carbon utilization. Furthermore, pea plants
had an almost identical nonxing phenotype inoculated with aap bra strains were severely
to the bacA mutant of S. meliloti on alfalfa (66). nitrogen starved with little difference in dry
However, bacA mutants of R. leguminosarum weights from uninoculated plants (84). The
bv. phaseoli or Rhizobium etli inoculated onto explanation for this was thought to involve
P. vulgaris (forming determinate nodules cycling of amino acids (130, 176), but it is now
lacking the cysteine-rich peptides) xed N2 known that branched-chain amino acids need
normally. Furthermore, BacA is not needed for to be supplied to bacteroids by the plant (130).
bacteroid formation by the broad host range Preventing branched-chain amino-acid uptake
Rhizobium NGR234 on a range of determinate by bacteroids by mutating bra and aap leads
and indeterminate hosts outside the clade of to amino acid starvation, causing a failure to
plants, to which M. truncatula and pea belong develop bacteroids fully. This phenomenon
(5). Hence, BacA only alters bacteroid devel- was named symbiotic auxotrophy because
opment in indeterminate nodules that produce R. leguminosarum only becomes auxotrophic
cysteine-rich peptides. Considering that muta- when in symbiosis with the plant and is caused
tion of bacA alters sensitivity to antimicrobial by the shutdown of amino acid synthesis
peptides, mutation of bacA may change the in bacteroids. Symbiotic auxotrophy may
uptake of cysteine-rich peptides directly, or it be energetically favorable for the plant, or
could change the uptake of peptides indirectly alternatively the specialized nature of bacteroid
by changing the lipid composition of the metabolism might restrict the supply of key
membrane. Mutation of bacA appears to have intermediates for synthesis of branched-chain
a particularly dramatic effect on the expression amino acids. What is clear is that bacteroids
of components of both the inner and outer have surrendered control over biosynthesis of
membrane in developing pea bacteroids, and essential compounds (Figure 6).
this would be expected to dramatically alter Bacteroids in indeterminate nodules behave
sensitivity to antimicrobial peptides (66). like organelles as demonstrated by the whole-
sale shut down of most aspects of cell growth
and division, synthesis of ribosomal proteins,
Bacteroids as Organelles in nucleic acid synthesis and repair, outer mem-
Indeterminate Nodules brane proteins, and peptidoglycan (10, 11,
R. leguminosarum has two broad-specicity 19, 21, 67). There is, of course, the specic
amino-acid transporters, AapJQMP and induction of nif and x genes that enable the
BraDEFGC, which belong to the ABC family reduction of N2 to ammonia, and this reaches
(58, 169). AapQ, AapM, BraD, and BraE its peak in mature bacteroids. The switch on of
are integral membrane proteins, AapP, BraF, ammonia synthesis by bacteroids (N2 xation)
and BraG are ATP-binding proteins, and AapJ is accompanied by the switch off of ammonia
and BraC are solute binding proteins. AapJ and assimilation into amino acids (reviewed in
BraC bind amino acids in the periplasm and 121). This is supported by the observation that
deliver them to the AapQMP and BraDEFG peas infected by bacteria mutated in both gltB
membrane complexes respectively for ATP- (glutamate oxoglutarate amidotransferase) and
dependent solute transport. R. leguminosarum aldA (alanine dehydrogenase) are unaltered
mutated in both aap and bra nodulated peas in N2 xation and plant growth, even though
but xed N2 at around 30% of wild-type this mutant cannot assimilate any ammonia

and must be provided with amino acids (109). to nutrient limitation. It is notable that rhizobia
Thus, mature indeterminate bacteroids can be have a dual specicity synthestase/hydrolase
considered to be ammoniaplasts whose role RelA protein that mediates this response and S.
is to release ammonia to the plant without meliloti relA mutants are defective for nodula-
assimilation. In this regard, there is an intrigu- tion (174). Similarly, an R. etli relA mutant has
ing mutant with a deletion in the N-terminal a 75% reduction in N2 -xation on beans and
uridylyltransferase domain of the ammonium- an autoregulatory loop with the nodule specic
sensing protein GlnD in S. meliloti, and this N (RpoN2) (106). Tiling arrays have now
causes severe nitrogen starvation in the plant been used to dissect the stringent response in R.
even though 15 N2 is reduced at wild-type rates etli and show that many genes and sRNAs are
(182). The precise fate of incorporated 15 N is upregulated in response to ppGpp during entry
unclear, although it appeared in shoots but was into stationary phase as expected (165). How-
lost within 24 h. This may represent an im- ever, unexpectedly, 223 genes were dependent
portant example of misregulation in bacteroids upon ppGpp in exponential phase. Suppressor
causing ammonia to be assimilated into a form mutants of the S. meliloti relA mutation mapped
of nitrogen that is not utilized by the plant. to the RNA polymerase subunits rpoBC and
The dependency of bacteroids on the plant is caused a global reduction in transcription, sug-
further illustrated by the provision by the plant gesting a tight coupling between an attenuated
of homocitrate, which is incorporated into the bacterial transcription rate and the ability to
FeMo cofactor of dinitrogen reductase in bac- form a successful symbiosis (175). However,
teroids (52). In free-living diazotrophs homo- the suppressors did not mediate changes in
citrate is synthesized from 2-ketoglutarate and transcription of specic sets of genes involved
acetyl-CoA by NifV, which is absent in most in symbiosis, although downstream mediators
rhizobia with notable exceptions being Azorhi- of the stringent response might do this.
zobium caulinodans and the recently sequenced
photosynthetic Bradyrhizobia (43, 78). They are Section Conclusions
notable because these species are rare among Overall we now appreciate that bacteroid devel-
the characterized rhizobia in being able to x opment and intracellular accommodation have
N2 in free-living culture. The provision of ho- to be tightly coupled to plant development, and
mocitrate by plants was discovered because mu- although in Figure 6 we show a static bacteroid
tation of the L. japonicus FEN1 gene blocked transporting and exporting many metabolites,
N2 xation even though infection by M. loti the reality is far more complex. Many cysteine-
occurred. FEN1 encodes homocitrate synthase rich peptides are actively secreted to developing
and expression of this plant gene or the Azoto- symbiosomes, and others may be secreted later.
bacter vinelandii nifV gene in M. loti could re- Metals may also be acquired very early in de-
store N2 xation in the fen1 mutant (52). Thus, velopment along with branched-chain amino
the plant supplies a key component of the nitro- acids. Cofactors such as homocitrate may also
genase complex (Figure 6) that most rhizobia be acquired at an early stage of development,
have lost the ability to synthesize and so have and these are necessary for N2 xation. Thus,
become dependent on a supply from the plant. the bacteroid becomes highly dependent on
Most housekeeping genes are switched off the plant cell for many aspects of metabolism,
in bacteroids, and selected genes such as those and therefore we can consider the bacteroid
encoding the TCA cycle are upregulated (67). as analogous to an organelle. It is intriguing to
This suggests the involvement of a global consider that such a state may be forced upon
transcriptional switch, such as that mediated by rhizobia by the host plant and may also reect
guanosine tetraphosphate (ppGpp), which is a level of control that ensures the bacteria
the effector molecule of the stringent response remain benecial to the plant.
134 Oldroyd et al.

CO2
Sucrose
Oxaloacetate PEP Hexoses
Malate H+
ATP
Dct? ADP + Pi Acetyl-CoA
Phloem
Xylem
DctA Aap FEN1
Homocitrate -ketoglutarate
Bra H+ ?
H+
A Ala H+
Ald
Pyr
Symbiosome
Symbiosome
?
membrane
Acetyl-CoA PHB
space
OAA Amino acid

e Nitrogenase pool Asn
and complex
TCA ATP GS/ H+ Glu
GOGAT ? AS
cycle GS
Aap N2 NH3 NH4+ Gln
Ile
Leu
Val Endoreduplicated
Bra chromosome
?
Sst1 BacA
Mol
SO4 2 ? MgtE ER
? ? Nucleus
MoO42 Dmt1 Golgi
Mg2+ NCR
Fe2+
SPC
Symbiotic plant cell
Figure 6
An idealized bacteroid in an indeterminate nodule. The bacteroid is shown already enclosed in a plant-derived symbiosome membrane
but many of the processes shown may only occur in infection threads or very early in bacteroid development. Plants supply
dicarboxylates; malate is shown as the main one, but there are large concentrations of succinate and fumarate. Bacteroids shut down
ammonia assimilation into amino acids and secrete ammonia to the plant. An obvious but overlooked feature is that transport systems in
the symbiosome and bacteroid membranes should be paired unless there is signicant modication of ligands in the symbiosome space.
Not all of the transport systems have been identied in one species: MgtE is a putative Mg2+ transporter in Rhizobium leguminosarum
and mutation of which causes a Fix phenotype (67); mutation of SST1, a sulfate transporter of Lotus japonicus, causes a Fix- phenotype
(72); divalent metal transporter (DMT)I is present in Glycine max symbiosome membranes but has not been shown to be essential for
N2 xation (62); likewise the Zn2+ transporter Zip1 (not shown) is present on G. max symbiosome membranes (104). Developing or
mature bacteroids may also become dependent on the plant for provision of amino acids (at least in pea and bean) as seen by the
requirement for Aap and Bra (130), whereas the FeMo Cofactor of dinitrogen reductase (enzyme II of nitrogenase) requires
homocitrate synthase in the plant (FEN1) but almost certainly has the same role in other nodules because of the absence of NifV in
most rhizobia (52). Cysteine-rich peptides (NCR) are only made in indeterminate nodules of plants such as alfalfa, Medicago truncatula
and pea but are lacking in L. japonicus and bean (98). Up to 400 different peptides are made at different stages of development,
suggesting the possibility of different targets.
SUMMARY POINTS
1. NF recognition at the root surface leads to nodule organogenesis in the root cortex, and
this requires a diffusible signal.
2. Cytokinin and auxin are key regulators of nodule initiation in the root cortex. However,
unlike initiation of the root apical meristem or lateral root meristems, high cytokinin
levels and low auxin levels are associated with nodule initiation.

3. NF perception activates a suite of responses in root-hair cells that ultimately redirects

polar growth, from an outward growing cell tip to an inward growing invagination.
4. Regulation of receptor-like kinases necessary for NF recognition appears to be critical
to allow rhizobial infection, and this process may be associated with the recruitment of
these receptors to membrane microdomains.
5. Some legume species enforce a high degree of bacterial development, associated with
bacteroids, and this occurs through a suite of cysteine-rich peptides that are secreted into
the symbiosome, where they are taken up by the bacteroid, possibly through the direct
or more probably indirect action of BacA.
6. Rhizobia in the symbiosome develop a high degree of dependence on the plant for many
aspects of metabolism, and this degree of metabolic exchange is analogous to an organelle.
DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We would like to thank Dr. Alex Bourdes for his help in preparing Figures 3 and 6 and our
funding sources: the European Research Council as a grant to G.E.D.O., the European Union for
a grant (MRTN-CT-2006-035546) to J.A.D. and G.E.D.O.; the BBSRC for providing a David
Philips Fellowship to J.D.M. (CA403A13B), a grant (E017045/1) to J.A.D., and a grant in aid to
all authors.
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The Rules of Engagement in the Legume-Rhizobial Symbiosis
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Sex in Fungi
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GE45CH07-Oldroyd ARI 5 October 2011 15:45

Giles E.D. Oldroyd, Jeremy D. Murray,

Annu. Rev. Genet. 2011. 45:11944 Keywords

The Annual Review of Genetics is online at Abstract

INTRODUCTION collectively referred to as rhizobia. Thus,

bacterial infection (116). Both nodule organo-

Bacterial infection recognition of the rhizobial signaling molecule

120 Oldroyd et al.

Nodulation in legumes evolved from the NODULE ORGANOGENESIS

www.annualreviews.org Legume-Rhizobial Symbiosis 121

122 Oldroyd et al.

Lateral root initiation

High cytokinin RR1

www.annualreviews.org Legume-Rhizobial Symbiosis 123

124 Oldroyd et al.

development. regulated by these transcription factors are not

revealed functions in the maintenance and

www.annualreviews.org Legume-Rhizobial Symbiosis 125

root-hair tip, and it continues to extend through

body until it joins the cell wall at the interface

126 Oldroyd et al.

The IT is surrounded by an array of microtubules

A Ca2+ gradient is needed for vesicle fusion

cell wall and matrix materials and signaling

Small G proteins localized at the apex may

Axial actin cables. Actin is probably required for

The vacuole controls cell turgor.

www.annualreviews.org Legume-Rhizobial Symbiosis 127

Microtubules and actin coordinately act in of clover by Rhizobium leguminosarum biovar

128 Oldroyd et al.

Table 1 Genes involved in rhizobial infection

ERN1 (100) IT initiation AP2/ERF TF Transcription

FLOT4 (53) IT initiation Flotillin Unknown

www.annualreviews.org Legume-Rhizobial Symbiosis 129

130 Oldroyd et al.

advanced state, and L. japonicus that normally

the capability to be infected via crack entry

www.annualreviews.org Legume-Rhizobial Symbiosis 131

132 Oldroyd et al.

www.annualreviews.org Legume-Rhizobial Symbiosis 133

134 Oldroyd et al.

OAA Amino acid

Symbiotic plant cell

www.annualreviews.org Legume-Rhizobial Symbiosis 135

3. NF perception activates a suite of responses in root-hair cells that ultimately redirects

136 Oldroyd et al.

www.annualreviews.org Legume-Rhizobial Symbiosis 137

Trends Plant Sci. 13:11520

of temperate legumes. Microbiol. Mol. Biol. Rev. 68:280300

138 Oldroyd et al.

process. Mol. Plant-Microbe Interact. 16:21725

www.annualreviews.org Legume-Rhizobial Symbiosis 139

in developing stem nodules of Sesbania rostrata. Mol. Plant-Microbe Interact. 15:16468

140 Oldroyd et al.

www.annualreviews.org Legume-Rhizobial Symbiosis 141

142 Oldroyd et al.

www.annualreviews.org Legume-Rhizobial Symbiosis 143

mutant. J. Bacteriol. 185:560210

system of Rhizobium leguminosarum bv. viciae 3841. J. Bacteriol. 191:154755

144 Oldroyd et al.

Volume 45, 2011 Contents

Comparative Genetics and Genomics of Nematodes: Genome

Uncovering the Mystery of Gliding Motility in the Myxobacteria

Beiyan Nan and David R. Zusman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p21