Cell Host & Microbe

Review

Root Nodulation: A Paradigm

for How Plant-Microbe Symbiosis
Influences Host Developmental Pathways
Guilhem J. Desbrosses1 and Jens Stougaard2,*
1Laboratory of Mediterranean and Tropical Symbiosis (LSTM) CC002, Université Montpellier 2, Place E. Bataillon, F-34095 Montpellier

Cedex 05, France

2Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark

*Correspondence: stougaard@mb.au.dk
DOI 10.1016/j.chom.2011.09.005

Legume plants have an exceptional capacity for association with microorganisms, ranging from largely
nonspecific to very specific interactions. Legume-rhizobial symbiosis results in major developmental and
metabolic changes for both the microorganism and host, while providing the plant with fixed nitrogen. A
complex signal exchange leads to the selective rhizobial colonization of plant cells within nodules, new
organs that develop on the roots of host plants. Although the nodulation mechanism is highly specific, it
involves the same subset of plant phytohormones, namely auxin, cytokinin, and ethylene, which are required
for root development. In addition, nodulation triggered by the rhizobia affects the development of the host
root system, indicating that the microorganism can alter host developmental pathways. Nodulation by
rhizobia is a prime example of how microorganisms and plants have coevolved and exemplifies how micro-
bial colonization may affect plant developmental pathways.

Introduction development of actinorhizal and legume root nodules (Gherbi

Besides anchoring plants in the soil, roots are essential for
mineral nutrition and water uptake. When exposed to fluctua-
tions in the soil environment such as drought or nitrate or phos-
phate availability, roots activate compensating mechanisms
ranging from changes in nutrient uptake ability to changes in
root architecture (Desnos, 2008). This plasticity enables the plant
to cope with adverse environmental conditions. Adding another
level of flexibility, roots also interact with their biotic environment.
Roots of most land plants develop beneficial interactions with
soil microorganisms in the form of arbuscular mycorrhiza and
for woody trees ectomycorrhiza. These ancient fungal symbi-
oses improve the plants water and nutrient supply, especially
the phosphate uptake (for review, see Parniske, 2008). In con-
trast to the majority of plants, legumes have the capacity to
interact with nitrogen-fixing soil bacteria collectively called
rhizobia and develop a beneficial root endosymbiosis. The plant
gains access to nitrogen fixed by the bacteria, while the microor-
ganism gets privileged access to plant-derived carbon sources.
Bacterial endosymbiosis is also found outside the legume
family. The actinomycete Frankia forms actinorhizal root nodules
on a number of host plants, such as Alnus glutinosa (alder tree)
and Casuarina glauca (Perrine-Walker et al., 2011). Interestingly,
these plants together with legumes belong to the same Eurosid 1
clade, in which the capacity for nodulation emerged several
times independently (Soltis et al., 1995). One or several unknown
preconditioning step(s) specifically acquired by an Eurosid 1
ancestor must have been essential for assembling the genetic
program controlling root nodule development (Markmann and
Parniske, 2009; Soltis et al., 1995). Actinorhizal root nodules
differ from legume nodules in that they possess a central
vascular system like roots (Perrine-Walker et al., 2011). Never-
theless, there is a substantial overlap in the gene-set controlling
et al., 2008).
Root nodules are lateral root organs, and like lateral roots
they influence the architecture of the root system. Both organs
are formed postembryogenically, and both are integrated
into the program governing development of the root system.
One example is the modification of the root system of the
model legume Lotus japonicus (Lotus) after inoculation with
Mesorhizobium loti. The primary root growth is slower in inocu-
lated plants compared to control plants (Wopereis et al., 2000).
This phenotype is even stronger in the hypernodulating mutant
har1-1 (Wopereis et al., 2000). Other examples are the Lotus
brush and crinkle mutants that show a defect in both root devel-
opment as well as reduced nodulation (Maekawa-Yoshikawa
et al., 2009; Tansengco et al., 2003). At the regulatory
level, availability of nitrate affects nodule formation, and in
Arabidopsis, nitrate regulates lateral root development both
locally and systemically (Desnos, 2008). Although poorly under-
stood, this apparent coordination of root and nodule develop-
ment gives raise to several questions. For example, are there
similarities in anatomy, ontogeny, or the developmental regula-
tion of nodule development and lateral root formation? Have
the genetic programs that govern nodulation co-opted part of
a pre-existing program for lateral root development rather than
evolving independently? How are the plant phytohormone auxin,
cytokinin, and ethylene pathways, which control root develop-
ment, involved in nodulation? In this review, we consider these
questions and discuss how legume-rhizobial symbiosis influ-
ences the root developmental pathways of the host plant.
The Cellular Origin of Roots and Nodules Are Different
Formation of a root nodule is a result of two tightly coordinated
processes: an organogenic process developing the nodule

348 Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
Review
organ and its tissues, and an infection process mediating bacte-
rial colonization (Madsen et al., 2010). Nodulation is inducible
and involves rapid activation of epidermal, cortical, and pericycle
cells (Figure 1A). The host-symbiont signal exchange is initiated
by secretion of flavonoids that are sensed by rhizobia. In
response, rhizobia synthesize lipochitooligosaccharides, called
Nod factors (NFs), triggering the nodule developmental process.
In Lotus, these NFs are perceived by epidermal cells via
membrane-localized LysM receptor kinases that most likely
distinguish the different NF decorations synthesized by different
rhizobia (Madsen et al., 2011; Radutoiu et al., 2003, 2007). This
selective perception explains why a particular legume is only
nodulated by a limited number of rhizobial strains (host speci-
ficity). In the best-characterized legumes, like Lotus and
Medicago truncatula (Medicago), this interaction occurs on a
subset of elongating root hairs cells in the ‘‘susceptible zone,’’
corresponding to the root differentiation zone in Arabidopsis
(Figure 1B). The tips of individual root hairs curl to form a niche
for a rhizobial microcolony that later colonize the infection thread
(IT) transgressing into the root cortex (for review, see Held et al.,
2010). Simultaneously, the underlying root cortex cells are dedif-
ferentiating and undergo rounds of mitosis to form a nodule
primordium. When an IT reaches a nodule primordium, rhizobia
are released into organelle-like symbiosomes, where they differ-
entiate into nitrogen-fixing bacteroids. Further growth, differenti-
ation, and infection result in nodules that are either spherical or
elongated depending on the legume species (Figures 2A–2D).
Indeterminate elongated nodules from Medicago have a
permanent meristematic (undifferentiated growth zone) activity
(Figures 2A and 2B), while the determinate spherical nodules of
soybean, common bean, and Lotus have a short-lived meristem
(Figures 2C and 2D). Curiously, indeterminate nodules with
continuous growth initiate from the inner cortex, while nodules
with determinate growth are founded in the middle/outer cortex.
Perception of the NFs by the Lotus NFR1 and NFR5 receptors
triggers the signal transduction that leads to both IT formation
Figure 1. Schematic Representation of Root
Development
(A) Legume root nodule development.
(B and C) Arabidopsis root development.
(A) In legumes the differentiation zone and the ‘‘susceptible
zone’’ sensitive to rhizobia overlap.
(B) The Arabidopsis root can be divided in three different
regions: meristematic zone, the transition zone, and the
elongation and differentiation zone (Scheres et al., 2002).
The meristematic zone defines the region where most cell
division occurs (pale blue box). The meristem is itself
composed of various types of stem cells. Cells in the
quiescent center (QC) rarely divide while the surrounded
stem cells frequently divide following a reiterated program.
According to their position, daughter cells differentiate
into the various root cell files comprising the tissues. In
the elongation and differentiation zone (pale green box)
cells formed by the meristem undergo differentiation
processes. Cells elongate and the vascular system is
formed mainly under the control of cytokinin.
(C) A radial section of an Arabidopsis primary root. In the
epidermis, trichoblast cells sits on top of two adjacent
cortical cells. They will elongate a root hair while atricho-
blast not. Polar auxin transport indicated by arrows.
Abbreviations: IAA, indole-3-acetic acid; CK, cytokinin;
C2H4, ethylene; QC, quiescent center.
and nodule organogenesis (Madsen et al., 2010). Subsequent
induction of the downstream events depends on calcium-medi-
ated activation of a central regulator, the calcium-calmodulin-
kinase, CCaMK (Figure 3), and activation of several downstream
genes such as the GRAS transcription factors NSP1 and NSP2
(Heckmann et al., 2006; Hirsch et al., 2009), as well as the
transcriptional regulator NIN. Gain-of-function mutations in the
CCaMK kinase gene uncouple infection from organogenesis,
resulting in spontaneous nodule formation in the absence of
rhizobia (Gleason et al., 2006; Tirichine et al., 2006). Autoactiva-
tion of the LHK1 cytokinin receptor (snf2 allele), which is located
downstream of CCaMK (and is known to be involved in root
development; see details below) also leads to spontaneous
nodule formation, allowing the root nodule organogenic process
to be more directly compared to the lateral root formation (Mur-
ray et al., 2007; Tirichine et al., 2007).
Primary and lateral root development in legumes is less well
characterized than in Arabidopsis. Certain tissues (e.g., cortex)
contain additional cell layers, and almost all epidermal cells elon-
gate into a root hair (Karas et al., 2005) (see below). Nevertheless,
the general tissue organization and zonation in legumes is
comparable. In this review, we will consider the Arabidopsis
root as a valid model for the uninoculated legume root. Lateral
roots result from reactivation of the cell cycle in auxin-primed
founder cells, located in the pericycle (Figure 1B). Priming of
the founder cells occurs in the meristematic zone (Dubrovsky
et al., 2008). At the transition into the differentiation zone, founder
cells undergo several rounds of mitosis that form a lateral root
primordium. Later, this primordium differentiates into a fully func-
tional lateral root meristem identical in structure and function to
the primary root meristem. For plants like tomato or Arabidopsis
grown on solid plates, lateral roots emerge at equal intervals and
alternate on each side of a root system (De Smet et al., 2007).
This depends on internal mechanisms such as oscillations of
transcription from a subset of genes in the meristem (Moreno-
Risueno et al., 2010) and on external stimuli such as gravity
Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc. 349

Figure 2. Legumes Develop Two Main Types of Nodules
Upon inoculation with their respective rhizobial microsymbionts, Medicago
develop elongated nodules (A) of the indeterminate type (B), while Lotus ja-
ponicus develop spherical nodules (C) of the determinate type (D).
(De Smet et al., 2007). A difference between legumes and
Arabidopsis is the development of the root epidermis. In
Arabidopsis, specific epidermal cells spanning two cortical cells
differentiate into root-hair cells (trichoblasts) (Dolan and Costa,
2001), while most epidermal cells have this capacity in Lotus
(Karas et al., 2005) (Figure 1C).
Auxin and Cytokinin Are Required for Nodule
Development
Rhizobia inoculation of legumes or NF application leads to local
changes in the accumulation of auxin (Mathesius et al., 1998; Pa-
cios-Bras et al., 2003), and accumulation of the putative influx
auxin transporter AUX1 transcripts has been observed (de Billy
et al., 2001). Some legumes also respond to auxin transport
inhibitors (Hirsch et al., 1989; Mathesius et al., 1998), and in
Medicago, local disruption of polar auxin transport (PAT) was de-
tected at the site where nodule primordia emerge. Endogenous
flavonoids were proposed to inhibit PAT as knockdown of
chalcone synthase (CHS) resulting in reduced level of flavonoids
was associated with reduced nodulation and reduced PAT
(Mathesius et al., 1998; Wasson et al., 2006). A decreased
number of nodules was also observed in transgenic soybean
lines with reduced level of flavonoids (isoflavone synthase
knockdowns), although reduced PAT was not observed in these
lines (Subramanian et al., 2006). Furthermore, normal nodulation
350 Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
Review
was restored by a B. japonicum strain synthesizing NFs in
response to lower levels of isoflavones, indicating that a lack of
NF synthesis caused the knockdown phenotype. PAT may
therefore be essential for development of indeterminate nodules
but not for determinate nodules (Subramanian et al., 2006).
Another complication is the clustering of nodules observed on
most legume species (Figures 2A and 2C). The individual
nodules in these clusters must be initiated within a short time
and a limited cellular space. This seems to call for more detailed
studies of the quantitative and qualitative aspects of auxin trans-
port and flow in the context of nodulation. How auxin synthe-
sized by bacteria affect nodule development will also need to
be addressed. Indeed, expression of a novel IAA biosynthesis
pathway in a Rhizobium leguminosarum bv. viciae strain resulted
in fewer but larger nodules (Camerini et al., 2008).
Cytokinin has long been thought to be involved in root endo-
symbiosis. Experiments applying cytokinin to roots of the
tropical legume Sesbania rostrata resulted in accumulation of
transcripts from the early nodulin Enod2 gene (Dehio and de
Bruijn, 1992). Later, a nonnodulating rhizobial strain expressing
a cytokinin biosynthetic gene from Agrobacterium was shown
to induce nodule like structures on alfalfa (Cooper and Long,
1994). Recently, the implication of cytokinin as a key molecule
for nodule development was unambiguously demonstrated by
genetic analysis of allelic Lotus mutants carrying gain-of-func-
tion (snf2) or loss-of-function (lhk1-1) mutations in the LHK1
cytokinin receptor gene (Murray et al., 2007; Tirichine et al.,
2007). The loss-of-function lhk1-1 mutants show a large number
of infection threads but produce very few nodules (Murray et al.,
2007). In contrast, the gain-of-function snf2 mutants produce
nodules spontaneously (Tirichine et al., 2007). This demon-
strated that cytokinin signaling is necessary and sufficient for
initiation of root nodule organogenesis but not for IT formation.
However, IT progression into the cortex appears to be directly
or indirectly affected by cytokinin signaling (Murray et al.,
2007). Surprisingly, the gain-of-function mutations in the LHK1
receptor result in local activation of cell division and sponta-
neous nodule formation rather than prolific cell division along
the entire root. Application of low levels of cytokinin to the entire
Lotus root system also leads to formation of localized nodule
primordia, suggesting the presence of a mechanism restricting
nodule cell activation to a limited number of foci (Heckmann
et al., 2011). A mechanism involving a balance of positive and
negative actions could lead to such positional response. A posi-
tive mechanism might involve overlapping signaling fields, and
there are reports hinting at such a phenomenon. Early work on
pea suggested uridine as a cell division factor acting together
with auxin and cytokinin (Smit et al., 1995). Although fairly spec-
ulative, a gradient of uridine located opposite to protoxylem
poles could act as positional cue promoting cytokinin- and/or
auxin-induced activation of the cell cycle. Alternatively, posi-
tional information could come from a cytokinin-modified auxin
gradient as observed in Arabidopsis (Dello Ioio et al., 2008).
Indeed, PAT is modified by cytokinin treatment of wild-type
Medicago roots but not in cytokinin receptor mutants (Plet
et al., 2011). This effect correlated with local modification of
the expression pattern of the efflux auxin transporter PIN (Plet
et al., 2011). A negative action could involve receptor-like-kinase
activity. In Arabidopsis, the receptor-like-kinase CRINKLY4
Cell Host & Microbe

Review

Figure 3. The Lotus Pathways Involved in Nodule Development and Infection

Perception of Nod factor (NF) by NFR1 and NFR5 receptors at the plasma membrane triggers two parallel pathways facilitating infection thread initiation and root
nodule organogenesis, respectively. The signal transduction pathway for organogenesis includes a leucine-rich repeat receptor kinase (SYMRK), cation channels
(CASTOR and POLLUX), nucleoporins (NUP85, NUP133, and NENA), a calcium calmodulin-dependent protein kinase (CCaMK/SNF1), and a nuclear-localized
coil-coil protein (CYCLOPS). Activation of the NFRs or SYMRK is believed to start transmission of a secondary signal to the nucleus releasing calcium spiking.
Calcium oscillations are decoded by the nuclear-localized CCaMK, while the nuclear membrane-localized CASTOR and POLLUX potassium channels may
regulate release of calcium from the nuclear envelope. The nucleoporins may import secondary signal(s), anchor potassium channel proteins, or transport NSP2
proteins. Double-mutant analysis has suggested a role for CYCLOPS-CCaMK complex in cross-signaling between the organogenic and infection pathways.
Further downstream, the putative transcriptional regulators of NIN, NSP1, and NSP2 are required for initiation of nodule organogenesis. Cytokinin signaling
mediated by the (LHK1) receptor is crucial for primordia formation. The NIN, NSP1, and NSP2 transcriptional regulators are also required for IT formation, and the
Nap1 and Pir1 genes encoding proteins of the SCAR/WAVE complex activating actin rearrangement and Cerberus encoding a putative E3 ubiquitin ligase are
needed for maintenance and progression of the infection thread. Epidermal responses are shown in pink boxes, and the blue box represents the cortical
response. Gene product marked with dark orange boxes constitute the common symbiosis pathway shared with arbuscular mycorrhizal endosymbiosis and
some of them may also play a role in the cortex. Adapted from Madsen et al. (2010).

(ACR4) restricts the dedifferentiation of pericycle cells in the early
events of lateral root primordia elongation (De Smet et al., 2008)
(Figure 4). In Lotus, the hypernodulating too much love mutant is
impaired in the perception of a signal that represses nodule
development (Magori et al., 2009). It is tempting to speculate
that a receptor-like-kinase such as ACR4 might restrict the
nodule primordia development.
Cytokinin and cytokinin-related molecules may also be
produced by rhizobia (Okazaki et al., 2007). That could be of
functional importance as a Bradyrhizobium sp. strain forming
nodules in absence of NFs relies on functional purine synthesis
(similar to cytokinin and cytokinin-related molecules) for func-
tional nodule development (Giraud et al., 2007). How the cyto-
kinin produced by rhizobacteria still located on the root
epidermis could affect cell division in the root cortex remains
to be solved.
Auxin and Cytokinin Signaling Are Important for Root
Development
The roles of these two phytohormones in Arabidopsis root devel-
opment (Figures 1B and 4) have been reviewed recently (Over-
voorde et al., 2010; Perilli et al., 2010). Auxin is distributed in
a gradient with a maximum localized around the quiescent
center (QC). This results mainly from polar transport through
a combination of influx transporter (AUX1) and efflux transporter
(PINs) activity. The gradient is an essential cue that spatially
defines the QC and consequently the fate of neighboring stem
cells. Conversion of the morphogenic gradient information at
the cellular level relies on AP2 type transcription factors such
as PLETHORA 1 to 4. PLETHORA protein levels are correlated
to auxin levels and their functions change according to concen-
tration. Another important player is the SHY2/IAA3 protein,
which is a repressor of Auxin Responsive Factors (ARF). In pres-
ence of auxin, the SCFTIR1 receptor binds to SHY2/IAA3 proteins
and forms a complex that targets ubiquitinated SHY2/IAA3
protein for degradation. This releases the activity of ARF proteins
and consequently triggers auxin action. When auxin is abundant,
SHY2 is degraded leading to auxin response (Figure 4).
Lateral root founders cells are primed by transient auxin
accumulation at the site of initiation in the meristematic zone
(Figure 3). Later, a local auxin accumulation dependent on
AUX1 and PIN activity together with the nuclear protein protein
ALF4 will switch founder cells toward a lateral root primordium.
Two auxin pathways will be activated: The Solitary Root
SLR/IAA14 Aux/IAA repressor will be degraded by SCFTIR1,
thus releasing the activity of ARF7 and ARF9 transcriptional

Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc. 351
 Cell Host & Microbe

Review

Figure 4. Primary and Lateral Root Development Requires Interaction between the Auxin and Cytokinin Pathways
High concentrations of auxin trigger a repression of the cytokinin pathway and activate auxin-responsive genes such as PLETHORA 1 to 4 (PL1 to 4) in order to
maintain cell divisions in the root meristem. Locally produced or transported cytokinin will repress the auxin signaling by stimulation of the expression of the
Aux/IAA repressor SHY2. This interaction will spatially define the size of the root meristem. SHY2 is a repressor of PIN auxin efflux transporter. Because SHY2 is
simultaneously degraded by auxin while its expression is stimulated by cytokinin, this may result in oscillations of PIN expression and thus building of local and
transient accumulation of auxin that could prime pericycle cells into lateral root founder cells. Molecular interactions in the lateral root is shown above the dashed
line while interactions in the primary root is shown below.

regulators. In parallel, degradation of the Bodenlos BDL/IAA12
auxin repressor alleviates repression of the Monopteros MP/
ARF5 transcriptional regulator. This auxin activation leads to
successions of anticlinal and periclinal cell divisions, the building
of an auxin maximum, and eventually the formation of a lateral
root meristem that will later emerge from the root. Temporal
priming of the lateral root founder cells in the meristematic
zone would be earlier than the induction of nodule cells which
occur in the differentiation zone of the legume root.
Cytokinins are essential for the control of the meristem size,
for lateral root development, and, during development of the
vascular system, for maintaining cells in a procambial state. In
Arabidopsis, cytokinins produced locally or distibuted via the
vascular system (Hirose et al., 2008) are perceived by the
AHK2, 3, and 4 receptors. Binding of cytokinin will result in phos-
phorylation of phosphotransfer proteins (AHP) and transfer to
the nuclei, where type B and type A Arabidopsis Response
Regulator (ARR) proteins are then phosphorylated. While type
B-ARR will mediate positive response to cytokinin, A-type ARR
will repress the signaling pathway. During root development,
cytokinins interact with the auxin pathway. Cytokinins stimulate
the transcription of the SHY2/IAA3 gene, resulting in increased
SHY2 protein abundance. SHY2 represses the expression of
PIN1, 3, and 7 auxin efflux transporters, as well as the major
cytokinin biosynthetic gene IPT5. Consequently, cytokinin
prevents the establishment of a PIN dependent auxin gradient
and simultaneously inhibits its own biosynthesis, probably
achieving homeostasis over time. The balance between cyto-
kinin and auxin controls SHY2 abundance and thus contributes
to defining the size of the root meristem. While auxin is positively
regulating lateral root development, cytokinin inhibition of PIN
auxin efflux transporter prevents the elongation of lateral roots
primordia without affecting the fate of founder cells. This effect
is opposite of the cytokinin promotion of nodule primordia.
Recent data from Lotus suggest that legume lateral roots may
also respond differently (Heckmann et al., 2011).
Ethylene, a Third Player in the Game
In roots, ethylene promotes root hair elongation (Tanimoto et al.,
1995) and interferes with the fate of root epidermal cells. High
concentrations of ethylene commit Arabidopsis atrichoblast to
form root hairs (Cao et al., 1999). Ethylene also promotes auxin
biosynthesis in roots (Stepanova et al., 2007), altering the auxin
root tip gradient (Ikeda et al., 2009). This results in increased
cell division and inhibition of cytokinin-dependent cell elonga-
tion/differentiation. One of the cellular effects is division of QC
cells leading to supernumerary but functional QC cells (Ortega-
Martı́nez et al., 2007).
In legumes, ethylene appears to be produced mostly in roots
and to be significantly induced by light (Hunter, 1993; Lee and

352 Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
Review
Larue, 1992). Simultaneously, the nodule number decreases
(Lee and Larue, 1992), suggesting that ethylene is a repressor
of nodulation. Treatment of pea plants with exogenous ethylene
represses nodule organogenesis as infection threads were
arrested in the inner cortex (Lee and Larue, 1992). This arrest
could reflect a role for ethylene in defining the radial epidermis-
cortex boundary in the susceptible zone (Guinel and Geil,
2002). Ethylene inhibition would need to occur very early in the
symbiosis-signaling pathway since calcium spiking in response
to NF treatment (Ehrhardt et al., 1996) is inhibited by addition
of an ethylene precursor (Oldroyd et al., 2001). Further evidence
for ethylene’s negative role has emerged from rhizobial studies.
Many rhizobacteria encode ACC deaminase (accD), which
catalyzes degradation of the ethylene precursor 1-aminocyclo-
propane-1-carboxylic acid (ACC), tuning down the plants
defense response (Glick, 2005). Consistent with this function,
rhizobial accD mutants induce fewer nodules compared to the
wild-type strain (Ma et al., 2003). Furthermore, increased expres-
sion of accD is observed during differentiation of Mesorhizobium
loti, suggesting a function during symbiosis (Uchiumi et al.,
2004).
Ethylene also influences the positioning of nodule primordia.
Nodule primordia are precisely located (i.e. opposite to proto-
xylem poles) on the root, and their number is tightly controlled
(Ding and Oldroyd, 2009). The ethylene-forming enzyme ACC
oxidase transcripts are more abundant in cells facing proto-
phloem poles, positions where nodules are unlikely to develop
(Heidstra et al., 1997). Additional ethylene effects on nodule
meristems and infection were inferred from semiaquatic le-
gumes such as Sesbania rostrata that can develop both determi-
nate nodules by crack entry and indeterminate root nodules by
root-hair IT infection depending on the ethylene concentration.
Together with gibberelin and H2O2, ethylene contributes to cell
death and creation of infection pockets in the root cortex charac-
teristic for crack entry (Lievens et al., 2005). This intercellular type
of infection mechanism has also been observed in Lotus (Karas
et al., 2005; Madsen et al., 2010), and studies of the nena mutant
infected by crack entry suggest that ethylene promotes crack
entry in Lotus (Groth et al., 2010).
Genetic support for the role of ethylene in legumes symbiosis
is relatively scarce. A genetic screen for components of the
nodulation pathway identified a Medicago mutant line (skl) with
about 10-fold increased nodule numbers (Oldroyd et al., 2001;
Penmetsa et al., 2008). A careful analysis of its hypernodulation
phenotype indicated that skl is insensitive to ethylene (Oldroyd
et al., 2001; Penmetsa et al., 2008). Further characterization of
skl alleles uncovered mutations in a homolog of Arabidopsis
ethylene-insensitive (EIN2), an essential component of ethylene
signaling, confirming ethylene’s role as a repressor of nodula-
tion, or alternatively that a decrease in EIN2 activity is required
for nodulation. In-parallel transgenic approaches conducted in
Lotus provided further evidence for ethylene as a negative regu-
lator of nodulation. Transgenic Lotus plants constitutively over-
expressing a mutated version of the melon ERS1 ethylene
receptor or the Arabidopsis etr1-1 that cannot bind ethylene
have reduced ethylene sensitivity. These transgenic lines had
increased numbers of infection threads (Lohar et al., 2009; Nukui
et al., 2004) and increased numbers of primordia, which were
often mislocated.
Using Genome and Transcriptome Resources
to Address the Role of Ethylene during Nodulation
The experimental results obtained so far indicate that the
ethylene story is far from complete. A genetic screen conducted
in soybean identified several lines showing strong ethylene
insensitivity (Schmidt et al., 1999). Surprisingly, these lines
showed reduced nodule numbers compared to the wild-type
(Schmidt et al., 1999). A similar genetic screen in Lotus identified
the enigma mutants showing a severe ethylene insensitivity
phenotype (Gresshoff et al., 2009). The enigma mutants appear
to be defective in a Lotus homolog of Arabidopsis EIN2 (Gressh-
off et al., 2009), and, as opposed to the Medicago skl mutants,
these mutants also display a significant reduction in nodule
number (Gresshoff et al., 2009).
One way to approach this complexity is to take a global view
with the genome and transcriptome resources available in an
attempt to define central genes and molecular mechanisms.
This is by no means a fail-safe approach, since genome informa-
tion in even the best-characterized organisms is regularly up-
dated. However, it can focus attention on interesting features.
Among the legumes, genome sequences are available from
Lotus (Sato et al., 2008), Medicago, and also soybean (Schmutz
et al., 2010). Using the currently available Lotus genome
resources, we identified a set of genes corresponding to the
Arabidopsis genes involved in ethylene biosynthesis, signaling,
and regulation (Figure 5). A quick view indicates that the Lotus
and Arabidopsis ethylene pathways are comparable (Figure 6)
but not necessarily identical.
Looking at the differentially regulated gene family of ACC syn-
thases (ACS), which mediate the penultimate rate-limiting step of
ethylene synthesis, as with Arabidopsis, we can identify three
Lotus subtypes (Figures 5 and 6). So far, the subtype 1 ACS1
and ACS2 have not been found in Lotus, while ACS6 is present
in two copies. In Arabidopsis, ACS6 is likely to be active during
hypoxia (Peng et al., 2005), while ACS1 is a nonfunctional subunit
that positively regulates ACS activity (Tsuchisaka et al., 2009).
These differences may suggest a unique regulation of ACS
activity in Lotus in the context of nodulation. The Arabidopsis,
subtype 2 ACS5 plays a key role during cytokinin-induced
ethylene emission (Vogel et al., 1998). Cytokinin also promotes
ethylene biosynthesis in pea (Lorteau et al., 2001), suggesting
a similar role for Lotus ACS5. Finally, the two ACS7 gene prod-
ucts, representing subtype 3, may, like the Arabidopsis ACS7,
interact with XBAT32, an E3 ubiquitin ligase, and affect lateral
root development by a mechanism involving ethylene biosyn-
thesis (Prasad et al., 2010). This level of posttranslational regula-
tion may therefore be conserved in Lotus.
Homologs of both Arabidopsis ETO1 and EOL were found in
Lotus, suggesting that ETO1/EOL-mediated posttranslational
mechanisms regulating ethylene biosynthesis may also be
conserved in Lotus. The Arabidopsis ETO1/EOL proteins interact
with the C-terminal domain of subtype 2 ACS and with cullin 3A
through their BTB (Broad complex/Tramtrack/Bric-à-brac) and
a TPR (Tetratricopeptide) domain. The resulting protein complex
interacts with ubiquitin ligase and targets subtype 2 ACS for
degradation (Christians et al., 2009).
In Lotus, there are two copies of EIN2. This distinguishes Lotus
since there seems to be only one copy in the Medicago genome
and three or more copies in soybean. In Arabidopsis, EIN2 is an
Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc. 353
 Cell Host & Microbe

Review

Ethylene biosynthesis Ethylene signaling Regulation of ethylene pathway

ljACS5 : Chr4(39901032-39911687) LjETR1 : Chr3(4672259-4669484) LjRTE1 : Chr5(3721148-3719496)
LjACS6a: Chr1(33636624-33632949) LjETR2 : Chr6(806387-803587) LjRAN1 : Chr4(31494614-31498998)
LjACS6b: Chr4(12132022-12133551) LjEIN4a: Chr1(65323091-65319323) LjXRN4 : Chr1(64832882-64839837)
LjACS7a: Chr2(13771873-13769909) LjEIN4b: Chr5(22933006-22936352) LjXRN4*: Chr5(23685268-23681717)
LjACS7b: Chr4(40865032-40867042) LjERS1 : Chr1(63288280-63291864) LjEBF2 : Chr4(27241605-27243482)
LjACS8 : Chr2(29248773-29247079) Missing: AtERS2 LjEBF1 : Chr5(6781599-6779680)
Missing:AtACS1, AtACS2, AtACS4, Missing: AtETP1 and AtETP2
AtACS9, AtACS11 LjCTR1 : Chr1(60036296-60028663)
*:truncated polypeptide
LjEIN2a: Chr1(58693229-58687139)
LjETO1a: Chr1(30466245-30468074) LjEIN2b: Chr5(10193466-10186945)
LjETO1b: Chr2(34162381-34171688)
LjEOL1: Chr5(17749346-17753071) LjEIN3 : Chr2(39272736-39270913)
Missing: AtEOL2 LjEIL3 : Chr3(23123320-23122388)
LjEILx2: Chr4(37820171-37819195)
LjEILx3: Chr5(30881423-3088225)
Missing: AtEIL1, AtEIL2

LjERF1 : Chr6(560153-559521)

Figure 5. Lotus Genes Defining the Ethylene Pathway

Biosynthesis: Arabidopsis has 11 ACS genes mediating ethylene synthesis. Only six were found in the current version of the Lotus genome. Subtype 1 ACS is
represented by two copies of ACS6 and subtype 2 ACS by only ACS5 and ACS8, while two ACS7 genes represent subtype 3. The overall number of ACS genes
may thus be lower in Lotus. In Lotus, ETO1 is duplicated, and one EOL1 homolog was found. Overall, this is the same number as in Arabidopsis.
Signaling: The Lotus and Arabidopsis genomes encode an equal number of ethylene receptors. However, while homologs of ETR1 and ERS1 receptors can be
found, an ERS2 homolog was not yet identified. In contrast, EIN4 is present in two copies. Like in Arabidopsis, there is one copy of CTR1 in Lotus, but EIN2 is
present in two copies. Homologs of ETP1 and ETP2 have so far not been identified. While in Arabidopsis there is EIN3 and five homologs (EIL1 to 5), only one
homolog of EIN3 and one EIL3 homolog was found in Lotus.
Regulation: A homolog for each of EBF1, EBF2, and XRN4 was found. A truncated version of XRN4 was also found (*).

essential component in the ethylene-signaling pathway as
a loss-of-function EIN2 mutation leads to complete insensitivity
to ethylene (Guzmán and Ecker, 1990). It is tempting to speculate
that in Lotus EIN2 might have acquired a specific function in
termination of the determinate nodule meristem, where the pres-
ence of multiple EIN2 copies would have an impact. Deregulated
repression of the additional EIN2 copies might lead to fewer
nodules and explain the opposing phenotypes of enigma
(Gresshoff et al., 2009) and Medicago skl mutants (Penmetsa
et al., 2008). Curiously, the two F-box proteins ETP1 and ETP2
coding genes, which regulate the abundance of EIN2 by a post
translational mechanism in Arabidopsis (Qiao et al., 2009), are
either missing or too diverged to be detected by bioinformatics
in Lotus (Figure 6), soybean, and Medicago. The EIN2 node
may therefore be regulated differently in legumes.
There are also marked differences for the transcriptional regu-
lators EIN3 and EIN3-like coding genes (Binder et al., 2007),
which seem to have a narrower genetic basis in Lotus. This
would suggest additional functions compared to the Arabidopsis
regulators. One of the targets of EIN3 is the regulation of the
AP2-type transcription factor ERF1 that in turn regulates expres-
sion of ethylene downstream responsive genes (Solano et al.,
1998). We can identify one homolog of ERF1 in Lotus (Figure 6).
Finally, regulation of the ethylene-signaling pathway in Lotus
appears similar to that in Arabidopsis. We have identified
a homolog for each of the two F-box proteins EBF1 and EBF2
that negatively regulates the EIN3/EIL1 polypeptide (Potuschak
et al., 2003). We have also found homologous copies of the
XRN4 ribonuclease that posttranscriptionally regulates EBF1
and EBF2 transcripts (Potuschak et al., 2006) (Figure 5).
A substantial number of gene expression experiments in
legumes provide insight on transcriptional activity and transcrip-
tome databases are accessible (Benedito et al., 2008; Severin
et al., 2010). Taking advantage of the Lotus transcriptome data-
base (Høgslund et al., 2009), the transcript level of the genes
involved in ethylene synthesis, perception signaling, and regula-
tion in uninoculated roots can be compared to transcript levels in
roots inoculated for 1 day, 3 days, 7 days, and 21 days. Overall,
the transcript levels suggest differential gene regulation possibly
reflecting the role of ethylene at different stages of symbiosis
development. One example of a testable prediction is the poten-
tial activation of ethylene biosynthesis and signaling pathway

Figure 6. Alignment of the Lotus and

Arabidopsis Ethylene Pathways
The ethylene pathway defined by mining the
genome of Lotus is very similar to the Arabidopsis
pathway (reviewed in Stepanova and Alonso,
2009). Positive action is represented by green
arrows, while negative action is shown with red
arrows. The names of genes present in both plants
are in black, while genes absent in Lotus are in
gray. For clarity, the ACS gene family is described
according to their three subtypes.

354 Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
Review
1 day after inoculation. Enhanced transcript levels of LjACS7b,
LjETR2, LjERS1, LjEBF2, and LjERF1 may suggest an activation
of ethylene biosynthesis and signaling at this stage. Interestingly,
a recent functional analysis indicates that LjERF1 has a positive
role during early stages of nodulation that is dependent on the
presence of NFs (Asamizu et al., 2008).
Symbionts and Pathogens Converge on Common
Host-Perception Systems
Endosymbioses with soil fungi represents yet another beneficial
plant-microbe interaction. Both endomycorrhizal fungi and
rhizobia secrete lipochitin-oligosaccharide signal molecules
that are perceived by the plant (Maillet et al., 2011; Radutoiu
et al., 2003). Legumes have apparently developed different
receptors for the mycorrhizal and the rhizobial lipochito-oligo-
saccharide signals. Several lines of evidences show that the
LysM receptor-like kinases like NFR1 and NFR5 are required
for perception of NFs, and a LysM receptor-like kinase has
also been implicated in perception of the mycorrhizal lipochito-
oligosaccharide (Op den Camp et al., 2011). Downstream,
a shared signal transduction pathway encoded by at least eight
genes is then involved in mycorrhizal accommodation or devel-
opment of the nodule organ that distinguish rhizobial symbiois
(Figure 3). An Arabidopsis LysM receptor-like kinase, CERK1,
homologous to the Lotus NFR1, was also shown to be involved
in triggering innate immunity in response to chitin oligomers
from pathogens (Miya et al., 2007). The similarity between the
kinase domains of the NFR1 and CERK is remarkably high,
and domain-swap experiments indicate that only a few amino
acid substitutions are sufficient to confer symbiotic signaling
on the CERK kinase domain (Nakagawa et al., 2011). Pathogens
and symbionts have obviously evolved different mechanisms to
influence plant growth but appear to have converged on the
same type of perception system within the host.
Conclusions and Future Perspectives
Like lateral roots, nodules are inducible lateral root organs. Phe-
notyping of legume mutants like har1, brush, crinkle affecting
both nodulation and root formation (Maekawa-Yoshikawa
et al., 2009; Tansengco et al., 2003; Wopereis et al., 2000) as
well as the hybrid structures occasionally observed in bean or
clover (Ferraioli et al., 2004; Morris and Djordjevic, 2006) suggest
that functions involved in lateral root formation may have been
co-opted for nodule development. Further supporting this
notion, purified NF positively affects the Medicago root architec-
ture by augmenting lateral root emergence (Maillet et al., 2011;
Oláh et al., 2005). However, there are also observations pointing
at the differences. Nodules and lateral root differ in origin and
anatomy. Legume root nodules originate mainly from cortex cells
although pericycle cells are also activated, while lateral roots
originate from pericycle cells. Nodules induced by rhizobia
have a peripheral vasculature while the vasculature of lateral
roots is central. Furthermore, root nodules are not formed at
equal distances on opposite sites like lateral roots of Arabidopsis
(De Smet et al., 2007). Finally, the indeterminate nodule meristem
appears to resemble the dome shaped shoot apical meristem,
and there is currently no evidence for a quiescent stem cells
niche and nodule cells appear not to be deposited in cell files
like root cells (Figures 1 and 2). There is an obvious conservation
of anatomy and developmental overlap between the main root
and lateral roots but such ontogeny is not equally obvious for
nodules. To some extent the cellular structure of nodules points
more in the direction of the shoot for similarities. This suggests
we should keep an open mind in relation to the source of genes
recruited for nodulation.
Despite these differences, lateral root formation and nodule
organogenesis result from meristem activity initiated by dediffer-
entiation of cells located next to the vascular system (pericycle;
inner/outer cortex). Recruitment of genes would predict an over-
lap in the genetically defined pathways governing lateral root
formation and nodulation. So far, the genetic analysis of the
pathways controlling initiation of nodule development (Figure 3)
and initiation of lateral root formation (Figure 4) has not produced
an obvious overlap. The smoking gun is missing, which may indi-
cate that the two genetic programs governing the early stages of
development differ. At later stages the three phytohormones,
auxin, cytokinin, and ethylene, are major regulators of both
lateral root and nodule development. Again, the genetics of
nodulation show a surprising paucity of mutants in the hormonal
pathways, but gene redundancy and neofunctionalization may
be limiting the genetic analysis. The sequencing and annotation
of legume genomes together with the corresponding transcript
databases are therefore likely sources for predicting at least
some of the underlying molecular mechanisms and for designing
informative experiments.
Regarding ethylene, three key points will be important to
address. First, is ethylene repression dependent on the presence
of cells posed for division or an active meristem and therefore
different in Lotus (forming determinate nodules) and Medicago
(indeterminate nodules)? Analysis of the respective plant
genomes identifies similar gene sets but there may be important
differences for example in gene copy numbers. Second,
ethylene is also a stress hormone affecting wound responses
and defense against pathogens. One of the challenges is to
determine how much of the ethylene response is specific to
rhizobial infection compared to the response to pathogens.
This borderline between the symbiotic response and the defense
response is virtually undescribed and is one of the challenges for
the future. Finally, the ethylene signaling pathway is interacting
with the auxin pathway, which itself is interacting with the cyto-
kinin pathway. This high level of interconnectivity will be a major
challenge that reverse-genetics tools will be important to help
overcome.
Both nodule and lateral root development is under the regula-
tion of auxin, cytokinin, and ethylene. In root, auxin is keeping
meristematic cells dividing and forming a bidirectional gradient
of concentration. Cytokinin contributes to the differentiation of
the root cells especially the vasculature, suggesting that they
are accumulated in and around the vascular system. In close
interaction with auxin, cytokinin defines the size/length of the
meristem. Ethylene is regulating the QC and mostly controlling
root hair elongation together with auxin. It is tempting to specu-
late that this spatial distribution provides cues for regulation of
the inner root (cytokinin and auxin) and the outer root (auxin
and ethylene). The polarized transport of auxin could contribute
to the coordination of the development of the inner and the outer
cell layers. Could the same cues be operating in nodules?
Formation of the nodule primordium is resulting from an as yet
Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc. 355

unclear interaction between auxin and cytokinin, while ethylene
is mostly acting on the epidermis controlling the infection
processes. Pushing that model to its limit would suggest that
an auxin gradient between the epidermis toward the cortex
would contribute to synchronize epidermal and cortical events
during the development of a nodule primordium. Local accumu-
lation of auxin in the first dividing cells of Lotus nodules, detected
by a GH3 reporter construct, is consistent with this notion
(Pacios-Bras et al., 2003), but immunolocalization of individual
PIN transporters after spot inoculation is needed for a detailed
description of the changes in auxin flow associated with recog-
nition of rhizobia/NFs and activation of the nodulation path-
way(s). A subsequent molecular dialog with the mechanisms
that positions lateral roots founder cells (prebranch sites) is
a tantalizing possibility. Recent work in Arabidopsis suggests
that prebranching depends on cyclic gene expression and that
the resulting oscillation occurs in the meristematic zone. Further-
more, it was shown that positioning of these prebranch sites
could be shifted by application of auxin (Moreno-Risueno
et al., 2010). Although there are circumstantial observations (Lar-
kin et al., 1996), this mechanism for determining prebranching
sites has not yet been demonstrated in legumes. A molecular
dialog with the nodule induction mechanism would nicely
account for the positioning of nodules in time and space. Activa-
tion of predetermined sites of competence would also explain
the development of temporal and spatially separated sponta-
neous nodules in the snf2 mutants (Tirichine et al., 2007) and
the distinct primordia formation after cytokinin application to
whole Lotus roots (Heckmann et al., 2011).
While the initial cellular events and genetic programs between
lateral roots and a nodules are different, the regulatory roles of
the main hormones involved may be shared or overlap, offering
a possible explanation for how invading microorganisms can
influence host development.
ACKNOWLEDGMENTS
G.D. was supported by the Aarhus University Foundation and the Conseil
National des Universités CNU-66. The Danish National Research Foundation
funded this work. The authors would like to thanks Anne Lau-Heckman, Kath-
arina Markmann, Simona Radutoiu, and Stig Uggerhøj Andersen for careful
reading of the manuscript and a wealth of constructive comments.
REFERENCES
Asamizu, E., Shimoda, Y., Kouchi, H., Tabata, S., and Sato, S. (2008). A posi-
tive regulatory role for LjERF1 in the nodulation process is revealed by system-
atic analysis of nodule-associated transcription factors of Lotus japonicus.
Plant Physiol. 147, 2030–2040.
Benedito, V.A., Torres-Jerez, I., Murray, J.D., Andriankaja, A., Allen, S., Kakar,
K., Wandrey, M., Verdier, J., Zuber, H., Ott, T., et al. (2008). A gene expression
atlas of the model legume Medicago truncatula. Plant J. 55, 504–513.
Binder, B.M., Walker, J.M., Gagne, J.M., Emborg, T.J., Hemmann, G.,
Bleecker, A.B., and Vierstra, R.D. (2007). The Arabidopsis EIN3 binding F-
Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene
signaling. Plant Cell 19, 509–523.
Camerini, S., Senatore, B., Lonardo, E., Imperlini, E., Bianco, C., Moschetti, G.,
Rotino, G.L., Campion, B., and Defez, R. (2008). Introduction of a novel
pathway for IAA biosynthesis to rhizobia alters vetch root nodule development.
Arch. Microbiol. 190, 67–77.
Cao, X.F., Linstead, P., Berger, F., Kieber, J., and Dolan, L. (1999). Differential
ethylene sensitivity of epidermal cells is involved in the establishment of cell
pattern in the Arabidopsis root. Physiol. Plant. 106, 311–317.
356 Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
Review
Christians, M.J., Gingerich, D.J., Hansen, M., Binder, B.M., Kieber, J.J., and
Vierstra, R.D. (2009). The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act
collectively to regulate ethylene biosynthesis in Arabidopsis by controlling
type-2 ACC synthase levels. Plant J. 57, 332–345.
Cooper, J.B., and Long, S.R. (1994). Morphogenetic rescue of Rhizobium
meliloti nodulation mutants by trans-zeatin secretion. Plant Cell 6, 215–225.
de Billy, F., Grosjean, C., May, S., Bennett, M., and Cullimore, J.V. (2001).
Expression studies on AUX1-like genes in Medicago truncatula suggest that
auxin is required at two steps in early nodule development. Mol. Plant Microbe
Interact. 14, 267–277.
De Smet, I., Tetsumura, T., De Rybel, B., Frey, N.F., Laplaze, L., Casimiro, I.,
Swarup, R., Naudts, M., Vanneste, S., Audenaert, D., et al. (2007). Auxin-
dependent regulation of lateral root positioning in the basal meristem of
Arabidopsis. Development 134, 681–690.
De Smet, I., Vassileva, V., De Rybel, B., Levesque, M.P., Grunewald, W., Van
Damme, D., Van Noorden, G., Naudts, M., Van Isterdael, G., De Clercq, R.,
et al. (2008). Receptor-like kinase ACR4 restricts formative cell divisions in
the Arabidopsis root. Science 322, 594–597.
Dehio, C., and de Bruijn, F.J. (1992). The early nodulin gene SrEnod2 from
Sesbania rostrata is inducible by cytokinin. Plant J. 2, 117–128.
Dello Ioio, R., Nakamura, K., Moubayidin, L., Perilli, S., Taniguchi, M., Morita,
M.T., Aoyama, T., Costantino, P., and Sabatini, S. (2008). A genetic framework
for the control of cell division and differentiation in the root meristem. Science
322, 1380–1384.
Desnos, T. (2008). Root branching responses to phosphate and nitrate. Curr.
Opin. Plant Biol. 11, 82–87.
Ding, Y., and Oldroyd, G.E. (2009). Positioning the nodule, the hormone
dictum. Plant Signal. Behav. 4, 89–93.
Dolan, L., and Costa, S. (2001). Evolution and genetics of root hair stripes in the
root epidermis. J. Exp. Bot. 52 (Spec Issue), 413–417.
Dubrovsky, J.G., Sauer, M., Napsucialy-Mendivil, S., Ivanchenko, M.G., Friml,
J., Shishkova, S., Celenza, J., and Benková, E. (2008). Auxin acts as a local
morphogenetic trigger to specify lateral root founder cells. Proc. Natl. Acad.
Sci. USA 105, 8790–8794.
Ehrhardt, D.W., Wais, R., and Long, S.R. (1996). Calcium spiking in plant root
hairs responding to Rhizobium nodulation signals. Cell 85, 673–681.
Ferraioli, S., Tatè, R., Rogato, A., Chiurazzi, M., and Patriarca, E.J. (2004).
Development of ectopic roots from abortive nodule primordia. Mol. Plant
Microbe Interact. 17, 1043–1050.
Gherbi, H., Markmann, K., Svistoonoff, S., Estevan, J., Autran, D., Giczey, G.,
Auguy, F., Péret, B., Laplaze, L., Franche, C., et al. (2008). SymRK defines
a common genetic basis for plant root endosymbioses with arbuscular mycor-
rhiza fungi, rhizobia, and Frankiabacteria. Proc. Natl. Acad. Sci. USA 105,
4928–4932.
Giraud, E., Moulin, L., Vallenet, D., Barbe, V., Cytryn, E., Avarre, J.C., Jaubert,
M., Simon, D., Cartieaux, F., Prin, Y., et al. (2007). Legumes symbioses:
absence of Nod genes in photosynthetic bradyrhizobia. Science 316, 1307–
1312.
Gleason, C., Chaudhuri, S., Yang, T., Muñoz, A., Poovaiah, B.W., and Oldroyd,
G.E. (2006). Nodulation independent of rhizobia induced by a calcium-acti-
vated kinase lacking autoinhibition. Nature 441, 1149–1152.
Glick, B.R. (2005). Modulation of plant ethylene levels by the bacterial enzyme
ACC deaminase. FEMS Microbiol. Lett. 251, 1–7.
Gresshoff, P.M., Lohar, D., Chan, P.K., Biswas, B., Jiang, Q., Reid, D., Fergu-
son, B., and Stacey, G. (2009). Genetic analysis of ethylene regulation of
legume nodulation. Plant Signal. Behav. 4, 818–823.
Groth, M., Takeda, N., Perry, J., Uchida, H., Dräxl, S., Brachmann, A., Sato, S.,
Tabata, S., Kawaguchi, M., Wang, T.L., and Parniske, M. (2010). NENA, a Lotus
japonicus homolog of Sec13, is required for rhizodermal infection by arbuscu-
lar mycorrhiza fungi and rhizobia but dispensable for cortical endosymbiotic
development. Plant Cell 22, 2509–2526.
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
Cell Host & Microbe
Review
the roles of ethylene in the establishment of these two symbioses. Can. J.
Microbiol. 80, 695–720.
Guzmán, P., and Ecker, J.R. (1990). Exploiting the triple response of Arabidop-
sis to identify ethylene-related mutants. Plant Cell 2, 513–523.
Heckmann, A.B., Lombardo, F., Miwa, H., Perry, J.A., Bunnewell, S., Parniske,
M., Wang, T.L., and Downie, J.A. (2006). Lotus japonicus nodulation requires
two GRAS domain regulators, one of which is functionally conserved in
a non-legume. Plant Physiol. 142, 1739–1750.
Heckmann, A.B., Sandal, N., Bek, A.S., Madsen, L.H., Jurkiewicz, A., Nielsen,
M.W., Tirichine, L., and Stougaard, J. (2011). Cytokinin induction of root nodule
primordia in Lotus japonicus is regulated by a mechanism operating in the root
cortex. Mol. Plant Microbe Interact., in press. Published online July 19, 2011.
Heidstra, R., Yang, W.C., Yalcin, Y., Peck, S., Emons, A.M., van Kammen, 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.
Held, M., Hossain, M.S., Yokota, K., Bonfante, P., Stougaard, J., and Szczy-
glowski, K. (2010). Common and not so common symbiotic entry. Trends Plant
Sci. 15, 540–545.
Hirose, N., Takei, K., Kuroha, T., Kamada-Nobusada, T., Hayashi, H., and
Sakakibara, H. (2008). Regulation of cytokinin biosynthesis, compartmentali-
zation and translocation. J. Exp. Bot. 59, 75–83.
Hirsch, A.M., Bhuvaneswari, T.V., Torrey, J.G., and Bisseling, T. (1989). Early
nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport
inhibitors. Proc. Natl. Acad. Sci. USA 86, 1244–1248.
Hirsch, S., Kim, J., Muñoz, A., Heckmann, A.B., Downie, J.A., and Oldroyd,
G.E. (2009). GRAS proteins form a DNA binding complex to induce gene
expression during nodulation signaling in Medicago truncatula. Plant Cell 21,
545–557.
Høgslund, N., Radutoiu, S., Krusell, L., Voroshilova, V., Hannah, M.A., Goffard,
N., Sanchez, D.H., Lippold, F., Ott, T., Sato, S., et al. (2009). Dissection of
symbiosis and organ development by integrated transcriptome analysis of
lotus japonicus mutant and wild-type plants. PLoS ONE 4, e6556.
Hunter, W.J. (1993). Ethylene production by root nodules and effect of ethylene
on nodulation in Glycine max. Appl. Environ. Microbiol. 59, 1947–1950.
Ikeda, Y., Men, S., Fischer, U., Stepanova, A.N., Alonso, J.M., Ljung, K., and
Grebe, M. (2009). Local auxin biosynthesis modulates gradient-directed planar
polarity in Arabidopsis. Nat. Cell Biol. 11, 731–738.
Karas, B., Murray, J., Gorzelak, M., Smith, A., Sato, S., Tabata, S., and
Szczyglowski, K. (2005). Invasion of Lotus japonicus root hairless 1 by
Mesorhizobium loti involves the nodulation factor-dependent induction of
root hairs. Plant Physiol. 137, 1331–1344.
Larkin, P.J., Gibson, J.M., Mathesius, U., Weinman, J.J., Gartner, E., Hall, E.,
Tanner, G.J., Rolfe, B.G., and Djordjevic, M.A. (1996). Transgenic white clover.
Studies with the auxin-responsive promoter, GH3, in root gravitropism and
lateral root development. Transgenic Res. 5, 325–335.
Lee, K.H., and Larue, T.A. (1992). Ethylene as a possible mediator of light- and
nitrate-induced inhibition of nodulation of Pisum sativum L. cv Sparkle. Plant
Physiol. 100, 1334–1338.
Lievens, S., Goormachtig, S., Den Herder, J., Capoen, W., Mathis, R., Hedden,
P., and Holsters, M. (2005). Gibberellins are involved in nodulation of Sesbania
rostrata. Plant Physiol. 139, 1366–1379.
Lohar, D., Stiller, J., Kam, J., Stacey, G., and Gresshoff, P.M. (2009). Ethylene
insensitivity conferred by a mutated Arabidopsis ethylene receptor gene alters
nodulation in transgenic Lotus japonicus. Ann. Bot. (Lond.) 104, 277–285.
Lorteau, M.A., Ferguson, B.J., and Guinel, F.C. (2001). Effects of cytokinin on
ethylene production and nodulation in pea (Pisum sativum) cv. Sparkle. Phys-
iol. Plant. 112, 421–428.
Ma, W., Guinel, F.C., and Glick, B.R. (2003). Rhizobium leguminosarum biovar
viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation
of pea plants. Appl. Environ. Microbiol. 69, 4396–4402.
Madsen, L.H., Tirichine, L., Jurkiewicz, A., Sullivan, J.T., Heckmann, A.B., Bek,
A.S., Ronson, C.W., James, E.K., and Stougaard, J. (2010). The molecular
network governing nodule organogenesis and infection in the model legume
Lotus japonicus. Nat Commun 1, 10.
Madsen, E.B., Antolı́n-Llovera, M., Grossmann, C., Ye, J., Vieweg, S., Brog-
hammer, A., Krusell, L., Radutoiu, S., Jensen, O.N., Stougaard, J., and Par-
niske, M. (2011). Autophosphorylation is essential for the in vivo function of
the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling
in cooperation with Nod factor receptor 5. Plant J. 65, 404–417.
Maekawa-Yoshikawa, M., Müller, J., Takeda, N., Maekawa, T., Sato, S., Ta-
bata, S., Perry, J., Wang, T.L., Groth, M., Brachmann, A., and Parniske, M.
(2009). The temperature-sensitive brush mutant of the legume Lotus japonicus
reveals a link between root development and nodule infection by rhizobia.
Plant Physiol. 149, 1785–1796.
Magori, S., Oka-Kira, E., Shibata, S., Umehara, Y., Kouchi, H., Hase, Y., Ta-
naka, A., Sato, S., Tabata, S., and Kawaguchi, M. (2009). Too much love,
a root regulator associated with the long-distance control of nodulation in
Lotus japonicus. Mol. Plant Microbe Interact. 22, 259–268.
Maillet, F., Poinsot, V., André, O., Puech-Pagès, V., Haouy, A., Gueunier, M.,
Cromer, L., Giraudet, D., Formey, D., Niebel, A., et al. (2011). Fungal lipochi-
tooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469,
58–63.
Markmann, K., and Parniske, M. (2009). Evolution of root endosymbiosis with
bacteria: How novel are nodules? Trends Plant Sci. 14, 77–86.
Mathesius, U., Schlaman, H.R., Spaink, H.P., Of Sautter, C., Rolfe, B.G., and
Djordjevic, M.A. (1998). Auxin transport inhibition precedes root nodule forma-
tion in white clover roots and is regulated by flavonoids and derivatives of chitin
oligosaccharides. Plant J. 14, 23–34.
Miya, A., Albert, P., Shinya, T., Desaki, Y., Ichimura, K., Shirasu, K., Narusaka,
Y., Kawakami, N., Kaku, H., and Shibuya, N. (2007). CERK1, a LysM receptor
kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc. Natl. Acad.
Sci. USA 104, 19613–19618.
Moreno-Risueno, M.A., Van Norman, J.M., Moreno, A., Zhang, J., Ahnert, S.E.,
and Benfey, P.N. (2010). Oscillating gene expression determines competence
for periodic Arabidopsis root branching. Science 329, 1306–1311.
Morris, A.C., and Djordjevic, M.A. (2006). The Rhizobium leguminosarum bio-
var trifolii ANU794 induces novel developmental responses on the subterra-
nean clover cultivar Woogenellup. Mol. Plant Microbe Interact. 19, 471–479.
Murray, J.D., Karas, B.J., Sato, S., Tabata, S., Amyot, L., and Szczyglowski, K.
(2007). A cytokinin perception mutant colonized by Rhizobium in the absence
of nodule organogenesis. Science 315, 101–104.
Nakagawa, T., Kaku, H., Shimoda, Y., Sugiyama, A., Shimamura, M., Takana-
shi, K., Yazaki, K., Aoki, T., Shibuya, N., and Kouchi, H. (2011). From defense to
symbiosis: limited alterations in the kinase domain of LysM receptor-like
kinases are crucial for evolution of legume-Rhizobium symbiosis. Plant J.
65, 169–180.
Nukui, N., Ezura, H., and Minamisawa, K. (2004). Transgenic Lotus japonicus
with an ethylene receptor gene Cm-ERS1/H70A enhances formation of infec-
tion threads and nodule primordia. Plant Cell Physiol. 45, 427–435.
Okazaki, S., Hattori, Y., and Saeki, K. (2007). The Mesorhizobium loti purB
gene is involved in infection thread formation and nodule development in Lotus
japonicus. J. Bacteriol. 189, 8347–8352.
Oláh, B., Brière, C., Bécard, G., Dénarié, J., and Gough, C. (2005). Nod factors
and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root
formation in Medicago truncatula via the DMI1/DMI2 signalling pathway. Plant
J. 44, 195–207.
Oldroyd, G.E., Engstrom, E.M., and Long, S.R. (2001). Ethylene inhibits the
Nod factor signal transduction pathway of Medicago truncatula. Plant Cell
13, 1835–1849.
Op den Camp, R., Streng, A., De Mita, S., Cao, Q., Polone, E., Liu, W., Ammir-
aju, J.S., Kudrna, D., Wing, R., Untergasser, A., et al. (2011). LysM-type mycor-
rhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia.
Science 331, 909–912.
Ortega-Martı́nez, O., Pernas, M., Carol, R.J., and Dolan, L. (2007). Ethylene
modulates stem cell division in the Arabidopsis thaliana root. Science 317,
507–510.
Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc. 357

Overvoorde, P., Fukaki, H., and Beeckman, T. (2010). Auxin control of root
development. Cold Spring Harb Perspect Biol 2, a001537.
Pacios-Bras, C., Schlaman, H.R., Boot, K., Admiraal, P., Langerak, J.M., Stou-
gaard, J., and Spaink, H.P. (2003). Auxin distribution in Lotus japonicus during
root nodule development. Plant Mol. Biol. 52, 1169–1180.
Parniske, M. (2008). Arbuscular mycorrhiza: the mother of plant root endosym-
bioses. Nat. Rev. Microbiol. 6, 763–775.
Peng, H.P., Lin, T.Y., Wang, N.N., and Shih, M.C. (2005). Differential expres-
sion of genes encoding 1-aminocyclopropane-1-carboxylate synthase in
Arabidopsis during hypoxia. Plant Mol. Biol. 58, 15–25.
Penmetsa, R.V., Uribe, P., Anderson, J., Lichtenzveig, J., Gish, J.C., Nam,
Y.W., Engstrom, E., Xu, K., Sckisel, G., Pereira, M., et al. (2008). The Medicago
truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of
symbiotic and pathogenic microbial associations. Plant J. 55, 580–595.
Perilli, S., Moubayidin, L., and Sabatini, S. (2010). The molecular basis of cyto-
kinin function. Curr. Opin. Plant Biol. 13, 21–26.
Perrine-Walker, F., Gherbi, H., Imanishi, L., Hocher, V., Ghodhbane-Gtari, F.,
Lavenus, J., Benabdoun, F.M., Nambiar-Veeti, M., Svistoonoff, S., and Lap-
laze, L. (2011). Symbiotic signaling in actinorhizal symbioses. Curr. Protein
Pept. Sci. 12, 156–164.
Plet, J., Wasson, A., Ariel, F., Le Signor, C., Baker, D., Mathesius, U., Crespi,
M., and Frugier, F. (2011). MtCRE1-dependent cytokinin signaling integrates
bacterial and plant cues to coordinate symbiotic nodule organogenesis in
Medicago truncatula. Plant J. 65, 622–633.
Potuschak, T., Lechner, E., Parmentier, Y., Yanagisawa, S., Grava, S., Koncz,
C., and Genschik, P. (2003). EIN3-dependent regulation of plant ethylene
hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2. Cell
115, 679–689.
Potuschak, T., Vansiri, A., Binder, B.M., Lechner, E., Vierstra, R.D., and Gens-
chik, P. (2006). The exoribonuclease XRN4 is a component of the ethylene
response pathway in Arabidopsis. Plant Cell 18, 3047–3057.
Prasad, M.E., Schofield, A., Lyzenga, W., Liu, H., and Stone, S.L. (2010).
Arabidopsis RING E3 ligase XBAT32 regulates lateral root production through
its role in ethylene biosynthesis. Plant Physiol. 153, 1587–1596.
Qiao, H., Chang, K.N., Yazaki, J., and Ecker, J.R. (2009). Interplay between
ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers
ethylene responses in Arabidopsis. Genes Dev. 23, 512–521.
Radutoiu, S., Madsen, L.H., Madsen, E.B., Felle, H.H., Umehara, Y., Grønlund,
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.
Radutoiu, S., Madsen, L.H., Madsen, E.B., Jurkiewicz, A., Fukai, E., Quist-
gaard, E.M., Albrektsen, A.S., James, E.K., Thirup, S., and Stougaard, J.
(2007). LysM domains mediate lipochitin-oligosaccharide recognition and
Nfr genes extend the symbiotic host range. EMBO J. 26, 3923–3935.
Sato, S., Nakamura, Y., Kaneko, T., Asamizu, E., Kato, T., Nakao, M., Sasa-
moto, S., Watanabe, A., Ono, A., Kawashima, K., et al. (2008). Genome struc-
ture of the legume, Lotus japonicus. DNA Res. 15, 227–239.
Scheres, B., Benfey, P., and Dolan, L. (2002). Root Development. In The Ara-
bidopsis Book, C.R. Somerville and E.M. Meyerowitz, eds. (Rockville, MD:
American Society of Plant Biologists), p. e0101.
Schmidt, J.S., Harper, J.E., Hoffman, T.K., and Bent, A.F. (1999). Regulation of
soybean nodulation independent of ethylene signaling. Plant Physiol. 119,
951–960.
Schmutz, J., Cannon, S.B., Schlueter, J., Ma, J., Mitros, T., Nelson, W., Hyten,
D.L., Song, Q., Thelen, J.J., Cheng, J., et al. (2010). Genome sequence of the
palaeopolyploid soybean. Nature 463, 178–183.
358 Cell Host & Microbe 10, October 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
Review
Severin, A.J., Woody, J.L., Bolon, Y.T., Joseph, B., Diers, B.W., Farmer, A.D.,
Muehlbauer, G.J., Nelson, R.T., Grant, D., Specht, J.E., et al. (2010). RNA-Seq
Atlas of Glycine max: a guide to the soybean transcriptome. BMC Plant Biol.
10, 160.
Smit, G., de Koster, C.C., Schripsema, J., Spaink, H.P., van Brussel, A.A., and
Kijne, J.W. (1995). Uridine, a cell division factor in pea roots. Plant Mol. Biol. 29,
869–873.
Solano, R., Stepanova, A., Chao, Q., and Ecker, J.R. (1998). Nuclear events
in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-
INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev. 12,
3703–3714.
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.
Stepanova, A.N., and Alonso, J.M. (2009). Ethylene signaling and response:
where different regulatory modules meet. Curr. Opin. Plant Biol. 12, 548–555.
Stepanova, A.N., Yun, J., Likhacheva, A.V., and Alonso, J.M. (2007). Multilevel
interactions between ethylene and auxin in Arabidopsis roots. Plant Cell 19,
2169–2185.
Subramanian, S., Stacey, G., and Yu, O. (2006). Endogenous isoflavones
are essential for the establishment of symbiosis between soybean and
Bradyrhizobium japonicum. Plant J. 48, 261–273.
Tanimoto, M., Roberts, K., and Dolan, L. (1995). Ethylene is a positive regulator
of root hair development in Arabidopsis thaliana. Plant J. 8, 943–948.
Tansengco, M.L., Hayashi, M., Kawaguchi, M., Imaizumi-Anraku, H., and Mur-
ooka, 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.
Tirichine, L., Imaizumi-Anraku, H., Yoshida, S., Murakami, Y., Madsen, L.H.,
Miwa, H., Nakagawa, T., Sandal, N., Albrektsen, A.S., Kawaguchi, M., et al.
(2006). Deregulation of a Ca2+/calmodulin-dependent kinase leads to sponta-
neous nodule development. Nature 441, 1153–1156.
Tirichine, L., Sandal, N., Madsen, L.H., Radutoiu, S., Albrektsen, A.S., Sato, S.,
Asamizu, E., Tabata, S., and Stougaard, J. (2007). A gain-of-function mutation
in a cytokinin receptor triggers spontaneous root nodule organogenesis.
Science 315, 104–107.
Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S., and
Theologis, A. (2009). A combinatorial interplay among the 1-aminocyclopro-
pane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis
thaliana. Genetics 183, 979–1003.
Uchiumi, T., Ohwada, T., Itakura, M., Mitsui, H., Nukui, N., Dawadi, P., Kaneko,
T., Tabata, S., Yokoyama, T., Tejima, K., et al. (2004). Expression islands clus-
tered on the symbiosis island of the Mesorhizobium loti genome. J. Bacteriol.
186, 2439–2448.
Vogel, J.P., Woeste, K.E., Theologis, A., and Kieber, J.J. (1998). Recessive and
dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis
confer cytokinin insensitivity and ethylene overproduction, respectively.
Proc. Natl. Acad. Sci. USA 95, 4766–4771.
Wasson, A.P., Pellerone, F.I., and Mathesius, U. (2006). Silencing the flavonoid
pathway in Medicago truncatula inhibits root nodule formation and prevents
auxin transport regulation by rhizobia. Plant Cell 18, 1617–1629.
Wopereis, J., Pajuelo, E., Dazzo, F.B., Jiang, Q., Gresshoff, P.M., De Bruijn,
F.J., Stougaard, J., and Szczyglowski, K. (2000). Short root mutant of Lotus
japonicus with a dramatically altered symbiotic phenotype. Plant J. 23,
97–114.