International Review of Cell and Molecular Biology) Volume 316 - Leguminous Plant

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Leguminous Plants: Inventors of

Root Nodules to Accommodate
Symbiotic Bacteria
Takuya Suzaki1, 2, *, a, Emiko Yoro1, 2, a and
Masayoshi Kawaguchi1, 2, *
1
National Institute for Basic Biology, Okazaki, Japan
2
School of Life Science, Graduate University for Advanced Studies, Okazaki, Japan
*Corresponding authors: E-mail: tsuzaki@nibb.ac.jp; masayosi@nibb.ac.jp
Contents
1. Introduction 2
2. Nodule Developmental Process 3
2.1 Morphology of Nodules 3
2.2 Positive Regulation of Nodulation 4
2.2.1 A signaling pathway to induce nodulation 4
2.2.2 Cytokinin receptors 5
2.2.3 Downstream of cytokinin receptors 6
2.2.4 DNA topoisomerase VI 7
2.3 Negative Regulation of Nodulation: Autoregulation of Nodulation (AON) 10
2.3.1 Concept of AON 10
2.3.2 Molecular basis of AON 11
3. Rhizobial Invasion Process 16
3.1 Initial Responses and Root Hair Deformation 16
3.2 IT Initiation and IT Membrane Characterization 18
3.3 IT Growth and Elongation 29
3.4 Cytoplasmic Bridge or Preinfection Thread (PIT) in Cortex 30
3.5 Bacterial Release 30
3.6 Cross Talk between Rhizobial Infection and Nodule Development Pathways 31
3.7 Regulatory Mechanisms for Rhizobial Infection 32
3.8 Intercellular Rhizobial Invasion 33
4. Concluding Remarks and Future Perspectives 34
Acknowledgments 35
References 35
a
These authors contributed equally to this work.
International Review of Cell and Molecular Biology, Volume 316
2015 Elsevier Inc.
j
ISSN: 1937-6448
http://dx.doi.org/10.1016/bs.ircmb.2015.01.004 All rights reserved. 1
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2 Takuya Suzaki et al.
Abstract
Legumes and a few other plant species can establish a symbiotic relationship with
nitrogen-xing rhizobia, which enables them to survive in a nitrogen-decient environ-
ment. During the course of nodulation, infection with rhizobia induces the dedifferen-
tiation of host cells to form primordia of a symbiotic organ, the nodule, which prepares
plants to accommodate rhizobia in host cells. While these nodulation processes are
known to be genetically controlled by both plants and rhizobia, recent advances in
studies on two model legumes, Lotus japonicus and Medicago truncatula, have pro-
vided great insight into the underlying plant-side molecular mechanism. In this chapter,
we review such knowledge, with particular emphasis on two key processes of nodula-
tion, nodule development and rhizobial invasion.
1. INTRODUCTION
Root nodule symbiosis is a mutualistic interaction observed between
mainly leguminous plants and nitrogen-xing soil rhizobia, in which plants
can obtain xed atmospheric nitrogen, and provide rhizobia with photosyn-
thate as a carbon source. Although root nodule symbiosis is accomplished by
successive regulatory processes that are controlled by plants and rhizobia,
formation of a symbiotic organ, the nodule, is obviously the key event for
establishment of the symbiosis. During nodulation, rhizobial infection resets
and alters the fate of differentiated root cortical cells, and the activated
cortical cells start to divide to form nodule primordia. At the initial stage
of nodulation, host plants precisely recognize their symbiotic partners and
allow them to invade root tissue. The rhizobial invasion process starts
from the tip of the root hair, and rhizobia then invade dividing cortical cells
through a plant-derived specialized intracellular tube-like structure, termed
the infection thread (IT). Successful rhizobial invasion is indispensable for
further nodule development. Traditional histological observation or
biochemical approaches to analyze nodule development and rhizobial infec-
tion have been performed with many leguminous plants including Medicago
sativa, Pisum sativum, Vicia species, Astragalus sinicus, and Trifolium repens. Mu-
tants and genes involved in a number of nodulation regulatory processes
have been identied by both forward and reverse genetic approaches in
two model legumes, Lotus japonicus and Medicago truncatula. Although the
two plants have differences in the morphology of their nodules (see Section
2.1), many studies have indicated that there is basically no major difference
between the two plants in terms of the molecular mechanisms involved in
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Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria 3
root nodule symbiosis. Thus, in this review, we summarize past and recent
studies mainly from these two legumes. We rst focus on the nodule devel-
opmental process and then shift to the rhizobial invasion process.
2. NODULE DEVELOPMENTAL PROCESS

2.1 Morphology of Nodules
Nodules are basically categorized as two major and morphologically
distinct types, indeterminate and determinate nodules (Figure 1; Brewin,
1991; Ferguson et al., 2010). In indeterminate nodules formed in such plants
as M. truncatula and P. sativum, denition of nodule zones is well established:
there are the nodule meristem, the rhizobial infection zone, and the nitrogen
xation zone from the apical to basal parts of the nodules (Figure 1A). The
activity of the nodule meristem enables the semipermanent growth of the
nodule. In the infection zone, rhizobia start to invade host cells and
rhizobia-colonized cells constitute a nitrogen xation zone. It seems that
cells of the infection zone require endoreduplication, a replication process
of the nuclear genome in the absence of cell division, for their differentiation
to rhizobia-colonized cells (Foucher and Kondorosi, 2000; Kondorosi and
Kondorosi, 2004; Vinardell et al., 2003). In determinate nodules, such as
those in L. japonicus and Glycine max, the nodule zone is not clearly
distinguished. Like indeterminate nodules, however, there are enlarged
Figure 1 Nodule morphology. (A) Indeterminate nodule formed in Medicatgo trunca-

tula. (B) Determinate nodule formed in Lotus japonicus. (C) Nodule formed in L. japonicus
vag1 mutants. The size of the nodules formed in vag1 mutants is largely indistinguish-
able from those of the wild-type, but the vag1 nodule has fewer rhizobia-colonized cells
and more rhizobia-infected cells (as yet uncolonized). Cells of epidermis, nodule
meristem, rhizobial infection zone, and nitrogen-xation zone are highlighted in green
(gray in print versions), pink (light gray in print versions), light blue (dark gray in print
versions), and blue (black in print versions), respectively.
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rhizobia-colonized cells in the inner region of the nodule, where symbiotic

nitrogen xation occurs, and relatively small rhizobia-infected (as yet
uncolonized) cells in the surrounding region (Figure 1B). In determinate
nodules, the activity of the nodule meristem appears to cease during nodule
development, although the identity and location of the nodule meristem
remain elusive. Thus, the difference in the activity of the nodule meristem
can determine the ultimate nodule shape. Furthermore, the difference in the
origin of nodule cells might be an additional element affecting morphology
of nodules. During nodule initiation in M. truncatula, rhizobial infection in-
duces cell division of the inner cortex and pericycle, whereas cell division of
the outer cortex seems to be predominantly activated in L. japonicus
(Szczyglowski et al., 1998; Timmers et al., 1999; Xiao et al., 2014). Identi-
cation of factors that regulate the identity and determinacy of nodules will
undoubtedly provide greater insights into the diversication and evolution
of nodule morphology.
2.2 Positive Regulation of Nodulation

2.2.1 A signaling pathway to induce nodulation
In L. japonicus root epidermis, rhizobially produced lipochitin oligosaccha-
rides, called Nod factors, directly bind to the LysM receptor-like kinases
NOD FACTOR RECEPTOR 1 (NFR1) and NFR5 (Broghammer
et al., 2012; Madsen et al., 2003; Radutoiu et al., 2003), which leads to
the activation of a downstream epidermal signaling cascade. This signaling
pathway involves SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK),
nucleoporins, and cation channel proteins (CASTOR and POLLUX)
(Groth et al., 2010; Imaizumi-Anraku et al., 2005; Kanamori et al., 2006;
Saito et al., 2007; Stracke et al., 2002). These components of the signaling
cascade seem to be well conserved in M. truncatula, although this plant has
a solo channel protein DOES NOT MAKE INFECTIONS 1 (DMI1), a
putative POLLUX ortholog (Venkateshwaran et al., 2012). The ultimate
output of this signaling pathway is considered to cause transient increases
in cytosolic calcium levels dened as calcium spikes, and it is believed that
CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE
(CCaMK) decodes the calcium signals (Gleason et al., 2006; Lvy et al.,
2004; Miller et al., 2013; Shimoda et al., 2012; Tirichine et al., 2006).
CYCLOPS, a nuclear coiled-coil protein, is a direct phosphorylation target
of CCaMK (Yano et al., 2008). All these proteins other than NFR1 and
NFR5 are additionally involved in the regulation of other plantesoil
microbe systems, such as present in a wide range of symbioses between plants
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and arbuscular mycorrhizal (AM) fungi (Kouchi et al., 2010). As the estab-
lishment of AM symbiosis is estimated to be more ancient than root nodule
symbiosis, root nodule symbiosis might have developed partly through the
co-opting of some genes involved in AM symbiosis (Parniske, 2008). While
recognition of Nod factors by their receptor at the epidermis is crucial for the
induction of the downstream signaling cascade that leads to the initiation of
nodule development, it appears that rhizobia continue producing Nod
factors in host cells after the formation of nodules (Roux et al., 2014; Sharma
and Signer, 1990). Recently, M. truncatula NOD FACTOR PERCEP-
TION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE 3
(LYK3), which are respectively NFR5 and NFR1 orthologs, were shown
to be involved in establishing intracellular infection of rhizobia in nodules,
as described below (see Section 3.5).
Two GRAS-type transcription factors, NODULATION SIGNALING
PATHWAY 1 (NSP1) and NSP2, and an RWP-RK type transcription fac-
tor NODULE INCEPTION (NIN) seem to act downstream of calcium
spikes (Kal o et al., 2005; Kouchi et al., 2010; Murakami et al., 2006;
Schauser et al., 1999; Smit et al., 2005). In plants expressing a gain-of-
function mutation of CCaMK or phosphorylated CYCLOPS, spontaneous
nodules that are morphologically similar to rhizobia-colonized nodules are
formed in the absence of rhizobia (Gleason et al., 2006; Singh et al.,
2014; Tirichine et al., 2006). CYCLOPS acts as a transcriptional activator
and directly binds the NIN promoter (Singh et al., 2014). In addition, NU-
CLEAR FACTOR-Y (NF-Y) subunit genes have been identied as direct
targets of NIN (Soyano et al., 2013). While expression of NIN and NF-Y
genes is induced by rhizobial infection, constitutive expression of either of
these genes seems to be sufcient to induce cortical cell division (Soyano
et al., 2013). Thus, based on these ndings, a hierarchical transcriptional acti-
vation model is proposed (Singh et al., 2014), in which, upon decoding of
calcium spikes, CCaMK phosphorylates CYCLOPS. Phosphorylated
CYCLOPS then directly activates NIN expression, and NIN initiates
cortical cell proliferation through direct activation of NF-Y subunit genes.
Moreover, in indeterminate nodules of M. truncatula, the NF-Y subunit
gene (formerly called HAP2-1) is expressed in nodule meristems and is
needed for further nodule development (Combier et al., 2006).
2.2.2 Cytokinin receptors

Two phytohormones, cytokinin and auxin, have pivotal roles in the regu-
lation of cell proliferation and differentiation. Genetic analyses of cytokinin
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receptors, L. japonicus LOTUS HISTIDINE KINASE 1 (LHK1) and M.

truncatula CYTOKININ RESPONSE 1 (MtCRE1), have detailed the
role of cytokinin signaling in the regulation of nodule organogenesis
(Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007).
The two histidine kinases belong to a single clade that also contains Arabidop-
sis HISTIDINE KINASE 4. In L. japonicus, loss-of-function mutation in
LHK1 causes a signicant reduction in the number of nodules, but does
not completely suppress nodulation (Murray et al., 2007). Recently, it
was shown that two other cytokinin receptors, designated LHK1A and
LHK3, have redundant function with LHK1 in the regulation of nodule
organogenesis (Held et al., 2014): in lhk1 lhk1a lhk3 triple mutants, nodula-
tion is abolished. In addition, spontaneous nodulation is induced in sponta-
neous nodule formation 2 (snf2) mutants, in which cytokinin signaling seems
to be constitutively activated by the LHK1 gain-of-function mutation
(Tirichine et al., 2007). Thus, these ndings indicate that activation of
cytokinin signaling is necessary and sufcient as a trigger for nodule
organogenesis. Consistent with this, external application of cytokinin to
L. japonicus roots can induce spontaneous nodulation (Heckmann et al.,
2011). Since snf2-mediated spontaneous nodulation is not affected by the
mutation of either of the genes positioned upstream of calcium spikes
(Madsen et al., 2010; Tirichine et al., 2007), activation of cytokinin signaling
occurs after the induction of calcium spikes. In M. truncatula, nodulation is
almost completely compromised by the Mtcre1 single mutation (Plet et al.,
2011), suggesting that, unlike L. japonicus, M. truncatula may have no obvious
functional redundancy among cytokinin receptors. It remains unknown
whether MtCRE1 has a conserved ability with LHK1 with respect to
inducing nodulation, because we are unaware of reports investigating
gain-of-function effects of MtCRE1 on nodulation.
2.2.3 Downstream of cytokinin receptors

Cytokinin signal transduction is based on a phosphorelay cascade similar to
bacterial two-component systems (Heyl and Schmulling, 2003), where after
the perception of the signal by its receptor, B-type response regulators (RRs)
with DNA-binding activity regulate the expression of cytokinin primary
response genes. In M. truncatula, MtRR1 is identied as a B-type RR
possibly involved in the control of nodulation, although its genetic role is
unknown (Gonzalez-Rizzo et al., 2006). Among the potential target genes
of MtRR1, NSP2 has been identied (Ariel et al., 2012). In addition, it
seems that expression of a microRNA targeting NSP2 (miR171) is also
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induced in an MtCRE1-dependent manner (Ariel et al., 2012). Thus, it is

probable that cytokinin signaling has a dual mode for regulating NSP2
expression: it can directly activate NSP2 and repress its expression through
activation of miR171 expression. This ne-tuning mechanism for NSP2
expression may be important for nodule organogenesis.
Several observations indicate that NIN is positioned downstream of the
cytokinin receptor. First, snf2-dependent spontaneous nodulation is masked
by the nin mutation (Tirichine et al., 2007). Second, external application of
cytokinin to the root can induce NIN expression in an LHK1-dependent
manner (Heckmann et al., 2011; Soyano et al., 2014). NIN activation by
cytokinin is severely attenuated by the lhk1 single mutation (Soyano et al.,
2014); thus, LHK1A and LHK3 may not be required for the regulation of
NIN. In addition, it is unknown how cytokinin signaling is involved in
the hierarchical transcription cascade involving CYCLOPS, NIN, and
NF-Y. Activation of NIN or NF-Y cannot produce full-sized nodule-like
structures as are produced by the expression of phosphorylated CYCLOPS.
It is possible that cytokinin signaling is required for CYCLOPS-mediated
nodulation. To verify this, it will be important to investigate the effect of
phosphorylated CYCLOPS expression in cytokinin receptor mutants.
In terms of auxin involvement in nodule organogenesis, an auxin
response is activated during cortical cell division and is also induced during
snf2-dependent spontaneous nodule development (Suzaki et al., 2012). In
M. truncatula, application of an auxin-transport inhibitor to roots induces
formation of pseudonodules in the absence of rhizobia (Rightmyer and
Long, 2011). In addition, accumulation of MtPIN proteins, which are
putative auxin efux carriers, appears to be negatively regulated by cytokinin
signaling (Plet et al., 2011). Thus, cytokinin signaling may have a role in the
establishment of a localized auxin response in cortical cells through the
control of auxin transport. To clarify the genetic role of auxin in nodule
organogenesis, it will undoubtedly be important to characterize the nodula-
tion phenotypes of mutants involved in auxin biosynthesis, transport, and
signaling.
2.2.4 DNA topoisomerase VI

In a recent screen for suppressor mutants of snf2, we identied a gene named
VAGRANT INFECTION THREAD 1 (VAG1) in L. japonicus (Suzaki
et al., 2014). The vag1 mutation suppresses snf2-dependent spontaneous
nodulation as well as normal rhizobia-induced nodulation. Nodulation is
abolished in the severe alleles of vag1. Thus, it is reasonable to conclude
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that VAG1 is an essential regulator for nodule organogenesis. VAG1 en-

codes a protein orthologous to Arabidopsis ROOT HAIRLESS 1 (RHL1),
which functions as a subunit of DNA topoisomerase VI (Sugimoto-Shirasu
et al., 2005; Suzaki et al., 2014). DNA topoisomerase VI is a subclass of type
II topoisomerases and is considered to decatenate replicated chromosomes in
archaea (Corbett and Berger, 2003). In Arabidopsis, loss-of-function mutants
of genes encoding components of DNA topoisomerase VI have defects in
ploidy-dependent cell growth (Breuer et al., 2007; Hartung et al., 2002;
Kirik et al., 2007; Sugimoto-Shirasu et al., 2005, 2002; Yin et al., 2002).
The number of highly endoreduplicated cells is signicantly reduced in
the Arabidopsis mutants. These ndings suggest that plant DNA topoisomer-
ase VI has a role in promoting endoreduplication, although its biochemical
function remains elusive.
As mentioned above (see Section 2.1), endoreduplication results in the
formation of enlarged rhizobia-colonized cells in mature nodules. In
L. japonicus, there are enlarged rhizobia-colonized cells in the inner region
of the nodule, and smaller rhizobia-infected (as yet uncolonized) cells in
the surrounding region (Figure 1B). In vag1 nodules, the number of these
small rhizobia-infected cells is higher, whereas the number of rhizobia-
colonized cells is lower (Figure 1C; Suzaki et al., 2014). This suggests that
the vag1 mutant has a defect in differentiation from small rhizobia-infected
cells to enlarged rhizobia-colonized cells. Given that these small rhizobia-
infected cells require endoreduplication for their differentiation to rhizobia-
colonized cells, as is the case in M. truncatula (Kondorosi and Kondorosi,
2004), the vag1 nodule phenotype suggests that VAG1 is most likely involved
in the endoreduplication associated with this cell differentiation. Further-
more, in vag1, it seems that this large number of small rhizobia-infected cells
(most considered to be diploid) can be produced by somehow enhancing cell
division. Therefore, it is probable that the defect in DNA topoisomerase VI
does not affect the DNA replication required for cell division. This is consis-
tent with the observation that Arabidopsis rhl1 cells can proliferate mitotically
as efciently as wild-type cells (Sugimoto-Shirasu et al., 2005).
Analyses focusing on nuclear size found the emergence of a few cortical
cells with enlarged nuclei during initiation of cortical cell division (Suzaki
et al., 2014). In the vag1 mutants, these potentially endoreduplicated cells
are not observed and subsequent cortical cell division is severely compro-
mised. Thus, it is possible that the DNA topoisomerase VI encoded by
VAG1 is involved in the endoreduplication of cortical cells, which can
trigger the onset of cortical cell division (Figure 2). In L. japonicus, a positive
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role of DNA topoisomerase VI in nodule organogenesis was recently

conrmed by the identication of a gene, SUNERGOS1 (SUNER1),
which encodes a predicted subunit A of DNA topoisomerase VI (Yoon
et al., 2014). The nodulation defects in suner1 are weaker than those of
vag1, but may be due to the difference in the mutation site observed in
the respective mutants. Interestingly, the suner1 mutation is temperature sen-
sitive; thus, there may be a temperature-dependent mechanism that controls
the activity of DNA topoisomerase VI.
Localized endoreduplication of cortical cells seems to occur downstream
of cytokinin signaling (Suzaki et al., 2014). In Arabidopsis root development,
cytokinin signaling controls endocycle onset by directly upregulating the
expression of CCS52A1 (Takahashi et al., 2013), a necessary and sufcient
factor for the progress of endocycling (Baloban et al., 2013; Cebolla et al.,
Figure 2 Initiation of cortical cell division mediated by VAG1. In Lotus japonicus wild-
type plants, rhizobial infection may cause endoreduplication of a few cortical cells,
which may trigger initiation of the division of surrounding cells. In contrast, endoredu-
plication is suppressed and subsequent cortical cell division is not activated in vag1
mutants. In addition, elongation of mutant infection threads (ITs) is blocked at the
epidermalecortical interface.
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1999); the induced endoreduplication appears to act to stop cell division of

the root meristem. Thus, the action of this endoreduplication is different
from that induced during early nodule development. As described above
(see Section 2.2.3), a preferential auxin response at the sites of incipient
nodule primordia occurs downstream of the cytokinin signaling pathway
(Suzaki et al., 2012). In addition, such an auxin response seems to be unaf-
fected by the vag1 mutation (Suzaki et al., 2014). Thus, it is probable that the
endoreduplication of cortical cells is induced downstream of cytokinin and
auxin signaling. This notion is consistent with a previous observation that
exogenous application of cytokinin and auxin increases the ploidy level of
cortical explants from pea (Libbenga and Torrey, 1973).
Overall, DNA topoisomerase VI may be a prerequisite regulator for the
control of two key nodule developmental processes: the rst is related to the
onset of nodule organogenesis during early nodule development and
the second is associated with the differentiation of rhizobia-colonized
infected cells in late nodule development. In order to reveal the regulatory
relationship among DNA topoisomerase VI, endoreduplication and nodule
organogenesis, further experiments need to be performed. These will
involve the elucidation of the molecular and biochemical function of
DNA topoisomerase VI in endocycling and the identication of other endo-
cycle-related genes involved in the developmental process.
2.3 Negative Regulation of Nodulation: Autoregulation of

Nodulation (AON)
2.3.1 Concept of AON
While nodulation is benecial to plants, formation of an excessive number
of nodules can be disadvantageous because photosynthate, normally used for
plant growth, seems to be consumed for nodule development. In fact, the
growth of plants with an excessive number of nodules, described below, is
severely impaired, especially in the presence of rhizobia. To balance the
prot and cost caused by nodulation, plants have a genetic mechanism
termed AON, which strictly controls the number of nodules through
systemic long-distance signaling between root and shoot (Caetano-Anolles
and Gresshoff, 1991; Oka-Kira and Kawaguchi, 2006). In a proposed model
of the AON, rhizobial infection induces the production of putative signaling
molecules called root-derived signals in the root, which are mobile and
transmitted from root to shoot. The perception of the signals by their recep-
tor(s) in shoots activates the generation of putative secondary signals, also
called shoot-derived inhibitors (SDIs). It is postulated that the SDIs are
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also mobile and transported from shoot to root, and there inhibit nodule
development to restrict unnecessary nodulation.
2.3.2 Molecular basis of AON

2.3.2.1 Signaling from root to shoot in AON
L. japonicus HYPERNODULATION ABERRANT ROOT FORMA-
TION 1 (HAR1) was the rst identied AON-related gene in plants
(Kawaguchi et al., 2002; Krusell et al., 2002; Nishimura et al., 2002;
Wopereis et al., 2000). The har1 mutation markedly increases the number
of nodules. Grafting analysis between wild-type and har1 plants indicates
that malfunction of HAR1 in shoots causes the hypernodulation because
the hypernodulating phenotype is observed only when har1 plants are
used for the shoot. HAR1 encodes a putative leucine-rich repeat recep-
tor-like kinase (LRR-RLK), and phylogenetic analysis reveals that HAR1
is orthologous to Arabidopsis CLAVATA1 (CLV1), which is involved in
negative regulation of the stem cell population in the shoot apical meristem
(SAM) (Clark et al., 1997; Krusell et al., 2002; Nishimura et al., 2002).
In Arabidopsis, CLV3, a member of the CLAVATA3/ENDOSPERM
SURROUNDING REGION (CLE) family peptides, acts as a ligand of
CLV1 (Fletcher et al., 1999; Ogawa et al., 2008). CLV3 and CLV1 are,
respectively, expressed in the stem cell region and at the inner region of
the SAM (Clark et al., 1997; Fletcher et al., 1999): the short-distance
ligandereceptor communication is essential for SAM homeostasis. Based
on these ndings, an obvious next step in searching for potential HAR1
ligands focused on L. japonicus CLE genes. Among 39 CLE genes examined,
Okamoto and colleagues found that expression of two CLE genes, desig-
nated CLE-ROOT SIGNAL 1 (CLE-RS1) and CLE-RS2, is strongly
induced in roots upon rhizobial infection (Okamoto et al., 2009). While
the timing and levels of expression of the CLE-RS genes seem to be
adequately controlled during nodulation, constitutive activation of the
respective genes results in strong inhibition of nodule development
(Okamoto et al., 2009). Although no loss-of-function phenotype of CLE-
RS1/2 has yet been characterized, in M. truncatula, the simultaneous knock-
down of MtCLE12 and MtCLE13, functional counterparts of the CLE-RS
genes, increases the number of nodules (Mortier et al., 2012). Thus, it seems
reasonable to conclude that the CLE-RS1/2 genes have a negative role in
nodulation. Since constitutive CLE-RS1/2 expression has no effect on
nodulation in the har1 mutant (Okamoto et al., 2009), HAR1 is required
for the CLE-RS1/2-mediated inhibition of nodulation.
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Recently, biochemical analysis has determined the mature form of CLE-

RS2, which is a posttranslationally arabinosylated glycopeptide derived from
the CLE domain that contains 12 amino acids (Okamoto et al., 2013). Chem-
ically synthesized mature CLE-RS peptides can directly bind to HAR1, and
application of these peptides to shoots is sufcient to inhibit nodulation.
Importantly, the CLE-RS2 glycopeptide is detected from xylem sap in shoots
of plants that express the CLE-RS2 gene only in roots (Okamoto et al., 2013).
These data indicate that the CLE-RS2 glycopeptide is transported from root
to shoot through the xylem and acts as a ligand of HAR1 in the shoot
(Figure 3). The ligandereceptor interaction seems to occur in phloem tissues
of the leaf, where HAR1 is expressed (Nontachaiyapoom et al., 2007).
Overall, CLE-RS2 seems to meet the criteria for the root-derived signal
that works in AON.
With respect to posttranslational modication of CLE-RS protein, hy-
droxyproline (Hyp) O-arabinosyltransferase (HPAT) has been recently
identied in Arabidopsis as a key enzyme that catalyzes the transfer of the
L-arabinose to the hydroxyl group of Hyp residues (Ogawa-Ohnishi
et al., 2013). It seems that HPAT mediates Hyp O-arabinosylation of diverse
peptide families, and the amount of Hyp-arabinosylated CLE2 is decreased
in hpat mutants. Thus, it is probable that HPAT is involved in Hyp arabino-
sylation of CLE family peptides. The HPAT-mediated Hyp arabinosylation
of target peptides seems to occur in the Golgi complex. Interestingly, Ara-
bidopsis HPAT family proteins have similarity to M. truncatula ROOT
DETERMINED NODULATION 1 (MtRDN1) (Schnabel et al., 2011).
The Mtrdn1 mutation causes an increase in the number of nodules, and graft-
ing experiments suggest that the genotype of Mtrdn1 in roots is responsible
for the hypernodulating phenotype. Moreover, although constitutive
expression of MtCLE13 suppresses nodulation in wild-type pea, the inhib-
itory effects appear to be abolished in the pea nod3 mutant, which is defec-
tive in a gene orthologous to MtRDN1 (Osipova et al., 2012). These results
suggest that the LjHPAT protein (as yet unidentied) may be involved in
the Hyp arabinosylation of CLE-RS1/2 (Figure 3).
In L. japonicus, the klavier (klv) mutation confers a hypernodulating
phenotype, as is the case in har1 (Miyazawa et al., 2010; Oka-Kira et al.,
2005). Double mutant and grafting analysis indicate that KLV and HAR1
function in the same genetic pathway in the shoot. KLV encodes an
LRR-RLK belonging to a different subfamily than HAR1, which is orthol-
ogous to Arabidopsis RECEPTOR-LIKE PROTEIN KINASE 2, which has
a role in the regulation of SAM homeostasis (Kinoshita et al., 2010;
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Figure 3 Molecular basis of autoregulation of nodulation (AON). (1) Nodulation

signaling pathway, particularly downstream of cytokinin receptors, which ultimately
activate NIN expression. The NIN transcription factor activates CLE-RS1/2 expression
through direct binding to their promoter. (2) It is likely that the CLE-RS1/2 peptides
are posttranslationally modied with triarabinoside, a reaction mediated by an enzyme
similar to HPAT in the Golgi apparatus. These modied CLE-RS peptides are transported
to the xylem by an unidentied mechanism. (3) These peptides are transmitted from
roots to shoots and directly bind to HAR1 or a putative HAR1-KLV receptor complex
in the phloem of leaf cells. (4) Downstream of the CLE-RS/HAR1 signaling pathway, acti-
vated IPT3 produces cytokinin, which is transported to roots through phloem tissue. (5)
Shoot-derived cytokinin is directly or indirectly involved in proteasome-mediated
degradation of an unidentied positive regulator of nodule organogenesis. TML may
act as a component of the SCF complex. The site of AON action seems to be down-
stream of cytokinin signaling.
Miyazawa et al., 2010). Moreover, KLV physically interacts with HAR1, at

least in leaves of Nicotiana benthamiana (Miyazawa et al., 2010). In addition to
hypernodulation, klv mutants exhibit pleiotropic nonsymbiotic defects such
as an enlarged SAM, disordered leaf vasculature, and late owering. These
nonsymbiotic abnormalities are not observed in har1 mutants. Thus, KLV
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may interact with a receptor distinct from HAR1 in the control of such non-
symbiotic developmental processes.
2.3.2.2 Signaling from shoot to root in AON

Given that the production of SDI is under the control of CLE-RS1/2-
HAR1 signaling, it is possible that the amount of SDI is higher in plants
that constitutively express CLE-RS1 or CLE-RS2. Recently, we focused
on the amount of phytohormones in the shoots of these plants and found
that the level of iPRPs, intermediates of cytokinin biosynthesis, is signi-
cantly upregulated by CLE-RS expression (Sasaki et al., 2014). Rhizobial
infection also increases the iPRP level in shoots in a HAR1-dependent
manner. These results indicate that cytokinin production in shoots is
controlled by CLE-RS1/2-HAR1 signaling during nodulation. Remark-
ably, cytokinin application to the shoot suppresses nodule development in
both wild-type and har1 plants. We further demonstrate that cytokinin
can be transported from shoot to root in L. japonicus. Thus, these data suggest
that shoot-derived cytokinin has an SDI-like capacity to systemically sup-
press nodulation (Figure 3).
In the cytokinin biosynthetic pathway, synthesis of iPRPs is catalyzed by
isopentenyltransferase (IPT) (Takei et al., 2001). Among the L. japonicus IPT
genes examined, the expression of LjIPT3 is signicantly induced in shoots
by rhizobial infection in a HAR1-dependent manner (Sasaki et al., 2014).
Promoter-GUS analysis has revealed that LjIPT3 is expressed in the phloem
tissues of the leaf, with an expression pattern overlapping with HAR1
(Nontachaiyapoom et al., 2007). In addition, Ljipt3 knockout lines form
an increased number of nodules, and, conversely, constitutive LjIPT3
expression reduces nodule number (Sasaki et al., 2014). Thus, LjIPT3 work-
ing in shoots seems to have a role in the negative regulation of nodulation
(Figure 3).
Although the above ndings on the role of shoot-derived cytokinin
contribute to our understanding of events downstream of CLE-RS1/2-
HAR1 signaling, we cannot conclude that shoot-derived cytokinin is an
SDI on the basis of currently available data. It is possible that a second
messenger induced by cytokinin acts as a genuine SDI. As mentioned above
(see Section 2.2.2), cytokinin acts as a positive regulator of nodule develop-
ment (Frugier et al., 2008; Suzaki et al., 2013a). In fact, three cytokinin
receptors identied in L. japonicus are all involved in the positive regulation
of nodule organogenesis (Held et al., 2014). In addition, the role of LjIPT3
in nodulation may be complicated because LjIPT3, which works in the root,
ARTICLE IN PRESS
seems to have a positive role in nodulation (Chen et al., 2014). Thus, it

remains unclear how shoot-derived cytokinin inhibits nodulation. To
address this question, elucidation of the site of action of shoot-derived
cytokinin is crucial: this may involve the characterization of unidentied
cytokinin receptors and the detailed function of LjIPT3.
2.3.2.3 Site of AON action in the root

L. japonicus too much love (tml) is another hypernodulating mutant (Magori
et al., 2009; Takahara et al., 2013). Double mutants and grafting analysis
indicate that TML and HAR1 function in the same genetic pathway. In
addition, the results of an inverted-Y grafting approach suggest that TML
acts in roots downstream of CLE-RS1/2-HAR1 signaling (Magori et al.,
2009). Importantly, shoot-applied cytokinin does not affect nodulation in
tml mutants (Sasaki et al., 2014). Thus, TML is a prerequisite for shoot-
derived cytokinin to negatively affect nodulation. TML encodes a Kelch
repeat-containing F-box protein (Takahara et al., 2013). Generally, F-box
proteins are a component of the so-called SCF complex of SKP1, CULLIN,
and E3 ubiquitin ligase, which is involved in the degradation of a target
protein (Hua and Vierstra, 2011). In the SCF complex, TML may interact
with a target protein that has a positive role on nodulation (Figure 3).
The identication of the target protein of TML will contribute further to
our understanding of the site of AON action.
Since the har1, klv, or tml mutation enhances snf2-mediated sponta-
neous nodulation (Miyazawa et al., 2010; Takahara et al., 2013; Tirichine
et al., 2007), AON seems to act downstream of LHK1 (Figure 3). As
described above (see Section 2.2.1), NIN is a positive regulator of nodule
organogenesis working downstream of LHK1. However, some studies
have discussed the possibility of NIN having a negative role in nodulation
because nin mutants show excessive root-hair curling and spatially
expanded expression of ENOD11, a nodulation marker (Marsh et al.,
2007; Schauser et al., 1999). In addition, constitutive expression of NIN
not only induces spontaneous cortical cell division in the absence of
rhizobia, but also suppresses normal nodulation in the presence of rhizobia
(Soyano et al., 2013; Yoro et al., 2014). Recently, Soyano and colleagues
revealed that CLE-RS1 and CLE-RS2 are direct targets of NIN, and NIN
has the ability to systemically downregulate its own expression in a HAR1-
dependent manner (Figure 3; Soyano et al., 2014). Thus, NIN can create a
negative feedback loop to modulate its own expression through CLE-
RS1/2-HAR1 signaling. Clarication of the detailed spatiotemporal
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control of NIN expression will undoubtedly contribute to our understand-

ing of the site of AON action.
From a morphological point of view, a recent study focusing on the
cortical cell division pattern has shown that initiation of cortical cell division
can occur in plants that constitutively express CLE-RS1/2 genes (Suzaki
et al., 2012). The researchers also found vestiges of cortical cell division,
implying premature arrest during the course of cell division. These observa-
tions suggest that AON has a negative regulatory effect on cortical cell
division after its initiation.
3. RHIZOBIAL INVASION PROCESS

3.1 Initial Responses and Root Hair Deformation
How can rhizobia sense where host plant roots exist in the rhizo-
sphere? It is believed that rhizobia recognize avonoids or betaines secreted
from the host plant, which then induce nod genes expression in rhizobia and
the subsequent production of Nod factor. Some avonoids directly bind
NodD transcription factors to activate transcription of the nod gene cluster
(Honma et al., 1990; Peck et al., 2006). Many avonoids have been identi-
ed as inducers of nod genes in rhizobia (Cooper, 2004). The nod gene
cluster encodes approximately 25 proteins that are essential for stepwise
Nod factor production.
Next, Nod factor produced by rhizobia induces multiple initial responses
of host plants that are essential for rhizobial invasion. Rhizobia are
entrapped by the tightly curled root hair, also known as a shepherds crook
(Figure 4(2)). Nod factor itself can induce elongation and deformation of
root hair cells. Among several growth stages of root hairs (Karas et al.,
2005), it is thought that growth-terminating root hairs, which have a dense
cytoplasmic region at the tip, are the most sensitive to Nod factor (Sieberer
and Emons, 2000). At least 90 min after Nod factor application, the tip starts
to swell, and a new outgrowth develops from the site of swelling. These
processes are accompanied by cytoplasmic streaming, reorientation of the
ER, and movement of the nucleus and vacuole (Gage, 2004; Miller et al.,
2000; Sieberer and Emons, 2000). Furthermore, spot application of Nod
factor is sufcient to induce fully curled root hair formation accompanied
by MtENOD11 expression (Esseling et al., 2003).
Root hair growth is thought to conserve a mechanism observed in pollen
tubes (Hepler et al., 2001). In addition, some of the machinery for tip
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Figure 4 Rhizobial infection and invasion in root epidermis and cortical cells. (1) Rhizo-
bial attachment to root hair and accumulated Nod factor induce initial responses
involving root hair deformation and curling. (2) At the tightly curled root hair, cell
wall degradation occurs and invagination of the IT membrane in the root hair cell is
induced. (3) IT elongation occurs, which is accompanied by cytoplasmic streaming
and nuclear movement. (4) During rhizobial infection ((1)e(3)), in the root cortical cells,
a cytoplasmic bridge or preinfection thread (PIT) is formed to guide elongating ITs. (5)
When an IT reaches the newly divided cortical cell, the IT membrane collapses and
rhizobia are released into the cortical cell to form a specialized nitrogen-xing organ-
elle, the symbiosome.
growth may support IT membrane organization and inner growth of the IT

inside the root hair (Gage, 2004). The rst evidence is that Golgi-derived
vesicles that deliver cell wall components and extracellular matrix highly
accumulate at the outgrowing apex of the root hair (Miller et al., 2000;
Ridge, 1995). Second, a calcium gradient is formed at the tip of root hairs
as well as pollen tubes (Esseling et al., 2003; Gage, 2004; Hepler et al.,
2001; Peck et al., 2006), and Nod factor can induce calcium inux and
calcium spikes in root hairs (Miwa et al., 2006; Shaw and Long, 2003).
Calcium inux phenotypes correlate well with root hair deformability of
several non-nodulation mutants, thereby conrming an important role of
Nod factor-dependent calcium inux in root hair deformation (Miwa
et al., 2006). Third, reactive oxygen species (ROS) are produced during
rhizobial infection: this is supported by observations of rhizobial- or Nod
factor-dependent induction of a putative peroxidase gene, MtRIP1 (Cook
et al., 1995; Peng et al., 1996; Ramu et al., 2002), as observed at the tip
of root hairs and pollen tubes (Foreman et al., 2003; Liu et al., 2009). In
contrast to ROS production induced by Nod factor or putative calcium
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inux, ROS accumulation is signicantly decreased by Nod factor treatment

through downregulation of MtRBOH2/3 (membrane-associated NADPH
oxidases). This ROS reduction is required for root hair deformation (Lohar
et al., 2007). The authors point out that the rapid calcium inux and ROS
reduction can be distinct physiological responses, because they are not
temporally correlated. In addition, the abolishment of ROS reduction in
Nod factor receptor mutants suggests a requirement for Nod factor in this
process, as described below (see Section 3.3; Lohar et al., 2007).
Several studies have identied key genes that are required for these Nod
factor-dependent initial events (Table 1(A); Lohar et al., 2007; Miwa et al.,
2006). Severe impairment of root hair deformation is observed in mutants
for the Nod factor receptors NFR1/NFP and NFR5/LYK3/HCL (Ben
Amor et al., 2003; Limpens et al., 2003; Madsen et al., 2003; Radutoiu
et al., 2003). Intriguingly, a variety of mutant alleles of the Nod factor recep-
tor show extensive or extreme root hair deformation (Table 1(C),(F); Smit
et al., 2007). These observations indicate that a slightly different degree of
Nod factor signaling directly affects root hair deformation. Similarly,
symrk/dmi2 mutants show aberrant swelling and branching of root hairs
(Table 1(F); Catoira et al., 2000; Miwa et al., 2006; Stracke et al., 2002).
This can be explained by a contribution of SYMRK to modulation of
Nod factor-dependent signal transduction, based on a recent study that
SYMRK lacking an MLD domain interacts with NFR5, as further discussed
below (see Section 3.6).
3.2 IT Initiation and IT Membrane Characterization

After root hair deformation, rhizobia start to invade dividing cortical cells
through IT (Figures 4 and 5). At the initial step of IT organization, degra-
dation of the cell wall of the root hair is required for rhizobial entry. Strong
candidates for degradation of the plant-side cellulose are polygalacturonases
(PGs) (Munoz et al., 1998; Rodriguez-Llorente et al., 2004). The expression
patterns of PGs in both the initial growth stages of ITs and pollen tubes indi-
cate that the mechanism for IT growth may be co-opted from that of pollen
tube growth, as described above. Alternatively, this degradation could be
achieved by rhizobially produced cellulase (Laus et al., 2005; Robledo
et al., 2008).
The IT membrane originates from invagination of the plant cell
membrane, or in other words, the lumen of the IT harboring rhizobia is to-
pologically outside of the root cell (Brewin, 2004; Gage, 2004). However,
the components of the IT lumen seem to be different from normal cell wall
Table 1 Plant genes or mutants involved in rhizobial infection
Nodulation phenotype
Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria

Nod/low Nod/Nod/ Remarks (gene function,
Genes/mutants Gene products Nod other observations) References
(A) Impared in root hair deformation

LjNFR1/MtLYK3/ Nod factor Nod Phenotype differs Limpens et al. (2003),
MtHCL receptor slightly between Radutoiu et al.
alleles (see below) (2003), Smit et al.
(2007).
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LjNFR5/MtNFP/ Nod factor Nod Ben Amor et al. (2003),
PsSYM10 receptor Madsen et al. (2003),
Radutoiu et al.
(2003), Ovtsyna et al.
(2005), Rival et al.
(2012).
(B) IT membrane localized
LjFLOT4 Plant otillin-like See LjFLOT2/4-RNAi Localized to the tip of Haney and Long
protein growing IT in the (2010), Haney et al.
presence of rhizobia, (2011).
colocalized with
MtLYK3
AtPIP2;1 Aquapolin e Used for live cell Fournier et al. (2008).
imaging of IT in root
hair
MtDMI2 Receptor-like Nod Limpens et al. (2005).
kinase
MtSYMREM1 Plant-specic Low Nod Lefebvre et al. (2010).
remorin protein
19
(Continued)
Table 1 Plant genes or mutants involved in rhizobial infectiondcont'd
20
(C) Impared in IT elongation
LjNIN/MtNIN/ RWP-RK TF Nod Root hair curling Schauser et al. (1999),
PsSYM35 occurs, but no IT Borisov et al. (2003),
formed Marsh et al. (2007),
Soyano et al. (2013).
Nod
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MtERN/MtBIT1/ ERF TF Distended infection foci Middleton et al. (2007).
Mtpld and branched IT in
root hair
LjNPL (Ljnpl-2/ Pectate lyase Nod (white nodule) involved in cell wall Xie et al. (2012).
Ljitd1) degradation,
expressed in pollen
LjPIR1 SCAR/WAVE Nod (white nodule) Oversized nodule Yokota et al. (2009).
components formed, participate in
cytoskeleton
rearrangements
LjNAP1/MtRIT SCAR/WAVE Nod (white nodule) Participate in Yokota et al. (2009),
components cytoskeleton Miyahara et al.
rearrangements, (2010).
impaired in trichome
Takuya Suzaki et al.

and root hair growth
LjARPC1 ARP2/3 Nod (white nodule) Participate in Hossain et al. (2012).
components cytoskeleton
rearrangements,
impaired in trichome
and root hair growth
LjFLOT2/4-RNAi Plant otillin-like Low Nod Flotillin exist in lipid Haney and Long
protein raft (2010).

LjCERBERUS/ E3 ubiquitin ligase Low Nod IT arrest occurs Kuppusamy et al.
MtLIN (2004), Yano et al.
(2009).
LjCYCLOPS/ Coiled coil Low Nod IT arrest occurs, and Messinese et al. (2007),
MtIPD3 domain, TF functions as a Yano et al. (2008),
CCaMK interactor Horvath et al. (2011),
Ovchinnikova et al.
(2011).
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MtRPG Long-coiled coil Low Nod Localized to nuclear, Arrighi et al. (2008).
protein mediate proteine
protein interaction
MtVPY MSP domain, Low Nod Involved in AM Murray et al. (2011).
Ankyrin repeat symbiosis and
membrane trafcking
MtLATD/MtNIP NRT1 transporter Low Nod Disorganization of root Bright et al. (2005),
meristem, decreased Yendrek et al.
lateral root number (2010).
MtNF-YA1 CCAAT-binding TF Low Nod Thicker, bulbous, and Laloum et al. (2014),
(MtHAP2-1), branched IT Laporte et al. (2014).
NF-YA2
PvNF-YC-RNAi CCAAT-binding TF Low Nod Decreased IT, IT arrest Eugenia Zanetti et al.
occurs (2010).
PvSIN1-RNAi GRAS TF Low Nod IT arrest occurs Battaglia et al. (2014).
Mthcl-4/Mtlyk3-4 Nod factor receptor Low Nod Extensive root hair Smit et al. (2007).
(weak allele) deformation, big sac-
like infection
(Continued)
21
22
Ljsymrk-14 (specic Receptor-like Low Nod IT arrest occurs Kosuta et al. (2011).
allele of symrk) kinase
Ljalb1/Ljsym74 N.D. Low Nod Abnormally enlarged Imaizumi-Anraku et al.
ARTICLE IN PRESS
IT, impaired in (2000), Kawaguchi
vascular bundle et al. (2002), Yano
differentiation et al. (2006).
(D) No or low IT with no or low infected nodules
LjCCaMK/MtDMI3/ Calmodulin- Nod Catoira et al. (2000),
PsSYM9/PsSYM30 dependant Lvy et al. (2004),
protein kinase Mitra et al. (2004),
Miwa et al. (2006).
LjSYMRK/MtDMI2/ Receptor-like Nod Exaggerated swelling Catoira et al. (2000),
PsSYM19 kinase and branching of root Stracke et al. (2002),
hair, also involved in Bersoult et al. (2005),
AM symbiosis Limpens et al. (2005),
Ovtsyna et al. (2005).
LjNSP1/MtNSP1, GRAS TF Nod Tsyganov et al. (2002),

LjNSP2/MtNSP2/ Kalo et al. (2005),
PsSYM7 Smit et al. (2005),
Heckmann et al.
(2006), Murakami
et al. (2006).
LjCASTOR, Ion channels Nod/low Nod Ane et al. (2004),
LjPOLLUX/ Edwards et al. (2007),

MtDMI1/PsSYM8 Imaizumi-Anraku
et al. (2005), Peiter
et al. (2007), Riely
et al. (2007).
LjNUP85 Nucleoporin Nod/low Nod Tempature-sensitive Saito et al. (2007),
phenotype Madsen et al. (2010).
LjNUP133 Nucleoporin Nod/low Nod Tempature-sensitive Kanamori et al. (2006),
phenotype Madsen et al. (2010).
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MtCRE1, MtCRE1-2, Cytokinin Low Nod Homologous to Gonzalez-Rizzo et al.
and MtCRE3 receptors LjLHK1, LjLHK1A, (2006), Plet et al.
LjLHK3 (2011).
LjnsRING-RNAi RING-H2 nger Low Nod Shimomura et al.
domain (2006).
MtROP9-RNAi Rac1 small Low Nod Involved in ROS Kiirika et al. (2012).
G protein production,
pathogenic
interactions, and leaf
shape
MtSINA E3 ubiquitin Low Nod Analyzed by Den Herder et al.
ligase overexpression of (2008).
dominant negative
type SINA
LjNENA Related to Low Nod Groth et al. (2010).
nucleoporins
Ljbrush N.D. Low Nod Disorganized root Maekawa-Yoshikawa
meristem, decreased et al. (2009).
lateral root number
23
(Continued)
24
Ljlot1 N.D. Low Nod Ooki et al. (2005).
Ljcrk/Ljsym79 N.D. Low Nod Kawaguchi et al.
(2002), Tansengco
et al. (2003),
Tansengco et al.
(2004), Yano et al.
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(2006).
Ljitd3,Ljitd4 N.D. Low Nod Lombardo et al. (2006).
Mtapi N.D. Low Nod Teillet et al. (2008).
proSYMRK:: Receptor-like Low Nod Less IT is canceledby Antolin-Llovera et al.
LjSYMRK-DMLD in kinase proUB::LjSYMRK- (2014).
symrk-3 DMLD
(E) Impared in bacterial release
MtLATD/MtNIP NRT1 transporter Low Nod Disorganized root Veereshlingam et al.
meristem, decreased (2004), Bright et al.
lateral root number (2005), Yendrek
et al. (2010).
MtIPD3 Coiled coil Low Nod IPD3 localized to the Horvath et al. (2011),
domain, TF nucleus of infection Ovchinnikova et al.
zone inside nodule (2011).
Nod

MtDMI2/SrSYMRK Receptor-like Capoen et al. (2005),
kinase Limpens et al. (2005).
MtNF-YA1 (MtHAP2- CCAAT- Low Nod Expressed in Combier et al. (2006).
1), NF-YA2 binding TF meristematic zone of
nodule, nodule
developmental arrest
occurs
MtEFD AP2/ERF TF Nod Verni et al. (2008).
MtSYMREM1-RNAi Plant-specic Low Nod Retarded bacterial Lefebvre et al. (2010).

remorin protein release
MtNFP-RNAi Nod factor Low Nod Nodule-specic Moling et al. (2014).
receptor knockdown using
promoter
MtENOD12,
aberrant infection
zone appears
(F) Increased early infection events with low or no nodulation
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Mthcl-1/Mthcl-2/Mthcl- Nod factor Nod Extensive root hair Smit et al. (2007).
3 (strong alleles) receptor deformation
LjSYMRK/MtDMI2 Receptor-like Nod Exaggerated swelling Catoira et al. (2000),
kinase and branching of root Stracke et al. (2002),
hair, also involved in Miwa et al. (2006).
AM symbiosis
Ljnin RWP-RK TF Nod Excessive root-hair Schauser et al. (1999),
curling Marsh et al. (2007).
Ljpir1 SCAR/WAVE Nod (white nodule) Increased infection foci Yokota et al. (2009).
components
Ljnap1 SCAR/WAVE Nod (white nodule) Increased infection foci Yokota et al. (2009).
components
Mtkce(Mtlin-4; specic E3 ubiquitin ligase Low Nod Increased infection foci, Guan et al. (2013).
allele of Mtlin) nodule vascular
bundles are centrally
localized
Mtnf-ya1-1 CCAAT-binding Low Nod Increased infection foci, Laporte et al. (2014).
TF most infections were
present in epidermis
25
not in cortex
(Continued)
26
(G) Hyperinfection mutants with low or no nodulation
Ljdaphne (specic allele RWP-RK TF Nod Yoro et al. (2014).
of Ljnin)
Nod/low Nod
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Ljvag1 Subunit of Misguided IT Suzaki et al. (2014).
topoisomerase VI
Ljsuner1 Subunit of Low Nod Misguided IT Yoon et al. (2014).
topoisomerase VI
Ljlhk1(Ljhit1) Cytokinin receptor Low Nod LjLHK1, LjLHK1A, Murray et al. (2007),
and LjLHK3 Held et al. (2014).
redundantly
functions in cell
division in cortex
MtCRE1, MtCRE1-
2, and MtCRE3 are
homologs in Mt
MtSYMREM1-RNAi Plant-specic Low Nod Highly branched IT Lefebvre et al. (2010).
remorin protein
MtLATD/MtNIP NRT1 transporter Low Nod Ramied IT inside Veereshlingam et al.

nodule (2004).
Ljccamk-14 (specic Calmodulin- Nod (white nodule) Larger microcolonies Liao et al. (2012).
allele of Ljccamk) dependant
protein kinase
(H) Hyperinfection mutants with hypernodulation
Mtsickle Ethylene signaling Nod Involved in ethylene Penmetsa and Cook
protein EIN2 response (1997), Penmetsa
et al. (2003).
Mtefd AP2/ERF TF Nod Verni et al. (2008).
MtPUB1-RNAi in E3 ubiquitin Nod Identied as a MtLYK3 Mbengue et al. (2010).
Mtlyk3-4 ligase interactor,
overexpression of
ARTICLE IN PRESS
MtPUB1 suppres
nodulation.
Abbreviations (gene/mutant names): Nod factor receptor 1 (NFR1); LysM receptor kinase 3 (LYK3); Hair curling (HCL); Nod factor receptor 5 (NFR5); Nod
factor perception (NFP); Symbiosis (SYM); Flotillin (FLOT); Plasma membrane intrinsic protein (PIP); Does not infection 1/2/3 (DMI1/2/3); Symbiotic remorin 1
(SYMREM1); Nodule inception (NIN); ERF required for nodulation (ERN); Branching infection threads 1 (BIT1); Poodle (pld); Nodulation pectate lyase;
infection-thread decient (itd); 121F-specic p53 inducible RNA (PIR1); Nck-associated protein 1 (NAP1); Actin-related protein component 1 (ARPC1); Required
for infection thread (RIT); Lumpy infections (LIN); Interacting protein of DMI3 (IPD3); Rhizobium-directed polar growth (RPG); Vapyrin (YPY); Lateral root
organ-defective (LATD); Numerous infections and polyphenolics (NIP); Nuclear Factor-Y (NF-Y); Heme-Activated protein 2-1 (HAP2-1); Scarecrow-like13
Involved in Nodulation (SIN1); Symbiosis receptor-like kinase (SYMRK); aberrant localization of bacteria inside nodule 1 (alb1); Ca2/calmodulin-dependent
protein kinase (CCaMK); Nodulation signaling pathway 1/2 (NSP1/2); Cytokinin Response (CRE); Lotus histidine kinase (LHK); Nodule-specic RING-H2 nger
protein (nsRING); r-related GTPases of plants (ROP); Seven in absentia (SINA); low nodulation and trichome distortion (lot1); crinkle (crk); altered nodule pri-
mordium invasion (api); Ethylene response factor required for nodule differentiation (EFD); knocks but cant enter (kce); vagrant infection thread 1 (vag1); sunergos 1
(suner1); Plant U-box E3 ubiquitin ligase 1 (PUB1).
Abbreviations (etc.): Lotus japonicus (Lj); Medicatgo truncatula (Mt); Pisum sativum (Ps); Phaseolus vulgaris (Pv); Sesbania rostrata (Sr); Infection thread (IT); Not determined
(N.D.); transcription factor (TF); Ethylene Responsive Factor (ERF); SCAR/WAVE (suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein); ARP (actin-
related protein); Major sperm protein (MSP); NRT (nitrate transporter); GRAS (GAI, RGA and SCR members); Really interesting new gene (RING); arbuscular
mycorrhizal (AM); ubiquitin (UB); non-nodulation (Nod); decreased nodulation (low Nod); normal number of nodules (Nod); increased or hypernodulation
(Nod).
27
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(A) (B) (C) (D) (E)
Figure 5 IT formation in Lotus japonicus root inoculated with Mesorhizobium loti

that expresses DsRED. (A) A confocal image of IT. (B, C) Magnied images of (A). (D)
A bright-eld image of (C). (E) A merged image of (C) and (D). Red (gray in print ver-
sions) signal represents rhizobia. Arrow and arrowhead indicate root hair membrane
and IT membrane, respectively. Bars 10 mm.
components. Electron microscopic observation of ITs suggests that the IT

membrane is resistant to plant cell-wall degrading enzymes such as Driselase
(Higashi et al., 1986). Another electron microscopic study using antibodies
against cell wall components found that MAC265, which was later identi-
ed as an antibody to an extensin-like glycoprotein, is enriched in the lumen
of the IT (Rae et al., 1992; Rathbun et al., 2002). In addition, xyloglucan,
unesteried pectin, and methyl-esteried pectin are in the IT wall but not in
the IT lumen (Rae et al., 1992). Moreover, IT arrest and uninfected nodules
are observed in the L. japonicus npl (nodulation pectate lyase) mutant,
demonstrating a requirement for pectate lyase in rhizobial penetration,
putatively mediated by cell wall degradation (Xie et al., 2012). Other plant
glycoproteins potentially involved in cell wall rearrangement during rhizo-
bial infection have been identied (Brewin, 2004).
Although the components of the IT membrane are largely unknown, a
few IT-membrane-localized proteins have been discovered (Table 1(B)).
Considering the identication of MtFLOT2/4 (plant otillin-like protein)
and MtSYMREM1, lipid raft, cholesterol-rich, and detergent-resistant
membrane microdomain may play a key role in IT membrane organization
(Haney and Long, 2010; Lefebvre et al., 2010). A FLOT4-GFP fusion pro-
tein is localized to the tip of the growing root hair and IT membrane upon
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rhizobial inoculation. Furthermore, rapid colocalization of LYK3 and

FLOT4 in the root hair is dependent on rhizobial infection (Haney et al.,
2011). DMI2 and AtPIP2;1 are also reportedly IT-membrane-localized
proteins (Fournier et al., 2008; Limpens et al., 2005).
3.3 IT Growth and Elongation

The impairment of IT progression in pir1, nap1/rit, and arpc1 mutants suggests
that IT growth is controlled by the cytoskeleton. The responsible genes
participate in stabilizing actin bundles via SCAR/WAVE or ARP2/3 com-
plex formation and are also necessary for trichome and root hair formation
(Hossain et al., 2012; Miyahara et al., 2010; Yokota et al., 2009). RAC/
ROP-like small GTPases, which are usually found in a variety of tip-growing
cells, seem to be localized to the IT membrane. MtROP9-RNAi transgenic
roots exhibit an extreme swelling of root hairs, delay in rhizobial infection,
and uninfected nodulation, implicating them in IT growth (Kiirika et al.,
2012). RAC/ROP-like small GTPases are also associated with RBOH activ-
ity and ROS generation, as described above (see Section 3.1; Kiirika et al.,
2012; Lohar et al., 2007). In addition, exocytosis or membrane trafcking
may provide the driving force for IT elongation, as shown by the identica-
tion of a role for VAPYRIN, which contains a major sperm protein (MSP)
domain and an ankyrin repeat domain (Murray et al., 2011). Human MSP
domain-containing protein interacts with an SNARE complex and functions
in exocytosis-dependent secretion. Overall, these ndings indicate mecha-
nistic similarities between IT growth and other tip growth involving root
hairs and pollen tubes (Campanoni and Blatt, 2007; Hepler et al., 2001).
This possible molecular link among ITs, root hairs, and pollen tubes is further
suggested by the L. japonicus crinkle mutant phenotype: the mutant has defects
in all these tip-growing cells (Tansengco et al., 2003, 2004).
Other genes involved in IT elongation or IT formation are listed in Table
1(C)e(D). In particular, when special observations of ITs were performed in
the original paper, they are categorized as impaired in IT elongation, with
special remarks (Table 1(C)). However, the individual molecular mechanisms
and their roles in IT elongation remain largely obscure. In addition, most of
these genes are also involved in nodule development (see Nodulation
phenotype column). This indicates the complexity of the two signaling
pathways, rhizobial infection in the epidermis and cell division in the cortex,
as discussed below (see Section 3.6).
Recent advances in live cell imaging of IT formation in root hair cells
together with mobility of the nucleus, ER, other organelles, and
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microtubules have provided new insights into the dynamic morphology of

ITs in vivo (Fournier et al., 2008; Perrine-Walker et al., 2014a,b). For
instance, the speed of the growing IT is approximately 4e5 mm per hour,
and the frequent appearance of a rhizobial-free zone behind the tip of the
growing IT indicates that invagination of the IT membrane precedes rhizo-
bial colonization (Figure 4(3); Fournier et al., 2008). In contrast, currently it
is experimentally difcult to trace IT growth in the inner cortex due to the
technical barriers to imaging deeper focal planes.
3.4 Cytoplasmic Bridge or Preinfection Thread (PIT) in

Cortex
Although this review has focused on epidermal events necessary for rhizobial
infection (3.2e5), during this process, root cortical cells are simultaneously
activated and seem to prepare for acceptance of rhizobial invasion
(Figure 4(4)). In response to rhizobial inoculation or Nod factor application,
the nuclei and cytoplasm of the cortex are relocalized to the center of the
cells and are aligned in a way to guide the IT, a formation called a cytosolic
bridge or PIT, as it occurs in preparation for the usual cell division
(Figure 4(4); Niwa et al., 2001; Timmers et al., 1999; van Brussel et al.,
1992). Interestingly, although no IT or nodule formation is induced in
M. sativa inoculated with the R. meliloti nodFL double mutant, nuclear
activation and cortical and pericycle cell division are induced in a wider
region of the roots (Ardourel et al., 1994; Timmers et al., 1999). These
cortical cells have nuclei that are surrounded by accumulated starch granules.
This phenomenon is considered one of the cortical reactions occurring prior
to rhizobial invasion, but the role of starch accumulation in cell activation
remains a mystery. The activated cortical cells specically express HISTONE
H4, a marker for S-phase during the cell cycle, indicating reentry to the
cell cycle for nodule organogenesis (Foucher and Kondorosi, 2000; Yang
et al., 1994).
3.5 Bacterial Release

In the nal stage of infection, rhizobia are released into newly divided
cortical cells. Endocytosis occurring at the tip of the IT allows rhizobia to
form a special organelle-like structure termed a symbiosome, where symbi-
otic nitrogen xation takes place (Figure 4(5)). In this process, collapse of the
cellulose sheath at the apex of the IT enables rhizobia to directly interact
with the host plasma membrane (Figure 4(5); Brewin, 2004). This interac-
tion might be mediated by plant glycoproteins and glycolipids in the
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symbiosome fraction (Perotto et al., 1991). Several mutations involved in

this bacterial release have been identied (Table 1(E)). Recently, it was
shown that NFP and LYK3, Nod factor receptors, form a heteromeric com-
plex and are localized to two cell layers between the meristem and infection
zone of the nodule (Moling et al., 2014). Proper rhizobia release in the
nodule is signicantly impaired by nodule-specic knockdown of NFP.
In indeterminate nodules, the symbiosome becomes enlarged in parallel
with bacterial differentiation, which results in nodule differentiation, and the
size or morphology of the indeterminate nodule is affected by mutation of
genes involved in rhizobial release (Combier et al., 2006; Horvath et al.,
2011; Limpens et al., 2005; Vernie et al., 2008). It seems that both rhizobia-
and plant-side factors are required for rhizobial differentiation (Jones et al.,
2007). For example, bacA and lpsB, involved in LPS production in rhizobia,
are essential for rhizobial survival inside the host cell. On the plant side,
secretion of nodule-specic cysteine-rich (NCR) peptide, processing of
which is putatively mediated by DNF1, encoding a predicted subunit of
the signal peptidase complex, is required for further bacterial differentiation
(Van de Velde et al., 2010; Wang et al., 2010). The NCR peptide family
exists only in inverted repeat-lacking clade (IRLC) legumes including
Medicago species, P. sativum, Vicia species, and T. repens, but not in non-
IRLC legumes such as L. japonicus.
3.6 Cross Talk between Rhizobial Infection and Nodule

Development Pathways
As briey mentioned above, two signaling pathways, rhizobial infection in
the epidermis and cell division in the cortex, are tightly coupled, which
has made it difcult to understand the two processes separately. Recently,
however, the situation has been partially resolved by several reports. Madsen
et al. performed large-scale phenotyping of non-nodulation mutants in the
presence of the snf1 mutation, a gain-of-function mutation in CCaMK
(Madsen et al., 2010). The introduced snf1 mutation rescues defects of IT
formation in symrk, nup85, nup133, castor, and pollux, but not those of
pir1, nap1, cyclops, cerberus, nsp1, or nsp2. Thus, CCaMK has the ability to
induce rhizobial infection in the epidermis. The responsible genes of
all unrescued mutants seem to work downstream of CCaMK (Madsen
et al., 2010).
Another approach focuses on tissue-dependent requirements for NFP
and DMI3 using an epidermis- or cortex-specic promoter (Rival et al.,
2012), and has led to the conclusion that (1) epidermal NFP is sufcient
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to induce cell division in the cortex, (2) epidermal DMI3 but not NFP is
sufcient for IT formation in the root hair and for activating cell division,
and (3) epidermal and cortical expression of DMI3 is required for both pro-
cesses. This part of the conclusion is supported by another study using a
similar tissue-specic promoter system (Hayashi et al., 2014).
SYMRK may play an important role as a hub for coordinating the two
signaling pathways, for rhizobial infection and nodulation. Unlike typical
non-nodulation symrk mutants, a unique symrk-14 mutant forms uninfected
white nodule bumps with many arrested ITs (Table 1(D); Kosuta et al.,
2011). SYMRK consists of a cytosolic kinase domain and an ectodomain
encompassing a malectin-like domain (MLD), GDPC motif, and three
LRRs. Recent work revealed that the MLD domain is released by a cleavage
reaction (Antolin-Llovera et al., 2014); symrk-14 has a mutation within the
GDPC motif, causing a defect in MLD release. Furthermore, a truncated
SYMRK that lacks the MLD domain (SYMRK-DMLD) is able to interact
with NFR5, but is rapidly degraded. Thus, SYMRK-DMLD is required for
IT formation, and control of SYMRK protein turnover may be essential for
balancing the signal transduction pathways of infection and cell division.
3.7 Regulatory Mechanisms for Rhizobial Infection

To date, hyperinfection mutants that have been isolated show more early
infection events, root hair curling and infection foci (Figure 4(2)), and
hyper-IT formation (Table 1(F)e(H)). Unlike AON, although the effects
of excessive rhizobial invasion on plant growth are not yet known, these
mutant phenotypes suggest the existence of a genetic mechanism that reg-
ulates the extent of rhizobial infection. Some researchers have noticed
that more root hair deformation or infection foci tend to appear in several
less-nodulating mutants (Table 1(F)). These increased early infection events
may be caused by failure of infection, suggesting that successful IT formation
may inhibit rhizobial invasion near a preexisting IT. The rst observation of
hyper-IT formation was in the lhk1 mutant, which has a defect in a gene that
encodes a cytokinin receptor (see Section 2.2.2; Murray et al., 2006, 2007).
Very recently, we isolated another two hyper-IT formation mutants in
L. japonicus, daphne (Yoro et al., 2014) and vag1 (see Section 2.2.4; Suzaki
et al., 2014). As these mutants show no or low nodulation, their hyper-IT
phenotype might be due to the failure of nodule development.
In mutants of daphne, which is a unique allele of nin, expression of NIN
in both epidermis and cortex is disorganized, and thereby epidermal infec-
tion seems to be enhanced by a malfunction of NIN working in the cortex.
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Moreover, strong suppression of excessive ITs by overexpression of NIN

indicates the possibility of a NIN-mediated IT inhibition pathway (Yoro
et al., 2014). Consistent with this assumption, in the lhk1 mutant, the inoc-
ulation-dependent expression of NIN is lower than in wild-type (Murray
et al., 2007). VAG1 encodes a subunit of DNA topoisomerase VI potentially
involved in endoreduplication for reinitiating cell division in the root cortex
(see Section 2.2.4; Suzaki et al., 2014). These hyper-IT mutant phenotypes
prompt exploration of the mechanism by which newly divided cortical cells
repress IT formation during early nodule development. This will be consid-
ered as a mechanism for cross talk between infection and nodulation (see
Section 3.6).
On the other hand, another type of hyper-IT M. truncatula mutants, sickle
and efd, form an increased number of nodules (Table 1(G)). In this case,
hyperinfection may be a cause of hypernodulation. Sickle is less sensitive
to ethylene (Penmetsa and Cook, 1997; Penmetsa et al., 2003). Ethylene it-
self can downregulate calcium spiking in root hairs and following rhizobial
infection (Lohar et al., 2009; Oldroyd et al., 2001). In particular, accumula-
tion of ethylene near the protoxylem pole negatively regulates both infec-
tion and cortical cell division, a process mediated by the specic spatial
expression of aminocyclopropane-1-carboxylic acid oxidase (Heidstra
et al., 1997). Therefore, nodules in sickle expand with an aberrant radial
pattern. This inhibition by ethylene is clearly independent from the AON
pathway (Penmetsa et al., 2003). In contrast to the negative role of ethylene
in infection and cell division, EFD has positive roles in nodule differentiation
and bacterial release in indeterminate nodules.
Another negative regulator of rhizobial infection, MtPUB1, was found
by screening for LYK3-interacting proteins (Mbengue et al., 2010).
MtPUB1 is a phosphorylation target protein of MtLYK3. MtPUB1-RNAi
in lyk3 (a weak allele of lyk3) shows increased IT formation together with
an increased number of nodules. In contrast, overexpression of MtPUB1
delays nodulation. Thus, MtPUB1 seems to negatively regulate rhizobial
infection downstream of Nod-factor signaling.
3.8 Intercellular Rhizobial Invasion

The machinery of IT formation could have been co-opted from that of pollen
tube development and root hair elongation, as described above (see Sections
3.1e3.3). In any case, rhizobial invasion through intracellular IT formation
includes complex, step-by-step reactions and is tightly linked with cortical
cell division and strictly controlled (see Sections 3.4e3.7). Some legumes,
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such as Sesbania rostrata, Chamaecrista species, and Aeschynomene sensitiva

(Giraud et al., 2007; Sprent, 2007)), probably have simpler invasion systems,
namely, a root hair-independent intercellular or intracellular invasion mode
(also known as crack entry) (Held et al., 2010). Sesbania rostrata has a dual-
mode invasion system: this species adopts both a root hair-independent crack
entry and a root hair-dependent invasion, depending on whether the soil is
ooded or dry (Goormachtig et al., 2004). Interestingly, some L. japonicus
mutants, in which cortical cell proliferation occurs in the absence of an
epidermal calcium spike, seem to adopt such a root hair-independent invasion
system (Suzaki and Kawaguchi, 2014). Further characterization of this inva-
sion system may contribute to the understanding of the evolution of the
invasion process that leads to IT formation.
4. CONCLUDING REMARKS AND FUTURE

PERSPECTIVES
As we have shown in this review, recent advances in molecular genetic
and biochemical studies have furthered our understanding of the key mech-
anisms involved in nodulation, which fundamentally encompasses nodule
developmental and rhizobial invasion processes. Generally, plants have the
capacity to form new organs from differentiated cells, and nodulation may
be a form of such de novo organogenesis triggered by an external stimulus.
Thus, determination of the mechanisms controlling nodule organogenesis
not only will be of interest to researchers studying plantemicrobe interac-
tions, but also will have a more general relevance to investigation of spatio-
temporal regulatory mechanisms in differentiated cells.
With respect to the origin of root nodule symbiosis, the most plausible
current model suggests that it might have developed through the co-opting
of genes involved in AM symbiosis (see Section 2.2.1). The so-called com-
mon symbiosis pathway genes predominantly function in the rhizobial inva-
sion process, implying that other mechanisms involved, for example, in
plant-rhizobia initial recognition or nodule organogenesis cannot be
explained by an analogy to AM symbiosis. Accommodation of rhizobia is
accomplished by rigid host-symbiont recognition through Nod factor
receptors, which seem to prevent ineffective or pathogenic microbes from
invading host root tissues. Recently, it was shown that Nod factor can
suppress the innate immunity induced by bacterial agellin, even in the
nonlegume Arabidopsis, and that this suppression is mediated by Nod factor
receptor homolog AtLYK3 (Liang et al., 2013). Thus, an important question
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is how the Nod factor receptor has evolved as a regulator that facilitates such
a tight host-symbiont recognition. In addition, the innovation of nodule
organogenesis might have been achieved by co-opting of collaborative
genes that were originally involved in other developmental processes
involving SAM homeostasis and lateral root formation (see Section 2.3.2;
Couzigou et al., 2012; Suzaki et al., 2013b). Future studies focusing on
Nod factor signaling in a variety of plantemicrobe interactions and
conserved mechanisms in the regulation of nodulation and other organs
will undoubtedly shed new light on the evolutionary origin of root nodule
symbiosis.
ACKNOWLEDGMENTS
We thank Momoyo Ito for her help in preparation of the gures; Makoto Hayashi for
providing Mesorhizobium loti expressing DsRED. This work is supported by MEXT/JSPS
KAKENHI, Japan (25114519 to T.S. and 25291066, 22128006 to M.K), and JSPS Grant-
in-Aid for JSPS Fellow (25-3940 to E.Y.).
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Leguminous Plants: Inventors of

2. NODULE DEVELOPMENTAL PROCESS

Figure 1 Nodule morphology. (A) Indeterminate nodule formed in Medicatgo trunca-

rhizobia-colonized cells in the inner region of the nodule, where symbiotic

2.2 Positive Regulation of Nodulation

2.2.2 Cytokinin receptors

receptors, L. japonicus LOTUS HISTIDINE KINASE 1 (LHK1) and M.

2.2.3 Downstream of cytokinin receptors

induced in an MtCRE1-dependent manner (Ariel et al., 2012). Thus, it is

2.2.4 DNA topoisomerase VI

that VAG1 is an essential regulator for nodule organogenesis. VAG1 en-

role of DNA topoisomerase VI in nodule organogenesis was recently

1999); the induced endoreduplication appears to act to stop cell division of

2.3 Negative Regulation of Nodulation: Autoregulation of

2.3.2 Molecular basis of AON

Recently, biochemical analysis has determined the mature form of CLE-

Figure 3 Molecular basis of autoregulation of nodulation (AON). (1) Nodulation

Miyazawa et al., 2010). Moreover, KLV physically interacts with HAR1, at

2.3.2.2 Signaling from shoot to root in AON

seems to have a positive role in nodulation (Chen et al., 2014). Thus, it

2.3.2.3 Site of AON action in the root

control of NIN expression will undoubtedly contribute to our understand-

3. RHIZOBIAL INVASION PROCESS

growth may support IT membrane organization and inner growth of the IT

inux, ROS accumulation is signicantly decreased by Nod factor treatment

3.2 IT Initiation and IT Membrane Characterization

Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria

(A) Impared in root hair deformation

Takuya Suzaki et al.

Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria

Takuya Suzaki et al.

Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria

Takuya Suzaki et al.

Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria

Takuya Suzaki et al.

(A) (B) (C) (D) (E)

Figure 5 IT formation in Lotus japonicus root inoculated with Mesorhizobium loti

components. Electron microscopic observation of ITs suggests that the IT

rhizobial inoculation. Furthermore, rapid colocalization of LYK3 and

3.3 IT Growth and Elongation

microtubules have provided new insights into the dynamic morphology of

3.4 Cytoplasmic Bridge or Preinfection Thread (PIT) in

3.5 Bacterial Release

symbiosome fraction (Perotto et al., 1991). Several mutations involved in

3.6 Cross Talk between Rhizobial Infection and Nodule

3.7 Regulatory Mechanisms for Rhizobial Infection

Moreover, strong suppression of excessive ITs by overexpression of NIN

3.8 Intercellular Rhizobial Invasion

such as Sesbania rostrata, Chamaecrista species, and Aeschynomene sensitiva

4. CONCLUDING REMARKS AND FUTURE

autoregulation genes interchangeably drive phloem-specic expression in transgenic

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