Actinorhizal root nodule symbioses: what is signalling telling on the origins of nodulation?

Actinorhizal root nodule symbioses: what is signalling telling onthe origins of nodulation?Sergio Svistoonoff 1, Valerie Hocher1 and Hassen Gherbi1

Available online at www.sciencedirect.com

ScienceDirect

Two groups of bacteria are able to induce the formation of

nitrogen-fixing nodules: proteobacteria called rhizobia, which

associate with Legumes or Parasponia and actinobateria from

the genus Frankia which are able to interact with �220 species

belonging to eight families called actinorhizal plants. Legumes

and different lineages of actinorhizal plants differ in bacterial

partners, nodule organogenesis and infection patterns and

have independent evolutionary origins. However, recent

technical achievements are revealing a variety of conserved

signalling molecules and gene networks. Actinorhizal

interactions display several primitive features and thus provide

the ideal opportunity to determine the minimal molecular toolkit

needed to build a nodule and to understand the evolution of

root nodule symbioses.

Addresses

Institut de Recherche pour le Developpement (IRD), Unite mixte de

recherche DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier

Cedex 5, France

Corresponding author: Svistoonoff, Sergio ([email protected])1 Present address: Institut de Recherche pour le Developpement (IRD),

Laboratoire des Symbioses Tropicales et Mediterraneennes, TA A-82/J,

Campus International de Baillarguet, 34398 Montpellier Cedex 5,

France.

Current Opinion in Plant Biology 2014, 20:11–18

This review comes from a themed issue on Biotic interactions 2014

Edited by Makoto Hayashi and Martin Parniske

1369-5266/$ – see front matter, # 2014 Elsevier Ltd. All rights

reserved.

http://dx.doi.org/10.1016/j.pbi.2014.03.001

IntroductionRoot nodule symbiosis (RNS) is a remarkable adaptation

which enables plants to acquire nitrogen directly from the

atmosphere. These symbioses involve the development

of specialised organs, the root nodules, in which nitrogen-

fixing bacteria are accommodated intracellularly and pro-

vided with a suitable environment for the efficient con-

version of atmospheric nitrogen into ammonia and its

transfer to the plant. By far the most widely studied RNS

is that involving rhizobia and legumes, and particularly

two model species, Lotus japonicus and Medicago truncatula[1]. However RNS is not limited to legumes: rhizobia can

also form nodules on Parasponia sp. (Cannabaceae) and a

diverse group of plants known as actinorhizal is able to

develop nodules in symbiosis with a very different kind of

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bacteria, the actinobacterium Frankia. Little is known

about the signalling mechanisms involved in the for-

mation of nodules in actinorhizal plants or Parasponia,

partly because most of these plants are woody shrubs or

trees which are unsuitable for genetic approaches, and

because of the recalcitrance of Frankia to stable genetic

transformation [2]. However, recent technical progress

including the sequencing of several Frankia genomes

[3��], transcriptome studies [4,5��,6��,7,8��], and efficient

plant transformation and gene silencing procedures in

Parasponia and several actinorhizal species [9–12] now

make it possible to better understand these atypical RNS.

Here we focus on the early steps of actinorhizal nodula-

tion and show that very diverse interactions in terms of

plants, bacterial partners and infection mechanisms often

recruited similar signalling molecules and orthologous

sets of genes (Box 1).

Pre-infection signallingThe existence of a molecular dialogue involving molecules

related to legume flavonoids and rhizobial lipochitooligo-

saccharides called Nod factors (NFs) has long been pos-

tulated for actinorhizal RNS. On the plant side, the

secretion of signalling molecules by plant roots was

recently examined in the Casuarina species (Fagales) in

which exposure of the corresponding Frankia to root

exudates was shown to increase its growth rate, to cause

changes in its surface properties, and to favour the infection

and nodulation process [15��]. Among the molecules pre-

sent in Casuarina root exudates are flavonoids, whose role

was studied in Casuarina glauca. Genes involved in the

biosynthesis of flavonoids are upregulated during the early

steps of nodulation in Casuarina [16] and silencing of the

chalcone synthase gene, the first committed step of the

flavonoid biosynthetic pathway has strong negative

impacts on nodulation [17��], indicating that flavonoids

play a critical role in the Casuarina-Frankia interaction.

The role of flavonoids has also been studied in Myrica gale(Fagales), where they were shown to promote Frankiagrowth and nitrogen fixation only in compatible strains

[18,19]. On the bacterial side, Frankia signalling molecules

were partially purified using bioassays based on root hair

deformation of Alnus glutinosa (Fagales) and nodulation

kinetics of Discaria trinervis (Rosales). Surprisingly these

factors had different biochemical properties compared to

NFs and canonical NodA genes which are indispensable for

the production of NFs in rhizobia were not found in the

genome of the corresponding Frankia strains [4,20–22,23��]. Genes distantly related to the other rhizobial

Nod genes are present in Frankia but they are not clustered


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12 Biotic interactions 2014

Box 1 Actinorhizal nodulation: origin and diversity

Actinorhizal RNS involve �220 species belonging to eight families of

the orders, Fagales, Cucurbitales and Rosales which, together with

Fabales, comprise the so-called nitrogen-fixing clade (=Fabids),

indicating that all plants able to form RNS share a recent common

ancestor. However, the aptitude to form nodules is not an ancestral

character: the distribution of nodulating clades among Fabids points to

at least nine independent acquisitions of this trait in different lineages

of Fabids [13]. The anatomy and development of the nodule are among

the main features which differentiate legume from non-legume RNS.

Legume nodules are novel organs initiated by cortical cell divisions and

have a peripheral vascular bundle, whereas in actinorhizal plants and

Parasponia, nodules are modified lateral roots with a central

vasculature and are initiated by cell divisions in the pericycle.

Actinorhizal Fagales are characterised by range of relatively advanced

features reminiscent of model legumes: a complex root hair infection

process involving the deformation of root hairs and the formation of

infection threads and the involvement of cortical cell divisions which

give rise to a transient symbiotic organ, the prenodule. In addition the

corresponding Frankia strains belong to the same phylogenetic group

and generally can only nodulate actinorhizal Fagales. On the other

hand, actinorhizal Rosales and probably actinorhizal Cucurbitales are

infected through the more primitive intercellular infection pathway

which does not involve root hair deformation or cortical cell divisions

and are able to interact with Frankia strains belonging to phylogen-

etically distant groups [14] (Figure 1).

within a symbiotic island and are not upregulated under

symbiotic conditions [4,23��], suggesting that they are not

involved in the synthesis of signalling molecules. In C.glauca, we tried a different approach by designing a bioas-

say based on plant genes expressed specifically at early

stages of RNS. In legumes, two genes have been widely

used as markers of NF recognition: MtENOD11 [24] and

Nodule INception (=NIN) [25]. A ProMtENOD11:GUS fusion

was introduced in C. glauca. Although ProMtENOD11 was

active in infected plant cells, it could not be activated at

pre-infection stages or by cell-free extracts from Frankia,

indicating that the transcriptional up-regulation of ProM-tENOD11 preceding microbial infection is not conserved

between M. truncatula and C. glauca [26]. Using a tran-

scriptomic approach, we were able to identify CgNIN, the

putative ortholog of NIN in C. glauca [6��,27]. We observed

that ProCgNIN was active a few hours after inoculation with

Frankia or with a cell-free supernatant obtained from a

Frankia culture (Svistoonoff et al., unpublished). We are

currently using a bioassay based on plants expressing a

ProCgNIN:GFP fusion to identify Frankia signalling mol-

ecules.

Signalling related to infectionLike in rhizobial symbioses, successful mutual recognition

is followed by the penetration of symbiotic bacteria into

plant tissues. Among the signalling molecules which may

play a role at this stage, the phytohormone auxin is one of

the best characterised in actinorhizal symbioses. In C.glauca, the inhibition of auxin influx using naphthoxyacetic

acid (1-NOA) has a negative effect on nodulation [28].

Auxins are produced by Frankia and have been detected in


Frankia-infected cells. Infected cells also express an auxin

influx carrier (CgAUX1) whereas a PIN1-like auxin efflux

carrier is present in surrounding uninfected cells. Compu-

ter simulations indicate that this specific pattern of trans-

porter activity leads to auxin accumulation in infected cells,

where auxins are assumed to induce changes in gene

expression, cell metabolism, or in the cell wall properties

necessary for infection by Frankia [28–30]. One of the

genes targeted by the auxin signalling pathway could be

Cg12, a Casuarina gene encoding a subtilisin-like protease

(=subtilase), which is specifically expressed during the

infection by Frankia but not during the formation of ecto

or endo mycorrhizae [31,32]. When a ProCg12:GFP con-

struct was introduced in M. truncatula, GFP expression was

detected in cells infected by rhizobia [33]. Similarly, the

expression of the M. truncatula gene MtENOD11 in

infected cells is conserved in Casuarina [26] indicating

that the two symbioses share common gene regulation

mechanisms during bacterial infection. Similar results were

obtained with ProMtENOD11:GUS and ProCg12:GFP con-

structs in Discaria (Rosales), an actinorhizal plant infected

through the primitive intercellular pathway [10]. Interest-

ingly these promoters were activated in cells surrounding

the intercellular Frankia hyphae indicating that intercel-

lular and intracellular infection pathways share molecular

components. Subtilases similar to Cg12 and expressed

specifically in infected tissues are present in Alnus [34]

and Discaria (Dt12, Svistoonoff et al., unpublished results)

and can be found in M. truncatula [35] and L. japonicus[36,37] suggesting that these subtilases perform a specific

function related to infection shared by all RNS [32]. Using

comparative transcriptomics, we recently identified genes

upregulated in actinorhizal, rhizobial, and arbuscular

mycorrhizal (AM) symbioses and, remarkably, we found

that the majority of these genes encode subtilases and

other proteases. This suggests that all RNS recruit parts of

the ancient mechanisms used by plants to accommodate

AM fungi [8��].

Signal transduction pathwaysSignalling pathways involved in Legume RNS are begin-

ning to be well understood [1]. Remarkably, these path-

ways also have much in common with the ancient

programme allowing plants to form AM. Transcriptomic

approaches have shown that most genes belonging to this

common signalling pathway (CSP) are expressed during

actinorhizal nodulation in two Fagales (Alnus and Casuar-ina) and Datisca (Cucurbitales) [5��,6��,8��] but detailed

functional characterisation has only been undertaken of

two genes: SymRK and CCaMK. SymRK is needed for

actinorhizal nodulation in Fagales [38] and Cucurbitales

[11]. SymRK genes from actinorhizal plants and closely

related species are able to fully complement the L. japo-nicus symrk mutant; the characteristic presence of three

LRR motifs in these genes — as opposed to two LRR

motifs in non-Rosid SymRK homologs — was assumed to

be one of the evolutionary events which enabled Fabids to


Actinorhizal signalling and the origins of nodulation Svistoonoff, Hocher and Gherbi 13

Figure 1

Casuarinaceae

Betulaceae

Myricaceae

Datiscaceae

Coriariaceae

Elaeagnaceae

Rhamnaceae

Fabaceae60 MY

FAGALES

CUCURBITALES

ROSALES

FABALES

Cannabaceae

Casuarina (18/18)Allocasuarina (54/59)Ceuthostoma (2/2)Gymnostoma (18/18)Alnus (47/47)

Comptonia (1/1)Myrica (28/60)

Datisca (2/2)

Coriaria (16/16)

Elaeagnus (35/45)Hippophae (2/3)Shepherdia (2/3)Adolphia (1/1)Colletia (4/17)Discaria (5/10)Kentrothamnus (2/2)Retanilla (2/3)Telguenea (1/1)Trevoa (2/6)Ceanothus (31/55)

Parasponia

Frankia clade III"Elaeagnus "

Rhizobia

Rosaceae Dryas (1/3)Purshia (2/4)Cowania (1/25)Cercocarpus (4/20)Chamaebatia (1/2)

39 MY

55 MY

23 MY

>700 genera> 20.000 sp.

88 MY

88 MY

65 MY

39 MY

39 MY

**

Frankia clade II"Uncultured"

ROOT HAIR - C+P

INTERCELLULAR (?)- P

INTERCELLULAR1 - P

ROOT HAIR - C

CRACK ENTRY - C

INTERCELLULAR- C

CRACK ENTRY- C+P

ORDER

Frankia clade I"Alnus "

*

*

*

*

*

*

*

FAMILY GENUS

L

INFECTIONMECHANISM

NODULEANATOMY

Current Opinion in Plant Biology

Phylogeny and diversity of RNS. Phylogenetic relationships between plant families containing species able to form root nodule symbioses and the

corresponding bacterial strains. Solid arrows show the broad correspondence between plant orders and Frankia clades; dashed arrows show

exceptional associations. Circles indicate the putative evolutionary origins of actinorhizal (pink) or rhizobial nodulation (red). Dates correspond to the

oldest fossil evidence for each family. Genera in which the common symbiosis signalling pathway was shown to be involved in nodulation are in bold;

red labels specify the availability of transcriptomic resources. Numbers in parenthesis indicate the total number of species and the number of species

known to be nodulated. Schematic views of infection mechanisms and the anatomy of nodules are shown for each group. Asterisks indicate the first

cell divisions involved in the nodulation process which occur in the cortex (C), the pericycle (P) or both tissues (C + P). When present, infection threads

are shown in blue. Nodule apical meristems are in grey and tissues colonised by Frankia or rhizobia are in pink and red respectively. 1Actinorhizal

Rosales and probably actinorhizal Cucurbitales are infected through the intercellular infection pathway. L. lenticels, typical of actinorhizal

Cucurbitales.Modified after [13,14,46��,53–56].

www.sciencedirect.com Current Opinion in Plant Biology 2014, 20:11–18


Figure 2

Legume symbioses Actinorhizal symbioses

Gene name Expression innodule vs root

Mutantphenotype

Species Expression innodule vs root

NFR1 / LYK3 root nod-/inf-(for Lyk3) Ag, Cg*, Dt*, Dg similarNFR5 / NFP similar nod- Cg*, Dt*, Dg similar

SymRK / DMI2 similar nod-/myc- Ag, Cg*, Dg* similar

CASTOR / POLLUX / DMI1 nod nod-/myc- Cg / Dg nod / nd

NUP133 root nod-/myc- Cg / Dg similar / nd

CCaMK / DMI3 nod nod-/myc- Ag, Cg*, Dt* / Dg similar / nd

IPD3 / CYCLOPS nod nod- (S)/myc- Cg / Dg similar / ndCRE1 / HK1 similar nod- Cg / Dg root / nd

NSP1 nod nod-/myc- Ag / Dg similar / nd

NSP2 nod nod-/myc- Dg nd

ERN1 similar inf- Cg* / Dg similar / nd

ERF1 root Ag similarNIN nod nod- (IT, NP)- Cg*, Dg nod

NF -Y Complex nod Ag, Cg / Dg similar / nd

SYMREM1 nod inf- Ag, Cg, Dg nod

CERBERUS/LIN nod inf-/myc- Cg / Dg nod / similar

RIT/NAP1 similar inf- Dg similar

PIR1 similar inf- Dg ndVAPYRIN nod inf-/myc- Cg, Dg nod

RPG nod inf- Ag nod

PUB1 nod Cg, Dg root

HMGR1 root Ag, Cg / Dg nod / nd

LATD/NIP similar nod- Ag, Cg similar


Actinorhizal putative orthologues of legume genes encoding proteins involved in Legume RNS. –: defective; nod: nodule enhanced; myc�: defective in

mycorhiza; inf: infection; S: symbiosome; IT: infection thread; NP: nodule primordium; nd: not determined; Ag: Alnus; Cg: Casuarina glauca; Dg:

Datisca glomerata; Dt: Discaria trinervis. *: functional characterisation available or in progress.

Modified from [1,5��,6��,43].

accommodate symbiotic N2-fixing bacteria [11,39]. An

essential role in nodulation was also demonstrated for

CCaMK in Casuarina [40��]. Genes from the CSP were

also recruited for nodulation in Parasponia [12] indicating

that each independent acquisition of nodulation in differ-

ent lineages of Fabids made use of the same symbiotic

signalling pathway. An intriguing question regarding genes

of the CSP is the involvement of the LysM receptors,

which are responsible for the specific recognition of rhi-

zobial NFs in model legumes and in Parasponia [12,25]: a

role of actinorhizal LysM receptors in actinorhizal nodula-

tion would be a sign that Frankia, like rhizobia, uses

chemically related to NFs as signalling molecules.

The involvement of other signalling networks not related

to AM is not well understood. In legumes, many mutants

whose nodulation ability is affected can still form AM [1].


Putative orthologs of the corresponding genes have been

found in actinorhizal Fagales and Cucurbitales (Figure 2).

Among these genes is NIN, a transcription factor crucial

for Legume nodulation [1,27]. CgNIN and DgNIN, the

putative orthologs of NIN are expressed in nodules of

Casuarina and Datisca [5��,6��,8��] and we recently

showed that the CgNIN is essential for nodule formation

in Casuarina and is able to complement a legume nin

mutant for nodule organogenesis (Svistoonoff et al.,unpublished) suggesting that even signalling pathways

which are not derived from the ancient AM symbioses

were recruited by lineages of nodulators which have

independent evolutionary origins [13].

Signalling during nodule organogenesisAs mentioned above, a fundamental difference between

actinorhizal plants and legumes is nodule organogenesis:



cortical cell divisions give rise to nodules with a periph-

eral vasculature in legumes whereas actinorhizal nodules

originate from the pericycle and develop a central vas-

cular bundle. Auxin is a central player in lateral root

formation and is therefore expected to be equally import-

ant in actinorhizal root-like nodules. Treatments with

exogenous auxins lead to the formation of thick lateral

roots resembling nodules in actinorhizal Fagales [32,41]

and, as mentioned above, an auxin influx inhibitor per-

turbs the formation of nodules in Casuarina [28]. Because

classical markers of auxin perception are not active in

actinorhizal nodules in Casuarina [30], it is difficult to

provide evidence linking auxins with the early steps of

nodule organogenesis. Furthermore, given that nodule

organogenesis is concomitant with bacterial infection, it

was difficult to distinguish between the molecular mech-

anisms specifically involved in one of these two pro-

cesses. However spontaneous nodulation, which has

recently been achieved in a variety of gain-of-function

mutants or overexpressors in model legumes [42–44]

provides a powerful new tool to study the specific path-

ways involved in nodule formation. Among the genes

involved, CCaMK appears to play a pivotal role in several

interactions: autoactive forms of CCaMK induce the

formation of spontaneous nodules in model legumes

but also in Parasponia [12], actinorhizal Fagales and

Rosales [40��]. The same genetic pathway based on the

activation of CCaMK was therefore used to trigger

nodule organogenesis in all RNS despite the striking

differences in origin and anatomy described above.

Spontaneous nodulation was also described in legumes

in a mutant of the cytokinin receptor CRE1/LHK [44],

and overexpression of the above mentioned NIN tran-

scription factor and one of its targets, NF-YB triggers

the formation of bumps reminiscent of nodule primor-

dia [43]. In model legumes these three proteins are

involved in the same pathway the main outcome of

which is the activation of cortical cell divisions

mediated by cytokinins [43]. We have shown that

NIN is necessary for Casuarina nodulation (Svistoonoff

et al., unpublished results) and that homologs of, NINand NF-YB are present in Casuarina [6��], where this

pathway is possibly involved in the formation of pre-

nodules which originate from cortical cell divisions. The

presence of these genes in Datisca [5��] is more intri-

guing because prenodules are not formed in actinorhizal

Cucurbitales and cortical cells are not involved in

nodule organogenesis. Overexpressors and gain-of-func-

tion versions of these genes could help to clarify the role

of this pathway related to cytokinin in actinorhizal

nodule organogenesis.

Concluding remarksCompared to model legumes, our knowledge of actinor-

hizal plants and Parasponia is sparse and limited to a few

species. Nevertheless, a broad trend can be inferred from

promoter studies, the functional characterisation of sym-


biotic genes, and the transcriptomic data described in this

paper: the recruitment of similar signalling molecules and

homologous sets of genes through interactions which

have independent evolutionary origins and differ at many

levels. As we have seen, this ‘deep homology’ between

plants able to form nodules [13] goes beyond the CSP and

possibly reflects evolutionary constraints. In other words,

the evolution of Fabids created an excellent toolkit to

build nitrogen-fixing nodules which was used each time

the ability to nodulate was acquired. This counters a

common misconception in the literature: not only leghae-

moglobins but many other genes usually only associated

with legume nodules are a feature of the whole Fabids

clade. However, if many key determinants are shared,

how can the variety of RNS be explained? One way is to

consider the different RNS as a series of snapshots of the

same evolutionary sequence affecting several characters

(Figure 3). In this series, advanced interactions are typi-

fied by model legumes; primitive interactions by actinor-

hizal Rosales and Cucurbitales, and between the two are

actinorhizal Fagales, Parasponia and various atypical

legume species like Arachis, Sesbania, Mimosa or Aeschy-nomene harbouring mixtures of advanced, intermediate,

and ancestral traits. The ‘snapshot’ hypothesis is sup-

ported by recent studies showing that ancestral traits like

intercellular infection or lateral root-like nodules are

present even in advanced model legumes and become

apparent — often at very low frequencies — in mutants

[45��,46��]. This view offers the exciting opportunity to

go back in time to understand how the ancestors of model

legumes successively improved their ability to nodulate.

The most primitive group of plants and symbionts is

perhaps one of the most interesting, since this is the

closest state to the original asymbiotic condition in which

only the minimal signalling toolkit was used to accom-

modate symbiotic bacteria. Among the groups of plants

listed in Figure 3, actinorhizal Rosales, which are infected

intercellularly without the involvement of transcellular

infection threads, is the group showing ancestral charac-

teristics for all criteria. The intermediate group is also of

particular interest to understand transitional stages: for

instance, prenodules, which are present in Parasponia and

Fagales, are considered to be a simple ancestral version of

legume nodules [47]; and in contrast to model legumes,

Parasponia appears to use the same receptor to recognise

both rhizobial and AM signalling molecules [12]. Also in

this group is Aeschynomene which is able to recognise

rhizobia without the intervention of NFs [48]. Another

interesting observation regarding non-legume RNS is the

prevalence of trees and shrubs (25 out 26 genera,

Figure 1). One possible explanation is the slower

mutation rate generally observed in woody species com-

pared to herbaceous species [49]: as a consequence,

ancient traits that predispose Fabids for nodulation are

more likely to remain functional in trees. Conversely, as

previously suggested [13,50], this hypothesis also

indicates that the basic toolkit needed for nodulation is



Figure 3

Evolutionary sequence

Ground state Intermediate Advanced

Cri

teri

a

Infection Intercellular Crack entry Root hair

Rosales Ø, Cucurbitales ‡ Fagales ‡Mimosa Ø, Lotus mutants Ø Aeschynomene Ø, Parasponia

‡, Arachis ‡, Sesbania ‡1,Neptunia‡, Lotus mutants ‡

Model legumes‡,Sesbania‡1

Tissuesinvolved

Pericycle Pericycle & cortex (prenodule) Cortex

Rosales2 & Cucurbitales FagalesMedicago mutant Parasponia All legumes

Specificity Poor Intermediate High

Rosales & Cucurbitales, Myrica Alnus CasuarinaParasponia, Vigna Arachis, Aeschynomen e2 Most Legumes,

Aeschynomene3

NF signalling Independent of NFs NF

? ?Aeschynomene3, Lotus mutants Most Legumes,

Parasponia

N2 fixation Free living bacteria Fixation threads Bacteroids

Frankia All Actinorhizal plantsAzorhizobium, Bradyrhizobium,β-rhizobia

Parasponia, Chamaecrista Model legumes,Aeschynomene


Evolution of RNS according to different criteria. Different types of RNS were classified as ground state, intermediate or advanced according to five

criteria: the infection mechanism, the tissues where the first cell divisions occur, the strain specificity, the involvement of NFs, and the structures in

which nitrogen fixation occurs. Examples are given for each criterion. Model legumes exhibit advanced characteristics for all criteria. Intermediate or

ancestral traits are naturally present in some non-model legumes, Parasponia and actinorhizal plants. Intermediate or ancestral traits can also be

artificially obtained in mutants of model legumes. Symbols indicate the presence (^) or absence (Ø) of infection threads. 1Sesbania is infected by crack

entry when plants are cultivated in hydroponics. 2Except in Ceanothus griseus where cortical cell divisions occur but do not become infected.3Aeschynomene species which do not rely on NFs for nodulation have narrower strain specificity than NF-dependent species.

Based on [14,46��,48,57].

probably functional in many non-nodulating Fabids trees.

Powerful genomics and metabolomics tools are now

available to test this hypothesis by comparing closely

related species which differ in their ability to nodulate

[51�]. In this respect, we believe the neglected Rosales

deserve increased attention for two main reasons: first,

this is the group in which the most primitive forms of

rhizobial and actinorhizal RNS are represented and sev-

eral genome sequences of non-nodulating Rosales, in-

cluding trees [52], are now available. Second, RNS

evolved three to five times independently within this

order [13] suggesting that non-nodulating Rosales form

one of the easiest groups to target in order to achieve the

old ambition of transferring the ability to form RNS.


AcknowledgementsIRD, the French National Research Agency (ANR-2010 BLAN-1708-01,ANR-12-BSV7-0007-01) and the United States Department of Agriculture(USDA NIFA 2010-65108-20581) provided financial support. We thank A.Champion, L. Laplaze, E. Giraud and L. Wall for helpful discussions andcritical review of the manuscript.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest

�� of outstanding interest

1. Oldroyd GED: Speak, friend, and enter: signalling systems thatpromote beneficial symbiotic associations in plants. Nat RevMicrobiol 2013, 11:252-263.


http://refhub.elsevier.com/S1369-5266(14)00029-6/sbref0005




2. Kucho K, Kakoi K, Yamaura M, Iwashita M, Abe M, Uchiumi T:Codon-optimized antibiotic resistance gene improvesefficiency of transient transformation in Frankia. J Biosci 2013,38:713-717.

3.��

Tisa LS, Beauchemin N, Gtari M, Sen A, Wall LG: What stories canthe Frankia genomes start to tell us? J Biosci 2013, 38:719-726.

This paper describes the conclusions drawn from the analysis of elevenFrankia genomes, most notably regarding genes similar to rhizobial NodA/B/C: genes similar to NodA are absent; genes distantly related to NodB/C are present but did not cluster in a symbiotic island like in mostrhizobial genomes.

4. Alloisio N, Queiroux C, Fournier P, Pujic P, Normand P, Vallenet D,Medigue C, Yamaura M, Kakoi K, Kucho K: The Frankia alnisymbiotic transcriptome. Mol Plant Microbe Interact 2010,23:593-607.

5.��

Demina IV, Persson T, Santos P, Plaszczyca M, Pawlowski K:Comparison of the nodule vs. root transcriptome of theactinorhizal plant Datisca glomerata: actinorhizal nodulescontain a specific class of defensins. PLOS ONE 2013,8:e72442.

This paper provides a detailed list of genes expressed during actinorhizalnodulation in Datisca (Cucurbitales). Together with Ref. [5��], it revealsthat putative ortologues of most genes involved in rhizobial RNS arepresent in several actinorhizal species, suggesting thus that all RNS arelargely based on the same molecular toolkit.

6.��

Hocher V, Alloisio N, Auguy F, Fournier P, Doumas P, Pujic P,Gherbi H, Queiroux C, Da Silva C, Wincker P et al.:Transcriptomics of actinorhizal symbioses reveals homologsof the whole common symbiotic signaling cascade. PlantPhysiol 2011, 156:700-711 See annotation to Ref. [5��].

7. Mastronunzio JE, Tisa LS, Normand P, Benson DR: Comparativesecretome analysis suggests low plant cell wall degradingcapacity in Frankia symbionts. BMC Genomics 2008, 9:47.

8.��

Tromas A, Parizot B, Diagne N, Champion A, Hocher V, Cissoko M,Crabos A, Prodjinoto H, Lahouze B, Bogusz D et al.: Heart ofendosymbioses: transcriptomics reveals a conserved geneticprogram among arbuscular mycorrhizal, actinorhizal andlegume-rhizobial symbioses. PLOS ONE 2012, 7:e44742.

A comparative analysis of gene expression between Casuarina, Medi-cago and rice allowed the identification of a common set of genesinduced in AM, rhizobial and actinorhizal endosymbioses and providean illustration of how these three different symbioses recruit similar sets ofgenes. Genes encoding proteases compose the biggest class.

9. Gherbi H, Nambiar-Veetil M, Zhong C, Felix J, Autran D, Girardin R,Vaissayre V, Auguy F, Bogusz D, Franche C: Post-transcriptionalgene silencing in the root system of the actinorhizal treeAllocasuarina verticillata. Mol Plant Microbe Interact 2008,21:518-524.

10. Imanishi L, Vayssieres A, Bogusz D, Franche C, Wall LG,Svistoonoff S: Transformed hairy roots of Discaria trinervis: avaluable tool for studying actinorhizal symbiosis in the contextof intercellular infection. Mol Plant Microbe Interact 2011,24:1317-1324.

11. Markmann K, Giczey G, Parniske M: Functional adaptation of aplant receptor-kinase paved the way for the evolution ofintracellular root symbioses with bacteria. PLoS Biol 2008,6:e68.

12. Op den Camp R, Streng A, De Mita S, Cao Q, Polone E, Liu W,Ammiraju JSS, Kudrna D, Wing R, Untergasser A et al.: LysM-typemycorrhizal receptor recruited for rhizobium symbiosis innonlegume Parasponia. Science 2011, 331:909-912.

13. Doyle JJ: Phylogenetic perspectives on the origins ofnodulation. Mol Plant Microbe Interact 2011, 24:1289-1295.

14. Pawlowski K, Demchenko KN: The diversity of actinorhizalsymbiosis. Protoplasma 2012, 249:967-979.

15.��

Beauchemin NJ, Furnholm T, Lavenus J, Svistoonoff S, Doumas P,Bogusz D, Laplaze L, Tisa LS: Casuarina root exudates alter thephysiology, surface properties, and plant infectivity of Frankiasp. strain CcI3. Appl Environ Microbiol 2012, 78:575-580.

This paper demonstrates that exposure of Frankia to root exudates fromCasuarina has several physiological effects and favours the nodulationprocess.


16. Auguy F, Abdel-Lateif K, Doumas P, Badin P, Guerin V, Bogusz D,Hocher V: Activation of the isoflavonoid pathway inactinorhizal symbioses. Funct Plant Biol 2011, 38:690-696.

17.��

Abdel-Lateif K, Vaissayre V, Gherbi H, Verries C, Meudec E,Perrine-Walker F, Cheynier V, Svistoonoff S, Franche C, Bogusz Det al.: Silencing of the chalcone synthase gene in Casuarinahighlights the important role of flavonoids during nodulation.New Phytol 2013, 199:1012-1021.

This paper describes how nodulation of Casuarina is affected when thechalcone synthase gene is silenced pointing to a role for flavonoids in theearly stages of actinorhizal nodulation.

18. Popovici J, Comte G, Bagnarol E, Alloisio N, Fournier P, Bellvert F,Bertrand C, Fernandez MP: Differential effects of rare specificflavonoids on compatible and incompatible strains in theMyrica gale–Frankia actinorhizal symbiosis. Appl EnvironMicrobiol 2010, 76:2451-2460.

19. Popovici J, Walker V, Bertrand C, Bellvert F, Fernandez MP,Comte G: Strain specificity in the Myricaceae–Frankiasymbiosis is correlated to plant root phenolics. Funct Plant Biol2011, 38:682-689.

20. Ceremonie H, Debelle F, Fernandez MP: Structural andfunctional comparison of Frankia root hair deforming factorand rhizobia Nod factor. Can J Bot 1999, 77:1293-1301.

21. Gabbarini L, Wall L: Analysis of nodulation kinetics in Frankia–Discaria trinervis symbiosis reveals different factors involvedin the nodulation process. Physiol Plant 2008, 133:776-785.

22. Gabbarini L, Wall L: Diffusible factors involved in earlyinteractions of actinorhizal symbiosis are modulated by thehost plant but are not enough to break the host range barrier.Funct Plant Biol 2011, 38:671-681.

23.��

Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N,Bagnarol E, Bassi CA, Berry AM, Bickhart DM, Choisne N:Genome characteristics of facultatively symbiotic Frankia sp.strains reflect host range and host plant biogeography.Genome Res 2007, 17:7-15 See annotation to Ref. [3��].

24. Boisson-Dernier A, Andriankaja A, Chabaud M, Niebel A,Journet EP, Barker DG, de Carvalho-Niebel F: MtENOD11 geneactivation during rhizobial infection and mycorrhizalarbuscule development requires a common AT-rich-containing regulatory sequence. Mol Plant Microbe Interact2005, 18:1269-1276.

25. Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y,Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N: Plantrecognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 2003, 425:585-592.

26. Svistoonoff S, Sy MO, Diagne N, Barker DG, Bogusz D, Franche C:Infection-specific activation of the Medicago truncatulaENOD11 early nodulin gene promoter during actinorhizal rootnodulation. Mol Plant Microbe Interact 2010, 23:740-747.

27. Schauser L, Roussis A, Stiller J, Stougaard J: A plant regulatorcontrolling development of symbiotic root nodules. Nature1999, 402:191-194.

28. Peret B, Swarup R, Jansen L, Devos G, Auguy F, Collin M, Santi C,Hocher V, Franche C, Bogusz D: Auxin influx activity isassociated with Frankia infection during actinorhizal noduleformation in Casuarina. Plant Physiol 2007, 144:1852-1862.

29. Peret B, Svistoonoff S, Lahouze B, Auguy F, Santi C, Doumas P,Laplaze L: A role for auxin during actinorhizal symbiosesformation? Plant Signal Behav 2008, 3:34-35.

30. Perrine-Walker F, Doumas P, Lucas M, Vaissayre V,Beauchemin NJ, Band LR, Chopard J, Crabos A, Conejero G,Peret B et al.: Auxin carriers localization drives auxinaccumulation in plant cells infected by Frankia in Casuarinaactinorhizal nodules. Plant Physiol 2010, 154:1372-1380.

31. Laplaze L, Ribeiro A, Franche C, Duhoux E, Auguy F, Bogusz D,Pawlowski K: Characterization of a Casuarina nodule-specificsubtilisin-like protease gene, a homolog of Alnus ag12. MolPlant Microbe Interact 2000, 13:113-117.

32. Svistoonoff S, Laplaze L, Auguy F, Runions J, Duponnois R,Haseloff J, Franche C, Bogusz D: cg12 expression is specifically




























































































































linked to infection of root hairs and cortical cells duringCasuarina and Allocasuarina verticillata actinorhizal noduledevelopment. Mol Plant Microbe Interact 2003, 16:600-607.

33. Svistoonoff S, Laplaze L, Liang J, Ribeiro A, Gouveia MC, Auguy F,Fevereiro P, Franche C, Bogusz D: Infection-related activation ofthe cg12 promoter is conserved between actinorhizal andlegume-rhizobia root nodule symbiosis. Plant Physiol 2004,136:3191-3197.

34. Ribeiro A, Akkermans AD, van Kammen A, Bisseling T,Pawlowski K: A nodule-specific gene encoding a subtilisin-likeprotease is expressed in early stages of actinorhizal noduledevelopment. Plant Cell 1995, 7:785-794.

35. Benedito VA, Torres-Jerez I, Murray JD, Andriankaja A, Allen S,Kakar K, Wandrey M, Verdier J, Zuber H, Ott T et al.: A geneexpression atlas of the model legume Medicago truncatula.Plant J Cell Mol Biol 2008, 55:504-513.

36. Verdier J, Torres-Jerez I, Wang M, Andriankaja A, Allen SN, He J,Tang Y, Murray JD, Udvardi MK: Establishment of the Lotusjaponicus gene expression atlas (LjGEA) and its use to explorelegume seed maturation. Plant J Cell Mol Biol 2013, 74:351-362.

37. Takeda N, Sato S, Asamizu E, Tabata S, Parniske M: Apoplasticplant subtilases support arbuscular mycorrhiza developmentin Lotus japonicus. Plant J 2009, 58:766-777.

38. Gherbi H, Markmann K, Svistoonoff S, Estevan J, Autran D,Giczey G, Auguy F, Peret B, Laplaze L, Franche C et al.: SymRKdefines a common genetic basis for plant root endosymbioseswith arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria. Proc Natl Acad Sci U S A 2008, 105:4928-4932.

39. Markmann K, Parniske M: Evolution of root endosymbiosis withbacteria: how novel are nodules? Trends Plant Sci 2009, 14:77-86.

40.��

Svistoonoff S, Benabdoun FM, Nambiar-Veetil M, Imanishi L,Vaissayre V, Cesari S, Diagne N, Hocher V, de Billy F, Bonneau Jet al.: The independent acquisition of plant root nitrogen-fixingsymbiosis in fabids recruited the same genetic pathway fornodule organogenesis. PLOS ONE 2013, 8:e64515.

This paper describes the characterisation of the Casuarina CCaMK gene, itsability to complement the corresponding legume mutant, and its involve-ment in the actinorhizal nodulation. The paper also shows that the activationof CCaMK is sufficient to trigger spontaneous nodule formation not only inC. glauca but also in the Discaria trinervis (Rosales) indicating that eachindependent acquisition of nodulation used the same pathway based on theactivation of CCaMK to trigger nodule organogenesis.

41. Hammad Y, Nalin R, Marechal J, Fiasson K, Pepin R, Berry AM,Normand P, Domenach AM: A possible role for phenyl aceticacid (PAA) on Alnus nodulation by Frankia. Plant Soil 2003,254:193-205.

42. Tirichine L, James EK, Sandal N, Stougaard J: Spontaneous root-nodule formation in the model legume Lotus japonicus: a novelclass of mutants nodulates in the absence of rhizobia. MolPlant Microbe Interact 2006, 19:373-382.

43. Soyano T, Kouchi H, Hirota A, Hayashi M: NODULE INCEPTIONdirectly targets NF-Y subunit genes to regulate essentialprocesses of root nodule development in Lotus japonicus.PLoS Genet 2013, 9:e1003352.

44. Tirichine L, Sandal N, Madsen LH, Radutoiu S, Albrektsen AS,Sato S, Asamizu E, Tabata S, Stougaard J: A gain-of-function


mutation in a cytokinin receptor triggers spontaneous rootnodule organogenesis. Science 2007, 315:104-107.

45.��

Guan D, Stacey N, Liu C, Wen J, Mysore KS, Torres-Jerez I,Vernie T, Tadege M, Zhou C, Wang Z et al.: Rhizobial infection isassociated with the development of peripheral vasculature innodules of Medicago truncatula. Plant Physiol 2013, 162:107-115.

This paper shows that model legumes have kept ancient traits whichbecome visible when mutations disrupt particular signalling pathways:here mutations in lin-4 lead to the formation of nodules with a centralvascular bundle similar to Parasponia or actinorhizal nodules.

46.��

Madsen LH, Tirichine L, Jurkiewicz A, Sullivan JT, Heckmann AB,Bek AS, Ronson CW, James EK, Stougaard J: The molecularnetwork governing nodule organogenesis and infection in themodel legume Lotus japonicus. Nat Commun 2010, 1:10 Seeannotation to Ref. [45��].

47. Laplaze L, Duhoux E, Franche C, Frutz T, Svistoonoff S,Bisseling T, Bogusz D, Pawlowski K: Casuarina prenodule cellsdisplay the same differentiation as the corresponding nodulecells. Mol Plant Microbe Interact 2000, 13:107-112.

48. Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre J-C,Jaubert M, Simon D, Cartieaux F, Prin Y et al.: Legumessymbioses: absence of nod genes in photosyntheticbradyrhizobia. Science 2007, 316:1307-1312.

49. Smith SA, Donoghue MJ: Rates of molecular evolution arelinked to life history in flowering plants. Science 2008, 322:86-89.

50. Swensen SM: The evolution of actinorhizal symbioses:evidence for multiple origins of the symbiotic association. AmJ Bot 1996, 83:1503-1512.

51.�

Geurts R, Lillo A, Bisseling T: Exploiting an ancient signallingmachinery to enjoy a nitrogen fixing symbiosis. Curr Opin PlantBiol 2012, 15:438-443.

This review is mainly focused on symbiotic signalling in Parasponia, theonly non-legume able to form nitrogen fixing root nodules with rhizobiawhich shares many similarities with actinorhizal plants.

52. Jung S, Ficklin SP, Lee T, Cheng C-H, Blenda A, Zheng P et al.: TheGenome Database for Rosaceae (GDR): year 10 update.Nucleic Acids Res 2013, 42:D1237-D1244.

53. Soltis DE, Smith SA, Cellinese N, Wurdack KJ, Tank DC,Brockington SF, Refulio-Rodriguez NF, Walker JB, Moore MJ,Carlsward BS et al.: Angiosperm phylogeny: 17 genes, 640 taxa.Am J Bot 2011, 98:704-730.

54. Swensen SM, Benson DR: Evolution of actinorhizal host plantsand Frankia endosymbionts. In Nitrogen-Fixing ActinorhizalSymbioses. Edited by Pawlowski K, Newton WE. Springer;2008:73-104.

55. Wall LG: The actinorhizal symbiosis. J Plant Growth Regul 2000,19:167-182.

56. Dawson JO: Ecology of actinorhizal plants. In Nitrogen-FixingActinorhizal Symbioses. Edited by Pawlowski K, Newton WE.Springer; 2008:199-234.

57. Sprent JI: Evolving ideas of legume evolution and diversity: ataxonomic perspective on the occurrence of nodulation. NewPhytol 2007, 174:11-25.





































































































Actinorhizal root nodule symbioses: what is signalling telling on the origins of nodulation?

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