Chapter 45

Lipochitooligosaccharide Perceptionand the Basis of Partner Recognitionin Root Endosymbioses

Julie Cullimore and Clare GoughLaboratoire des Interactions Plantes-Microorganismes (LIPM), INRA, CNRS, France

45.1 INTRODUCTION

Root endosymbioses play key roles in plant nutrition in,both, agronomic and natural ecosystems. Of most impor-tance are the arbuscular mycorrhizal (AM) symbiosis, inwhich the fungus improves the uptake of nutrients by theplant (particularly phosphorus and nitrogen), and secondlythe Rhizobium–legume (RL) symbiosis in which Rhizobiabacteria fix dinitrogen allowing legumes to grow inde-pendently of a mineral nitrogen source (Harrison, 2005;Oldroyd et al., 2011; see Chapters 43, 44). Although thesesymbioses seem quite different, studies on legumes haveshown that the establishment of the AM and the RL sym-bioses require a set of common plant genes, constitutingthe common symbiotic pathway (CSP), thus supportingthe hypothesis that the more recent RL symbiosis evolvedfrom the more ancient AM symbiosis (Parniske, 2008; seeChapter 43).

A major difference between the two symbiosesis their degree of partner specificity. AM fungi arephylogenetically conserved, all belonging to a singlephylum, the Glomeromycota. Studies suggest that thesefungi show little host specificity and are able to formmycorrhiza on a wide range of terrestrial plants, includingdicots, monocots, pteridophytes and some bryophytes,and liverworts (Harrison, 2005). However, the planthost shows specificity for this one taxonomic group offungi. In contrast, Rhizobia are phylogenetically diverse,including species from many different taxonomic classes

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

of both the alpha- and beta-proteobacteria (see Chapter44; Masson-Boivin et al., 2009; Gyaneshwar et al., 2011),and each Rhizobia has a restricted host specificity,nodulating either a few (narrow host-range Rhizobia)or many (broad host-range Rhizobia) legume species(Denarie et al., 1996; Perret et al., 2000). Moreover, someRhizobia that nodulate legumes can also nodulate tropicaltrees of the genus Parasponia (Streng et al., 2011). Onthe plant side some legumes are very strict regardingtheir nodulating partner whereas others can be nodulatedby various Rhizobia strains and species. Thus, for the RLsymbiosis a large variation in specificity is seen on, both,the plant and microbial sides. As the two symbioses aregenetically and evolutionarily linked, the question arisesof the molecular basis of partner specificity.

For Rhizobia, secreted lipochitooligosaccharidic sig-nals (LCOs) are involved in a signal exchange required forestablishing most RL symbioses. Genetic studies on Rhi-zobia identified a set of genes (the nod genes), inducedby plant factors, which are essential for nodulating theirlegume hosts. These genes specify the production of LCOs(the Nod factors), which are generally composed of 4–5N-acetyl glucosamines (GlcNAc) with an N-acyl chain onC2 of the terminal nonreducing sugar. Variation in thestructure of the acyl chain and in chemical substitutionson the sugar residues are characteristic of the Nod fac-tors produced by different rhizobial species and strains(Denarie et al., 1996; Perret et al., 2000; D’Haeze andHolsters, 2002; Fig. 45.1). Nod factors stimulate many

483

484 Chapter 45 Lipochitooligosaccharide Perception and the Basis of Partner Recognition

(a) Generic LCO

Microbe LCO substitutionsR1 R2 R3 R4 R5 R6 n

Sinorhizobium meliloti C16 :2 ∆2E∆9Z – – H, Ac 1, 2Rhizobium leguminosarum bv.viciae

C18 :1∆11ZC18 :4∆2E∆4E∆6E∆11Z

H, Ac * 1, 2

Mesorhizobium loti Me C18 :1∆11Z Cb, H Cb, H 4-O-AcFuc 2Bradyrhizobium japonicum C18 :1∆11Z 2-O-MeFuc 2Rhizophagus irregularis C18 :1∆9Z

C16 :0H, S 1, 2

*Ac in nodX strains

(b) Major LCOs produced by some symbiotic microbes

R4O

NR1

R10R9

CR8

OR7

C CH3O

CO

OO

NH

OR6CO

OR3O

O

OR5

R2

O

O NH

OH

OO

HONH

OH

O

CCH3O

n O

NodL

NodF NodE

NodHNodXNodZ

S

Figure 45.1 (a). Structure of an LCO showing sites of chemical substitutions and the enzymes involved in rhizobial Nod factor synthesis; n isgenerally 1 or 2 leading to substituted tetrameric and pentameric LCOs. The enzymatic activities of the genes discussed in the text are NodLand NodX (6-O-acetyl-transferases), NodH (sulfotransferase), NodZ (fucosyl transferase), NodE (β-ketoacyl-synthase), and NodF (acyl carrierprotein); (b) Chemical substitutions in the major LCOs produced by selected Rhizobia and by an AM fungus. The double bonds in the acylchains are designated E-trans and Z-cis. The substitutions are Me (methyl), Cb (carbamoyl), Ac (acetyl), S (sulfuryl), Fuc (fucosyl) groups.

symbiotic responses, including root hair deformation, spe-cific gene induction, calcium spiking, as well as lateralroot formation. Although some photosynthetic Rhizobiado not contain nod genes and thus have found a wayof circumventing the Nod factor signaling step, gener-ally most Rhizobia depend on nod genes and Nod fac-tor production for establishing symbioses with legumes(Masson-Boivin et al., 2009; see Chapter 44). Recentlyan AM fungus (Rhizophagus irregularis) has been shownto produce LCOs (the Myc-LCOs) that have a similar gen-eral structure to Nod factors (Fig. 45.1) and that stimulatemycorrhization in several different plant families (Mail-let et al., 2011; see Chapter 43). Similar to Nod factors,the Myc-LCOs also stimulate lateral root formation (Mail-let et al., 2011; Gough and Cullimore, 2011).

Studies on Rhizobia have shown that Nod factors playa key role in partner recognition and specificity. The nodgenes are induced by host plant compounds, thus pro-viding a first level of specificity of interaction betweensymbiotic partners. Secondly, the presence of differentgene and allelic variation in different rhizobial speciesleads to species and strain-specific variation in the level,structure, and variety of different Nod factors produced.In this way, although Rhizobia strains each produce apopulation of Nod factors, their structures range frombeing highly related to chemically diverse. Studies on nodgene mutants suggest that particular chemical substitu-tions are associated with responses on particular legumesand that generally broad host-range Rhizobia produce a

wide range of Nod factor structures whereas narrow host-range Rhizobia produce fewer, more specific structures(Denarie et al., 1996; Perret et al., 2000; D’Haeze andHolsters, 2002). For AM fungi, it is not yet clear if Myc-LCO structure is involved in the wide host-range of thesefungi, because the LCOs from only one species have beenstudied (Maillet et al., 2011) and because the coenocyticnature and lack of genetic tools in these fungi (Sandersand Croll, 2010) make genetic analysis of the role of Myc-LCOs in host range difficult to establish.

On the plant side it has been speculated that partnerspecificity would be controlled by the receptors forthe symbiotic signals. In 2003, receptors required forNod factor perception were identified through geneticapproaches (Gough and Cullimore, 2011). These recep-tors are lysin-motif-receptor-like kinases (LysM-RLKs),which are plasma membrane proteins with an extracellu-lar region containing three divergent LysMs (Fig. 45.2).LysM domains are involved in the perception of Glc-NAc containing ligands (Buist et al., 2008), includingchitooligosaccharides (COs) and peptidoglycan in plants(Hamel and Beaudoin, 2010; Gough and Cullimore, 2011;Willmann et al., 2011). Although some LysM proteins(called LYMs) are not associated with other domains,plants are unique in that most LysMs are associated withan intracellular kinase domain to form the LysM-RLKs.Plants contain a variable number of LysM-RLK genesthat can be divided by phylogenetic analysis into twosubfamilies, which are termed the LYK (LysM-I or

45.2 LysM Receptors and Rhizobium Specificity 485

Figure 45.2 Structure of plant symbiotic LysM-RLKs. LysM-RLKs form two well-supported clades, named the LYR and LYK subfamilies.The three predicted LysM domains within each protein are highly divergent and each of them is generally more highly conserved betweenorthologs in different legumes than between paralogs. Generally the intracellular regions of LYR proteins lack the glycine-rich loop and theactivation segment of active kinases, as those predicted in the LYK proteins. These predictions are supported by functional studies on MtNFP,LjNFR5, MtLYK3, and LjNFR1 (Arrighi et al., 2006; Klaus-Heisen et al., 2011; Madsen et al., 2011). The stars in LysM2 represent Leu118 ofLjNFR5 and Leu154 of MtNFP implicated in the control of infection in L. japonicus and M. truncatula , respectively.

LYS-I) and the LYR (LysM-II, Lys-II) families (Goughand Cullimore, 2011). Generally the LYR proteins arepredicted to contain an inactive kinase domain whereasthe LYK proteins contain an active kinase domain. Inthe best-studied legumes (Medicago, Pisum, Lotus, andGlycine) a LysM-RLK from each subfamily is requiredfor nodulation and infection (Fig. 45.2). Recently, studieson the AM symbiosis implicate a LYR-type LysM-RLKin mycorrhization of Parasponia andersonii and inMyc-LCO responses in Medicago truncatula (Op denCamp et al., 2010; Maillet et al., 2011). Thus, geneticstudies suggest that establishment of, both, the AM andRL symbioses require LysM-RLKs which act as LCOreceptors and that these proteins could be involved in dis-criminating different LCOs (Hamel and Beaudoin, 2010;Gough and Cullimore, 2011).

The symbiotic LysM-RLKs act upstream of genesof the CSP, which is required for establishing, both,the AM and RL symbioses (Fig. 45.3). These genesinclude a plasma membrane located leucine-rich-repeatreceptor-like kinase (called SYMRK or DMI2), whichis required to activate calcium spiking in the nucleus.As this RLK is not required for certain very early Nodfactor responses and can be interchanged with potentiallyorthologous proteins from other plant species, it appearsnot to be involved in partner specificity (Parniske, 2008).Calcium spiking is decoded by a calcium–calmodulin-dependent protein kinase (called DMI3 or CCamK)and its interacting protein (called IPD3 or CYCLOPS)leading to gene expression and the preparation forinfection in the epidermal cells and, in the case of the

RL symbiosis, to activation of cortical cell divisions(CCD) leading to nodulation (Chapter 43; Fig. 45.3).Infection by Rhizobia also requires LysM-RLKs andgenerally involves the production of infection threads(ITs) through which the Rhizobia enter the plant (Oldroydand Downie, 2008). The genetic pathway leading toIT formation is dependent on but can be geneticallyseparated from the CSP, although both pathways mustbe coupled, via DMI3/CCamK and IPD3/CYCLOPS, toproduce infected nitrogen-fixing nodules (Oldroyd andDownie, 2008; Madsen et al., 2010). A scheme of therole of the LysM-RLKs in symbiotic signaling in the twomodel legumes, M. truncatula and Lotus japonicus isshown in Figure 45.3.

Here, we review the evidence from studies onfour legume plant genera (Medicago, Pisum, Lotus,and Glycine) and on Parasponia that LysM-RLKs areinvolved in the specificity of LCO and partner recognitionleading to the establishment of the RL and AM symbioses.

45.2 LysM RECEPTORS ANDRHIZOBIUM SPECIFICITY

45.2.1 MedicagoThe genus Medicago contains over 80 recognizedspecies but most attention has been focused on theagronomically important tetraploid species Medicagosativa (alfalfa/luzerne) and the diploid model legumeM. truncatula. These species are very stringent for

486 Chapter 45 Lipochitooligosaccharide Perception and the Basis of Partner Recognition

(a) (b)

Figure 45.3 Model of endosymbiotic LCO perception and signal transduction in (a) M. truncatula and (b) L. japonicus . The model shows therole of LysM-RLKs (green and gray) and their articulation with downstream signaling leading to nodulation (red pathways) and mycorrhization(blue pathways). Nod factor perception by the LysM-RLKs leads to activation of the common symbiotic pathway (yellow components) via thesymbiotic LRR-RLK (MtDMI2/LjSYMRK) and also of the rhizobial infection pathway. Current evidence suggests a two-receptor model of Nodfactor perception in M. truncatula leading to activation of these two pathways whereas in L. japonicus a single receptor is hypothesized. In theCSP, calcium spiking is decoded by a calcium calmodulin kinase (MtDMI3/LjCCamK) and its interacting protein (MtIPD3/LjCYCLOPS),which play a role in coordination with the rhizobial infection pathway. Other components of the pathways are not shown for simplicity.Activation of the CSP leads to specific gene expression, CCD and nodulation and is also required for mycorrhization. Note that although NFP isinvolved in perception of Myc-LCOs (Maillet et al. 2011), it is not essential for mycorrhization, leading to the hypothesis that anotherLysM-RLK is involved in Myc factor perception. As MtNFP, as LjNFR5, has an inactive kinase domain, it is hypothesized to interact with aLYK-type protein. RLKs with active kinase domains are denoted by stars.

their symbiotic partner, forming nitrogen-fixing nodulesonly with Sinorhizobium meliloti and Sinorhizobiummedicae strains. However, some other Rhizobia strainsappear able to form small ineffective nodules on alfalfa,and these Rhizobia may compete with effective strainsfor nodulation. S. meliloti produces a range of verycharacteristic Nod Factors, which are all sulfated onthe reducing sugar. Many are also 6-O-acetylated onthe terminal nonreducing sugar and the major acylchain seems to be C16:2 (D’Haeze and Holsters, 2002Fig. 45.1). Studies on S. meliloti mutants have shown theimportance of the sulfate in host specificity as a nodHmutant, producing nonsulfated Nod factors, is incapableof nodulating alfalfa but gains the capacity to nodulatevetch (Denarie et al., 1996). Recently, studies of an acid-tolerant “Oregon-like” strain, which is closely related toRhizobium mongolese and forms ineffective nodules onalfalfa, has surprisingly shown that a nodH mutant iscapable of nodulation on this host, although with a delay(Del Papa et al., 2007). This strain produces a largervariety of non-sulfated Nod Factors than a S. melilotinodH mutant, including ones that are N-methylated onthe terminal nonreducing sugar (Tejerizo et al., 2011)and it will be interesting to establish whether particular

modifications are able to compensate for the lack of thesulfate group.

In terms of the mechanisms of Nod Factor perception,studies on S. meliloti mutants led to the suggestion thatNod factors are perceived by two receptors in Medicagowith differing ligand specificity. The first is a signalingreceptor which requires sulfated Nod factors and leadsto epidermal responses such as root hair deformation,expression of the early nodulin gene ENOD11 and alsoCCD (Fig. 45.3). As these responses can be initiated bymutants in the nodFL genes, this putative receptor can beactivated with Nod factors lacking the O-acetate and witha C18:1 acyl chain. The second is an entry receptor thatleads to infection, which is more stringent for the Nodfactor structure as infection does not occur with nodFLmutants (Ardourel et al., 1994; Limpens et al., 2003).Analysis of symbiotic mutants in M. truncatula identifiedtwo LysM-RLK genes that are required for nodulationand different Nod factor responses and which has largelysubstantiated the 2-receptor hypothesis (Gough and Cul-limore, 2011). MtNFP is a LYR gene required for allNod factor responses, infection, and nodulation whereasMtLYK3 is not required for early responses (such as roothair deformation, ENOD11 expression, and initial CCD),

45.2 LysM Receptors and Rhizobium Specificity 487

but is required for tight root hair curling, IT formation,and for progression of CCD. MtNFP and MtLYK3 there-fore appear to be components of the signaling and entryreceptors respectively (Fig. 45.3). MtNFP is likely to bealso part of the entry receptor as RNAi knock down plantsof MtNFP show early Nod Factor responses and ITs thatdo not form normally (Arrighi et al., 2006).

As MtNFP is required for signaling responses whichare highly specific for sulfated Nod factors, it waspredicted that this receptor would be specific for sulfatedfactors. In comparison, its ortholog in pea, PsSYM10,would be expected to be specific for non-sulfated factors,as this legume is nodulated by Rhizobia-producingnon-sulfated Nod factors. However, domain swapexperiments have shown that the extracellular regionsof MtNFP/PsSYM10 do not discriminate sulfated/non-sulfated Nod factors, respectively, in M. truncatulaas predicted from their roles in the plant hosts: theextracellular region from either receptor (when fusedto the MtNFP transmembrane and kinase-like domain)leads to only sulfated Nod factor activation of ENOD11expression in M. truncatula (Bensmihen et al., 2011).This result suggests that the specific perception of sul-fated Nod Factors in M. truncatula could involve anothercomponent which has yet to be identified. Moreover,although a domain swap of the extracellular region ofPsSYM10 for that of MtNFP leads to signaling responsesin M. truncatula, it does not function in infection-threaddevelopment thus confirming the role of MtNFP in infec-tion and suggesting that MtNFP is activated differentlyfor the infection process. Dissection of the requirementfor this process identified Leu154 in the LysM2 domainas the critical residue (Bensmihen et al., 2011). Thisamino acid residue is predicted by molecular dockingstudies to be situated in the part of the protein whichinteracts with the acyl chain (Mulder et al., 2006;Bensmihen et al., 2011).

MtLYK3 is clearly involved in partner specificity asRNAi knock down plants, or a weak Mtlyk3 mutant showspoorer nodulation with rhizobial nodL and nodFE mutants(Limpens et al., 2003; Smit et al., 2007), which pro-duce Nod Factors affected in O-acetylation of the ter-minal reducing sugar and in the structure of the acylchain, respectively. This specificity could partly be dueto the ability of the kinase domain of MtLYK3 to inter-act with an E3 ubiquitin ligase protein, named PUB1(Mbengue et al., 2010). RNAi knock down plants of PUB1show better nodulation by nodL and nodFL mutants thanwild-type plants suggesting that PUB1 negatively controlsthe specificity of nodulation by S. meliloti mutants. Themechanism of action of PUB1 in this response is currentlybeing investigated.

MtLYK3 is part of a cluster of 7 LYK genes onchromosome 5 and knock down by RNAi suggests

that MtLYK4 is also involved in partner specificity asplants inoculated with a nodFE mutant show a defect ininfection (Limpens et al., 2003). Although no role wasidentified for two other LYK genes in the cluster, it ispossible that this approach does not sufficiently inactivatethe genes to reveal a phenotype. Analysis of the genomeof M. truncatula revealed at least 7 LYR and 10 LYKgenes (Arrighi et al., 2006) and it is possible that morewill be discovered on complete sequencing of the genome(Young et al., 2011). Many of these LysM-RLK genes areexpressed in roots and nodules suggesting that they mayplay symbiotic roles. However it is possible that they maybe involved in perception of other GlcNAc-containingligands and in this respect it is noteworthy that theLYM2, but not the LYM1 gene, has been shown to bindCOs (Fliegmann et al., 2011). To date, direct Nod factorbinding has not been reported to MtNFP and MtLYK3,despite progress in expressing and characterizing theproteins (Klaus-Heisen et al., 2011; Lefebvre et al., 2012).However, a biochemical approach has identified threeNod Factor Binding Sites (NFBSs) in M. truncatula thatdo not depend on MtNFP (Hogg et al., 2006). Whetherthe binding proteins in these sites are LysM proteinsand whether these sites are involved in the specificity ofperception of symbionts remains to be seen.

45.2.2 PisumPisum sativum (pea) and related legumes such as Viciasativa (vetch) are nodulated by strains of Rhizobiumleguminosarum bv. viciae. Studies on the Nod factorsproduced by this Rhizobium showed that they arepredominately chitooligo-tetramers or pentamers, often6-O-acetylated on the terminal nonreducing sugar andacylated with various fatty acids including C18:4 orC18:1 (D’Haeze and Holsters, 2002; Fig. 45.1). Theproduction of the trans-double bonds is conferred byNodE as a nodE mutant produces Nod factors withonly C18:1 chains. Both nodL dependent O-acetylationand nodE dependent C18:4 acylation are important forthe Nod factors to initiate CCD in pea and, moreover,the nodE gene is a major determinant of specificity fornodulation of pea versus clover (Bloemberg et al., 1995).

By analysis of pea symbiotic mutants, genes wereidentified encoding proteins which by sequence and syn-teny analysis are the probable orthologs of M. truncatulaMtNFP (PsSYM10) and MtLYK3 (PsSYM37). Analysesof these mutants showed that PsSYM10, as MtNFP, isrequired for all symbiotic responses whereas PsSYM37,as MtLYK3, is not involved in initial responses butis required for infection (Walker et al., 2000; Mad-sen et al., 2003; Zhukov et al., 2008). These studiesare compatible with a two-receptor model of Nod factorperception as in M. truncatula, although partial genetic

488 Chapter 45 Lipochitooligosaccharide Perception and the Basis of Partner Recognition

redundancy in PsSYM37 function could explain the lackof an apparent role of PsSYM37 in initial responses(Zhukov et al., 2008). Studies using a nodE mutantrevealed considerable variation in the ability of this strainto nodulate different pea cultivars suggesting naturalvariation in the requirement for the C18:4 acyl chain(Li et al., 2011). By analyzing recombinant inbred linesderived from a cross between cultivars differing in theirnodulation with the nodE strain, followed by Quanti-tative Trait Locus (QTL) mapping, this phenotype wasshown to be linked to the PsSYM37 gene. Sequencingof this gene in the inbred lines and in various peacultivars identified certain polymorphisms in the LysMdomains associated with the variable response to nodENod factors (Li et al., 2011). Thus, partner specificityanalysis has identified that the M. truncatula/P. sativumMtLYK3/PsSYM37 orthologs are involved in the responseto Rhizobia producing Nod factors with acyl chainscontaining trans-double bonds.

An interesting strain-specific nodulation of pea hasbeen reported that concerns the requirement of certainMiddle East cultivars of pea, for example Afghanpeas, to be nodulated by local R. leguminosarum bv.viciae strains whereas they are resistant to nodulationby European strains. Further analysis revealed that theblock in nodulation by European strains is at the levelof infection rather than in initial epidermal and corticalresponses (Geurts et al., 1997). Studies on the bacterialpartner showed that nodulation by certain strains wasdue to the presence of the nodX gene that specifiesproduction of Nod factors which are 6–O acetylated onthe reducing sugar. The O-acetate is not required per se assubstitution by a fucose group using the nodZ gene fromBradyrhizobium japonicus will also allow nodulationof Afghan peas (Ovtsyna et al., 1998). On the plantside, genetic analysis identified a locus, Sym2, in whichthe Sym2A allele allows nodulation by nodX containingstrains. Mapping of the Sym2 gene and synteny analysisled to the discovery of the M. truncatula cluster of genescontaining MtLYK3, as mentioned before. The sequenceof the corresponding region in pea is still not publishedbut analysis of LysM-RLK genes expressed in pea rootsidentified not only the PsSYM37 gene but also a gene,PsK1, that maps to the same region (Zhukov et al., 2008).Sequencing of different European and Afghan cultivarsrevealed differences in the extracellular regions of PsK1but not of PsSYM37 between the two groups, suggestingthat PsK1 may correspond to Sym2 (Zhukov et al., 2008).However, this hypothesis awaits genetic confirmation,particularly as this region could contain other LysMgenes as in M. truncatula.

In this work, it was noted that a point mutation inthe PsSYM37 mutant, leading to a Leu77Phe amino acidchange in LysM1, leads to a low level of nodulation by

a Rhizobium strain carrying nodX but not by a Euro-pean strain (Zhukov et al., 2008). This result suggeststhat changes in the structure or conformation of the LysMdomain can lead to changes in the specificity of perceptionof Nod factors leading to changes in partner specificity.

Another phenomenon identified in pea is the abilityof certain European R. leguminosarum bv. viciae strainsto inhibit nodulation of Afghan peas by strains carry-ing nodX. This phenomenon is referred to as competitivenodulation blocking (Cnb) and could be of agronomicimportance as nonnodulating Rhizobia could prevent thedevelopment of an effective symbiosis. Genetic and Nodfactor analyses suggest that Cnb is due largely to thehigher amounts of Nod factors produced by the blockingstrains, particularly in relation to the amount produced bythe natural nodX containing strains (Hogg et al., 2002).On the plant side, the Sym2 region seems to be partiallyinvolved in the blocking phenotype, suggesting the roleof LysM-RLKs in perceiving inappropriate Nod factorsand blocking nodulation by a strain carrying the correctNod factors. Because of the likely complexity of the Sym2region it is not clear whether the same LysM-RLK isinvolved in, both, blocking and compatible Nod factorperception.

45.2.3 LotusThe major symbiont of Lotus spp. is Mesorhizobiumloti that produces pentameric Nod factors substitutedwith an acetylated fucose at C6 of the reducing sugar, acarbamoyl group, an N-methyl group, and mainly C16:0and C18:1 acyl chains at the nonreducing end (Fig. 45.1).The roles of Nod factor structure in host specificity havebeen studied using M. loti mutants and four Lotus spp.:L. japonicus, Lotus filicaulis, Lotus corniculatus, andLotus burttii (Rodpothong et al., 2009). These studiesshowed that the presence of the carbamoyl group onthe nonreducing end is not necessary for nodulation,while M. loti mutants producing Nod factors without theacetylated fucose on the reducing end have a host-rangephenotype, forming mainly uninfected nodule primordiaon L. filicaulis and L. corniculatus, and delayed noduleson L. japonicus and L. burttii. Defects in IT formationand the delayed induction of the NIN gene are associatedwith the delayed nodulation phenotype on L. japonicus.In addition to M. loti, two other rhizobial species havebeen studied on Lotus spp.. These rhizobia are calledBradyrhizobium sp. (Lotus) and R. leguminosarumbv. viciae strain DZL. Similar to M. loti, both strainsproduce pentameric Nod factors substituted with anacetylated fucose at C6 of the reducing sugar moiety,but Nod factors differ from those of M. loti in thesubstitutions at the nonreducing end sugar; R.l. viciaeDZL Nod factors are characterized by C18:1 and C18:4

45.2 LysM Receptors and Rhizobium Specificity 489

acyl chains and absence of the N-methyl group, andNod factors produced by Bradyrhizobium sp. (Lotus)have two carbamoyl groups. On L. japonicus, bothR.l. viciae DZL and Bradyrhizobium sp. (Lotus) formmostly infected, but ineffective nodules (Bek et al., 2010;Radutoiu et al., 2003; Radutoiu et al., 2007), indicatinga certain stringent requirement of L. japonicus for thenonreducing end substitutions of Nod factors. However,as the acetylated fucose of M. loti Nod factors is impor-tant for normal nodulation on this Lotus spp., it seemsthat L. japonicus Nod factor receptors could recognizeNod factor substitutions at both ends of the molecule.

In L. japonicus, LysM-RLKs so far identified as hav-ing symbiotic roles are LjNFR1 and LjNFR5, orthologs ofMtLYK3 and MtNFP, respectively (Figs. 45.2 and 45.3).Ljnfr1 and Ljnfr5 mutants are all deficient for Nodfactor responses, rhizobial infection, and nodulation,and the fact that they present the same phenotypeshas led to the hypothesis of an NFR1-NFR5 receptorcomplex for Nod factor perception (Madsen et al., 2003;Radutoiu et al., 2003). Using complemented L. japonicusmutants, however, it has been shown that nodulationof Ljnfr1 plants transformed with LjNFR1 is severelyimpaired with M. loti Nod factor mutants compared to thewild-type strain, while this difference was not seen withLjnfr5 plants transformed with LjNFR5, suggesting thatperception of modified Nod factors is particularly sensi-tive to LjNFR1 expression levels or to LjNFR1-mediatedsignaling (Rodpothong et al., 2009). LjNFR1 and LjNFR5have clearly been shown to control symbiont-host rangeas their transfer into M. truncatula enables two Lotusrhizobial species, M. loti and R.l. viciae DZL, to nodulatetransgenic M. truncatula roots (Radutoiu et al., 2007).With R.l. viciae DZL, more ITs and nodules are formedbut neither strain becomes endocytosed into host cells ofnodules to form symbiosomes.

Unlike L. japonicus, the closely-related Lotus spp.L. filicaulis cannot be nodulated by R.l. viciae DZL. How-ever, when LjNFR1 and LjNFR5 were transferred intoL. filicaulis, these transgenic roots could be nodulated byR.l. viciae DZL, providing more evidence for the role ofthese LysM-RLK genes in the control of symbiont host-range specificity (Radutoiu et al., 2007). This differentialnodulation phenotype of R.l. viciae DZL was exploitedto study the role of the LysM domains of LjNFR1 andLjNFR5 in symbiont specificity. With R.l. viciae DZL,complementation for nodulation was efficient in Ljnfr1mutants transformed with a chimeric LfNFR1–LjNFR1construct bearing the three LysM domains of LfNFR1and the kinase domain of LjNFR1, while an equivalentchimeric construct for NFR5, LfNFR5–LjNFR5, conferredlow nodulation capacity on Ljnfr5 mutant plants. Thischimeric LfNFR5–LjNFR5 construct could be modified torestore complementation efficiency with R.l. viciae DZL to

the same level as with the entire LjNFR5 gene, by replac-ing a single L. filicaulis amino acid residue with the cor-responding residue from L. japonicus (L118K) in LysM2of the chimeric construct. These results suggest that theleucine–lysine difference in the amino acid residue 118of LysM2 of NFR5 is largely responsible for the dif-ferent responsiveness of L. filicaulis and L. japonicus toR.l. viciae DZL. This amino acid residue is also predictedto be in a Nod factor-binding site on LysM2 of NFR5and could participate in recognizing structural featuresspecific to the nonreducing end of M. loti Nod factors(Radutoiu et al., 2007).

Analysis of L. japonicus and Lotus pedunculatus inoc-ulated with each other’s symbionts, which are M. loti andBradyrhizobium sp., respectively, led to the conclusionthat the amino acid differences between NFR1 and NFR5of these two Lotus spp. are expendable for recognition ofcertain different Nod factor structural features and initia-tion of the first steps of nodulation, but not for infection(Bek et al., 2010). Furthermore, early recognition of Rhi-zobia was not affected by exchanging the LysM domainsof NFR1 and NFR5 from L. japonicus and L. peduncu-latus, but infection was never complemented, suggestingthat these LysM-RLKs alone are unlikely to be the majorcomponents determining the infection phenotypes in Lotusroots (Bek et al., 2010).

Taken together, these studies show that native LysMdomain sequences are critical for an efficient infectionprocess but not for initial steps of Nod factor perceptionwithin Lotus spp.. Furthermore, the role of specific LysMsequences in the infection process is linked to Nod factorstructural variations at the nonreducing end.

As in M. truncatula, the L. japonicus genome encodesa relatively large LysM-RLK gene family of at least 17members (Lohmann et al., 2010). Analysis of synony-mous and nonsynonymous substitutions revealed distinctregions in the LysM domains of these proteins where pos-itive selection may have shaped ligand interaction. ForLjNFR5, one of these regions coincides with the predictedNod factor-binding groove and the L118 area implicatedin specificity of Nod factor recognition. Nine L. japonicusLysM-RLK genes are upregulated after rhizobial inocula-tion, suggesting that, in addition to LjNFR1 and LjNFR5,other L. japonicus LysM-RLK genes might be involved inestablishment of the nitrogen-fixing symbiosis with rhizo-bia (Lohmann et al., 2010).

Another study in Lotus has addressed whetherthe kinase domains of symbiotic LysM-RLKs playany roles in host specificity (Nakagawa et al., 2011).This work showed that chimeric LjNFR1-MtLYK3 andLjNFR1-PsSYM37 constructs consisting of LjNFR1LysM domains fused to the kinase domains of MtLYK3and PsSYM37, respectively, fully complement an Ljnfr1mutant for nodulation with M. loti, indicating that

490 Chapter 45 Lipochitooligosaccharide Perception and the Basis of Partner Recognition

the kinase domains of these proteins are functionallyconserved between legume spp. and do not contribute tohost specificity. As the complete MtLYK3 or PsSYM37did not complement for nodulation, this shows that theexternal LysM regions have diverged more recently forrecognizing different Rhizobia symbionts.

45.2.4 GlycineGenetic analysis in soybean (Glycine max) is complicatedby its allotetraploid nature. Furthermore, as a result ofpartial genome duplication before tetraploidization, thereare two copies of each of the LysM-RLKs implicatedin Nod factor perception in soybean. These are calledGmNFR1α, GmNFR1β, GmNFR5α, and GmNFR5β

(Fig. 45.2). Within pairs of proteins there is a highlevel of homology, for example 89% identity betweenGmNFR1α and GmNFR1β. The different phenotypesof GmNFR1α and GmNFR1β mutants suggest thatthe functions of GmNFR1α and GmNFR1β havediverged following genome duplication. Indeed, Gmnfr1α

mutants are completely deficient for nodulation, while aGmNFR1β mutant that is predicted to be a knock out isnot affected for nodulation, and 80-fold overexpressionof GmNFR1β does not complement Gmnfr1α mutants(Indrasumunar et al., 2011). However, if Gmnfr1α

mutants are inoculated with high titers of a soybeansymbiont, Bradyrhizobium japonicum CB1809, thenGmNFR1α symbiotic deficiency is partially suppressed(Indrasumunar et al., 2011). Also, Bradyrhizobium elkaniican nodulate the rj1 mutant of GmNFR1α, and, interest-ingly, this rhizobial species produces more Nod factorstructures than B. japonicum, for example, includingtetrameric Nod factors whereas B. japonicum Nod factorsare pentameric (Stacey et al., 1995). Taken together,these data suggest that GmNFR1α governs nodulationby B. japonicum, but in its absence GmNFR1β playsa role in partner specificity by allowing nodulation bystrains producing either high Nod factor concentrationsor specific Nod factor structures.

The functionality of the GmNFR5α and GmNFR5β

genes of soybean has been shown by the ability of eachgene to individually complement Gmnfr5α mutants fornodulation (Indrasumunar et al., 2010). These mutantswere derived from the wild-type soybean genotypes Braggand Williams that interestingly have a natural mutationin GmNFR5β, corresponding to insertion of the GmRE-1retro-element. These results show that GmNFR5α alonepermits nodulation with B. japonicum but the functionof GmNFR5α is likely to be partially redundant withGmNFR5β. There is no data on whether these genes playa role in partner specificity.

45.3 LysM RECEPTORS AND THEAM SYMBIOSIS

As the CSP is essential for both the RL and AM symbiosesand is activated by rhizobial LCOs, it was hypothesizedthat AM fungi could activate this pathway in a similarmanner. Following this rationale, it was recently shownthat a LYR gene of P. andersonii (PaNFP), which is verysimilar to M. truncatula NFP (Fig. 45.2), is required forestablishment of both symbioses: RNAi knock down ofPaNFP leads to smaller nodules lacking intracellular fix-ation threads with Rhizobia and to an AM fungal interac-tion with R. irregularis in which arbuscules fail to form(Op den Camp et al., 2010). Thus in this unusual, nodu-lating nonlegume, the same LysM-RLK PaNFP controlsthe intracellular invasion of both Rhizobia and AM fungi,but does not play a role in partner specificity. As G.intraradices has recently been shown to produce LCOs,similar to Nod factors, it is reasonable to believe that therole of this protein in both symbioses is through the per-ception of LCOs.

The LCOs produced by R. irregularis (the Myc-LCOs) are composed of chitin oligomer tetra- andpentamers with predominantly C16:0 and C18:1 (oleicacid) acyl chains and are either 6–O sulfated (S-Myc-LCOs) or not (NS-Myc-LCOs) on the reducing sugar(Maillet et al., 2011; Fig. 45.1). These molecules beingproduced in low quantities by G. intraradices, S- andNS-Myc-LCO analogs were synthesized and shown tostimulate lateral root development in M. truncatula ina CSP-dependent manner, and stimulate mycorrhizationin, both, a legume and nonlegumes. In M. truncatula,NFP is involved in the lateral root stimulation by both Sand NS-Myc-LCOs (Maillet et al., 2011). As the formermolecules are very similar to the Nod factors of S.meliloti, their effect on M. truncatula could be due tostimulation of the Nod factor receptor (MtNFP) and Nodfactor pathways. The NS-Myc-LCOs stimulate lateralroot development via the CSP but this is independentof a downstream Nod signaling gene. Surprisingly, thisresponse is partly dependent on MtNFP, particularly atlow NS-Myc-LCO concentrations. This suggests thatanother LysM-RLK could be involved in the perceptionof NS-Myc-LCOs from AM fungi. The redundancy inMyc-LCO perception is also suggested by the fact thatnfp mutants are Myc+ (Gough and Cullimore, 2011).

Recently the draft sequence of the M. truncat-ula genome was published and comparison to otherlegumes suggests that legumes underwent a wholegenome duplication (WGD) event ∼58 million years ago(Young et al., 2011). In M. truncatula only remnants ofthis event remain, but one of them concerns the MtNFPgene and its homolog MtLYR1. Expression analysissuggests that MtNFP is expressed predominantly during

45.4 Conclusion 491

nodulation whereas MtLYR1 is specifically expressedduring mycorrhization leading to the suggestion that asingle ancestral gene participating in both interactions(such as PaNFP) underwent subfunctionalization of theduplicated genes following the WGD, leading to eachgene performing one function. This may be an oversim-plification as MtNFP can clearly partly control perceptionof synthetic Myc-LCOs, but it will be interesting to seewhether MtLYR1 plays a role in mycorrhization.

45.4 CONCLUSION

In this review of studies on four legumes genera, it is clearthat LysM-RLKs are key players in the perception of LCOsignals leading to establishment of the RL symbiosis; andrecent work on Parasponia also shows that a LysM-RLKcontrols establishment of the AM symbiosis. As the AMsymbiosis evolved at least 400 million years ago, it seemsthat Rhizobia acquired the ability to mimic the signalsproduced by AM fungi, allowing them to be perceivedby the plant and to activate an ancestral signaling path-way, the CSP, via LysM-RLKs. As discussed in Chapter43, for Rhizobia to form a symbiosis only with legumesand Parasponia, it is hypothesized that a genetic eventoccurred late in plant evolution predisposing the nodulat-ing clade of plants to be able to form root nodule sym-bioses. Currently the nature of this event is unknown. Thecuriosity of the two symbioses is that AM fungi remainedpromiscuous in being able to form mycorrhiza with awide range of terrestrial plants whereas there appears tohave been coevolution in the Rhizobium–legume sym-bioses leading to different symbiont-partner specificities.This coevolution has involved specialization of LYR/LYKgenes on the one hand and nod genes on the other hand inorder to perceive and produce different Nod factor signals.

LysM-RLK proteins activate not only the CSP, butalso a specific rhizobial infection pathway that is depen-dent on the CSP (Fig. 45.3). A current question concernswhether there is a single receptor (complex) whose acti-vation leads to, both, signaling and infection, or whetherseparate signaling and entry receptors are involved. Stud-ies on the four legumes suggest that a single LYR-typeLysM-RLK (MtNFP, PsSYM10, LjNFR5, and GmNFR5)controls both pathways, while a LYK-type LysM-RLKcontrols both pathways in Lotus and soybean (LjNFR1 andGmNFR1), but is specifically implicated in the infectionprocess in Medicago and pea (MtLYK3 and PsSYM37).This could suggest that there is a single receptor in thetwo legumes that produce determinate nodules (Lotus andsoybean) and two receptors in Medicago and Pisum thatproduce indeterminate nodules. However, several linesof evidence suggest many similarities between the two

groups. Both, MtNFP and LjNFR5, show different struc-tural requirements in the LysM2 domain for activationof infection compared to early signaling responses, sug-gesting that this LYR-type protein leads to differentialactivation of the two pathways in both groups of legumes.Also MtLYK3, PsSYM37, and LjNFR1 are all involvedin the discrimination of modified Nod factors, consistentwith the LYK-type protein playing a role in partner speci-ficity in both groups of legumes. In Medicago and Pisumthis LYK-type protein is involved in infection by Rhizo-bia producing Nod factors in which the acyl chains havetrans-double bonds and it has been pointed out that theGalegoid group of legumes (including M. truncatula andP. sativum) are all nodulated by Rhizobia-producing Nodfactors with this structural feature (Yang et al., 1999). Thissuggests that the LYK-type gene in the Galegoid groupof legumes evolved the ability to perceive this specificstructure, followed by adaptation and/or acquisition of thenodE gene by the rhizobial symbionts. Moreover, there isevidence that other genes in the MtLYK3/PsSYM37 regionof M. truncatula/P. sativum are important for responses todifferent Rhizobia strains suggesting an additional, recentlegume species-specific evolution of partner specificitythrough gene duplication in this region. This is supportedby the presence of more LYK genes (seven of them) inthis cluster in M. truncatula (Limpens et al., 2003) com-pared to only three in L. japonicas (Lohmann et al., 2010).All this work suggests that the notion of a single or twoNod factor receptors may be an oversimplification andreceptor complexes comprising one or more LysM-RLKs,probably with other proteins, may be the reality.

Clearly analysis of other members of the LysM-RLKfamilies is needed to establish whether they also play rolesin specificity of nodulation. Also, as nod genes and someLYR/LYK genes are expressed throughout nodulation, itwill be interesting to assess whether they are involved inlater stages leading to strain-specific symbiosome forma-tion and nitrogen fixation. Finally it should be pointedout that some of these genes are likely to be involvedin CO perception as the Arabidopsis thaliana and riceCO receptor CERK1 is closely related to the symbioticLYK protein (MtLYK3/PsSYM37/LjNFR1). Although theextracellular LysM domain of CERK1 seems to be spe-cific for CO perception, just small changes in its kinasedomain allows it to substitute for the kinase domain ofLjNFR1 in symbiotic signaling (Nakagawa et al., 2011).Therefore the recognition of friend and foe may involvevery subtle changes in signal output by a LYK protein,leading to symbiotic or defense responses respectively.

To study the role of LCOs/LysM-RLKs in host speci-ficity in the AM symbiosis we are currently lacking infor-mation on, both, the microbial and plant side. For AMfungi we do not yet know whether other AM fungi pro-duce LCOs and whether the conservation of a structure or

492 Chapter 45 Lipochitooligosaccharide Perception and the Basis of Partner Recognition

the ability to produce many different structures would beimportant for activating different hosts. The production ofboth, sulfated and non-sulfated LCOs, by R. irregulariscould suggest that different LCOs may be important fordifferent plant hosts, but this remains to be demonstrated.Clearly, analysis of Myc-LCOs from different membersof the Glomeromycota followed by analysis of symbioticresponses on different plant hosts would help to answerthis question. Calcium spiking is a rapid and sensitivetest that could be exploited for such studies. Secondly,on the plant side we do not know whether the recogni-tion of different AM fungi is because of the conservationof an ancestral LysM-RLK gene (probably of the LYR-type) or the evolution of different receptors for differentMyc-LCOs. The fact that a LysM-RLK has not yet beenidentified in forward genetic approaches on mycorrhiza-tion in model plant species may suggest that there isredundancy in Myc factor perception. Whether a LYK-type gene is involved in mycorrhization also needs to beestablished.

Biochemical and structural data are currently lackingto determine the mechanistic role of LysM-RLKs insymbiont-partner specificity. Although biochemical datasuggest that LysM proteins involved in CO perceptiondirectly bind COs (Gough and Cullimore, 2011), no suchdata is available, at the time of writing, for the symbioticreceptors and LCO binding. Data on mutants and naturalvariation in the LysM domains of M. truncatula, P.sativum, and L. japonicus are consistent with structuralfeatures of Nod factors being directly recognized bythe structure of the LysM domains, particularly LysM2.Molecular modeling and LCO docking studies suggest thateach LysM domain could bind an LCO molecule raisingthe question of a potential cooperativity of these domainsin LCO perception. However, several tantalizing data,especially on M. truncatula, suggest that other proteinscould be involved in the specificity of Nod factor recogni-tion and signal transduction and hence that the symbioticLysM-RLKs may be part of multiprotein complex(es).Advances in the expression of LysM-RLK proteins(Madsen et al., 2010; Lefebvre et al., 2012), in the iden-tification of their protein partners (Mbengue et al., 2010;Ke et al., 2012), and in the synthesis of various LCOsfor receptor–ligand binding studies (Maillet et al., 2011)will clearly help elucidate this point.

In this review, we have focused on the evidence thatsuggests a role of LysM proteins in partner recognition andspecificity in endosymbioses, through perceiving micro-bial LCO signals. This mechanism is almost universal inthe RL symbiosis as almost all Rhizobia studied to datecontain nod genes and rely on them for establishing thesymbiosis. For AM fungi, the sequencing of the genomesand the study of LCOs from other members of the Glom-eromycota will help determine whether LCO signaling is

universal during establishment of this symbiosis. How-ever, the question of whether LCO signaling is essentialfor establishing the AM symbiosis will be very difficult toanswer as AM fungi are not amenable to genetic analysis.The lack of nod genes in some photosynthetic Rhizobiawhich form nitrogen-fixing nodules on legumes from thegenus Aeschynomene, shows that it is possible to circum-vent a LCO recognition step to establish endosymbiosis(Masson-Boivin et al., 2009). Study of this exception tothe Rhizobium rule will provide information on the evolu-tion of the RL symbioses and the diversity of mechanismsof recognition and activation of downstream signaling.

In addition to LCO/LysM-RLK recognition, it isclear that other recognition mechanisms are superimposed(Wang et al., 2012) in order to establish endosymbioses.For example, cell surface components and derivatives(exo- and lipo-polysaccharides and cyclic-β-glucans) inRhizobia play an important role in infection and on theplant side legume lectins have been shown to be involvedin partner specificity. Also, some Rhizobia have a typeIII secretion system (T3SS) that leads to the injection ofprotein effectors into the host and some of these effectorsare involved in host range (Deakin and Broughton, 2009).On the plant side, the discovery that a soybean genethat restricts nodulation with certain Rhizobia strains,encodes a TIR-NBS-LRR protein homologous to a groupof plant disease resistance (R) genes (Yang et al., 2010),suggests a mechanism for the perception of such type IIIeffectors (Wang et al., 2012). Thus not surprisingly plantsand their root endosymbionts may employ a panoply ofmechanisms to ensure the efficient establishment of sym-biosis and to safeguard their intimate intracellular nichefrom exploitation by potentially pathogenic marauders.

ACKNOWLEDGMENTS

We acknowledge funding of recent work on LCOsignaling in our group by the Agence Nationale de laRecherche (contracts NodBindsLysM, SYMPASIGNALand MycSignalling) and the Marie Curie Actions of theEuropean Community (contract NODPERCEPTION).Work in our group has been done in the Laboratoire desInteractions Plantes-Microorganismes (LIPM), part ofthe “Laboratoire d’Excellence (LABEX) entitled TULIP(ANR-10-LABX-41)”. We thank Sandra Bensmihen forcritical reading of the manuscript.

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