Receptor-Like Kinases Sustain Symbiotic Scrutiny1[OPEN]...Update on Receptor-Like Kinases in Symbiosis Receptor-Like Kinases Sustain Symbiotic Scrutiny1[OPEN] Chai Hao Chiu,2 and Uta

Update on Receptor-Like Kinases in Symbiosis

Receptor-Like Kinases Sustain Symbiotic Scrutiny1[OPEN]

Chai Hao Chiu,2 and Uta Paszkowski3

Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom

ORCID IDs: 0000-0002-2840-0407 (C.H.C.); 0000-0002-7279-7632 (U.P.).

Plant receptor-like kinases (RLKs) control the initiation, development, and maintenance of symbioses with beneficialmycorrhizal fungi and nitrogen-fixing bacteria. Carbohydrate perception activates symbiosis signaling via Lysin-motif RLKsand subsequently the common symbiosis signaling pathway. As the receptors activated are often also immune receptors inmultiple species, exactly how carbohydrate identities avoid immune activation and drive symbiotic outcome is still not fullyunderstood. This may involve the coincident detection of additional signaling molecules that provide specificity. Because of themetabolic costs of supporting symbionts, the level of symbiosis development is fine-tuned by a range of local and mobile signalsthat are activated by various RLKs. Beyond early, precontact symbiotic signaling, signal exchanges ensue throughout infection,nutrient exchange, and turnover of symbiosis. Here, we review the latest understanding of plant symbiosis signaling from theperspective of RLK-mediated pathways.

Plants interact with a plethora of other organisms,and these symbioses range from detrimental to benefi-cial in outcomes and from transient to persistent interms of stability. For durable relationships wheresymbiosis is reestablished every generation, the abil-ity to recruit, maintain, and terminate symbiotic rela-tionships involves genetically encoded componentsthat sense and orchestrate signaling, metabolic, anddevelopmental changes in both parties to achieve mu-tually beneficial outcomes. Among these components,receptor-like kinases (RLKs) transduce external andendogenous signals and activate the correspondingsignaling processes.Here, we focus on the roles of plant RLKs in initiating,

establishing, limiting, and maintaining distinct stepsof symbiotic interactions, specifically in root endo-symbioses where the genetic underpinnings are wellunderstood. Box 1 provides a primer on the functionsand stages of the two plant symbioses with arbuscularmycorrhizal fungi (AMF) and with nitrogen-fixingbacteria. At each stage, we highlight emerging themesand the range of pathways identified.With this, we aimto complement the suite of recent reviews of plantsymbioses from the perspective of transcriptional reg-ulation (Diédhiou and Diouf, 2018; Pimprikar andGutjahr, 2018), phytohormone regulation (Fonouni-Farde et al., 2016; Liu et al., 2018; Müller and Harrison,2019), nutrient exchange (Udvardi and Poole, 2013;

Chiu and Paszkowski, 2019), and evolution (Martinet al., 2017; Strullu-Derrien et al., 2018).

THE COMMON SYMBIOSIS SIGNALING PATHWAY

From the diverse microorganisms in the soil, planthosts are able to recruit beneficial symbionts into theroot. Both AMF and nitrogen-fixing bacteria require acore signaling pathway that orchestrates the signalingand developmental changes necessary for symbiontaccommodation. Elucidation of plant symbiotic sig-naling pathways began with forward genetic screens inmodel legumes such as Medicago truncatula (Medicago)and Lotus japonicus (Lotus). Several mutants defective

1This work was supported by a Gates Cambridge Scholarship toC.H.C., the Biotechnology and Biological Sciences Research Council(grant no. BB/P003419/1 to U.P.), and the Bill and Melinda GatesFoundation (grant no. OPP1028264 to U.P.).

2Author for contact: [email protected] author.C.H.C. wrote the article and drew the figures; U.P. edited the

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in nodulation were also defective in arbuscular my-corrhizal symbiosis (AMS), leading to the concept ofa common symbiosis signaling pathway (CSSP). RLKactivation upon ligand recognition converges on theCSSP to activate symbiosis signaling. This ancienttoolkit evolved for AMS and was hypothesized to becoopted by the nodulating clade for root nodule sym-biosis (RNS; Parniske, 2000, 2008; Oldroyd, 2013). TheCSSP begins with SYMBIOSIS RECEPTOR KINASE(SYMRK/DMI2/NORK), a malectin domain-leucine-rich repeat (MLD-LRR) RLK that activates perinuclearcalcium oscillations (hereafter Ca21 oscillations) in-volving Ca21 channels on the nuclear envelope (e.g.DOESN’T MAKE INFECTION1 [DMI1]/CASTOR/POLLUX; Kim et al., 2019). These Ca21 oscillations ac-tivate a CALCIUM AND CALMODULIN-DEPEN-DENT KINASE (CCaMK/DMI3) that interacts withand phosphorylates CYCLOPS/INTERACTING PRO-TEIN OF DMI3 (IPD3), a transcriptional regulator,launching symbiosis signaling. In addition, the nu-clear pore complex components of NUCLEOPORIN85(NUP85), NUP133, and NENA in Lotus are also CSSPcomponents upstream of Ca21 oscillations, but theirsymbiotic defects are temperature sensitive (permissiveat lower temperatures). Readers are directed to previ-ous articles (Parniske, 2008; Oldroyd, 2013; MacLeanet al., 2017) for comprehensive reviews of the CSSP.

Nonetheless, it is important to point out that com-pared with strong nodulation defects, intraradical col-onization as well as arbuscule development is possiblein CSSP mutants, as is observed in Lotus symrk andccamkmutants (Demchenko et al., 2004; Parniske, 2008),that CSSP-independent signaling pathways occur(Kosuta et al., 2003; Kistner et al., 2005; Gutjahr et al.,2008; Camps et al., 2015), and that species-specific dif-ferences exist for mutant phenotypes. CYCLOPS is acase in point, where strong AM phenotypes in Lotusand rice (Oryza sativa) contrast starkly against reducedbut successful arbuscule development in Medicagoipd3/ipd3-like double mutants (Lindsay et al., 2019).Collectively, these and other data show that CYCLOPSis sufficient but not necessary for symbiosis signaling(Pimprikar et al., 2016). The clearest demonstration of aCSSP-independent pathway comes from Lotus LACKOF SYMBIONT ACCOMODATION (LjLAN), whichencodes for a MEDIATOR complex protein and regu-lates both AMS and RNSwhile displaying normal Ca21oscillations to Nod factor. Ljlan is impaired in infectionthread formation but low numbers of intercellular in-fection lead to some nodule development, whereasLjlan/cyclops was completely defective in nodule for-mation (Suzaki et al., 2019).

The existence of CSSP puts symbiosis evolution intoperspective and offers the tantalizing possibility of en-gineering nitrogen-fixing symbiosis into crops byidentifying components and regulatory mechanismsthat evolved to enable RNS (Mus et al., 2016). Yet theCSSP also raises an unresolved conundrum: how dorhizobia bacteria and AMF both activate CSSP but yetspecify different symbiotic outcomes in their host?

Bifurcating downstream transcriptional regulation (e.g.by transcription factors NODULATION SIGNALINGPROTEIN1 [NSP1]/REQUIRED FOR ARBUSCULARMYCORRHIZA1, previously suggested to specify RNS/AMS, respectively [Oldroyd, 2013]) is now revised withevidence demonstrating a role for NSP1 in AMS (Liuet al., 2011; Delaux et al., 2013; Takeda et al., 2013; vanZeijl et al., 2015; Floss et al., 2017), leaving the specificitydeterminants unresolved.

LYSIN-MOTIF RLKS ACTIVATE THE CSSP

Symbiosis signaling initiates with microbial carbo-hydrate perception by plant plasma membrane (PM)-localized RLKs, the very first step of symbiotic scrutiny.In the symbiosis between rhizobia bacteria and legumesor Parasponia, this step is especially stringent, as lip-ochitooligosaccharides (LCOs, also known as Nod fac-tors) from compatible rhizobia bind to and activatetheir cognate RLKs. These Lysin-motif (LysM)-RLKscontain three LysM domains in the extracellular do-main capable of binding oligosaccharides containingb1,4-glycosidic bonds. Forward genetic screens fornodulation-defective mutants and subsequent biochemi-cal characterization identified Lotus NOD FACTORRECEPTOR1 (LjNFR1) and LjNFR5 to be necessary fordirectly binding and perceivingMesorhizobium lotiNodfactors and activating the downstream signaling cas-cades for infection and nodule organogenesis (Madsenet al., 2003; Radutoiu et al., 2003; Broghammer et al.,2012). LjNFR1 and LjNFR5 bind Nod factors at nano-molar ranges, concentrations at which physiological re-sponses are triggered. Loss of either LjNFR1 or LjNFR5abolished infection or nodule organogenesis. MedicagoLYSM DOMAIN RECEPTOR KINASE3 (MtLYK3) andNOD FACTOR PERCEPTION (MtNFP) are similarlyrequired for nodulation. However, Mtlyk3-RNAi ormutants still retain some Nod factor responses, notablyCa21 oscillations. MtLYK3 therefore has been proposedto be an entry receptor that regulates rhizobia infection(Catoira et al., 2000; Wais et al., 2000; Amor et al., 2003;Limpens et al., 2003; Smit et al., 2007). This model alsoholds true for pea (Pisum sativum), where PsSYM10(LjNFR5/MtNFP homolog; Madsen et al., 2003) as wellas PsSYM37 and PsK1 (MtLYK3 homologs; Zhukovet al., 2008; Kirienko et al., 2018, 2019) are involved inthe perception of Rhizobium leguminosarum. Figure 1highlight the roles of the LysM-RLKs involved as wellas a phylogenetic relationship of some of these recep-tors with their most recent gene names. Importantly,the transfer of LjNFR1 and LjNFR5 into Medicago en-abled the Lotus symbiont M. loti to infect and developnodules onMedicago (a nonhost; Radutoiu et al., 2007),demonstrating that the perception of specifically deco-rated LCOs by its cognate LysM-RLKs in a receptorcomplex may determine symbiosis specificity in RNS.

In addition to LjNFR1/5, a new Nod factor receptorwas recently described. LjNFRe is closely related toLjNFR1 and is hypothesized to ensure robust epidermal

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Nod factor signaling at the susceptible zone of Lotusroots (Murakami et al., 2018). Ljnfre mutants developfewer nodules, consistent with its role in inducingNODULE INCEPTION (LjNIN) expression in the epi-dermis. Like LjNFR1, LjNFRe has an active kinasedomain, binding affinity for LCOs, and also phos-phorylates and requires NFR5 for activating epidermalNIN expression. Furthermore, replacing the kinase do-main activation segments of LjNFRe with that ofLjNFR1 is sufficient to change the signaling output tothat of NFR1, enabling cortical NIN induction andnodule development, showing that the activation seg-ment determines signaling specificity between NFR1and NFRe (Murakami et al., 2018).Together, these Nod factor signaling components

present a paradigmwhereby a LysM-RLKwith functional,

active kinase domain (LjNFR1/MtLYK3) forms a com-plex with an RLK with an inactive, pseudokinase do-main (LjNFR5/MtNFP), probably requiring the formerto transphosphorylate the latter to generate a signalingresponse (Madsen et al., 2011; Antolín-Llovera et al.,2014; Murakami et al., 2018). These two types alsocorrespond to the two LysM-RLK clades present in landplants. Why a pseudokinase is necessary for signalingremains to be fully appreciated, but the additional in-volvement of LjSYMRK in phosphorylating LjNFR5may point to the formation of heterotypic complexes ordifferent separable, distinct receptor complexes duringRNS as well as AMS.In contrast to RNS, where Nod factor perception ex-

plains most of host-symbiont specificity, the nature,range, and exact suite of ligand(s) for AMS are hitherto

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Figure 1. LysM-RLKs in symbioses and beyond. A, LysMReceptorswith characterised roles in symbiosis or immunity. Nod-factorsactivate receptor complexes that comprise both active kinases (blue) and pseudokinases (red), with homologues identified inLotus,Medicago and Pea. Only the pseudokinase LysM-RLK is known for P. andersonii, a species of the only non-legume genusthat engages in root nodule symbiosis with Rhizobia. A similar receptor complex may be employed for AMF perception; indeed,numerous active kinases have been identified to activate symbiosis signaling in response to AMF, whereas the identities ofpseudokinases are less clear. Knockouts of NFR5/NFP pseudokinases have wild-type colonization, and only have a quantitativecontribution in the case of Mtnfp/cerk1 double mutants. However, PaNFP/SlLYK10 may be required for full arbuscule devel-opment. Some receptors (e.g. MtLYR3) have been characterized biochemically to be a high-affinity LCO receptor, but theirsymbiotic roles are not reported, or potentially redundant (e.g. LjLYS11) and are excluded in (A). While CERK1/LYK1/LYK9orthologs are required for both immunity and AMS in some species, some species-specific differences exists (e.g. for tomato,Lotus). And while Nod-factors (LCOs) and short-chain chitin oligomers (CO4 and CO5) are non-immunogenic (with respect toreactive oxygen species [ROS] production and MAPK activation) symbiosis signaling elicitors; peptidoglycan and long-chainchitin oligomers (CO8) can activate both immune and symbiosis signaling. Note that superscript numbers indicate: 1,MtLYK3 isnot equivalent to LjNFR1 sensu stricto, sinceMtlyk3 retains some nod-factor responses, especially Ca21-oscillations whereas allnod-factor responses are abolished in Ljnfr1; 2, RNA-silencing was used, hence non-specific silencing of other LysM-RLKs cannotbe ruled out in RNAi lines except in SlLYK12/1/10where it was examined; and 3, Different AMS phenotypes in different reports.Ljnfr1 andMtlyk3 had weak phenotypes at early-timepoints and at low spore inoculums but not in two other separate studies. B,Unrooted maximum likelihood phylogenetic tree of full length LysM-RLKs containing active kinases with clear, characterizedroles in immunity and/or symbiosis. The two most recent gene names are included, as different publications may refer to samegenes with different names. For each node, bootstrap values are shown as percentages based on 1000 repetitions. Note thatbranch lengths are not proportional to the number of substitutions per site. Species prefixes are: At, Arabidopsis thaliana; Lj, Lotusjaponicus;Ma,Musa acuminata;Mt,Medicago truncatula;Os,Oryza sativa; Pa, Parasponia andersonii; Ph, Petunia hybrida; Ps,Pisum sativum; Sl, Solanum lycopersicum.


Chiu and Paszkowski



elusive. AMF produce simple LCOs and short-chainchitin oligomers (CO4 and CO5), both of which elicitsymbiotic outputs via the CSSP, often shown by acti-vation of Ca21 oscillations and increased lateral rootformation (Maillet et al., 2011; Genre et al., 2013; Sunet al., 2015). Exogenous application of LCOs increasedroot length colonization by AMF, consistent with itsrole as a positive regulator of symbiosis (Mailletet al., 2011). However, apart from LCOs and CO4 andCO5, the elicitors capable of activating Ca21 oscillationsand symbiotic gene expression may be wider thanexpected. Feng et al. (2019) demonstrated that CO8 andpeptidoglycan (PGN), typically considered pathogen-associated molecular patterns, are also capable of elicit-ing symbiotic signaling outputs. Coordinated perceptionof LCO and CO8/PGN attenuated the immunity out-puts of ROS production and MAPK activation andsynergistically boosted symbiotic gene expression,thereby suggesting that AMS might involve coordi-nated perception of microbe-associated molecularpatterns via LysM-RLKs and activating symbiosis sig-naling via SYMRK. Thus, redundancy at both ligandand receptors could explain (1) the lack of LysM-RLKmutants where symbiosis with AMF is abolished and(2) the broad host range of AMF.While the exact nature of signaling molecules re-

mains to be identified, it is clear that carbohydrateperception by LysM-RLKs activates symbiosis signal-ing in AMS. The requirement for LysM-RLKs has re-cently been elucidated in various plant species. Firstdemonstrated in rice, similar work in pea, Medicago,tomato (Solanum lycopersicum), and banana (Musa acu-minata) all revealed that the LysM-RLK CHITINELICITOR RECEPTOR KINASE1 (CERK1; or equiva-lent homologs) is involved in signal perception of AMF-derived chitinaceous ligands, since cerk1 null and/orcerk1-RNAi had reduced colonization (Miyata et al.,2014; Zhang et al., 2015, 2019; Leppyanen et al., 2017;Chiu et al., 2018; Liao et al., 2018; Feng et al., 2019;Gibelin-Viala et al., 2019). Importantly, symbiosis is notabolished, showing that CERK1 is necessary but notessential for AM symbiosis. In all cases (except for pea,where microscopy data are lacking), arbuscule devel-opment appears normal.Furthermore, in rice, where the LysM-RLK family is

less expanded vis-à-vis the Fabaceae, Oscerk1 mutantslack detectable epidermal Ca21 oscillations in responseto CO4 and germinated spore exudates, a cocktail ofsignal molecules from naïve AMF spores (Carotenutoet al., 2017). Nevertheless, intraradical colonization andarbuscule formation in Oscerk1 mutants (Miyata et al.,2014; Zhang et al., 2015; Chiu et al., 2018) suggest thateither symbiosis development can be achieved inde-pendent of Ca21 oscillations or that yet unobservedoscillations occur under prolonged signal exchangeemploying redundant LysM-RLKs or SYMRK.As such, in contrast to their essential role for symbi-

otic scrutiny in most RNS, the role of LCOs in AMsymbiosis is at present equivocal. Null mutants ofLjnfr5,Mtnfp displayed wild-type levels of colonization

(Maillet et al., 2011; Zhang et al., 2015; Feng et al., 2019).Contradictory observations were made for Ljnfr1 orMtlyk3 mutants, which either showed transiently re-duced colonization (Zhang et al., 2015) or no differencerelative to the wild type (Takeda et al., 2011; Feng et al.,2019). Normal colonization in mutants of LCO recep-tors may be due to the genetic redundancy present inthe Fabaceae, where the receptors have expanded. Forinstance, LjLYS11 encodes an LCO receptor closely re-lated to LjNFR5 that is induced during AM coloniza-tion. Yet, even triple mutants of Ljnfr1 nfr5 lys11 inLotus had no observable defects in AM colonization(Rasmussen et al., 2016). Also, whereas silencing Para-sponia andersonii NFP (PaNFP) did not prevent fungalcolonization, it blocked arbuscule development anddecreased nodulation frequency (Op den Camp et al.,2011). However, it cannot be excluded that closely re-lated LysM-RLKs may be silenced as well. WhetherPaNFP directly binds to or mediates LCO perceptionhas yet to be shown. In rice, where the LysM-RLKfamily is not neofunctionalized for RNS, knockout ofthe LjNFR5/MtNFP ortholog, OsNFR5/RLK2/MYR1,produced contrasting phenotypes. Homologous re-combination mutants showed normal AM symbiosis orCa21-spiking responses to germinated spore exudates(Miyata et al., 2016; Carotenuto et al., 2017). However,CRISPR-edited mutants of the same gene had reducedcolonization levels under low spore inoculum strength.Under high inoculation strength, osnfr5/rlk2/myr1 havewild-type-like colonization levels (He et al., 2019),suggesting that the CO4-binding function of OsNFR5/MYR1 is dispensable for symbiosis.On the other hand, LCO receptors of two sol-

anaceaeous species, petunia (Petunia hybrida) and to-mato, have been recently identified to be involved inAM symbiosis. Virus-induced gene silencing of the to-mato ortholog (SlLYK10) delayed fungal colonizationand resulted in abnormal arbuscule development(Buendia et al., 2016). Similarly, in a missense allele ofSllyk10-1, and in Phlyk10-1, a transposon-insertionmutant, the mutation/loss of LCO receptor quantita-tively reduced AM colonization and full arbuscule de-velopment in both species (Girardin et al., 2019).Importantly, both SlLYK10 and PhLYK10 can restoreRNS inMtnfp and Ljnfr5mutants (Girardin et al., 2019).This is consistent with the notion that the evolution ofRNS recruited existing components involved in AMS.Therefore, evidence so far suggests that LCO per-

ception by LysM-RLKs contributes to, but may not beessential for, AMS. This is supported by the observationthat reduction in AM colonization is further diminishedin the Mtcerk1 nfp double mutant relative to Mtcerk1mutants (Feng et al., 2019). Whether LCO perception byLysM-RLKs is necessary, or fully dispensable, for AMsymbiosis remains to be ascertained.Overall, carbohydrate signaling during AMS appears

to be more complex than expected, as symbiont-encoded proteins can also affect ligand binding andreceptor activation. Fungus-secreted LysM effectorspreviously described to be involved in subverting host





defense responses during chitin-triggered immunityhave been recently identified in AMF as well (Schmitzet al., 2019; Zeng et al., 2020). Rhizophagus irregularisSECRETED LYSM EFFECTOR (RiSLM) was capable ofbinding both COs and LCOs. Recombinant RiSLM re-duced chitin-triggered ROS production but allowed theactivation of symbiosis signaling. Consistent with itsrole as a positive regulator, host-induced gene silencingof RiSLM reduced AM colonization, although over-expression of RiSLM in Medicago did not increasecolonization (Zeng et al., 2020).

Whereas mutants of LysM-RLK show reduced sym-biosis, AMS is fully abolished when the a/b-hydrolasereceptor DWARF14-LIKE (D14L) is mutated (Gutjahret al., 2015; Liu et al., 2019). How D14L, the ancientparalog of the strigolactone receptor D14, fits with theCSSP in AM signaling remains to be demonstrated.Also unknown is the identity of the karrikin-like D14Lligand, of plant or fungal origin. Nonetheless, the es-sential function of D14L for AMS in rice is conserved inpetunia (Gutjahr et al., 2015; Liu et al., 2019), suggestingthat the critical role of D14L in symbiotic scrutiny issimilar inmonocotyledonous and dicotyledonous plantspecies.

MULTIFACETED LYSM RECEPTORS HAVE ROLESBEYOND SYMBIOSIS

An enigma with the chitin receptor is its role in acti-vating both defense and symbiotic responses in responseto fungal elicitors. This is the case in rice, Medicago, andpea (Miyata et al., 2014; Leppyanen et al., 2017; Gibelin-Viala et al., 2019). Like its Arabidopsis (Arabidopsisthaliana) counterpart, CERK1 is required for activatingimmunity outputs (ROS production and MAPK acti-vation; Miya et al., 2007; Wan et al., 2008) in both rootsand shoots, but it also mediates AM symbiosis in theroots. Nonetheless, ROS and MAPK assays are limitedin their specificity, as they are also triggered by damage,development, and early AMF perception. When ex-posed to pathogens, Oscerk1/Mtcerk1 mutants are moresusceptible to both fungal (rice blast, Magnaporthe ory-zae; Kouzai et al., 2014a) and also oomycete (in the caseof Mtcerk1) pathogens (Bozsoki et al., 2017; Gibelin-Viala et al., 2019). Yet immunity and symbiosis ap-pear to be separable in other species instead, showingthat species-specific differences and subfunctionaliza-tion of chitin receptors exist (Fig. 1). For example, inLjcerk6 mutants, where chitin-induced responses wereabolished, AM colonization was not affected (Bozsokiet al., 2017). Similarly, in tomato, whereas silencingSlLYK12 impaired AM colonization, silencing SlLYK1/Bti9 impaired ROS production and resistance to Pseu-domonas syringae (Zeng et al., 2012; Liao et al., 2018).

Symbiosis versus immunity signaling could be de-termined at the level of receptor/coreceptor complexcomposition. For instance, OsCEBiP, the high-affinityreceptor for CO8, is not involved in AM symbiosisbut in basal resistance against M. oryzae (Kaku et al.,

2006; Kishimoto et al., 2010; Kouzai et al., 2014b).However, the fact that CO8 and CERK1 signaling canactivate both defense and symbiotic signaling meansthat while exclusively pathogenic elicitors may activatedistinct immune receptor complexes, other elicitorsmay activate overlapping pathways for immunity andsymbiosis via a shared coreceptor and require othermicrobe-associated molecular pattern detection and/oreffector detection for signal integration before a fullimmune or symbiosis response is mounted. This isconsistent with observations that AMF transiently ac-tivates early defense responses that are attenuated(Harrison and Dixon, 1993; Kapulnik et al., 1996;García-Garrido and Ocampo, 2002; Liu et al., 2003;Campos-Soriano et al., 2010). It has been proposed thatthe changing ratio of immune-modulating CO4 andCO5/LCOs relative to immunogenic CO7-8 may berelevant (Schmitz and Harrison, 2014), but the sup-porting evidence is lacking.

On top of symbiosis signaling and chitin-triggereddefenses, OsCERK1 is also required for increased root-branching responses, specifically of large lateral roots,the preferred root type for AMF colonization (Chiuet al., 2018). The ultimate explanation for this re-sponse is perhaps that symbiont perception elicits anoverall increase in symbiotic interfaces available. Lat-eral root branching has been used as a biological assayfor Myc/Nod factors. In rice, this developmental re-sponse is however independent of CSSP and also in-dependent of the karrikin receptor D14L that isnecessary for presymbiotic dialogue and symbiotic ac-commodation (Gutjahr et al., 2009, 2015; Chiu et al.,2018). The developmental and intraradical coloniza-tion steps are thus genetically separable, at least in amonocot.

LCO PERCEPTION AND CSSP MAY NOT BE SPECIFICTO ENDOSYMBIOSIS SIGNALING

As Nod factors and potentially Myc factors, LCOshave been regarded as a nonimmunogenic symbioticsignal that connects membrane perception to CSSPfor endosymbiont accommodation. Before discussingsignaling events downstream of LysM-RLKs, it is im-portant to highlight that many nonendosymbiontsproduce similar signaling molecules that activate CSSP.Therefore, how specificity between different organismsin the rhizosphere is achieved is still unresolved.

LCO production is not an endosymbiont-exclusivetrait, as they have recently been demonstrated to alsobe produced by ectomycorrhizal (ECM) fungi, anotherecologically important plant-fungus symbiosis (Copeet al., 2019) but fundamentally not an endosymbioticassociation. The fungus Laccaria bicolor produces LCOsand elicits perinuclear Ca21 oscillations in Poplar tri-chorcarpa via PtCASTOR/POLLUX. Both castor/pollux-RNAi and ccamk-RNAi show slightly reduced ECMformation (Cope et al., 2019). However, a cautious in-terpretation is that interfering with CSSP affects both


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LCO and CO signaling, so whether ECM symbiosisrequires LCO recognition is not yet demonstrated. Asalluded to by this and earlier work, CSSP componentsare missing in some host species (e.g. Norway spruce[Picea abies]) and hence is likely not a conserved path-way required for ECM associations (Garcia et al., 2015;Cope et al., 2019). Moreover, compounds with nearlyidentical chemical and biological properties to LCOshave been reported to be produced by nitrogen-fixingmaize (Zea mays) root endophytes of Bacillus spp. (Lianet al., 2001) and nematodes (Weerasinghe et al., 2005).Finally, the LCO perception machinery is involved inother biotic interactions where Mtnfp mutants weremore susceptible to oomycete and fungal infections(Rey et al., 2013). Collectively, it appears that LCOproduction and perception in the rhizosphere may ex-tend beyond AMF and rhizobia.Downstream of LCO perception, Ca21 oscillations

are also not exclusively activated by fungal or rhizobialsymbionts, as glucan-chitosaccharide fractions fromAphanomyces euteicheswere reported to elicit perinuclearCa21 oscillations independent of MtNFP, MtDMI1, andMtDMI2 (Nars et al., 2013). However, the robustness ofCa21 oscillations in the traces, especially for the mu-tants, appears to be relatively low.More recently, it wasdemonstrated that endophytic Fusarium solani andSerendipita indica, as well as pathogenic Fusarium oxy-sporum, are capable of eliciting Ca21 oscillations (Skiadaet al., 2020). Clearly, beyond symbiotic AM/ECM fungiand rhizobia, nuclear Ca21 oscillations can also be ac-tivated by other microorganisms.In addition, the loss of LysM-RLK or CSSP signaling

could either suppress (Skiada et al., 2020) or promotecolonization by nonendosymbionts (Fernandez-Aparicioet al., 2010). There is also no obvious trend whether thiscorrelates with the intimacy of the pathogen lifestyle.However, the small quantitative nature of these effectsrelative to those of CSSP mutations on AMS/RNSsuggests that endophytes/pathogens rely less on theCSSP for host access. Further support is provided byM. oryzae, which leads an extended biotrophic lifestylein rice roots but is still capable of proliferating in rootsof rice CSSP mutants and d14l mutants (Marcel et al.,2010; Gutjahr et al., 2015), where AMF accommodationis abolished.Recently, next-generation sequencing has enabled

the profiling of bacterial and fungal communities in theroot microbiome (Zgadzaj et al., 2016; Thiergart et al.,2019; Xue et al., 2019). As demonstrated for Lotus mu-tants, the use of microbiome profiling, especially incomplex soils or controlled mesocosms, may allow amore refined understanding of whether the CSSP/LysM-RLK/autoregulatory mutants become more orless susceptible to pathogens/biotrophs of interestwhen challenged in a nearly native context.Moving out of the model legumes, LCOs are not nec-

essary for symbiosis signaling. In the rhizobia-legumesymbiosis between photosynthetic Bradyrhizobiumspp. and Aeschynomene sensitiva and Aeschynomeneindica, nodules can develop in both stems and roots of

the tropical tree without involving LCOs (Giraud et al.,2007) but still requiring the CSSP (Fabre et al., 2015).Similarly, in the actinorrhizal species Casuarina glauca,the unknown elicitors of Nod factor-independent sig-naling have been characterized to be heat stable, hy-drophilic, and chitinase resistant (Chabaud et al.,2016). For Nod factor-independent signaling, thetype III secretion system of Bradyrhizobium spp. hasbeen shown to be required for successful symbiosisdevelopment by delivering effectors that manipulatehost processes (Okazaki et al., 2016; Miwa andOkazaki, 2017).

RLK INTERACTIONS DOWNSTREAM OFLIGAND PERCEPTION

After ligand binding, downstream signaling eventsoccur to allow symbiont infection of the host. DuringRNS, concomitant production of lateral organs (nod-ules) is also required for infection and functionalnitrogen fixation. Intimate association is only achievedseveral days after the initial signal exchanges, so thespatiotemporal regulation of RLKs and their effect ontranscriptional networks are instrumental to under-standing symbiosis. To date, a few downstream com-ponents have been identified (Fig. 2), but there is not yetan overarching view of how these components link theRLKs to transcriptional regulation via the CSSP (Fig. 2).The localization and stability of LysM-RLKs is al-

tered upon ligand perception, which is important forallowing rhizobia infection to proceed. One of theemerging insights of receptor biology is their com-partmentalization at the PM into nanodomains/microdomains (Ott, 2017). RLKs in immunity signaling,such as Arabidopsis FLAGELLIN SENSING2 andBRASSINOSTEROID INSENSITIVE1, have been local-ized to submicron protein/lipid assemblies and arehypothesized to nucleate other signaling componentsthat change in size and composition dynamically, es-pecially upon stimuli (Bücherl et al., 2017). Thesemembrane nanodomains therefore could partitionRLKs into functionally distinct signaling units. Medi-cago MtLYK3, MtNFP, and MtDMI2 all are able to in-teract with SYMBIOTIC REMORIN1 (MtSYMREM1), amolecular scaffold protein (Tóth et al., 2012; Lianget al., 2018). Prior to Nod factor or rhizobia applica-tion, MtLYK3 has high lateral mobility at the PM, af-ter which it becomes immobilized in nanodomainslabeled by MtFLOTILLIN4 (MtFLOT4; Haney et al.,2011). MtSYMREM1 mediates the immobilization andstabilization of MtLYK3, reduces its endocytosis, andincreases its dwell time in the domain. This is hypoth-esized to recruit additional signaling components thatdetermine infection and control symbiosis output. Ge-netic evidence implicates MtFLOT4 and MtSYMREM1for successful infection, and cell biology revealsMtFLOT4/2 and actin to serve as a primary core ofthe nanodomain and MtSYMREM1 as a symbioticallyinduced secondary component (Liang et al., 2018).





Figure 2. Posttranslational modifications and interactions of LysM-RLKs determine symbiotic outcome. A, General themes ofreceptor regulation and activation towards symbiosis signaling. B, Known regulators of receptors. Signal cascades downstream ofRLKs is best understood in Lotus japonicus (Lj) and Medicago truncatula (Mt). Nod-factor perception by LjNFR1/5/e complexesactivate phosphorylation cascades and transduces signals to LjNiCK4, which then accumulates in the nucleus, positively regu-lating nodule organogenesis via an unknown mechanism. SYMRK activation can also directly activate transcription (e.g. viaLjSIP1 to activate LjNIN expression) or suppress MAPK signalling (via LjSIP2). RLKs are also regulated by ubiquitination (suchregulators denoted in gray), which may target RLKs to degradation via the 26S proteasome (e.g. MtPUB2), or alter RLK inter-actions/localization (e.g. LjSINA4, MtPUB1). Equally, they can be either positive or negative regulators of symbioses. LotusPUB13, SINA4, and SIE3 and Medicago PUB1 and PUB2 are E3 ubiquitin ligases, but can either promote or suppress symbiosis.Deubiquitination of K63-linked ubiquitin chains by LjAMSH1 is also required to coordinate infection and nodule organogenesis;although the differentially ubiquitinated targets are not clear. In Medicago, MtDMI2 was proposed to activate MtHMGR1 andhence the mevalonate pathway to produce a secondary messenger linking plasma membrane activation to perinuclear Ca21-oscillations; however genetic data is still elusive. Cell biology and genetics also revealed that creation of nanodomains as po-tential signaling clusters are instrumental for infection. MtFLOT2/4 and actin are primary nucleation sites, stabilising MtLYK3while symbiosis-inducedMtSYMREM1 can further recruitMtNFP, DMI2 to the cluster. With the numerous possible downstreamsignals, how each of these pathways are activated in space and time and controlled during symbiosis will be important to fullyunderstand the signaling processes in both RNS and AMS.


Chiu and Paszkowski



One possible member recruited for signaling down-stream of LCO perception may be Lotus RHIZOBIALINFECTION RECEPTOR-LIKE KINASE1 (LjRINRK1),an atypical receptor kinase first identified from amutant screen for infection thread defects. The full in-duction of early infection genes, including LjNIN, isindependent of LjRINRK1 kinase activity and maypoint to a possible role in a larger receptor complex incoordinating Nod factor signaling (Li et al., 2019).Downstream of LysM-RLKs, multiple signaling path-

ways emerge from SYMRK, ranging from the activationof the mevalonate pathway for a putative second-ary messenger for activating Ca21 oscillations (Keveiet al., 2007; Venkateshwaran et al., 2015), inhibitionof MAPKK activity (Chen et al., 2012), and impor-tantly to activation of LjNIN (Zhu et al., 2008; Wanget al., 2013), a transcriptional regulator that coordina-tes both infection and nodule organogenesis and re-cently was demonstrated to have recruited the lateralroot development pathway for nodule development(Schiessl et al., 2019). Because overexpression ofLjNFR5, LjNFR1, or especially LjSYMRK is sufficient forspontaneous nodule formation in the absence of sym-biont (Ried et al., 2014), it is unsurprising that thereis a multitude of regulatory mechanisms on the recep-tors to ensure appropriate activation only when thesymbiont is present. One notable mechanism is viathe cleavage of its MLD. When expressed in tobacco(Nicotiana tabacum) leaves, truncated LjSYMRK(ΔMLD)had increased affinity for LjNFR5, whereas the LRRdomain regulates its turnover (Antolín-Llovera et al.,2014). Whether overexpression of LjNFR1/LjNFR5/LjSYMRK or ectodomain cleavage/turnover of LjSYMRKalso affects AMS, or is specific to RNS, remains to beaddressed.Nod factor receptors and SYMRK have also been

revealed to be under tight posttranslational regulationby ubiquitination via Plant U-Box (PUB) proteins. Thereis an increasing body of work on the regulation ofRLKs, specifically MtLYK3, LjSYMRK/MtDMI2, andLjNFR5, by various PUBs. LjSYMRK, for example, istargeted by Lotus SEVEN IN ABSENTIA4 for degra-dation, negatively regulating RNS but not AMS (DenHerder et al., 2012). LjCERBERUS, an E3 ubiquitin li-gase, as well the deubiquitinating enzyme ASSOCI-ATED MOLECULE WITH THE SH3 DOMAIN OFSTAM (AMSH1) are also involved in both infectionand nodule organogenesis processes (Yano et al.,2009; Małolepszy et al., 2015). As a posttranslationalmodification, changes in the ubiquitination of RLKsor other signaling components by PUBs/E3 ligasesmay target them for degradation by the 26S protea-some. But as seen in the case with MtPUB1, ubiquiti-nation may not always lead to degradation butpossibly altered localization via endocytosis/vesicletrafficking or altered protein-protein interactions(Mbengue et al., 2010; Vernié et al., 2016). The impor-tance of ubiquitination as well as the signaling com-ponents modified in both symbioses still require a morecomplete analysis.

Downstream of membrane perception, the phos-phorylation cascades are also not well understood.Recently, a receptor-like cytoplasmic kinase (RLCK)was identified biochemically to act downstream ofLjNFR5. Lotus NFR5-INTERACTING CYTOPLASMICKINASE4 (LjNiCK4) phosphorylates LjNFR1 andLjNFR5 in vitro and relocates to the nucleus upon Nodfactor perception to positively regulate nodule orga-nogenesis (Wong et al., 2019). LjNiCK4 adds to thegrowing theme of RLK-RLCK combinations in othersignaling contexts (e.g. immunity and stress response;Liang and Zhou, 2018).Overall, complex signaling activation and regulation

occurs for receptors, especially for SYMRK, but howthis receptor functions to specify fungal and bacterialsymbioses remains to be fully dissected. Keeping Nodfactor-independent RNS in mind, the effector ErnA isdelivered to activate nodule development, and ectopicexpression in plant roots generated numerous lateralmeristem-like structures along the root length (Teuletet al., 2019). This could be a result of ErnA interactingwith RLK/SYMRK and the CSSP or of activating lateralorganogenesis independently.During RNS, symbiont recognition also activates the

expression of other LysM-RLKs for additional symbi-otic scrutiny by the plant host. Nod factor perception byLjNFR1/5 activates CSSP and induces the expression ofanother LysM-RLK, Lotus EXOPOLYSACCHARIDERECEPTOR3 (LjEPR3; Kawaharada et al., 2015, 2017).The surface exopolysaccharides (EPS) of rhizobia havebeen long studied for their roles in symbiosis. Like Nodfactors, EPS have strain-specific characteristics but areheteropolymers of eight to nine sugar residues. TheLjEPR3 extracellular domain directly binds EPS, andLjepr3 mutants show that EPS recognition is necessaryfor intracellular infection progression in epidermis,cortex, and into nodule primordia (Kawaharada et al.,2015, 2017).The continuation of both bacterial Nod gene expres-

sion and plant expression of LjNFR1/NFR5/EPR3 incortex cells of developing nodule primordia (but notmature ones), observations of Ca21 oscillations aheadof the growing infection thread (Sieberer et al., 2012),together suggest that Nod factor perception and CSSPactivation are involved beyond presymbiotic stages,through to infection and nodule development. Thisinsight is also demonstrated using promoters that en-rich for epidermal or cortical expression (Rival et al.,2012; Hayashi et al., 2014). Beyond RNS, the role ofEPR3-type LysM-RLKs remains enigmatic, as recentphylogenetic and phylogenomic surveys of LysM-RLKs identified the LjEPR3/MtLYK10 clade to be con-served in plant species capable of engaging in AMS,suggesting a possible function during AMS (Bravoet al., 2016; Buendia et al., 2018). The likely rice ortho-log, OsLYK1, is induced by AMF and expressed inarbusculated cells, but Oslyk1 mutants showed no de-fects in AMS (Roth et al., 2018). Thus, whether thevarious mechanisms of symbiotic scrutiny that are de-scribed for RNS also exist in AMS remains unknown.





RLKS AND SIGNALING NETWORKS ARE REQUIREDIN LATE STAGES OF SYMBIOSIS

Apart from vesicle trafficking and nutrient exchangein sustaining AM symbiosis development (for review,see Harrison and Ivanov, 2017; Chiu and Paszkowski,2019), we now also appreciate the existence of RLK-mediated signaling in symbiosis maintenance via noveland unknown signaling cascades at the periarbuscularmembrane. Rice ARBUSCULE RLK1 (OsARK1)/Medi-cago KINASE3 encodes a Ser/Thr RLK present in AMhost species (Bravo et al., 2016; Roth et al., 2018). Lo-calized to the periarbuscular membrane, OsARK1controls processes after arbuscule development and isrequired for sustaining fungal fitness during progress-ing symbiosis, as reflected by the compromised vesicleformation inOsark1mutants (Roth et al., 2018). Vesiclesemerge as lipid storage bodies and could be crucial forsubsequent rounds of infection affected in Osark1 mu-tants. Interestingly, the Osark1 phenotype is rescued inthe presence of a common mycorrhizal network whereAMF are supported by wild-type plants, suggestingthat OsARK1 is required for fungal vigor (Roth et al.,2018). The OsARK1 signaling mechanisms are still un-known. Whether kinase activity is required for itssymbiotic role, and whether it is required to suppressdefense signaling, also remain to be shown.

On the other hand, immune suppression is requiredfor the full development of functional nitrogen-fixingsymbiosis, especially when bacteria in infection threadsare released into nodules. In many nonfixing nodulat-ing mutants, strong immune responses lead to white,necrotic, nonfixing nodules, highlighting the need forimmune suppression for continued symbiosis. Medi-cago SYMBIOTIC CYSTEINE-RICH RLK (MtSymCRK)is the only Cys-rich RLK known to have a symbioticfunction so far (Berrabah et al., 2014), whereas the othermembers of the RLK family have known roles in de-fense, abiotic stress response, and programmed celldeath. Recently, it was demonstrated that MtSymCRKworks through ethylene signaling (Berrabah et al., 2018)as well as the CDPK-Rboh signaling axis to regulateimmune responses in nodules (Yu et al., 2018).

Secreted, Cys-rich peptides provide another mecha-nism for exercising host-symbiont compatibility, espe-cially for symbiotic function (N fixation). These peptidesare typically regarded as antimicrobial defense pep-tides (defensins). Nodule Cys-rich (NCR) peptidesconstitute an expanded, large gene family in invertedrepeat-lacking clade legumes. The small, white, non-fixing nodules in mutants lacking specific NCRs pointto an essential function in developing functional RNS(Horváth et al., 2015; Kim et al., 2015). The majority ofNCRs are targeted to the bacteria, where they are pro-posed to act in ensuring terminal differentiation ofthe symbionts into bacteroids. At the same time, rhi-zobial bacteria are capable of degrading NCRs, illus-trating a molecular dialogue at late symbiotic stages(Price et al., 2015). Excessive levels of NCRs are detri-mental to RNS; thus quantity, combination of NCRs,

and bacterial strains are important determinants ofsymbiotic outcome (Pan and Wang, 2017). However,other mechanisms of scrutiny at the stage of N fixationprobably exist, as NCRs appear to have arisen ininverted repeat-lacking clade legumes with indetermi-nate nodules (where the meristem is maintained andbacteroids are terminally differentiated), indepen-dently in dalbergoid legumes (e.g. Aeschynomene spp.;Czernic et al., 2015), and not in legumes with determi-nate nodules such as soybean (Glycine max), wherephytoalexin accumulation and hypersensitive responseappear to play a role (Parniske et al., 1990; Mergaertet al., 2003). The existence of symbiotic scrutiny up tothe late stages of symbiosis is perhaps driven by theneed to monitor and terminate nutritional exchanges.

Taken together, immune modulation for symbiosis isa recurring requirement from precontact signal ex-change to intimate endosymbiotic accommodation.

PEPTIDE LIGANDS AND COGNATE RLKS ENFORCEHOST CONTROL OF THE EXTENT OFSYMBIOSIS DEVELOPMENT

Because symbiosis with microorganisms comes at acost mostly but not limited to carbon, the extent ofmicrobial proliferation in situ needs to be balanced tothe nutrient demands of the plant. To achieve this,signal perception by RLKs also activates negative reg-ulatory mechanisms, which act systemically via an-other set of RLKs (Fig. 3).

Autoregulatory mechanisms involve systemic pep-tide transport and are common to both rhizobia-legumesymbiosis and AMS. RWP-RK-type transcriptionalregulators of the NIN/NIN-like protein (NLP) family,named after the conserved amino acid residues in theclade, activate the expression of root-derived CLAV-ATA3/EMBRYO SURROUNDING REGION (CLE) pep-tides. Nod factor perception activates LjNIN, whichtranscriptionally induces LjCLE-RS1,2. In the shoot,they activate the CLAVATA1-like LRR-RLKs Medi-cago SUPER NUMERIC NODULES1/Lotus HYPER-NODULATIONABERRANTROOT1/soybeanNODULEAUTOREGULATIONRECEPTORKINASE1 (MtSUNN1/LjHAR1/GmNARK1) that repress nodule formation inthe roots (Krusell et al., 2002, 2011; Nishimura et al.,2002; Searle et al., 2003). Ligand-receptor binding ofthe glycosylated LjCLE-RS2 peptide to LjHAR1 hasalso been demonstrated (Okamoto et al., 2013). Similarto the regulation of shoot apical meristem in Arabi-dopsis, autoregulation of nodulation also requiresMedicago and pea CLV2 and Medicago CORYNE, andin Lotus, another LRR-RLK, KLAVIER, is also involved(Miyazawa et al., 2010; Krusell et al., 2011; Crook et al.,2016), as summarized in Figure 3. In addition, signalingvia MtSUNN1/LjHAR1/GmNARK1 also integratesthe plant nitrate status (Jeudy et al., 2010; Reid et al.,2011; Okamoto and Kawaguchi, 2015). Using a forwardgenetics screen for loss of nitrate-suppression of nod-ulation in Lotus or a reverse genetic screen for NLP


Chiu and Paszkowski



mutants in Medicago, Lotus NITRATE UNRESPON-SIVE SYMBIOSIS1/NLP4 andMtNLP1 were identifiedto undergo nitrate-triggered nuclear accumulation,inducing their downstream CLE-RS peptides (Linet al., 2018; Nishida et al., 2018). Together, nitrate sta-tus and rhizobia presence work via RWP-RK regulators(NLPs) to induce specific and overlapping sets of CLEpeptides that activate shoot CLV1-like RLKs (Nishidaand Suzaki, 2018).One of the consequences of LjHAR1 activation is the

down-regulation ofmiR2111production, amobile shoot-to-root signal delivered via the phloem. OverexpressingmiR2111 resulted in hypernodulation (Tsikou et al.,2018), as it targets Lotus TML, a Kelch F-box proteinthat negatively regulates nodulation (Magori et al.,2009; Takahara et al., 2013). In uninfected roots, lowTML levels maintain susceptibility for rhizobia. In

symbiotic plants, root-derived CLE peptides activateLjHAR1 in the shoot, which reduces miR2111 abun-dance in shoots and roots, allowing LjTML to negativelyregulate RNS (Tsikou et al., 2018). Another consequenceof LjHAR1 signaling is shoot-derived cytokinins, whichalso inhibit nodulation (Sasaki et al., 2014).Meanwhile, the CLE-SUNN signaling module also

regulates AMS. As in RNS, plant phosphate status andAM colonization induce root-derived CLE peptidesthat negatively regulate AM symbiosis. Overexpressionof Pi-induced MtCLE33, or AM-induced MtCLE53, re-duced colonization levels by inhibiting the expressionof genes involved in SL biosynthesis and transport,leading to reduced SL levels (Müller et al., 2019). Ac-cordingly, the reduced AM colonization could beovercome by exogenous application of the synthetic SLanalog GR24. MtSUNN1 is required for CLE-mediated

Figure 3. Auto-regulation of nodulation and mycorrhizal symbioses share overlapping signalling pathways. Nod-factors activatea RWP-RK transcriptional regulator LjNIN which positively regulates nodule organogenesis, but also initiates long-distancenegative regulation by activating LjCLE-RS transcription. Nitrate sufficiency induces nuclear accumulation of paralogous NLPsLjNRSYM1 andMtNLP4 to transcriptionally activate overlapping sets of CLE-RS (red). How AMS activate CLE-RS transcription iscurrently unknown. In both cases, CLE peptides are transported to the shoot where they activate by direct binding (shown forLjCLE-RS2 to LjHAR1) or via possible co-receptors to activate CLAVATA1-like RLKs (LjHAR1/MtSUNN1/GmNARK/BdFON)MtCORYNE as well as LjCLV2/PsSYM28, which initiate less understood signaling cascades to generate a shoot-to-root signal.LjKLAVIER (KLV) is another LRR-RLK involved. LjHAR1 activation reducesmiR2111 levels, allowing TOOMUCH LOVE (TML) inthe roots to negatively regulate RNS. LjHAR1 also activates shoot cytokinin production, another shoot-to-root signal. Signalsdownstream of HAR1/SUNN/NARK/FON to inhibit AMS are unclear, except for its effect on reducing strigolactone (SL) bio-synthesis and export in the roots. In parallel, C-terminal encoded peptides (e.g.MtCEP1) also acts systemically on shootMtCRA2to positively regulate RNS via an unknown shoot-to-root signal, but this require common symbiosis signaling pathway andethylene receptor ETHYLENE INSENSITIVE 2. Species prefix as in Figure 1 except: Bd, Brachypodium distachyon. AON, auto-regulation of nodulation; AOM, autoregulation of mycorrhizal symbiosis.





signaling responses and is also involved in the sup-pression of AM symbiosis under phosphate sufficiency(Müller et al., 2019). Whether MtCLE33/53 directlybind to MtSUNN1 remains to be demonstrated, but acommonmode of systemic regulation is emerging fromthese recent findings (for review, see Kereszt et al., 2018;Müller and Harrison, 2019). As the CLE-SUNN/HARmechanism for autoregulation of AM colonization is pre-sent in Brachypodium distachyon (Müller et al., 2019) andbecause tomatoCLV2 is also involved in autoregulation ofAM colonization (Wang et al., 2018), it is tempting tospeculate that this host-control mechanism may predatethe evolution of RNS, and like the CSSP, this signalingpathway may have been coopted in the evolution of RNSto exert host control over the extent of symbiosis.

Independent of LjHAR1/MtSUNN/GmNARK, an-other peptide-LRR-RLK pathway exists in parallel, reg-ulating nodulation and root development. C-terminallyencoded peptides (CEPs) undergo extensive posttrans-lational modifications and have proposed roles in reg-ulating plant development and abiotic and bioticresponses (Taleski et al., 2018). MtCEP1 inhibits lateralroot emergence but promotes nodule formation viaseparate pathways downstream of its putative recep-tor, Medicago COMPACT ROOT ARCHITECTURE2(MtCRA2). MtCRA2 acts locally to limit lateral rootdevelopment, but its systemic activity positively regu-lates nodule formation. And while MtCEP1-MtCRA2regulation of lateral root development is independentof CSSP and Medicago ETHYLENE INSENSITIVE2,the regulation of nodule development requires thesesignaling components, suggesting that two differentpathways may exist downstream of MtCRA2 (Mohd-Radzman et al., 2016; Laffont et al., 2019).Moreover, theexpression of CEP peptides is also differentially regu-lated during AMS (de Bang et al., 2017), but their role inregulating AMS awaits further investigation.

CONCLUSION AND PERSPECTIVES

To perceive and engage with symbionts, plant rootsrecognize bacterial or fungal carbohydrate moleculesand orchestrate a series of signal exchanges before amutually beneficial symbiosis is established. Because ofthe rich understanding of the host signaling mecha-nisms relative to the symbiont, this review focused onthe roles of plant RLKs in recruiting and scrutinizingendosymbionts from precontact to termination of theassociation. It is still unclear how host-symbiont spec-ificity, especially for AMS, can be encoded in a rhizo-sphere where very similar carbohydrate molecules arelikely to be produced by different organisms. To date,the lack of genetically tractable AMF restricts our un-derstanding of the biosynthesis and relative impor-tance of important fungal signaling molecules, be theyLCOs, COs, or others. The most parsimonious hy-pothesis, based on the recent discoveries, might bethat the combinatorial and/or sequential perceptionof signaling molecules, including but not restricted to

carbohydrates, activates a set of host receptors forsymbiosis signaling. The present challenge (see Out-standing Questions) is to identify the range of signalsfor AMF to explain its broad host range and to explainhow plants integrate the signals downstream of per-ception and receptor activation. Moreover, althoughmultiple classes of RLKs have been identified to regu-late various stages of symbiosis, there is a lack of un-derstanding on their signalingmechanisms. At the levelof receptor complexes, membrane receptor domains,and phosphorylation/signal transduction cascade(s),we anticipate future discoveries to reveal shared anddiverging modes of receptor activities in the two moststudied plant root endosymbioses.Received October 29, 2019; accepted January 25, 2020; published February 13,2020.

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