Intracellular Catalytic Domain of Symbiosis ReceptorKinase Hyperactivates Spontaneous Nodulation inAbsence of Rhizobia1[W]
Sudip Saha2, Ayan Dutta2, Avisek Bhattacharya, and Maitrayee DasGupta*
Department of Biochemistry, University of Calcutta, Calcutta 700019, India
Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesisand intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of aSYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program inMedicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated thatTR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In thepresence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the noduleinterior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates noduleorganogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.
In rhizobia-legume symbiosis, the host plant respondsto rhizobial elicitation by developing root nodules andaccommodating the symbionts intracellularly in the nod-ule interior. At the molecular level, the response is initi-ated with the recognition of rhizobial Nod factors by Nodfactor receptors of the host plants (Limpens et al., 2003;Madsen et al., 2003; Broghammer et al., 2012). A com-patible interaction triggers the Sym pathway and gener-ates the calcium spikes as a signature of symbiotic signaltransduction (Ehrhardt et al., 1996; Sieberer et al., 2012;Oldroyd, 2013). Proteins acting upstream of Ca2+ spikingare Doesn’t Make Infections2 (DMI2), Symbiosis ReceptorKinase (SYMRK; Endre et al., 2002; Stracke et al., 2002),the cation channels DMI1, CASTOR, and POLLUX(Ané et al., 2004; Charpentier et al., 2008), and threenucleoporins (NUP85 and NUP133 [Kanamori et al., 2006;Saito et al., 2007] and NENA [Groth et al., 2010]). Anuclear calcium- and calmodulin-dependent proteinkinase (CCaMK) or DMI3 (Lévy et al., 2004; Mitra et al.,2004) decodes the Ca2+ spiking and phosphorylates a
transcription factor Interacting Protein of DMI3 (IPD3) orCYCLOPS (Messinese et al., 2007; Yano et al., 2008; Singhet al., 2014), which along with several other transcriptionfactors, like Nodulation Signaling Pathway1 (NSP1; Smitet al., 2005), NSP2 (Kaló et al., 2005), ERF Required forNodulation1 (Middleton et al., 2007), and Nodule In-ception (NIN; Schauser et al., 1999; Marsh et al., 2007),orchestrates the gene expression required for rhizobialinfection and nodule organogenesis.
Constitutively active forms of the cytokinin receptorLotus Histidine Kinase1 (LHK1; Tirichine et al., 2007),CCaMK (Gleason et al., 2006; Tirichine et al., 2006a),CYCLOPS (Singh et al., 2014), or NIN (Soyano et al., 2013)can result in spontaneous nodulation, showing that nod-ule organogenesis does not require rhizobial infection.In this model, CCaMK-induced nodule organogenesis ismediated by LHK1, because gain-of-function LHK1 in-duces spontaneous nodules in loss-of-function ccamk mu-tants (Tirichine et al., 2007; Hayashi et al., 2010; Madsenet al., 2010). CCaMK is placed upstream of all pathwaysrequired for this organogenic program, because autoactiveCCaMK versions can compensate for the loss of upstreamgenes, like SYMRK, CASTOR, POLLUX, or NUP85/NUP133, that are involved in calcium spike generation,indicating that the primary function of these genes is theactivation of CCaMK (Madsen et al., 2010).
In all tested eurosids, SYMRK contains three Leu-richrepeat (LRR) motifs, a malectin-like domain in the extra-cellular region, and a protein kinase catalytic domain inthe intracellular region (Markmann et al., 2008). Crossspecies complementation tests showed that SYMRKs fromeurosids, including nodulating and nonnodulating line-ages, can restore symbiosis in symrk null mutants, indi-cating that the extra cytoplasmic region of SYMRK doesnot mediate specificity in rhizobia-legume recognition(Gherbi et al., 2008; Markmann et al., 2008). In this report,
1 This work was supported by the Council of Scientific and Indus-trial Research, Government of India (grant no. 09/028[0830]/2010–EMR–I to S.S.); the University Grant Commission, Government ofIndia (grant nos. RFSMS/F.5–19/2007 BSR to A.D. and UGC/307/Jr. Fellow to A.B.); and the Centre of Excellence and Innovation inBiotechnology, Department of Biotechnology, Government of India(grant no. BT/01/CEIB/09/VI/10 to M.D.).
2 These authors contributed equally to the article.* Address correspondence to [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (http://www.plantphysiol.org) is: Maitrayee DasGupta ([email protected]).
[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.114.250084
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we show that the intracellular kinase domain of SYMRKfrom Arachis hypogaea (AhSYMRK-kd) hyperactivatesspontaneous nodulation in Medicago truncatula in the ab-sence of Sinorhizobium meliloti. Both CCaMK and ipd3/cyclops were required for triggering nodule organogene-sis in the absence of rhizobia. The effect was not specificfor SYMRK from A. hypogaea, because intracellular kinasedomain of SYMRK from M. truncatula (MtSYMRK-kd)could similarly autoactivate nodule organogenesis inM. truncatula. In the presence of S. meliloti, colonization ofnodules was rarely noted, leaving most of the nodulesempty. Thus, ligand-independent deregulated activationof the intracellular kinase domain of SYMRK is capable ofinducing nodule organogenesis in the absence of rhizobia,but the ectodomain of the receptor is important for propercolonization by S. meliloti.
RESULTS AND DISCUSSION
Cross Species Complementation of TR25 by AhSYMRK
Earlier, we reported isolation of AhSYMRK fromA. hypogaea, a basal legume that is supported by crackinvasion (Samaddar et al., 2013). It has 84% sequencesimilarity with SYMRK from an infection thread (IT)-supported legumeM. truncatula (MtSYMRK) and falls atthe point of divergence of legumes and nonlegumes ina distance tree (Supplemental Fig. S1). Here, we showAhSYMRK-mediated complementation of TR25, an symrkknockout mutant ofM. truncatula (Endre et al., 2002). Theempty vector-transformed TR25 roots did not show rhi-zobial colonization or nodulation upon inoculation withS. meliloti-expressing monomeric red fluorescent pro-tein (mRFP; Fig. 1A). Under identical conditions, 35S::AhSYMRK-GFP-transformed TR25 roots responded by ITformation (Fig. 1B; Supplemental Fig. S2) as well as de-velopment of nodules (Fig. 1, C–J), indicating qualitativerestoration of both organogenesis and rhizobial infectionin TR25 (Supplemental Table S1). Nodules harvested6 weeks after inoculation (WAI) were spherical in shape(Fig. 1C), whereas those harvested 8 WAI had the char-acteristic cylindrical shape of indeterminate nodules(Fig. 1D). In both cases, nodules remained white and in-effective, because functional restoration of symrk knockoutmutants demands its expression under native promoter(Limpens et al., 2005). The TR25/AhSYMRK nodules (11of 20) showed extensive growth of ITs in the central tissueof the nodule (Fig. 1E) with infrequent release of symbi-onts in some cells (Fig. 1, F and G). In a significant numberof nodules (9 of 20), intracellularization of the symbiontswas complete (Fig. 1, H and I), although the bacteroidsremained small and resembled freshly released S. meliloti(Fig. 1J; Supplemental Fig. S3), indicating that ectopic ex-pression of AhSYMRK in the entire nodule affects properdevelopment of symbiosomes. Wild-type M. truncatula(A17) roots expressing 35S::AhSYMRK-GFP, however,developed nodules (Fig. 1K, inset) with properly dif-ferentiated elongated symbiosomes (Fig. 1, L and M;Supplemental Fig. S3), suggesting that native expressionof endogenous SYMRK can dominate over the ectopically
expressed SYMRKs for proper development of nodulesand symbionts. Our observations add to the evidenceshowing that specificity of recognition of bacterial part-ners in rhizobia-legume or frankia-actinorhizal interac-tions is independent of the source of SYMRK as long asthe SYMRK proteins have a malectin-like domain andthree LRRs in their ectodomain (Gherbi et al., 2008;Markmann et al., 2008).
AhSYMRK-kd Hyperactivates the Nodule OrganogenicProgram in M. truncatula in the Absence of S. meliloti
Deregulated activation of CCaMK dispenses the re-quirement of SYMRK for triggering nodule organogenesisin the absence of rhizobia (Hayashi et al., 2010; Madsenet al., 2010). We reasoned that deregulated activation ofSYMRK could trigger the organogenic program by acti-vating CCaMK in the absence of S. meliloti. To check thispossibility, we complemented TR25 with AhSYMRK-kd(573–883) that has been shown to be active in vitro(Samaddar et al., 2013). AhSYMRK-kd is expected to havederegulated activity because of (1) loss of context of itsexpression in the absence of the transmembrane domainor (2) loss of its ectodomain- or juxtamembrane domain-imposed regulation (Oh et al., 2009; Antolín-Llovera et al.,2014).
As indicated in Supplemental Table S1, TR25 com-plemented with 35S::AhSYMRK-kd by Agrobacteriumrhizogenes-mediated transformation showed profuse nod-ulation in the absence of S. meliloti, with an average of28.2 6 5.22 (mean 6 SEM) nodules per root system. Underidentical conditions, there were 4.56 1.14 nodules per rootsystem in the S. meliloti-infected TR25 plants com-plemented with 35S::AhSYMRK. On average, AhSYMRK-kd-induced spontaneous nodulation was 6-fold more thanS. meliloti-induced nodulation in TR25 roots transformedwith AhSYMRK (Supplemental Fig. S4). This is in contrastto other instances of spontaneous nodulation events, wherethe efficiency of spontaneous nodulation was similar orlower than the S. meliloti-induced nodulation (Gleasonet al., 2006; Tirichine et al., 2006a, 2006b; Singh et al., 2014).
In most cases, the spontaneous nodules in TR25/AhSYMRK-kd roots grew as merged clusters (Fig. 2, A–D),causing distortion in the nodulated roots; however, inseveral instances, they were also found to grow as soli-tary spherical nodules (Fig. 2E). The distorted roots withexcessive nodulation indicated that the spontaneousnodulation in AhSYMRK-kd-transformed roots may lackcontrol over both nodule numbers and spatial position.In fact, spontaneously nodulated TR25 roots resembledS. meliloti-infected supernodulated M. truncatula roots insuper numeric nodule (sunn) or sickle mutants that definedistinct genetic pathways (Penmetsa and Cook, 1997;Penmetsa et al., 2003; Schnabel et al., 2005). SUNN en-codes an LRR receptor-like kinase and is a key com-ponent of autoregulation of nodulation, which regulatesnodule number by a long-distance negative feedbacksystem (Reid et al., 2011). The sickle mutant is defectivein ethylene perception, and insensitivity to ethylene is
Figure 1. Complementation of TR25 by AhSYMRK. TR25 roots were transformed with 35S::AhSYMRK-GFP and infected withS. meliloti expressing mRFP. A, Transgenic control roots lacking AhSYMRK cassette showing no nodules. GFP fluorescenceimage is shown. Bar = 2 mm. B, Root hairs of TR25/AhSYMRK 2WAI with S. meliloti in merged images of bright-field and mRFPfluorescence. ITs (arrow) can be seen inside the curled root hairs. Bar = 200 mm. C and D, Nodulated roots of TR25/AhSYMRK 6(C) and 8 (D) WAI shown as merged images of bright-field and mRFP fluorescence. Bottom shows enlarged views of sphericalnodules (C) and cylindrical nodules (D) with bright-field (bottom left) and merged images of GFP and mRFP fluorescence(bottom right). mRFP fluorescence in inner nodule tissue indicates the presence of S. meliloti. Bars in C, inset, and D = 2 mm.Bar in D, inset = 200 mm. E to M, Longitudinal sections (30 mm) of 6-week-old nodules formed on TR25/AhSYMRK (E–J) or A17/AhSYMRK (K–M) shown as merged images of red (S. meliloti expressing mRFP) and blue (Calcofluor; cell wall) fluorescence. E to G,TR25/AhSYMRK nodules where ITs (arrowheads) occupy the central tissue (E) with infrequent release of symbionts (F and G). H to J,TR25/AhSYMRK nodules where symbionts were released from ITs (H) without being differentiated into elongated symbiosomes(I and J). K to M, A17/AhSYMRK transgenic nodules. K, Inset is shown as the merged image of GFP and mRFP fluorescence.Nodule interior filled with symbionts (K) showing fully differentiated symbiosomes (L and M). Symbiosomes in J and M areindicated by double arrowheads. Bars in E, H, and K = 100 mm. Bars in F, I, and L = 20 mm. Bars in G, J, and M = 5 mm. Bar in K,inset = 1 mm.
Figure 2. Spontaneous nodule formation by overexpression of AhSYMRK-kd(573–883). A to F, TR25 roots were transformed with35S::AhSYMRK-kd-GFP. Both bright-field (top) and GFP fluorescence (bottom) images are shown for spontaneous nodules as eitherclusters (A–D) or solitary (E) in TR25/AhSYMRK-kd roots at 8 weeks after transplantation in vermiculite. F, No nodules in the presence
thought to be causal to its supernodulation phenotype(Penmetsa and Cook, 1997). These autoregulatory path-ways could be compromised under deregulated activationof SYMRK, leading to hyperactivation of spontaneousnodulation (Fig. 2, A–D). This is unlike spontaneousnodulation under deregulated activation of CCaMK,where nodule organogenesis seemed to be autoregulated(Tirichine et al., 2006a). Interestingly, SYMRK mutantswere identified as suppressors of hypernodulation inLotus japonicus, which already suggested SYMRK to havea possible role in regulating nodule numbers (Murrayet al., 2006).Nodule organogenesis was not observed in the ab-
sence of S. meliloti in full-length AhSYMRK-transformedTR25 roots, indicating that deregulated activity ofAhSYMRK-kd initiated the nodule organogenic programin TR25/AhSymRK-kd roots in the absence of any rhi-zobial trigger (Supplemental Table S1). To clarify whetherthe effect was caused by the enzymatic activity ofAhSYMRK-kd, we repeated these experiments with 35S::AhSYMRK-kd K625E, where the kinase was inactivated bymutation of the invariant ATP binding Lys (Samaddaret al., 2013). Spontaneous nodules did not develop onTR25/AhSYMRK-kd K625E roots (Fig. 2F), indicatingthat nodulation under rhizobia-free conditions was solelycaused by the deregulated catalytic activity of AhSYMRK-kd and not kinase-independent signals originating fromthe scaffold of the overexpressed kinase polypeptide.The spontaneous nodules on TR25/AhSYMRK-kd, both
solitary (n = 22) and merged (n = 30; Fig. 2, H and I),showed peripheral vascular bundles connected at theirproximal end to the root vasculature, which was similar toS. meliloti-infected nodules (n = 10) generated in TR25/AhSYMRK roots (Fig. 2G). Therefore, spontaneous nod-ules follow the genuine nodule organogenic program thatis elicited by rhizobia, but unlike rhizobia-induced nodules(Fig. 2G), they had an empty interior devoid of any bacteria(Fig. 2, H and I). Confocal images also revealed thebacteroid-free interior of the spontaneous nodules gener-ated in TR25/AhSYMRK-kd roots (Fig. 2, J–L) comparedwith S. meliloti-infected nodules in wild-type A17 roots,where the nodule interior was full of bacteroids (Fig. 2,M–O).To clarify whether the observed spontaneous nodulation
was specific for AhSYMRK-kd, we transformed TR25 with35S::MtSYMRK-kd(572–882)-GFP and cultivated them insterile agar plates in the absence of S. meliloti. As shown in
Figure 2P, MtSYMRK-kd, which has 91% sequence iden-tity with AhSYMRK-kd, could successfully trigger 6.960.97 spontaneous nodules per root system in TR25(Supplemental Table S2) with the characteristic tendencyof generating merged nodules (Fig. 2Q). This indicatesthat the property of triggering spontaneous nodulationby deregulated SYMRK activity is not a specific propertyof AhSYMRK-kd. Under this growth condition, TR25plants transformed with AhSYMRK-kd showed 7.0 61.22 nodules per root system (Fig. 2, R and S;Supplemental Table S2), showing the functional equiv-alence of AhSYMRK-kd and MtSYMRK-kd. It may berelevant to mention here that deregulated activity ofneither AhSYMRK-kd (Samaddar et al., 2013) norAhCCaMK-kd (Sinharoy and DasGupta., 2009) couldinduce spontaneous nodulation in A. hypogaea, wheredevelopment of aeschynomenoid nodules are thoughtto be coupled with endocytosis of symbionts (S. Saha,A. Dutta, and M. DasGupta, unpublished data).
To clarify whether AhSYMRK-kd could generate spon-taneous nodules in the presence of endogenous SYMRK, itwas overexpressed in the wild-type M. truncatula (A17)plant. An average of 3.4 6 0.51 spontaneous nodules de-veloped per root system (Fig. 2T; Supplemental Table S2),and the nodulation was also noted to occur in clusters(Fig. 2U), indicating that endogenous SYMRK does notinterfere with spontaneous nodule organogenesis. Thenumber of nodules per root systemwas consistently higherin TR25 compared with that noted in A17 (SupplementalFig. S5), suggesting a possible dominant negative effect ofendogenous SYMRK.
Finally, it was important to understand whether spon-taneous nodulation in the presence of autoactive SYMRKwas dependent on functional CCaMK. We, therefore,transformed 35S::AhSYMRK-kd-GFP in TRV25 (ccamknull) mutants of M. truncatula (Lévy et al., 2004) andcultivated the plants in the absence of S. meliloti. As in-dicated in Figure 2V, TRV25/AhSYMRK-kd roots failedto develop any spontaneous nodule, indicating that nod-ule organogenesis triggered by AhSYMRK-kd involves afunctional CCaMK. This observation was consistent withthe role of SYMRK in the activation of calcium spiking asan upstream member of the Sym pathway (Miwa et al.,2006) and the role of CCaMK as the central regulator ofnodule organogenesis (Hayashi et al., 2010; Madsen et al.,2010). Recently, it has been reported that nodule organo-genesis in the presence of autoactive CYCLOPS bypasses
Figure 2. (Continued.)of inactive kinase in TR25/AhSYMRK-kd (K625E)-GFP roots. Bars in A to D and F = 2 mm. Bar in E = 500 mm. G to O, Longitudinalsection of spontaneous nodules on TR25/AhSYMRK-kd roots (H–L) compared with infected nodules on TR25/AhSYMRK (G) or A17(M–O) roots. G and H, Toluidine blue-stained image. I, Bright-field image. J to O, Merged images of red (propidium iodide; DNA) andblue (Calcofluor; cell wall). Bars in G to I, J, and M = 100 mm. Bars in K and N = 20 mm. Bars in L and O = 5 mm. Arrowheadsindicate peripheral vascular bundles in nodules, and the arrow indicates ITs. ic, Infected cell containing bacteroids; iz, infected zone;n, nucleus. P to U, TR25 and A17 roots were transformed with 35S::MtSYMRK-kd-GFP or 35S::AhSYMRK-kd-GFP. Both bright-field(left) and GFP fluorescence (right) images are shown for spontaneous nodules that are either solitary (P, R, and T) or in clusters (Q, S,and U) scored 3 weeks after transplantation in sterile agar plates on TR25/MtSYMRK-kd roots (P and Q), TR25/AhSYMRK-kd roots(R and S), and A17/AhSYMRK-kd roots (T and U). V and W, TRV25 or ipd3-1 roots were transformed with 35S::AhSYMRK-kd-GFP.Both bright-field (left) and GFP fluorescence (right) images are shown. No nodules were observed on transformed roots of TRV25/AhSYMRK-kd (V) or ipd3-1/AhSYMRK-kd (W) under identical growth conditions in agar plates. Bars in P to W = 1 mm.
the requirement for CCaMK, suggesting that CCaMKmediated phosphorylation of CYCLOPS to activate nod-ule organogenesis (Singh et al., 2014). However, auto-active CCaMK can rescue nodule organogenesis in nullcyclops background, suggesting that alternative targets ofCCaMK are able to substitute for CYCLOPS in noduleorganogenesis (Yano et al., 2008). We, therefore, trans-formed 35S::AhSYMRK-kd-GFP in the ipd3-1 (cyclops null)mutant of M. truncatula (Horváth et al., 2011) to clarifywhether CYCLOPS could be bypassed for generatingspontaneous nodules by deregulated activation ofAhSYMRK-kd. As shown in Figure 2W, in the absence ofS. meliloti, we could not detect spontaneous nodules inipd3-1/AhSYMRK-kd plants. This was unlike the casewhere the autoactive form of CCaMK could generate
spontaneous nodules in cyclops mutant plants. Thus, ac-tivation of nodule organogenesis in the absence of rhizo-bia by deregulated activity of AhSYMRK-kd wasdependent on CCaMK and CYCLOPS, which functiondownstream to calcium spiking in the Sym pathway.
Early Nodulin11 Expression Was Triggered byAhSYMRK-kd
M. truncatula Early Nodulin11 (MtENOD11) is a mo-lecular marker for both early preinfection responses andlater infection-related processes occurring within root aswell as nodule tissues (Journet et al., 2001). To furtherassess the gain-of-function activity of AhSYMRK-kd, weintroduced both 35S::AhSYMRK and 35S::AhSYMRK-kdinto A17 containing pMtENOD11-GUS. In the absenceof rhizobia, AhSYMRK-transformed roots occasionallyshowed nonsymbiotic GUS expression in the root capwith no activity in the root hairs (Fig. 3, A–C). In con-trast, under the same conditions, almost 65% (13 of 21)plants transformed with AhSYMRK-kd showed scatteredGUS expression in lateral roots (Fig. 3, E and F), suggestingthat ENOD11 is activated by the deregulated activity ofAhSYMRK-kd. The GUS activity was limited to a zone ofepidermal cells associated with root hair outgrowth(Fig. 3G), which is similar to what is known as the normalexpression zone of ENOD11 in S. meliloti-infected or Nodfactor-treated roots (Journet et al., 2001). Additionally, inAhSYMRK-kd-overexpressed roots, ENOD11 expressionwas localized in the apex of the spontaneous nodules(Fig. 3H), which is identical to its expression zone inS. meliloti-induced nodules in plants overexpressingintact AhSYMRK (Fig. 3D). Thus, ectopic expressionof AhSYMRK-kd did not affect the restriction of ex-pression of ENOD11 within a strict pattern that wastriggered by S. meliloti. The similarity in expressionpattern of ENOD11 between S. meliloti-induced nodulesand AhSYMRK-kd-induced nodules confirms that thespontaneous nodules follow the same organogenic sig-naling pathway and that they are genuine nodules. Itshould be noted that spontaneous nodules inM. truncatula,triggered by deregulated activation of 35S::MtCCaMK, alsohave properly localized expression of ENOD11, indicatingthat ectopic expression of neither autoactive SYMRK (thiswork) nor autoactive CCaMK (Gleason et al., 2006) was ahindrance toward proper development of spontaneousnodules.
AhSYMRK-kd Supports IT-Mediated S. meliloti InvasionBut Fell Short of Releasing the Symbionts in theNodule Cells
TR25/AhSYMRK-kd roots responded by formationof root hair curls and ITs within 2 WAI with S. meliloti(Fig. 4, A and B). The number of ITs observed in TR25/AhSYMRK-kd roots was 1.8 times higher than thatobserved in TR25/AhSYMRK roots (SupplementalFig. S2). The deregulated activity of AhSYMRK-kd wassolely responsible for generating these features, because
Figure 3. Induction of ENOD11 by AhSYMRK-kd. 35S::AhSYMRK-GFPor 35S::AhSYMRK-kd-GFP was introduced in M. truncatula (A17)pMtENOD11-GUS lines. Nonsymbiotic pMtENOD11-GUS expressionin the absence of S. meliloti (A–C) and symbiotic GUS expression in thenodule apex in S. meliloti-infected nodules in pMtENOD11-GUS line/AhSYMRK roots (D). E to H, Scattered pMtENOD11-GUS expression inpMtENOD11-GUS line/AhSYMRK-kd roots in the absence of S. meliloti(E and F) with restricted expression in epidermal root hairs (G) or apicesof spontaneous nodules (H). Bars in A and E = 2 mm. Bars in B and F =500 mm. Bars in C and G = 50 mm. Bars in D and H = 1 mm.
Figure 4. Features of S. meliloti invasion and colo-nization in AhSYMRK-kd-transformed TR25 roots.TR25 roots were transformed with 35S::AhSYMRK-kd-GFP and infected with S. meliloti expressingmRFP. A and B, Root hairs at 2 WAI shown asmerged images of GFP and mRFP fluorescence; ITs(arrows) can be seen inside the curled root hairs (A).Magnified image showing the shepherds crook(double arrowhead) formation (B). Bars in A and B =100 mm. C to G, White nodules developed in TR25/AhSYMRK-kd roots at 6 WAI shown as bright-field(top) and merged (bottom) images of GFP and mRFPfluorescence. Numerous nodules in a root systemwith rare colonization of rhizobia (indicated by ar-rowhead; C), uninfected spherical nodules (D), rhi-zobial colonization in nodule apex (indicated byarrowheads; E and F), and nodule interior (G). Bar inC = 5 mm. Bars in D to G = 1 mm. H and I, Lon-gitudinal section of empty nodules developed inTR25/AhSYMRK-kd roots showing proper (H) andimproper (I) vasculature. Bright-field (H) and tolui-dine blue-stained (I) images. v, Vasculature. Bars inH and I = 100 mm. J to N, Longitudinal section ofnodules developed in TR25/AhSYMRK-kd roots withrhizobial colonization in the nodule apex (J–M) ornodule interior (N) shown as merged images of red(S. meliloti expressing mRFP) and blue (Calcofluor;cell wall). Magnified views of regions marked bysolid, dotted, and dashed boxes in J are shown in Kto M, respectively. ITs in L to N are indicated bystaggered arrowheads. Bars in J and N = 100 mm.Bar in K = 50 mm. Bars in L and M = 10 mm.
root hair curling or ITs are not detected in TR25 (Endreet al., 2002). Like nodule organogenesis, the rhizobialresponse features are also expected to be mediatedthrough CCaMK, because constitutive activity ofCCaMK dispenses the requirement of SYMRK for suc-cessful infection of rhizobia (Hayashi et al., 2010; Madsenet al., 2010). Whatever the case, it is clear that signalsrequired for triggering root hair curl or IT formation,which are primarily guided by membrane-bound re-ceptors, do not require SYMRK to be anchored in themembrane in its native location.
Efficiency of nodule organogenesis in TR25/AhSYMRK-kd roots remains similar in the presence ofS. meliloti (Supplemental Table S1). However, the phe-nomenon of merged nodule formation was rarely seen inthe presence of the symbiont, indicating that an S.meliloti-induced signal affects the proximity of nodule primordiageneration (Fig. 4C). S. meliloti-derived fluorescence wasundetectable in the majority of the nodules formed inTR25/AhSYMRK-kd roots, indicating that most nodulesare uncolonized by the symbionts (Fig. 4, C and D).Sectioning these nodules revealed their bacteroid-freeinteriors, but otherwise, they resembled genuine nod-ules with peripheral vascular bundles, like those seen inspontaneous nodules in the absence of S. meliloti (Fig. 4H).Intriguingly, in the presence of S. meliloti, a significantnumber of these empty nodules (23 of 30) had improperdevelopment of vasculature, indicating that the presenceof rhizobia adversely affected the progress of nodule or-ganogenesis (Fig. 4I). These observations are similar toobservations in cyclops/CCaMKT265D and cerberus/CCaMKT265D roots, where spontaneous nodules withgenuine nodule structure were generated in the absence ofrhizobia but epidermal arrest of rhizobial invasion ad-versely affected nodule organogenesis in its presence(Yano et al., 2008, 2009). Thus, similar to CYCLOPS andCERBERUS, the ectodomain of SYMRK seems to have arole in the concerted progression of S. meliloti infectionprocesses and nodule organogenesis.
We could detect S. meliloti in the nodule apex in only11% of nodules (Fig. 4, E and F), and we could rarely(in 2% of nodules) detect colonization in nodule interior(Fig. 4G), indicating an overall uncoupling of the nod-ule organogenic program with symbiont colonization inthe absence of the SYMRK ectodomain. Sectioning ofnodules (n = 20) with symbionts colonized in the apex(Fig. 4J; Supplemental Fig. S6A) revealed entangled roothairs ending in an infection patch in the epidermal layer(Fig. 4K; Supplemental Fig. S6B). The magnified imagesof nodule-associated ITs are shown in Figure 4L andSupplemental Figure S6C. From these huge infectionpatches, ITs were rarely found to expand into the cortexwithout proper intracellular release of the symbionts(Fig. 4M; Supplemental Fig. S6D). It is possible thatthese infection patches are the same as the infection pocketsnoted in the symrk-14 mutant of L. japonicus, where thesepockets are suggested to constitute an intermediate steptoward attempting successful intracellular infection ofnodule cortical cells (Kosuta et al., 2011). Such intermediateinfection pockets preceding colonization of the nodule
interior are also noted in nena-1mutants (Groth et al., 2010).In 2% of nodules where could we detect colonization ofS. meliloti in the nodule interior, sectioning (n = 10) revealedextensive IT formation throughout the nodule (Fig. 4N;Supplemental Fig. S6E), which is similar to what was notedin TR25/AhSYMRK roots (Fig. 1, E and F). However,unlike TR25/AhSYMRK nodules, symbionts were rarelyreleased from ITs into the nodule cells in AhSYMRK-kd-transformed roots, indicating an important role of SYMRKectodomain in intracellularization of rhizobia. Such exten-sive growth of ITs with rare or no release of symbionts issimilar to the aberrant cortical infection phenotypes ofSYMRK RNA interference lines in Sesbania rostrata orM. truncatula (Capoen et al., 2005; Limpens et al., 2005).Overall, we show that the unrestrained kinase activity ofSYMRK in TR25 can successfully restore the root hair curlsand epidermal ITs but that restoration of rhizobial coloni-zation in the nodule interior was highly compromised inthe absence of its ectodomain.
CONCLUSION
In conclusion, we have shown that deregulated ac-tivity of the intracellular kinase domain of SYMRK canactivate spontaneous nodulation in the absence of rhizobiabut that the ectodomain of SYMRK synchronizes the or-ganogenic process with the internalization of the symbiontin its presence. Unlike autoregulated spontaneous nodu-lation under constitutive activity of CCaMK, deregulatedactivation of SYMRK hyperactivates nodule organogenesisand seems to have overcome the controls of both nodulenumber and the spatial positioning (Fig. 2, A–D). Whetherthe hypernodulation is caused by loss of autoregulation ofnodulation through long-distance signaling (Nishimuraet al., 2002) or loss of the negative regulatory role of NIN(Marsh et al., 2007) or hormones, like ethylene (Penmetsaand Cook, 1997), remains to be clarified. Whatever theunderlying mechanism, it is clear that deregulated SYMRKactivity adversely affects the signaling networks that havea physiological control over nodule number and spatialposition.
MATERIALS AND METHODS
Plant and Rhizobial Strains
Medicago truncatula A17 and TRV25 seeds were from Jeanne Harris, TR25seeds were from Giles Oldroyd and Christian Roger, pMtENOD11-GUS seedswere from David G. Barker, TRV25 seeds and Agrobacterium rhizogenes strainMSU440 were from Douglas R. Cook, ipd3-1 seeds were from Peter Kalo, andpBHR-mRFP-Sinorhizobium meliloti 2011 was from Ton Bisseling and Erik Limpens.
Constructs
The full-length Arachis hypogaea SYMRK (Samaddar et al., 2013), AhSYMRK-kd(573–883), and M. truncatula SYMRK-kd(572–882) were cloned into pENTR-D-TOPO (Life Technologies) and recombined into pK7FWG2 using LR-Clonase(Life Technologies; Karimi et al., 2002), thereby generating 35S::AhSYMRK-GFP,35S::AhSYMRK-kd-GFP, and 35S::MtSYMRK-kd-GFP. AhSYMRK-kd(573–883) inpENTR-D-TOPO was used to generate AhSYMRK-kd K625E by site-directedmutagenesis and subsequently recombined into pK7FWG2, generating 35S::AhSYMRK-kd K625E-GFP (for details, see Supplemental Methods S1).
Generation of Composite M. truncatula Plants andScoring Nodulation
M. truncatula seedlings were transformed with indicated constructs inA. rhizogenes MSU440 following a standard procedure (Boisson-Dernier et al.,2001). Transgenic hairy roots were screened for GFP fluorescence, and compositetransgenic plants were transplanted in either agar plates containing bufferednodulation medium or vermiculite pots. Spontaneous nodulation was scoredafter 8 weeks in vermiculite pots or after 3 weeks in agar plates. For nodulation,S. meliloti expressing mRFP (Smit et al., 2005) was applied to plants 7 d aftertransferring to vermiculite. For observing ITs, plants were harvested 2 WAI, andfor scoring nodules, they were harvested 6 WAI (details in SupplementalMethods S1).
Images of whole-mount nodulated roots were captured using a Leica stereofluorescence microscope M205FA equipped with a Leica DFC310FX digitalcamera (Leica Microsystems). Histological assay for checking GUS expressionwas performed according to Sinharoy et al., 2009. For microscopy of thenodule interior, 30-mm sections of fresh nodules were generated with a rotarymicrotome (RM2235; Leica Microsystems). Sections were stained with toluidineblue (0.05% [w/v]; Lobachemie) and imaged under an Olympus IX71 micro-scope. For confocal microscopy, sections were stained with indicated stains andimaged using a Leica TCS SP5 II AOBS microscope (Leica Microsystems). Alldigital micrographs were processed using Adobe Photoshop CS5 (details inSupplemental Methods S1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogenetic position of AhSYMRK.
Supplemental Figure S2. ITs in TR25/AhSYMRK and TR25/AhSYMRK-kdroots 2 WAI with S. meliloti.
Supplemental Figure S3. Ultrastructure of S. meliloti-infected nodulesformed on AhSYMRK-transformed A17 or TR25 roots.
Supplemental Figure S4. Spontaneous nodule development in TR25 (non-nodulating symrk mutant of M. truncatula) by overexpression ofAhSYMRK-kd in plants grown in vermiculite.
Supplemental Figure S5. Spontaneous nodule development in A17 (thewild type) and TR25 (symrk mutant) of M. truncatula by overexpressionof AhSYMRK-kd or MtSYMRK-kd in plants grown in sterile agar plates.
Supplemental Figure S6. Ultrastructure of nodules developed on TR25/AhSYMRK-kd roots infected with S. meliloti.
Supplemental Table S1. Spontaneous nodule development in TR25 (non-nodulating symrk mutant of M. truncatula) by overexpression ofAhSYMRK-kd in plants grown in vermiculite.
Supplemental Table S2. Spontaneous nodule development in TR25 (symrkmutant), TRV25 (ccamk mutant), ipd3-1 (cyclops mutant), and A17 (thewild type) of M. truncatula by overexpression of AhSYMRK-kd orMtSYMRK-kd in plants grown in sterile agar plates.
Supplemental Methods S1. Methods in detail.
ACKNOWLEDGMENTS
We thank Jeanne Harris for Jemalong A17 and TRV25 seeds, Giles Oldroydand Christian Roger for TR25 seeds, Ton Bisseling and Erik Limpens forS. meliloti-harboring pBHR-mRFP, David G. Barker for pMtENOD11-GUS seeds,Douglas R. Cook for A. rhizogenes strains MSU440 and TRV25 seeds, Peter Kalofor ipd3-1 seeds, Alok Sil for the Olympus IX71 microscope, and Bannhi Das,Tridib Das, and Suman Ghosh for technical assistance.
Received September 9, 2014; accepted October 9, 2014; published October 10,2014.
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