Gatekeeper Tyrosine Phosphorylation of SYMRK Is Essential for Synchronizing the Epidermal and Cortical Responses in Root Nodule Symbiosis

Gatekeeper tyrosine phosphorylation of symbiosis receptor kinase is essential for guiding the infection threads through the epidermal-cortical barrier towards the nodule primordia during progress of root nodule symbiosis.

Abstract

Symbiosis receptor kinase (SYMRK) is indispensable for activation of root nodule symbiosis (RNS) at both epidermal and cortical levels and is functionally conserved in legumes. Previously, we reported SYMRK to be phosphorylated on “gatekeeper” Tyr both in vitro as well as in planta. Since gatekeeper phosphorylation was not necessary for activity, the significance remained elusive. Herein, we show that substituting gatekeeper with nonphosphorylatable residues like Phe or Ala significantly affected autophosphorylation on selected targets on activation segment/αEF and β3-αC loop of SYMRK. In addition, the same gatekeeper mutants failed to restore proper symbiotic features in a symrk null mutant where rhizobial invasion of the epidermis and nodule organogenesis was unaffected but rhizobia remain restricted to the epidermis in infection threads migrating parallel to the longitudinal axis of the root, resulting in extensive infection patches at the nodule apex. Thus, gatekeeper phosphorylation is critical for synchronizing epidermal/cortical responses in RNS.

The establishment of root nodule symbiosis (RNS) involves rhizobial invasion in the root epidermis and nodule organogenesis in the root cortical cells. In the epidermis, recognition of compatible rhizobia induces root hair curls in the host plant to entrap the rhizobia in an infection pocket (Gage, 2004). In these infection pockets or chambers, rhizobia multiply to form a microcolony that is believed to generate threshold levels of bacterial signal molecules required for initiation and progression of infection threads (ITs) through the epidermis into the cortex of the root (Fournier et al., 2015). A compatible host-symbiont interaction in the epidermis also leads to formation of nodule primordia in the cortex, where eventually the rhizobia is endocytically accommodated from the invading ITs to develop nitrogen-fixing symbiosomes in mature nodules. The epidermal and cortical processes are coordinated both spatially and temporally to ensure rhizobial accommodation in the subtending nodule primordia (Popp and Ott, 2011; Oldroyd, 2013; Miri et al., 2016).

At the molecular level, these host responses are initiated with the recognition of rhizobial Nod factors by two LysM receptor-like kinases (RLKs): NFR1 and NFR5 in Lotus japonicus and lysin motif domain-containing RLK-3 (LYK3) and NFP in Medicago truncatula (Limpens et al., 2003; Madsen et al., 2003; Broghammer et al., 2012). Recently, another Nod factor-induced LysM-RLK (exopolysaccharide receptor 3) was shown to monitor rhizobial EPS structures in Lotus, indicating a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals to ensure host symbiont compatibility (Kawaharada et al., 2015). Following the perception of Nod factors, Ca²⁺ influx at the tip of the root hair and Ca²⁺ spiking at the perinuclear region of the root hair are induced (Oldroyd, 2013). Components that function upstream of Ca²⁺ spiking include Symbiosis Receptor Kinase (SYMRK)/ Doesn’t Make Infections (DMI) 2 (Endre et al., 2002; Stracke et al., 2002), the nuclear envelope-localized cation channels, and components of the nucleopore complex. A nuclear calcium- and calmodulin-dependent protein kinase (CCaMK/DMI3; Lévy et al., 2004; Mitra et al., 2004) decodes the Ca²⁺ spiking and phosphorylates a transcription factor (TF) CYCLOPS/Interacting Protein of DMI3 (IPD3; Messinese et al., 2007; Singh et al., 2014), which along with several other TFs orchestrates the gene expression required for rhizobial infection and nodule organogenesis (Oldroyd, 2013; Suzaki et al., 2015).

SYMRK is a functionally conserved central component of symbiotic signaling that experienced structural diversification during evolution (Markmann et al., 2008). It contains an ectodomain composed of a malectin-like domain and a Leu-rich repeat region. Unlike the nfr mutants that lack most cellular and physiological responses, including induction of Ca²⁺ influx and Ca²⁺-spiking in response to rhizobia, the root hairs of symrk mutants respond with Ca²⁺ influx but not with Ca²⁺-spiking and do not develop ITs, indicating SYMRK to be positioned downstream of the NFRs (Miwa et al., 2006). Recently, LjSYMRK was shown to interact with NFR5 through its ectodomain, suggesting a role of SYMRK in initiating symbiotic signaling in concert with the NFRs (Antolín-Llovera et al., 2014a). Unlike the NFRs, specificity of recognition of bacterial partners in RNS is independent of the source of SYMRK (Gherbi et al., 2008; Markmann et al., 2008; Saha et al., 2014). SYMRK is indispensable at both epidermal and cortical levels and is important for coupling the epidermal/cortical events in RNS guided by a GDPC motif in its ectodomain (Kosuta et al., 2011; Antolín-Llovera et al., 2014a). The kinase activity of SYMRK is also vital for its role in symbiosis, as several SYMRK mutants have missense or nonsense mutations in the catalytic domain (Yoshida and Parniske, 2005). Several interactors are known for SYMRK that indicated this RLK to be nodal to various downstream cellular responses associated with inception of RNS (Antolín-Llovera et al., 2014b).

The majority of plant RLKs including SYMRK are distinguished by having Tyr in the “gatekeeper” position adjacent to the hinge region of their kinase domains (Shiu and Bleecker, 2001). This residue helps dictate the size of a hydrophobic pocket that forms in the DFG-out conformation of protein kinases (Hari et al., 2013; Sohl et al., 2015). Though the gatekeeper residue is important primarily because it is the architect of this deep catalytic cleft, several lines of evidence now indicate that protein kinases have adopted mechanisms of regulation mediated by their gatekeeper residues (Emrick et al., 2006; Chen et al., 2007; Azam et al., 2008). Most importantly, oncogenic activation of several receptor Tyr kinases (RTKs) has been shown to be due to changes in the gatekeeper (Azam et al., 2008). The importance of the gatekeeper Tyr residue is also emerging in the well-characterized RLKs like brassinosteroid-insensitive 1 (BRI1) and LYK3 or receptor-like cytoplasmic kinase like Botrytis-induced kinase 1 (BIK1), where structure-mimic substitutions of the gatekeeper affect either kinase activity or biological activity or both (Oh et al., 2009a, 2009b; Klaus-Heisen et al., 2011; Lin et al., 2014). RLKs are also shown to adopt an active conformation by forming a hydrogen-bonded triad between the invariant Lys-Glu salt-bridge and the gatekeeper Tyr (Bojar et al., 2014). But considering the contrasting effects of the gatekeeper substitution in RLKs, the involvement of the free hydroxyl group of gatekeeper Tyr in an H-bonding network cannot be considered as a quintessential signature of the activated state for all RLKs.

In an earlier report, we demonstrated SYMRK from Arachis hypogaea (AhSYMRK) to predominantly autophosphorylate on gatekeeper Tyr (Y670) both in vitro and in planta (Samaddar et al., 2013). Apart from SYMRK, autophosphorylation of gatekeeper Tyr is only evidenced in BRI1 (Oh et al., 2009b). Autophosphorylation on gatekeeper Tyr was not a prerequisite for AhSYMRK to be catalytically active, and hence the significance of gatekeeper phosphorylation remained elusive (Samaddar et al., 2013; Paul et al., 2014). Here we report the biochemical and biological importance of gatekeeper phosphorylation by demonstrating its role in autoactivation of AhSYMRK and progress of RNS. AhSYMRK with nonphosphorylatable residues in the gatekeeper position significantly increased or suppressed autophosphorylation on specific sites in the activation segment/αEF and β3-αC loop, indicating the identity of SYMRK’s gatekeeper residue to be important for determining the state of autoactivation of this RLK. This change of autophosphorylation profile with nonphosphorylatable gatekeepers led to complete loss of coordination between rhizobial invasion in the epidermis and nodule organogenesis in the cortex in RNS restricting the rhizobia in the epidermal-cortical interface.

RESULTS

Gatekeeper Tyr Defines a Distinct Autoactivation State of AhSYMRK

Gatekeeper Tyr (Y670) was identified previously as a potential site of autophosphorylation in AhSYMRK by mutagenesis and sequence-specific antibodies both in vitro and in planta (Samaddar et al., 2013). Here we first verified Y670 phosphorylation by mass spectrometry to reconfirm phosphorylation on this residue (Fig. 1A; Supplemental Table S1). To probe into the importance of gatekeeper phosphorylation, we substituted Y670 with another hydroxyl-containing amino acid residue like Thr (Y670T), nonphosphorylatable residues like Phe or Ala (Y670F, Y670A), or a phosphomimic like Glu (Y670E) and monitored the catalytic consequences of the modifications in recombinant AhSYMRK. The level of intrinsic autophosphorylation was higher in wild type and Y670T, but in vitro autophosphorylation activity was at least 4- to 5-fold higher in Y670F and Y670A (Fig. 1B). The phosphomimic substitution Y670E that permanently introduces a negative charge in the catalytic groove (Paul et al., 2014) drastically reduced both intrinsic and in vitro autophosphorylation activities of AhSYMRK but did not render the kinase catalytically dead like K625E. While pThr was detectable in all the mutants, including Y670E, pTyr was only detectable in wild type and Y670T, indicating that a phosphorylatable residue in the gatekeeper position was necessary for the kinase to autophosphorylate on Tyr residues and become dual specific. Gatekeeper phosphorylation-specific antibody α-pY670 (665–674) recognized AhSYMRK but did not react with its gatekeeper mutants like Y670F, Y670A, Y670E, or the catalytically dead mutant K625E, attesting to its specificity. But it was intriguing to note a feeble but consistent immunoblot cross-reactivity of α-pY670 (665–674) with Y670T, which we believe is due to phosphorylation on gatekeeper Thr (pT670) as suggested by MS analysis (Fig. 1B; Supplemental Table S2).

Figure 1.

Gatekeeper Y670 defines a distinct autoactivation state for AhSYMRK. A, AhSYMRK is autophosphorylated on gatekeeper Y670. Product ion spectrum of a doubly charged peptide ion at m/z 1103.5044 with the phosphosite assigned to be Y670 in the sequence 664-DQQILVYPFMSNGSLQNR-681. B, Autophosphorylation of gatekeeper mutants. Intrinsic phosphorylation detected by ProQ Diamond staining, in vitro AhSYMRK autophosphorylation activity detected by [³²P]γ-ATP (autorad), phosphorylation was detected by antiphosphothreonine (α-pThr), antiphosphotyrosine (α-pTyr), and modification specific (α-pY670: 665-674) antibodies, protein abundance visualized by Coomassie Brilliant Blue staining (CBB). The catalytically inactive K625E mutant is used as reference. C, Gatekeeper mutations significantly affect the autophosphorylation profile of AhSYMRK. An MS-based quantitation of specific phosphopeptides from recombinant AhSYMRK gatekeeper Y670 mutant proteins relative to WT (wild type, defined as 1). Phosphosites have been arranged N to C terminus and clustered under the typical secondary structural features of the kinase domain (Taylor and Kornev, 2011). Gatekeeper Y670/T670, S674, S677 (Supplemental Tables S2 and S3) have not been included due to different ionization efficiencies of these phosphopeptides. Phosphosites significantly affected due to Y670 substitutions have been marked with *. Activation loop (AL, red), P+1 loop (blue), αEF helix (orange), P+1 loop/ αEF (brown).

We then examined how the nature of the gatekeeper affected the relative levels of phosphorylation in each of the identified phosphorylation sites in AhSYMRK using label-free quantitative mass spectrometry (Ahmed et al., 2014). Only phosphopeptides that were detected in all experimental replicates of the wild type and Y670T/F/A mutants were subjected to quantitation (Supplemental Tables S2 and S3). Additionally, we quantified Y670E phosphorylation status for these sites only, which was either absent or barely detectable. The pY-containing peptides encompassing the activation segment/αEF helix were only detectable in wild type and Y670T and have been included in the analysis. Representative data plotted against the functional domains of protein kinases highlighted the sharp contrast in the autophosphorylation profile between AhSYMRK with phosphorylatable (Y670/Y670T) and nonphosphorylatable gatekeepers (Y670F/A; Fig. 1C). While autophosphorylation on most sites was relatively unaffected by gatekeeper substitution, in several sites autophosphorylation was selectively suppressed only in Y670F and Y670A. Those sites are located in the β3-αC loop (S631), activation segment (S754, T763), αEF helix (T773), and the αF-αG loop (S810). Besides, doubly phosphorylated peptides encompassing the P+1 loop and/or the αEF helix-containing pY766/pY771/pY772 were conspicuously absent in Y670F/A mutants, indicating this region to have a contrasting pY signature with nonphosphorylatable gatekeepers (Supplemental Table S3). As opposed to these sites, where autophosphorylation was compromised in Y670F/A, autophosphorylation in S757 residue in the activation loop showed a sharp increase in Y670F (approximately 48-fold) and Y670A (approximately 37-fold). Interestingly, Y670E polypeptide, which was barely active, showed an approximately 17-fold increase in autophosphorylation in S757 residue. Thus, two different autoactivation states with two distinct sets of autophosphorylation targets are defined in AhSYMRK with phosphorylatable or nonphosphorylatable gatekeepers.

Nonphosphorylatable Gatekeepers in AhSYMRK Arrested ITs in the Epidermis

Cross species complementation tests showed that SYMRKs from eurosids, including nodulating and nonnodulating lineages, can restore symbiosis in symrk null mutants (Gherbi et al., 2008; Markmann et al., 2008). Earlier we have demonstrated complementation of TR25, a symrk null mutant of M. truncatula by AhSYMRK (Saha et al., 2014). The same experimental conditions were used to determine the significance of gatekeeper Y670 phosphorylation in AhSYMRK in vivo, where we transformed TR25 with the native AhSYMRK and its gatekeeper mutants and monitored their ability to restore symbiotic features. Transformed roots inoculated with Sinorhizobium meliloti-mRFP were monitored for their colonization at 2 weeks after infection (WAI). Upon infection with S. meliloti, in both TR25/AhSYMRK and TR25/AhSYMRK-Y670T roots, microcolonies were observed within tightly curled root hairs and IT formation could be restored (Fig. 2, A and B). In TR25/AhSYMRK, the ITs were always a continuous tubular structure (Fig. 2A), whereas in TR25/AhSYMRK-Y670T, ITs sometimes developed sac-like structures (Fig. 2C) or revealed misdirected progression at the epidermal-cortical interface (Fig. 2D), but in most cases the ITs were found to progress normally (Fig. 2E). In contrast, in TR25/AhSYMRK-Y670F/A, the root hair curls were enlarged with diffused agglomerations (Fig. 2, F and J). In both cases the ITs contained extensive inflated sac-like structures, and this feature was more prominent in TR25/AhSYMRK-Y670F roots (Fig. 2, F–H and J-L). However, the most important distinguishing feature was the arrest of IT progression at the epidermal cortical interface or subepidermal cortex (Fig. 2, F–H, J and L). The misdirected ITs were noted to migrate parallel to the longitudinal axis of the root instead of ramifying and reaching the dividing cortical cells (Fig. 2, H and I, L and M). ITs were also found to be blocked within the root hair shaft but that was rare (Fig. 2K). Also, as compared to TR25/AhSYMRK and TR25/AhSYMRK-Y670T/A, the number of ITs was at least approximately 3 times higher in only TR25/AhSYMRK-Y670F (Fig. 2, N and O; Supplemental Fig. S1), which could be due to the plants’ attempt to compensate for the defect in IT propagation (more prominent in Y670F) by initiation of more ITs, as noted before in Lotus (Murray et al., 2007). In contrast to these observations, none of the epidermal features of rhizobial invasion were restored in TR25/AhSYMRK-Y670E roots, which is consistent with the drastic loss of catalytic activity in Y670E (Fig. 2P). In summary, with nonphosphorylatable gatekeepers in AhSYMRK, the epidermal/cortical barrier was insurmountable by ITs. This is a direct demonstration of the physiological consequences of the differential autophosphorylation profile of AhSYMRK with a phosphorylatable and a nonphosphorylatable gatekeeper.

Figure 2.

Effect of the gatekeeper mutation on progression of ITs. TR25 roots were transformed with 35S::AhSYMRK-GFP and gatekeeper mutants 35S::AhSYMRK-Y670T/F/A/E-GFP. Progress of IT was observed 2 WAI with S. meliloti-mRFP and is shown as merged images of bright field and mRFP fluorescence. IT progress in TR25/AhSYMRK (A), TR25/ AhSYMRK-Y670T (B-E), TR25/ AhSYMRK-Y670F (F-I), TR25/ AhSYMRK-Y670A (J-M). No S.meliloti colonization was observed in roots of TR25/AhSYMRK-Y670E (P). S. meliloti colonization in AhSYMRK-Y670F (N) and AhSYMRK-Y670A (O) roots. Scale bar: 200 μm (N-O), 100 μm (A, F, I-J, P), 50 μm (D-E, G, M), 20 μm (H, K-L) and 10 μm (B-C).

Nonphosphorylatable Gatekeepers in AhSYMRK Uncoupled Epidermal and Cortical Events in RNS

Our next objective was to monitor the gatekeeper substitution mutants of SYMRK for their ability to restore nodule organogenesis and rhizobial colonization in the cortex. By 6 WAI, nodules were visible with all the mutants, and their number was reproducibly higher (approximately 2–3 times) with all the gatekeeper substituted mutants (Supplemental Fig. S2; Supplemental Table S4). In TR25/AhSYMRK and TR25/AhSYMRK-Y670T, cortical ramification of ITs was restored, and the symbionts were released in the central infected zone of the developed nodule (n = 40; Fig. 3, A and B, J and K). However, some infected nodules in TR25/AhSYMRK-Y670T were distinct by having a small infection patch/pocket at the apex, a feature almost never observed in nodules developed in TR25/AhSYMRK roots (Fig. 3B). In at least 20% of TR25/AhSYMRK-Y670T nodules, the misdirected ITs remained restricted in apical pockets, leaving the nodules without a central infected zone (Fig. 3A inset, Figure 3, C and L). As opposed to TR25/AhSYMRK-Y670T, in TR25/AhSYMRK-Y670F/A nodules there was absolutely no ramification of ITs in the nodule interior (n = 60; Fig. 3, D-I, L). As a result, the nodules were always empty and the symbionts were strictly restricted to extensive infection patches on the apex. Moreover, the nodules developed in TR25/AhSYMRK-Y670F/A showed improper vasculature (Fig. 3, E and H), which is in accordance with the adverse effect on nodule organogenesis by epidermal arrest of rhizobial invasion as demonstrated in both Medicago and Lotus (Yano et al., 2009; Guan et al., 2013). This effect was not ephemeral, as the symbionts were detained in the infection pockets without any signs of IT propagation to the cortex even 8 WAI, and the nodule development never progressed further. Additionally, TR25/AhSYMRK-(573–883)-Y670F could profusely generate spontaneous nodules and these nodules developed with proper vasculature (Supplemental Fig. S3), as demonstrated earlier in TR25/AhSYMRK-(573–883; Saha et al., 2014; Saha and DasGupta, 2015). This confirms that the restricted autoactivation of AhSYMRK-Y670F can generate all the signals necessary for proper nodule organogenesis, and the improper vasculature noted in TR25/AhSYMRK-Y670F/A was indeed due to inhibitory signals generated by epidermal arrest of symbionts. Also the increase of nodule number with gatekeeper mutants is likely to be a downstream effect of the absence of mature nodules, where absence of suppression may have led to a recurrent initiation of new foci of cell divisions as proposed previously in Medicago (Kuppusamy et al., 2004). These results again show a correlation between the RNS features and the differential autophosphorylation profile of SYMRK with a phosphorylatable and a nonphosphorylatable gatekeeper.

Figure 3.

Effect of the gatekeeper mutation on nodule development and symbiont colonization. TR25 roots were transformed with 35S::AhSYMRK-GFP and gatekeeper mutants 35S::AhSYMRK-Y670T/F/A-GFP. Nodule development and S. meliloti colonization was observed 6 WAI. Nodule developed in TR25/AhSYMRK-Y670T (A-C), TR25/ AhSYMRK-Y670F (D-F), TR25/ AhSYMRK-Y670A (G-I), and TR25/ AhSYMRK (J-K). Nodulated roots (A, D, G, J) with insets showing enlarged view of nodules. Rhizobial accumulation at the nodule apex indicated by arrow. Longitudinal sections revealing S. meliloti colonized in nodule interior (B, K). Infection pocket (B) and extensive infection patches in nodule apex (C, E, H). Magnified view of infection pocket showing subepidermal rhizobial colonization (F and I). Vascular bundles indicated by arrowhead. (L) Percentage of S. meliloti colonization in the nodule interior or apex in TR25 plants transformed with indicated gatekeeper mutants. Images shown as merged images of bright-field and red (S. meliloti expressing mRFP) (A-C, d-E, G-H, J-K), bright-field (bottom left) and merged (bottom right) images of GFP and mRFP fluorescence (inset of A, D, G, J) or merged images of red and blue (Calcofluor, cell wall stain) fluorescence (F, I). Scale bar: 2 mm (A, D, G, J), 500 µm (inset of A, D, G, J), 100 µm (B-C, E, H, K), and 5 µm (F, I).

AhSYMRK with Nonphosphorylatable Gatekeepers Fails to Up-Regulate Expression of Genes Required for Epidermal-Cortical Coordination in RNS

Several factors have been evidenced in the literature to have a role in epidermal-cortical coordination in RNS (Popp and Ott, 2011): for example, TFs like CYCLOPS (Singh et al., 2014) and NIN (Soyano et al., 2013; Vernié et al., 2015), a nodule-specific remorin MtSYMREM1 (Lefebvre et al., 2010), the plasma membrane-resident ankyrin protein Vapyrin (Murray et al., 2011), the U-box containing E3 ubiquitin-ligase LIN (Kuppusamy et al., 2004), and the noncoding RNA ENOD40 (Crespi et al., 1994). In CYCLOPS mutant of Medicago (TE7), several nodules remain small (primordia), with ITs arrested at the nodule apex similar to what has been observed in presence of AhSYMRK with the nonphosphorylatable gatekeepers (Fig. 3, D-I; Benaben et al., 1995). LjCYCLOPS transactivates the NIN gene, a bifunctional membrane bound transcriptional coactivator (Singh et al., 2014). In both Medicago and Lotus, NIN is essential for IT initiation in the epidermis and nodule organogenesis in the cortex (Yoro et al., 2014; Fournier et al., 2015). MtSYMREM1 interacts with SYMRK and NFRs to mediate their assembly in membrane rafts, and its down-regulation leads to IT arrest in epidermal cells (Lefebvre et al., 2010). Both Vapyrin and LIN mutants in Medicago show impaired growth of ITs that are blocked in the epidermis (Kuppusamy et al., 2004; Murray et al., 2011). Finally, ENOD40, an early Nod factor-induced gene, has a role in both infection and nodule primordia initiation as demonstrated in Medicago (Crespi et al., 1994). Overall, all these genes appear to have a role in proper invasion of ITs into the dividing cells of the nodule primordia in the cortex.

We compared the expression of these genes in TR25/AhSYMRK-Y670T/F/A/E roots at 6 WAI. In TR25/AhSYMRK-Y670E, expression of the above symbiotic genes was barely detectable, which is in accordance with absence of any symbiotic response of these roots in presence of rhizobia (Fig. 4). Expression of LIN and MtENOD40 was relatively unaffected in TR25/AhSYMRK-Y670T/F/A, indicating their expression to be upstream or parallel to the pathway that leads to overcoming of the epidermal/cortical barrier. Vapyrin is the only factor that was almost equally affected in TR25/AhSYMRK-Y670T/F/A. Therefore, Vapyrin does not appear to be connected with the sharp contrast between TR25/AhSYMRK-Y670F/A and TR25/AhSYMRK-Y670T with respect to loss of cortical colonization. On the other hand, misdirected IT progression in the epidermis is a common feature that distinguishes TR25/AhSYMRK-Y670T/F/A from its wild-type counterpart, and this feature may be associated with low expression of Vapyrin. In TR25/AhSYMRK-Y670F/A, expression was significantly lower for MtNIN (approximately 6–9 fold), MtSYMREM1 (approximately 4-fold), and IPD3 (approximately 2-fold) as compared to TR25/AhSYMRK-Y670T, indicating these genes to be directly involved in synchronizing the epidermal and cortical events. This attests to the role of these factors in coupling the epidermal and cortical events as suggested previously and designates them to function downstream to SYMRK for the same.

Figure 4.

Effect of gatekeeper substitution on the expression of IPD3, NIN, SYMREM1, Vapyrin, LIN, and ENOD40. Expression of the mentioned genes in TR25/AhSYMRK-Y670T/F/A/E at 6WAI was measured relative to those in TR25/AhSYMRK roots. The expression level in TR25/AhSYMRK was set to 1. In all cases, MtACTIN2 was used as the reference gene. Histograms represent means of three biological replicates ± sem. Asterisks above bars indicate significant differences (P < 0.05).

DISCUSSION

Here, we demonstrate gatekeeper Tyr autophosphorylation as a hallmark of a distinct autoactivated state of AhSYMRK that generates vital phospho-cues for progress in symbiosis. AhSYMRK with nonphosphorylatable gatekeepers like Phe and Ala conspicuously up-regulate or suppress autophosphorylation in specific sites (Fig. 1C), notably in the activation segment/αEF and β3-αC loop, and in addition failed to support a coordinated epidermal and cortical response during establishment of RNS (Figs. 2 and 3).

The role of SYMRK in coordinating the epidermal and cortical responses was first noted in symrk-3 snf-1 double mutants of Lotus, where ITs were occasionally noted to be misdirected and swollen and unable to reach the nodule primordia (Madsen et al., 2010). Another example is symrk-14 in Lotus with a P386L substitution in the extracellular GDPC motif of SYMRK that prevents its ectodomain shedding (Kosuta et al., 2011; Antolín-Llovera et al., 2014a). Substitution in the GDPC motif does not affect IT formation or nodule organogenesis but restricts ITs to the epidermis. Our investigations reveal SYMRK with nonphosphorylatable gatekeepers to have a similar phenotypic output in Medicago (Figs. 2 and 3). Additionally, we show that the state of autoactivation differs with a nonphosphorylatable gatekeeper, where autophosphorylation on selective targets on activation segment/αEF and β3-αC loop are significantly affected or abolished (Fig. 1). The phenotypic overlap of symrk14 mutant of Lotus and our observations on TR25/AhSYMRK-Y670F/A in Medicago suggest SYMRK to have distinct autoactivation states based on the nature of its extracellular domains. SYMRK activity with its intact ectodomain triggers cortical cell division and the epidermal IT formation. Then ectodomain shedding switches SYMRK to another autoactivation state that enables gatekeeper phosphorylation and full-scale phosphorylation of activation segment/αEF and β3-αC loop of the receptor. Together, ectodomain shedding and gatekeeper phosphorylation could be a coupled switch that maintains a synchrony between epidermal and cortical events for precisely timing the crossing over of the epidermal-cortical barrier for endocytic accommodation of symbionts in the primordia. It remains to be seen whether and how the extracellular GDPC motif allosterically connects with the gatekeeper residue in the kinase domain to deliver the outputs required for a successful coordination of epidermal and cortical symbiotic features.

Several other mutants have indicated the epidermal-cortical interface to be an important checkpoint during the progress of RNS (Popp and Ott, 2011; Rival et al., 2012; Hayashi et al., 2014). Premature arrest of ITs in the epidermis and restriction of symbionts in infection patches in the nodule apex was observed in ccamk-14 (Liao et al., 2012), nap-1, pir-1(Yokota et al., 2009), and nena (Groth et al., 2010) of Lotus. In Medicago, similar phenotypes were observed in lyk3 (Smit et al., 2007), TE7 (Benaben et al., 1995), api (Teillet et al., 2008), and lin-1 mutants (Kuppusamy et al., 2004). The spreading of misdirected ITs in the epidermis as noted predominantly with gatekeeper substituted SYMRKs in Medicago (TR25/AhSYMRK-Y670F/A; Fig. 2) was previously noted in the vag-1 and suner1-1 mutants of Lotus (Suzaki et al., 2014; Yoon et al., 2014). The significance of CCaMK/DMI3 in epidermal-cortical crosstalk is illustrated in both Lotus and Medicago (Rival et al., 2012; Hayashi et al., 2014). Epidermal expression of CCaMK supported IT progression in the epidermis, but its cortical expression was essential for allowing the ITs to cross the epidermal/cortical barrier and triggering cortical cell division for nodule organogenesis. The Ca²⁺-responsive domains of CCaMK were indispensable for epidermal infections but dispensable for nodule organogenesis and cortical infection processes (Gleason et al., 2006; Shimoda et al., 2012; Hayashi et al., 2014). NIN is also a central player in coordinating epidermal/cortical responses in both Lotus and Medicago (Soyano et al., 2013; Vernié et al., 2015). Overexpressing NIN in either the epidermis or the cortex auto-activates cortical cell division, but unlike epidermal NIN, cortical NIN expression can promote the formation of nodule like-structures independent of Cre1 (Vernié et al., 2015). Since SYMRK functions at the upstream end of the SYM pathway, the phenotypic overlap of TR25/AhSYMRK-Y670F/A with the above mutants indicates SYMRK with its phospho-cues to be important for guiding the proper progression of the ITs. Additionally, expression of MtNIN, MtSYMREM1, and IPD3 was significantly affected in TR25/AhSYMRK-Y670F/A as compared to TR25/AhSYMRK-Y670T (Fig. 4), indicating that these factors have important roles in enabling the ITs to surmount the epidermal-cortical barrier. All these genes are therefore involved in the network that guides and guards the epidermal-cortical barrier.

The sites where autophosphorylation was affected with a nonphosphorylatable gatekeeper (Y670F/A) are all conserved in SYMRKs from other species (Fig. 1C; Supplemental Fig. S4), indicating the strategic importance of gatekeeper phosphorylation and suggesting the underlying mechanism of full-scale autoactivation of SYMRKs to be conserved. Even in other RLKs, the same sites or regions are known to be autophosphorylated, suggesting the autoactivating principles to be conserved beyond SYMRK (Supplemental Fig. S4). The residue S757, where autophosphorylation sharply increased in absence of a phosphorylatable gatekeeper (Fig. 1C), is also highly conserved in SYMRKs, suggesting its importance in symbiotic signaling. Phosphorylation on the invariant Thr (T763 in AhSYMRK/ T760 in LjSYMRK) in the activation loop was shown earlier to be important for SYMRK activity (Yoshida and Parniske, 2005; Samaddar et al., 2013). Since T763 autophosphorylation is drastically down-regulated in Y670F/A, it could be that the autoactivation with a nonphosphorylatable gatekeeper is actually independent of T763 phosphorylation in the activation segment. Such activation loop phosphorylation-independent activation is demonstrated in certain RTKs and RLKs (Dardick et al., 2012; Plaza-Menacho et al., 2014).

It remains to be understood as to how gatekeeper Tyr phosphorylation in the hinge region is involved with autophosphorylation in the distal and dynamic elements like the activation segment/αEF helix and β3-αC loop (Fig. 1C). However, the importance of hinge motions for kinase activation and allosteric communication between the activation loop and hinge has been suggested for some animal kinases (Hari et al., 2013; Sours et al., 2014). In the majority of RTKs, an inhibitory network of hydrogen bonds involving the gatekeeper residue is disengaged upon activation loop phosphorylation to activate the kinase (Chen et al., 2007). In ERK2, flexibility of the activation loop and its accessibility to autophosphorylation could be controlled over long distances by the nature of the gatekeeper through an intramolecular pathway of connectivity through interacting side chains (Emrick et al., 2006). A similar mechanism has been hypothesized for Arabidopsis (Arabidopsis thaliana) MPK6, where gatekeeper Tyr substitutions lead to constitutive activity and independence from upstream MAPK activation (Berriri et al., 2012). In tune with these examples, our results indicate SYMRK adopts such long-range allosteric interaction networks whereby it transmits the identity of the gatekeeper to distant locations within the core kinase domain for full-scale autoactivation of the kinase. Consistent with this proposition, we have already demonstrated that conformations of SYMRK distinctly differ based on whether it had Tyr or Phe as the gatekeeper (Paul et al., 2014). We hypothesize the phosphorylated or nonphosphorylated state of the gatekeeper Tyr to determine the autophosphorylation trajectory in SYMRK as described for the FGFR family of animal kinases and thereby define distinct autoactivation states (Furdui et al., 2006). Further experimentation is required for determining the state of phosphorylation of SYMRK with the initiation and progress of symbiosis to understand how well the in vitro phosphorylation studies reflect the trajectory of its autophosphorylation in planta. Such understanding would clarify the role of differential phosphoclusters of SYMRK in serving as a platform for ‘docking’ of effector proteins to transmit downstream signaling.

In conclusion, a phosphorylatable gatekeeper residue determines a distinct autoactivation state of SYMRK that differentially autophosphorylates and generates phospho-cues for coupling the epidermal and cortical responses of RNS. Since a gatekeeper Tyr is common in RLKs, a conserved mechanism of activation associated with gatekeeper phosphorylation could be of common occurrence in RLKs.

MATERIALS AND METHODS

Plant and Rhizobial Strains

Medicago TR25 seeds, Agrobacterium rhizogenes strain MSU440, and pBHR-mRFP-Sinorhizobium meliloti 2011 strain were used.

Constructs

pET28a-AhSYMRK-kd(573-883)(Samaddar et al., 2013) and its point mutations generated with the QuikChangeSite-Directed mutagenesis kit (Stratagene) were used to express the His₆-polypeptides in Escherichia coli strain BL21 (DE3). The full-length AhSYMRK and its gatekeeper mutants AhSYMRK-Y670T/F/A/E were recombined into pK7FWG2 using LR-Clonase (Life Technologies), thereby generating 35S::AhSYMRK-GFP and its corresponding Tyr residue mutants, for example 35S::AhSYMRK-Y670T/F/A/E-GFP (for details, see Supplemental Methods S1).

Kinase Assay, Phosphoamino Acid Analysis, and Immunoprecipitation

Kinase autophosphorylation assays, immunoblotting, immunoprecipitation and phosphostaining were performed as described previously (DasGupta, 1994; Samaddar et al., 2013; Paul et al., 2014).

Label-Free Quantitative Mass Spectrometry

The recombinant proteins were subjected to the kinase reaction with unlabeled ATP until phosphorylation was complete, followed by sample preparation and LC-MS/MS analysis as described in Supplemental Methods S1.

Phenotypic Analysis

Generation of composite Medicago truncatula plants and scoring nodulation, phenotypic analysis, and confocal microscopy were performed as described previously (Saha et al., 2014).

For detailed description of methods pertaining to construct generation, quantitative RT-PCR, primers used, see Supplemental Methods S1.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. ITs in TR25/AhSYMRK and TR25/AhSYMRK-Y670T/F/A roots 2 WAI with S. meliloti.

• Supplemental Figure S2. Nodules developed in TR25/AhSYMRK and TR25/AhSYMRK-Y670T/F/A roots 6 WAI with S. meliloti.

• Supplemental Figure S3. Spontaneous nodule formation by overexpression of AhSYMRK-(573–883) Y670F.

• Supplemental Figure S4. Conservation of phosphorylation sites in SYMRKs and other RLKs.

• Supplemental Table S1. Phosphorylation on gatekeeper Y670 of wild-type AhSYMRK-kd identified by LC-MS/MS.

• Supplemental Table S2. List of phosphorylation sites detected for AhSYMRK-kd and its gatekeeper mutants, identified by LC-MS/MS.

• Supplemental Table S3. List of phosphopeptides of AhSYMRK-kd and its gatekeeper mutants quantitated by LFQMS (represented in Fig. 1C).

• Supplemental Table S4. Restoration of RNS in TR25 by overexpression of AhSYMRK and AhSYMRK-Y670T/F/A/E.

• Supplemental Methods S1. Methods in detail.

Glossary

IT

infection thread

RNS

root nodule symbiosis

TF

transcription factor

WAI

weeks after infection