Cleavage of the SYMBIOSIS RECEPTOR-LIKE KINASE

Current Biology 24, 422–427, February 17, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.12.053 Report Cleavage of the SYMBIOSIS RECEPTOR- LIKE KINASE Ectodomain Promotes Complex Formation with Nod Factor Receptor 5

Meritxell Antolıń-Llovera,1 Martina K. Ried,1 leaf cells expressing L. japonicus SYMRK (Figures 1A and and Martin Parniske1,* 1B). SYMRK cleavage is thus either mediated by proteases 1Genetics, Faculty of Biology, University of Munich (LMU), present in both L. japonicus roots and N. benthamiana leaves Großhaderner Straße 4, 82152 Martinsried, Germany or, alternatively, is an intrinsic activity of the ectodomain. Upon expression in N. benthamiana leaf cells, most of the fluores- cently tagged protein was observed at the plasma membrane Summary (Figures S2 and S3). Moreover, most of the tagged protein as judged by protein blots (Figures 1, S1, and S4) is in the full- Plants form root symbioses with fungi and bacteria to length state. This observation, together with the observed gly- improve their nutrient supply. SYMBIOSIS RECEPTOR- codecoration of the MLD fragment, makes a cleavage at the LIKE KINASE (SYMRK) is required for phosphate-acquiring plasma membrane a likely scenario, but does not exclude arbuscular mycorrhiza, as well as for the nitrogen-fixing the possibility that it occurs already en route through the root nodule symbiosis of legumes [1] and actinorhizal plants secretory pathway. [2, 3], but its precise function was completely unclear. Here we show that the extracytoplasmic region of SYMRK, which The GDPC Motif Connects the LRRs with the MLD and Is comprises three leucine-rich repeats (LRRs) and a malectin- Critical for MLD Release and Symbiosis like domain (MLD) related to a carbohydrate-binding protein The symrk-14 mutant suffered from abnormal epidermal infec- from Xenopus laevis [4], is cleaved to release the MLD in the tion after inoculation with either arbuscular mycorrhiza (AM) absence of symbiotic stimulation. A conserved sequence fungi or rhizobia [10]. The symrk-14 mutation results in a motif—GDPC—that connects the MLD to the LRRs is P-to-L substitution within the GDPC motif (GDLC) and abol- required for MLD release. We discovered that Nod factor re- ishes MLD release (Figure 1A-B). The GDPC motif is located ceptor 5 (NFR5) [5–8] forms a complex with the SYMRK in a predicted loop connecting the MLD with the leucine-rich version that remains after MLD release (SYMRK-DMLD). repeats (LRRs) (Figure S2A) and is conserved in the majority SYMRK-DMLD outcompeted full-length SYMRK for NFR5 of MLD-LRR RLKs [10, 11]. In other proteins, the D-P bond interaction, indicating that the MLD negatively interferes is subject to autocatalytic hydrolysis of the peptide bond with complex formation. SYMRK-DMLD is present at lower [12, 13]. Therefore, the cleavage could occur within the amounts than MLD, suggesting rapid degradation after GDPC motif, due to autocatalytic self-processing or a yet un- MLD release. A deletion of the entire extracytoplasmic re- identified protease. Because the SYMRK-14 protein appeared gion increased protein abundance, suggesting that the to suffer from mislocalization (Figure S2B) and to determine LRR region promotes degradation. Curiously, this deletion the role of the GDPC motif in ectodomain release and symbi- led to excessive infection thread formation, highlighting osis, we generated additional mutant versions of the GDPC the importance of fine-tuned regulation of SYMRK by its motif, namely GAPC and RHPC, that are predicted to better ectodomain. preserve the secondary structure of the loop (Figure S2A). The free MLD fragment was not detectable in SYMRK(RHPC) Results and Discussion extracts from N. benthamiana, and only low amounts were present in SYMRK(GAPC) extracts (Figure 1B). The fluores- The Extracellular Domain of SYMRK Is Cleaved In Planta cence pattern derived from the wild-type and the RHPC and We discovered that the ectodomain of SYMBIOSIS RECEP- GAPC versions was consistent with their predicted endoplas- TOR-LIKE KINASE (SYMRK) is subject to proteolytic cleavage matic reticulum (ER) and plasma membrane localization (Fig- in planta; in addition to the microsome bound, full-length ure S2B). The GDLC and RHPC versions did not show MLD protein we consistently detected a smaller, soluble fragment release and were severely impaired in their symbiotic perfor- representing the released malectin-like domain (MLD) on pro- mance (Figure 2). In contrast, the GAPC version that released tein blots using a SYMRK antibody (Figure 1 and Figure S1 the MLD, although to a reduced extent, restored symbiosis to available online). The presence of N-glycans is likely to wild-type levels. These data indicate that the GDPC motif is account for a larger apparent size over the predicted sizes important for MLD release and for SYMRK function in for both SYMRK full-length and the MLD fragment (Figures symbiosis. S1B and S1C). Resistance of the N-glycans to PNGaseF treat- ment (Figures S1B and S1C) indicates the presence of a1,3- SYMRK-DMLD Undergoes Rapid Degradation fucose residues typically added in the Golgi apparatus, Upon MLD release, a C-terminal fragment lacking the MLD demonstrating that both SYMRK fragments are similarly pro- (SYMRK-DMLD) should be detectable in planta. A frag- cessed through the secretory pathway. In L. japonicus roots, ment matching the size of SYMRK-DMLD was observed in ectodomain cleavage was independent of symbiotic stimula- L. japonicus roots or N. benthamiana leaves expressing tion and also occurred with a kinase-dead mutant version SYMRK C-terminally tagged with mOrange or YFP (Figure 3 (symrk-10 [9]; Figures 1A and S1A). Similarly, cleavage was and S4), consistent with a cleavage at or in the vicinity of the observed in transiently transformed Nicotiana benthamiana GDPC motif. This fragment was consistently much less abun- dant than full-length SYMRK or the MLD (Figure 1, 3, S1, and S4), although cleavage products are produced at equal *Correspondence: [email protected] stoichiometry. SYMRK Cleavage Promotes NFR5 Interaction 423

A A control GDLC

GDPC (WT) RHPC

* *

GAPC bar= 0.5 mm **

B 90 primordia IT 30 20

Number per root system 0 Figure 1. The GDPC Motif Is Important for the Release of the SYMRK abcddb a c Malectin-like Domain GDPC GDLC RHPC GAPC Microsomal and soluble fractions from Lotus japonicus roots (A) or fraction- n= 20/23 1/19 2/21 19/21 ated (A) or crude (B) extracts from Nicotiana benthamiana leaves transiently expressing the indicated constructs (A and B) were analyzed by protein blot with an antibody recognizing the ectodomain of SYMRK and by Coomassie Figure 2. The GDPC Motif Is Important for SYMRK Functionality in blue (CB) staining. SYMRK full-length of w130 KDa (L. japonicus Symbiosis endogenous protein) or w155 KDa (tagged versions expressed in Transgenic roots of the symrk-3 null mutant expressing SYMRK variants N. benthamiana) is indicated with an arrowhead, while the (double) band carrying the amino acid sequence RHPC, GDLC (SYMRK-14 mutation), or representing the malectin-like domain (MLD) of 70–75 KDa is indicated GAPC instead of the wild-type GDPC motif were inoculated with with an arrow. Cross-reactive, nonspecific bands were observed in the Mesorhizobium loti MAFF 303099 DsRed. SYMRK variants (RFP-tagged at symrk-3 null mutant and did thus not originate from SYMRK. the C terminus) and control (RFP) were expressed under the control of the (A) Full-length SYMRK and the MLD fragment were detected in L. japonicus SYMRK promoter. roots and in N. benthamiana leaves expressing SYMRK [GDPC wild-type (A) Representative micrographs of transgenic roots 13 days after inocula- (WT)]. Although the full-length protein was detected in symrk-14 roots, tion. Fluorescent, DsRed-expressing bacteria are imaged in yellow. On the released MLD fragment was absent. GDPC (WT) was tagged with symrk-3 roots expressing the RFP protein (control), neither infection threads mOrange and NFR5 with YFP, both at the C terminus. (ITs) nor nodules developed. On symrk-3 roots expressing the GDPC (WT) or (B) The abundance of the MLD fragment was severely reduced in SYMRK GAPC variants, rhizobia were detected in root hairs in ITs (arrows). Bacteria- versions carrying variants of the GDPC motif. SYMRK variants are indicated derived fluorescence was also detected inside the root nodule cortex by the amino acid sequence replacing the GDPC motif. Versions were (asterisks), indicating qualitative restoration of organogenesis and infection. tagged at the C terminus with YFP [GDPC (WT), GDLC (SYMRK-14), and The SYMRK(GDLC) and SYMRK(RHPC) variants were unable to restore IT RHPC; left and middle panels] or mOrange [GDPC (WT), GAPC; right panel]. formation; instead, abnormal bacterial colonization occurred on the distal High-molecular-weight bands (asterisks) were observed in GDLC, RHPC, surface of otherwise uninfected primordia. and GAPC samples. These may originate from protein aggregation or (B) Box plot of the number of nodule organogenesis events (primordia) and from the formation of branched species. ITs per root system. None of the control roots showed nodule organogen- Numbers indicate molecular weight in kDa. See also Figure S1. esis or ITs. n indicates the number of roots systems with infection events per total number of scored roots. Different letters represent significant differences. All p values are <0.001. To address the functional consequences of the MLD See also Figure S2. release, we generated a SYMRK-DMLD construct that mimics the predicted residual SYMRK protein after loss of the MLD (Figure S3). Synthetic SYMRK-DMLD yielded very low protein Current Biology Vol 24 No 4 424

A B

Figure 3. SYMRK Deletion Versions Differ with Their Ability to Associate with NFR1 and NFR5 SYMRK or deletion versions of SYMRK (SYMRK-DMLD, SYMRK-DED, or SYMR-DKD) (A) or SYMRK and BRI1 (B) C-terminally tagged with mOrange (mOr) were coexpressed in N. benthamiana with NFR1, NFR5, or BRI1 C-terminally tagged with YFP and affinity bound with RFP magnetotrap (A) or GFP magnetotrap (B). The crude extracts (input) and immunoenriched (IP) fractions were subjected to protein blot (WB). Tagged proteins were detected with antiDsRed (mOr-tagged proteins) or antiGFP antibodies (YFP-tagged proteins). (A) NFR5 was strongly coenriched by SYMRK-DMLD and to a lesser extent by full-length SYMRK or SYMRK-DED. (B) NFR5 has strong affinity to endogenous SYMRK-DMLD (arrow). Cell suspensions of Agrobacterium tumefaciens bearing Transfer DNAs (T-DNAs) for transgenic expression of BRI1 and NFR5 were mixed 1:1:1 with cell suspensions bearing T-DNAs for transgenic expression of SYMRK (arrowhead). Three different cell-density ratios of A. tumefaciens carrying SYMRK relative to those carrying BRI1 or NFR5 were infiltrated (indicated by triangle; see Experi- mental Procedures). A. tumefaciens carrying NFR5 was replaced by infiltration buffer in the control samples (B). Only endogenous SYMRK-DMLD was coenriched with NFR5. Numbers indicate weight in kDa. See also Figures S3 and S4. amount in L. japonicus or N. benthamiana even when induced by the different LysM receptors—involved in root expressed under the control of strong promoters (Figure 3 nodule symbiosis or AM—converge on SYMRK to activate and S4). Since both the endogenous and the synthetic the appropriate symbiotic development but the mechanism SYMRK-DMLD are present at much lower concentration than is unclear. In a parsimonious model, SYMRK may directly the full-length protein or the MLD, we conclude that this engage in complex formation with NFR1 and/or NFR5. In order fragment experiences rapid turnover. Release of the MLD to test this, we immunopurified SYMRK-DMLD upon coex- therefore is associated with rapid degradation of the SYMRK- pression with different RLKs in N. benthamiana leaf cells, DMLD fragment. This fragment might provide a ‘‘degron’’ [14] including the NFRs and, as a specificity control, the Brassino- involved in self-clearance to reduce the amount of SYMRK at lide receptor 1 (BRI1) [19]. Neither BRI1 nor NFR1 copurified the plasma membrane. The ubiquitin E3 ligase SINA4 that tar- with SYMRK-DMLD (Figure 3A). Despite the low amount of gets SYMRK for degradation through the SYMRK cytoplasmic SYMRK-DMLD (input, Figure 3A) we observed a strong coen- domain may be involved in this process [15]. richment of NFR5, indicating a strong affinity between both Synthetic SYMRK-DMLD could only partially restore symbi- RLKs (Figure 3A). To further test the specificity of this interac- osis in the symrk-3 null mutant background. Infected root tion, we coexpressed NFR5 together with BRI1 and SYMRK. nodules occurred with a delay, and the number of infection Under these conditions, the endogenous SYMRK-DMLD frag- threads (ITs) was severely reduced (Figure 4). We hypothesize ment was massively enriched upon immunopurification of that the symbiosis defects displayed by SYMRKp:SYMRK- NFR5, at the expense of full-length SYMRK and BRI1 (Fig- DMLD roots may be due to suboptimal protein levels. Consis- ure 3B). We conclude that NFR5 interacts with SYMRK- tent with this idea, we observed ITs upon overexpression of DMLD and that the presence of the MLD region negatively SYMRK-DMLD via the L. japonicus ubiquitin promoter (Fig- interferes with SYMRK-NFR5 interaction. The MLD region ure 4). It is thus possible that the release of the MLD from may impede contact between the LRRs and the extracytoplas- SYMRK ensures appropriate timing and location of SYMRK- mic region of NFR5 because of a steric hindrance or diffusion DMLD production for full symbiotic competence. constraints.

NFR5 Preferentially Interacts with SYMRK-DMLD The SYMRK LRR Region Promotes Protein Turnover LysM-type receptor kinases, such as L. japonicus Nod factor We examined the contribution of different SYMRK domains to receptor 1 (NFR1) and NFR5, have been implicated in the the interaction with other RLKs. NFR1, NFR5, or BRI1 did not perception of (lipo)-chitooligosaccharide signals produced copurify with SYMR-DKD (construct lacking the kinase by rhizobia and AM fungi [16–18]. The signaling pathways domain; Figure S3), which is consistent with the incapacity of SYMRK Cleavage Promotes NFR5 Interaction 425

Figure 4. The SYMRK Ectodomain Controls SYMRK Function in Root Nodule Symbiosis Transgenic roots of the symrk-3 null mutant expressing RFP (control) or the indicated SYMRK deletion constructs, each with a C-terminal RFP tag and under the control of the SYMRK promoter (A) or with a C-terminal mOrange tag and under the control of the L. japonicus ubiquitin (Ub) promoter (B), were inoculated with M. loti MAFF303099 DsRed (A and B) or M. loti R7A lacZ (A). (A and B) Representative micrographs of transgenic roots 13 days after inoculation. The presence of R7A lacZ was detected by blue staining with X-Gal. Fluorescent, DsRed-expressing bacteria are imaged in yellow. (C) Box plot of the number of symbiotic events with R7A lacZ (nodules and primordia) or with MAFF303099 DsRed (IT) per root system. Under the control of the endogenous promoter, SYMRK-DMLD and SYMRK-DED were able to restore nodule organogenesis. SYMRKp:SYMRK-DED roots contained more ITs than SYMRKp:SYMRK roots. Roots expressing SYMRK-DMLD rarely formed ITs and carried significantly fewer mature nodules than roots transformed with the SYMRKp:SYMRK full-length construct. In contrast, Ub:SYMRK-DMLD roots contained mature infected nodules and ITs. None of the control roots showed organogenesis or infection events. n indicates the number of roots systems with nodules and primordia or with infection events per total number of scored roots. Different letters represent significant differences. All p values are <0.05. See also Figure S3. this construct to restore root symbiosis in the symrk-3 mutant dependent degradation, has a positive effect on protein abun- (data not shown). In N. benthamiana, immunopurification of dance (Figure 3A and S4)[15]. SYMRK-DED (construct lacking most of the ectodomain; Fig- ure S3) coenriched NFR1 and NFR5, but less NFR5 was recov- MLD Release Is Different from Animal Receptor ered when compared to SYMRK-DMLD, despite the higher Ectodomain Shedding and May Occur in a Large Number of amount of SYMRK-DED (Figure 3A). These results indicate Plant Receptor-like Kinases that the kinase domain and the LRRs both contribute to com- Here we discovered that the ectodomain of SYMRK orches- plex formation with NFR5. The deletion of the LRRs (SYMRK- trates signaling and stability through a mechanism involving DED) has a stabilizing effect on protein abundance (Figure S4). intramolecular cleavage. This phenomenon is reminiscent Therefore, the LRRs are essential for rapid protein turnover. of ectodomain shedding described for receptor tyrosine We conclude that the MLD region protects SYMRK from kinases (RTKs) of mammals [20–23]. Different from RTKs, how- degradation, while the exposed LRRs promote turnover. The ever, the cleavage site of SYMRK is positioned within and not reduced turnover of SYMRK-DED may be an explanation for at the stalk of the ectodomain. The ectodomain of SYMRK reg- the excessive number of ITs observed on symrk-3 mutant ulates at least two different aspects: (1) interaction with NFR5 roots expressing SYMRK-DED compared to those expressing and (2) protein stability. The MLD domain impedes interaction full-length SYMRK (Figure 4C). The deletion of the cytoplasmic with NFR5 in the absence of symbiotic stimulation and stabi- region (SYMR-DKD), which may promote ubiquitination- lizes SYMRK prior to the release of SYMRK-DMLD. It may Current Biology Vol 24 No 4 426

also be involved in ensuring proper posttranslational decora- Acknowledgments tion and subcellular localization or play additional roles mediated by its predicted sugar binding capability [4, 24]. After MLD The authors acknowledge Victor Gime´ nez-Oya for the homology modeling and molecular dynamics of the SYMRK ectodomain fragment, as well as release, the exposed LRR domain is promoting degradation of for fruitful discussion. This work was supported by the DFG priority program SYMRK and probably interaction with NFR5. Taken together SPP1212 ‘‘Microbial Reprogramming of Plant Cell Development.’’ with the demonstrated role of NFR5 in determining nod factor structural recognition [8] and direct binding of nod factor to the Received: August 9, 2013 ectodomain of NFR5 [5], our results open the possibility that Revised: November 25, 2013 SYMRK acts as a coreceptor and initiates symbiotic signaling Accepted: December 20, 2013 in concert with NFR5. After MLD release, the structure of Published: February 6, 2014 SYMRK resembles that of BAK1, a coreceptor of BRI1 and of the flagellin receptor Flagellin-insensitive 2 (FLS2). FLS2 or References BRI1 form alternative heterocomplexes with BAK1 upon ligand 1. Stracke, S., Kistner, C., Yoshida, S., Mulder, L., Sato, S., Kaneko, T., binding to initiate downstream signaling [25–28]. However, we Tabata, S., Sandal, N., Stougaard, J., Szczyglowski, K., and Parniske, already observed interaction in the absence of a ligand, M. (2002). A plant receptor-like kinase required for both bacterial and suggesting that the SYMRK-DMLD interaction may not be fungal symbiosis. Nature 417, 959–962. the critical stimulatory event in nod factor-mediated signal 2. Gherbi, H., Markmann, K., Svistoonoff, S., Estevan, J., Autran, D., Giczey, G., Auguy, F., Pe´ ret, B., Laplaze, L., Franche, C., et al. (2008). transduction. Cleavage-dependent interaction with the SymRK defines a common genetic basis for plant root endosymbioses adaptor protein MyD88 was observed for the mammalian with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria. Proc. Toll-like receptor 9 (TLR9), the extracytoplasmic region of Natl. Acad. Sci. USA 105, 4928–4932. which is cleaved distal to the plasma membrane. Similar to 3. Markmann, K., Giczey, G., and Parniske, M. (2008). Functional adapta- SYMRK and NFR5, only truncated TLR9 shows high affinity tion of a plant receptor-kinase paved the way for the evolution of intra- for MyD88 [29]. In the LRR I RLK family of Arabidopsis thaliana, cellular root symbioses with bacteria. PLoS Biol. 6, e68. 4. Schallus, T., Jaeckh, C., Fehe´ r, K., Palma, A.S., Liu, Y., Simpson, J.C., 41 out of 50 members possess an ectodomain with a MLD fol- Mackeen, M., Stier, G., Gibson, T.J., Feizi, T., et al. (2008). Malectin: a lowed by a GDPC motif and LRRs [11]. The GDPC motif is also novel carbohydrate-binding protein of the endoplasmic reticulum and present in members of the LRR V, the LRR IVX, and the LRR IV a candidate player in the early steps of protein N-glycosylation. Mol. groups of A. thaliana. Given our results, it is likely that ectodo- Biol. Cell 19, 3404–3414. main cleavage is not exclusive for SYMRK, but rather may be a 5. Broghammer, A., Krusell, L., Blaise, M., Sauer, J., Sullivan, J.T., feature of other MLD-GDPC-LRR containing proteins. Maolanon, N., Vinther, M., Lorentzen, A., Madsen, E.B., Jensen, K.J., et al. (2012). Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proc. Natl. Acad. Sci. Experimental Procedures USA 109, 13859–13864. 6. Madsen, E.B., Madsen, L.H., Radutoiu, S., Olbryt, M., Rakwalska, M., Protein Analysis Szczyglowski, K., Sato, S., Kaneko, T., Tabata, S., Sandal, N., and Homogenization buffer (HB) (50 mM HEPES [pH 7.5], 150 mM NaCl, 10 mM Stougaard, J. (2003). A receptor kinase gene of the LysM type is ethylenediaminetetraacetic acid, 10% sucrose, 2 mM dithiothreitol protease involved in legume perception of rhizobial signals. Nature 425, 637–640. inhibitors [Sigma, P9599], Pefabloc [Roche], and PhosStop [Roche]) was 7. Radutoiu, S., Madsen, L.H., Madsen, E.B., Felle, H.H., Umehara, Y., m added to pulverized tissue at a proportion of 1 l/mg frozen tissue with liquid Grønlund, M., Sato, S., Nakamura, Y., Tabata, S., Sandal, N., and nitrogen. Debris was discarded after centrifugation (10 min, 2,000 g, 4 C). Stougaard, J. (2003). Plant recognition of symbiotic bacteria requires The supernatant was ultracentrifuged (35 min, 250,000 g, 4 C). Subse- two LysM receptor-like kinases. Nature 425, 585–592. quently, the supernatant (soluble fraction) was TCA-acetone precipitated 8. Radutoiu, S., Madsen, L.H., Madsen, E.B., Jurkiewicz, A., Fukai, E., and homogenized with four times diluted SDS sample buffer (SB) (0.5 M Quistgaard, E.M., Albrektsen, A.S., James, E.K., Thirup, S., and Tris-HCl [pH 6.8], 40% glycerol, 7% SDS, 3% dithiothreitol, and a dash of Stougaard, J. (2007). LysM domains mediate lipochitin-oligosaccharide bromophenol blue) at one-tenth of the initial volume, and the precipitated recognition and Nfr genes extend the symbiotic host range. EMBO J. 26, fraction (microsomal fraction) was homogenized in HB-1% Triton X-100 at 3923–3935. one-tenth of the initial volume. After the addition of SB, the samples were 9. Yoshida, S., and Parniske, M. (2005). Regulation of plant symbiosis incubated for 10 min at 50 C prior to SDS-PAGE. receptor kinase through serine and threonine phosphorylation. J. Biol. For the obtainment of crude extracts under denaturing conditions, the Chem. 280, 9203–9209. m pulverized tissue was homogenized at a proportion of 100 l/mm diameter 10. Kosuta, S., Held, M., Hossain, M.S., Morieri, G., Macgillivary, A., of disk leaf tissue with HB containing 1% SDS, 5 M urea, and 2 M Thiourea, Johansen, C., Antolıń-Llovera, M., Parniske, M., Oldroyd, G.E., but no Pefabloc, and incubated for 1 hr at 37 C with agitation and centri- Downie, A.J., et al. (2011). Lotus japonicus symRK-14 uncouples the fuged (10 min, 16,000 g, room temperature), and the supernatant was taken. cortical and epidermal symbiotic program. Plant J. 67, 929–940. After addition of SB, the samples were incubated 10 min at 37 C prior to 11. Hok, S., Danchin, E.G., Allasia, V., Panabie` res, F., Attard, A., and Keller, SDS-PAGE. H. (2011). An Arabidopsis (malectin-like) leucine-rich repeat receptor- Coimmunopurification experiments are specified in the Supplemental like kinase contributes to downy mildew disease. Plant Cell Environ. Experimental Procedures. 34, 1944–1957. 12. Lidell, M.E., and Hansson, G.C. (2006). Cleavage in the GDPH sequence Statistics Analysis of the C-terminal cysteine-rich part of the human MUC5AC mucin. The numbers of infected structures per hairy root system did not follow Biochem. J. 399, 121–129. normal distributions. For the analysis, a Kruskal-Wallis test was applied 13. Osicka, R., Procha´ zkova´ , K., Sulc, M., Linhartova´ , I., Havlıćek, V., and followed by false discovery rate correction. The numbers of infected struc- Sebo, P. (2004). A novel ‘‘clip-and-link’’ activity of repeat in toxin (RTX) tures per hairy root system were represented with box plot graphs, which proteins from gram-negative pathogens. Covalent protein cross-linking th th th th plots the median, 10 ,25 ,75 , and 90 percentiles as vertical boxes by an Asp-Lys isopeptide bond upon calcium-dependent processing at with whiskers. Outliers are represented with round dots. an Asp-Pro bond. J. Biol. Chem. 279, 24944–24956. 14. Schrader, E.K., Harstad, K.G., and Matouschek, A. (2009). Targeting Supplemental Information proteins for degradation. Nat. Chem. Biol. 5, 815–822. 15. Den Herder, G., Yoshida, S., Antolıń-Llovera, M., Ried, M.K., and Supplemental Information includes Supplemental Experimental Procedures Parniske, M. (2012). Lotus japonicus E3 ligase SEVEN IN ABSENTIA4 and four figures and can be found with this article online at http://dx.doi.org/ destabilizes the symbiosis receptor-like kinase SYMRK and negatively 10.1016/j.cub.2013.12.053. regulates rhizobial infection. Plant Cell 24, 1691–1707. SYMRK Cleavage Promotes NFR5 Interaction 427

16. Genre, A., Chabaud, M., Balzergue, C., Puech-Page` s, V., Novero, M., Rey, T., Fournier, J., Rochange, S., Be´ card, G., Bonfante, P., and Barker, D.G. (2013). Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone. New Phytol. 198, 190–202. 17. Maillet, F., Poinsot, V., Andre´ , O., Puech-Page` s, V., Haouy, A., Gueunier, M., Cromer, L., Giraudet, D., Formey, D., Niebel, A., et al. (2011). Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469, 58–63. 18. Op den Camp, R., Streng, A., De Mita, S., Cao, Q., Polone, E., Liu, W., Ammiraju, J.S., Kudrna, D., Wing, R., Untergasser, A., et al. (2011). LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia. Science 331, 909–912. 19. Kim, T.W., and Wang, Z.Y. (2010). Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu. Rev. Plant Biol. 61, 681–704. 20. Carpenter, G. (2003). Nuclear localization and possible functions of receptor tyrosine kinases. Curr. Opin. Cell Biol. 15, 143–148. 21. Fortini, M.E. (2002). Gamma-secretase-mediated proteolysis in cell- surface-receptor signalling. Nat. Rev. Mol. Cell Biol. 3, 673–684. 22. Higashiyama, S., Nanba, D., Nakayama, H., Inoue, H., and Fukuda, S. (2011). Ectodomain shedding and remnant peptide signalling of EGFRs and their ligands. J. Biochem. 150, 15–22. 23. Kopan, R., and Ilagan, M.X. (2004). Gamma-secretase: proteasome of the membrane? Nat. Rev. Mol. Cell Biol. 5, 499–504. 24. Lindner, H., Mu¨ ller, L.M., Boisson-Dernier, A., and Grossniklaus, U. (2012). CrRLK1L receptor-like kinases: not just another brick in the wall. Curr. Opin. Plant Biol. 15, 659–669. 25. Heese, A., Hann, D.R., Gimenez-Ibanez, S., Jones, A.M., He, K., Li, J., Schroeder, J.I., Peck, S.C., and Rathjen, J.P. (2007). The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl. Acad. Sci. USA 104, 12217–12222. 26. Roux, M., Schwessinger, B., Albrecht, C., Chinchilla, D., Jones, A., Holton, N., Malinovsky, F.G., To¨ r, M., de Vries, S., and Zipfel, C. (2011). The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23, 2440–2455. 27. Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nu¨ rnberger, T., Jones, J.D., Felix, G., and Boller, T. (2007). A ﬂagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500. 28. Santiago, J., Henzler, C., and Hothorn, M. (2013). Molecular mechanism for plant steroid receptor activation by somatic embryogenesis coreceptor kinases. Science 341, 889–892. 29. Ewald, S.E., Lee, B.L., Lau, L., Wickliffe, K.E., Shi, G.P., Chapman, H.A., and Barton, G.M. (2008). The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456, 658–662.