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Proc Natl Acad Sci U S A. May 18, 2010; 107(20): 9452–9457.
Published online May 3, 2010. doi:  10.1073/pnas.1000675107
PMCID: PMC2889104
Plant Biology

A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides


Oligogalacturonides (OGs) released from the plant cell wall are active both as damage-associated molecular patterns (DAMPs) for the activation of the plant immune response and regulators of plant growth and development. Members of the Wall-Associated Kinase (WAK) family are candidate receptors of OGs, due to their ability to bind in vitro these oligosaccharides. Because lethality and redundancy have hampered the study of WAKs by reverse genetics, we have adopted a chimeric receptor approach to elucidate the role of Arabidopsis WAK1. In a test-of-concept study, we first defined the appropriate chimera design and demonstrated that the Arabidopsis pattern recognition receptor (PRR) EFR is amenable to the construction of functional and resistance-conferring chimeric receptors carrying the ectodomain of another Arabidopsis PRR, FLS2. After, we analyzed chimeras derived from EFR and WAK1. Our results show that, upon stimulation with OGs, the WAK1 ectodomain is capable of activating the EFR kinase domain. On the other hand, upon stimulation with the cognate ligand elf18, the EFR ectodomain activates the WAK1 kinase, triggering defense responses that mirror those normally activated by OGs and are effective against fungal and bacterial pathogens. Finally, we show that transgenic plants overexpressing WAK1 are more resistant to Botrytis cinerea.

Keywords: damage-associated molecular patterns, elongation factor tu receptor, pectin-mediated signaling, plant immunity, chimeric receptors

In both animal and plants, the extracellular matrix (ECM) separates the cell from the external environment and plays a fundamental role in filtering and interpreting external cues such as pathogen attack, wounding, or mechanical stress. In the ECM of vertebrates, a linear and structurally simple polysaccharide such as hyaluronan, a polymer of repeating disaccharides of D-glucuronic acid and D-N-acetylglucosamine linked via alternating β-1,4 and β-1,3 glycosidic bonds, contributes in maintaining cell and tissue integrity as well as regulating both the innate immune response and growth and development (1). Fragments released from hyaluronan upon mechanical damage or the action of specific enzymes are perceived by the leucine-rich repeat (LRR) Toll-like receptors 2 (TLR2) and 4 (TLR4) as signals of changes occurring in the ECM, triggering inflammatory responses and promoting recovery from tissue injury (13). Similarly, in plants the pectic polysaccharide homogalacturonan (HGA), a polyanionic linear polymer of 1,4-linked α-D-galacturonic acid residues, maintains wall integrity and cell–cell cohesion. This is due to the bridging by Ca++ of antiparallel HGA chains with continuous runs of dissociated and negatively charged carboxyl groups to form structures called “egg-boxes” (46). Fragments of HGA, named oligogalacturonides (OGs), function as danger signals and induce the expression of defense genes and proteins (5, 710), protecting plants against fungal diseases (11). Besides inducing defense responses, OGs also affect several aspects of plant growth and development (5, 12). Like hyaluronan fragments, OGs are regarded as host- or damage-associated molecular patterns (HAMPs or DAMPs) and their biological activity is related to their molecular size because OGs with a degree of polymerization (DP) between 10 and 15 are the most active (13, 14). Notably, the “egg box” conformation is necessary for the biological activity of OGs (5, 15).

The perception system for OGs is still unknown. Candidate receptors are some members of the wall-associated kinase (WAK) family, which in Arabidopsis thaliana includes five tightly clustered genes (WAK1WAK5) (16). WAKs display the typical eukaryotic Ser/Thr kinase signature and an extracytoplasmic domain (ectodomain) containing several EGF-like repeats. WAK1 and WAK2 bind in vitro to OGs and pectin through the N-terminal non-EGF portion of the ectodomain (15, 17, 18). Moreover, WAK2 has been recently shown to be required for the activation by pectin of numerous genes in protoplasts, including that encoding a vacuolar invertase (18). The binding of WAK1 in vitro to HGA, elicitor-active OGs and structurally related alginates occurs under conditions that are compatible with the formation of calcium-induced “egg box” structures (15, 19, 20). WAK1 is expressed in both juvenile and adult leaves, stems, and, at a lower extent, in siliques and flowers but not in roots and is induced by salicylic acid and its analog 2,2-dichloroisonicotic acid (INA), wounding, bacterial infection, and aluminum treatment (19, 2123). Notably, WAK1 is the only member of the family that is up-regulated in response to OGs (about twofold), whereas both WAK1 and WAK2 are slightly down-regulated by flg22 (7). Inducible silencing of individual WAK1 and WAK2, using gene-specific antisense transcripts, does not cause any phenotypic alteration, likely due to functional redundancy (19), whereas reduction of all WAK proteins by inducible expression of single antisense WAK transcripts determines loss of cell expansion and a dwarf phenotype (19, 24), as well as death upon salicylic acid treatment (21). Importantly, plants constitutively expressing WAK1 or WAK2 antisense transcripts could not be obtained, suggesting that loss of WAK function determines lethality (19). Lethality and redundancy therefore render the study of WAKs by reverse genetics very difficult, prompting the use of alternative approaches for the elucidation of their function.

The construction of chimeric receptors is a unique tool for studying the function of orphan or functionally redundant receptors and has been widely used in animal biology to provide a deep insight into the mechanisms of signal perception (25, 26). In plants, however, very little progress has been made using this approach. Apart from domain swaps between very closely related receptors (2730), the only example is a chimeric protein derived from the Arabidopsis brassinosteroid receptor BRI1 and comprising the extracytoplasmic [ectodomain, which includes the LRR domain and the extracytoplasmic juxtamembrane (eJM) region], the transmembrane (TM) and intracytoplasmic juxtamembrane (iJM) regions of this receptor fused upstream to the kinase domain of the resistance gene product Xa21 from rice. This chimera was reported to induce plant defense responses in response to brassinosteroids in rice cells (31).

In this work, we investigated whether WAKs perceive OGs as signal molecules. First, a test-of-concept study was undertaken to assess the possibility of obtaining functional plant chimeric receptors and devise an appropriate design for their construction. Specifically, we analyzed the amenability of the Arabidopsis EFR, a LRR receptor kinase for recognition of the microbe-associated molecular pattern (MAMP) EF-Tu and its derived peptide elf18 (32) as a recipient protein structure. EFR was chosen because it is functional when expressed in Nicotiana species (33), unlike the Arabidopsis FLS2, receptor for flagellin and its derived peptide flg22 (34). Next, we obtained chimeras between EFR and Arabidopsis WAK1 and demonstrated that WAK1 is capable to sense OGs in vivo and trigger a defense response that mirrors that normally activated by OGs.

Results and Discussion

Two chimeric proteins were constructed, both comprising the entire ectodomain of the Arabidopsis FLS2 and the iJM-kinase portion of EFR (Fig. 1A). Our design maintained the integrity of both the extracytoplasmic and the intracytoplasmic portions of the original receptors to allow a correct ligand-induced conformational change (35) and an unaltered interaction with the cytoplasmic partners (36), respectively. The two chimeras differed from each other in the junction point, located either immediately upstream of or within the EFR TM region. TM regions play an important role in the ligand-dependent dimerization and activation of several animal receptors (37, 38) and, in other cases, allow a dimerized inactive state that may be more readily switched to an active state by low concentrations of the ligand (39). In the design exemplified by the chimera named eJMC, similar to that used to study the Drosophila Toll-related receptors (25) and the rainbow trout TLR (26), the integrity of the EFR TM domain was maintained. The second chimeric receptor, named TMC, contained a hybrid FLS2/EFR TM domain and lacked a putative dimerization sequence GXXXG of EFR (Fig. S1); this sequence is absent in FLS2, which has been reported not to homodimerize (40). Both eJMC and TMC, as well as FLS2 and EFR as controls, were fused to GFP and placed under the control of the constitutive promoter CaMV 35S. Transient expression of the fluorescent proteins, here indicated simply as eJMC, TMC, FLS2, and EFR, was performed in Arabidopsis leaves infiltrated with Agrobacterium tumefaciens carrying the individual constructs. Western blot analysis using a GFP-specific polyclonal antibody revealed a specific band corresponding to a molecular mass of approximately 200 kDa for all of the four constructs (41). Confocal microscopy analysis showed comparable levels of fluorescence and a pattern compatible with a plasma membrane localization for all of the protein fusions; moreover dot blot analysis of total microsomal fractions and intercellular washing fluids (IWF) indicated a membrane localization of the GFP-tagged proteins (Fig. S2).

Fig. 1.
Constructs for the expression of the chimeric receptors. (A) The coding regions of EFR and FLS2 are labeled in white and gray, respectively, with the region corresponding to the signal peptide for translocation into the ER indicated in black. eJM and ...

The ability of the chimeric receptors to specifically activate downstream defense responses upon stimulation with flg22 was assessed in agroinfiltrated Nicotiana tabacum leaves by measuring ethylene production, which is normally induced by activated EFR (32). In the absence of elicitation, levels of ethylene were slightly higher in explants infiltrated with Agrobacterium carrying the empty vector than in the nonagroinfiltrated ones. In both types of explants, ethylene slightly increased in response to flg22 but not to elf18 (Fig. S3), indicating that tobacco, albeit weakly, responds to flg22 but, as expected, not to elf18, in agreement with previous results (34). On the other hand, elf18 induced a production of ethylene that was 3.8 times higher in EFR-expressing explants (EFR explants) than in FLS2 explants (Fig. 2A), confirming previous observations that Arabidopsis EFR is functional in tobacco (32). Significantly higher ethylene levels were induced by flg22 in tissues expressing eJMC than in those expressing EFR (negative control), whereas only a weak response to this elicitor was shown in tissues expressing TMC. This last result is in agreement with the observation that the Arabidopsis FLS2 did not produce ethylene in Nicotiana benthamiana (34).

Fig. 2.
Functional characterization of EFR/FLS2 chimeric receptors. (A) Induction of ethylene biosynthesis in agroinfiltrated tobacco leaves expressing the indicated fluorescent receptors. Excised infiltrated leaf sectors were stimulated for 2 h with the elicitor ...

Due to its higher activity, eJMC was chosen for stable expression in transgenic Arabidopsis ecotype Wassilewskija (Ws-0), a FLS2 natural mutant (33). Transformed T1 plants expressing fluorescent eJMC or FLS2 were selected by confocal microscopy analysis of adult rosette leaves. Two independent positive eJMC transgenic lines (numbers 1.4 and 2.6) and one FLS2 line showing comparable levels of fluorescence were chosen for further analyses. The expression of three genes potentially involved in pathogen resistance, i.e., a putative reticuline oxidase gene (RetOx; At1g26380), the cytochrome P450 gene ATCYP81F2 (At5g57220) and PAD3 (At3g26830), which encodes the last enzyme of the camalexin biosynthetic pathway (9, 11), was induced by flg22 in both FLS2 and eJMC transgenic plants but not in untransformed plants (Fig. 2B). Similarly, treatment with flg22 induced H2O2 production and callose deposition in both FLS2 and eJMC plants but not in untransformed Ws-0 plants, which instead responded to elf18 (Fig. 2C). Expression of eJMC also conferred resistance to Pseudomonas syringae pv tomato (Pst) DC3000, which carries a flagellin recognized by FLS2. Infected FLS2 or eJMC leaves showed a significantly slower and less severe development of disease symptoms than Ws-0 plants; the weaker symptoms correlated with a lower number of bacteria in both transgenic plants (Fig. 2D). Taken together, the results of Fig. 2 AC show that Ws-0 plants transformed with eJMC acquired responsiveness to flagellin and have no constitutive defense activation (see water controls in Fig. 2 B and C). Moreover, Fig. 2D shows that plant expressing eJMC are less susceptible to Pst DC3000.

Following the demonstration that eJMC is functional, the chimeric receptor approach based on EFR and the eJMC type of design was used to study whether WAK1, a 715 amino acid protein in its mature form, is involved in perception and transduction of the OG signal. To characterize the activity of WAK1, the chimeras WEG and EWAK were constructed (Fig. 1B). WEG comprised the WAK1 ectodomain fused to the TM-iJM-kinase portion of EFR and was aimed at testing the ability of the WAK1 ectodomain to perceive OGs and activate the EFR-derived kinase. The second chimera, EWAK, comprised the EFR ectodomain fused to the TM-iJM-kinase portion of WAK1 and was aimed at ascertaining whether activation by elf18 of the WAK1-derived kinase domain triggers defense responses mirroring those normally induced by OGs. This analysis is feasible because, whereas OG-induced responses include oxidative burst (9) but no ethylene production (42), EFR-triggered responses include both oxidative burst and ethylene production (32). Confocal microscopy analysis of agroinfiltrated leaf explants expressing fluorescent WEG, EWAK, or WAK1 showed in all cases fluorescence localized in proximity of the plasma membrane at levels similar to those observed with FLS2 and EFR. Western blot analysis on IWF proteins and total microsomal fractions indicated a membrane localization of the chimeras (Fig. S4).

The ability of WEG to activate downstream responses upon stimulation with OGs was analyzed in agroinfiltrated tobacco. Treatment with OGs induced a higher ethylene accumulation (2.4 times) in WEG-expressing explants than in negative controls, i.e., EFR-expressing tissues treated with OGs and WEG-expressing tissues treated with short and biologically inactive OGs (OG3-6) (Fig. 3A). Furthermore, the functionality of WEG was analyzed upon expression in transgenic Col-0 efr plants by monitoring the expression of two genes (At3g22270 and At4g37640) that are up-regulated by flg22 and elf18 but not by OGs (7, 32). No constitutive activation of the expression of these genes was observed in the transgenic plants (see water controls in Fig. 3B). After a 3-h treatment, both genes were up-regulated by elf18 and not by OGs in wild-type Col-0 and did not respond to elf18 in the efr mutant, as expected, whereas in WEG transgenic plants both genes were induced by OGs but not by other elicitors (Fig. 3B). These results show that WEG induces an EFR-type response upon sensing OGs.

Fig. 3.
Induction of defense responses by WAK1/EFR chimeric receptors. (A) Induction of ethylene biosynthesis in agroinfiltrated tobacco explants expressing EFR, WAK1, WEG, and EWAK. Explants were stimulated for 2 h with elf18 (10 μM), OGs (100 μg/mL), ...

Conversely, the EWAK chimera allowed us to investigate whether the WAK1 kinase domain activates the specific responses normally activated by OGs. Upon elicitation with elf18 of tobacco agroinfiltrated leaves, expression of both EWAK and EFR, but not of eJMC, was associated to a robust oxidative burst (Fig. 3C); on the other hand, although EFR-expressing explants treated with elf18 accumulated ethylene, tissues expressing EWAK or eJMC used as a negative control did not (Fig. 3A). Two hallmarks of the response to OGs, i.e., the occurrence of a robust oxidative burst and the absence of ethylene induction, were therefore observed upon specific activation of EWAK. As tobacco, like N. benthamiana, acquires responsiveness to elf18 only upon expression of EFR, and this receptor, but not FLS2, recognizes A. tumefaciens and restricts its growth (32), we investigated whether the expression of EWAK affects Agrobacterium survival in the tissues. A 30% decrease (P < 0.005) in the number of living Agrobacterium cells was observed 24 h after inoculation in tissues expressing EFR or EWAK, whereas no significant effect was observed in tissues expressing eJMC, WAK1, or WEG (Fig. 4A). These results indicate that Agrobacterium-induced activation of the WAK1-derived kinase of EWAK triggers defense responses that restrict colonization by this bacterium. The kinase of WAK1, like that of BAK1/SERK3 and WAKL22, is of the RD-type (Fig. S5), i.e., it carries a conserved arginine (R) immediately preceding the invariant aspartate (D) in the catalytically-active subdomain VI and required for the activation of the kinase through an autophosphorylation of a regulatory region termed the activation loop (43). The observation that, in a survey of the yeast, fly, worm, human, Arabidopsis, and rice kinomes, 12 of the 15 kinases known to function as pattern recognition receptors (PRRs) in innate immunity are of the non-RD-type, whereas the majority of the RD-type is involved in developmental processes, suggests that the RD motif may also characterize kinases that play a role in both defense and development.

Fig. 4.
Response to bacterial and fungal pathogens of plants expressing the chimeric receptors. (A) Growth of A. tumefaciens in Nicotiana tabacum tissues transiently expressing receptor proteins. Tobacco leaves were infiltrated with Agrobacterium carrying the ...

WAK1 and WEG, but not EWAK, plants showed increased resistance to Botrytis cinerea infection compared to wild-type controls (Col-0 or Col-0 efr) (Fig. 4B), in agreement with previous studies showing that OG perception plays a role in resistance against B. cinerea (11). Because none of the transgenic plants showed constitutive activation of defense responses, as determined by the analysis of H2O2 levels and callose deposits (Fig. S6), these data suggest on the one hand that both the WAK1 and the EFR kinase domains induce responses effective against this fungus and, on the other hand, that Botrytis does not activate the EFR ectodomain to an extent sufficient to reduce its growth.

All in all, using the chimeric receptors we were able to dissect the function of WAK1 and show that, upon stimulation with OGs, its ectodomain is able of intramolecularly transmitting the conformational change that activates the EFR kinase domain, whereas its kinase domain triggers defense responses that mirror those normally activated by OGs and are effective against fungal and bacterial pathogens. Interestingly, WAK1 is up-regulated upon perception of its own ligand, similarly to what has been shown for FLS2 and EFR. The elucidation of the ability of WAK1 of sensing OGs is an important step toward understanding the role of these oligosaccharides in plants. Important questions to be addressed are whether other WAK members can also perceive OGs and whether WAKs are the only class of receptors for these molecules. Moreover, the endogenous nature of the OGs and their ability to antagonize auxin suggest their role not only in the activation of the defense response upon pathogen attack or mechanical damage but also during growth and development.

In conclusion, we have shown that there is an appropriate design for the construction of plant chimeric receptor kinases and, furthermore, that it is possible to use EFR to construct receptors with ectodomains of different structural features and capable of activating defenses against pathogens in both Arabidopsis and tobacco. Our chimeric receptor design allowed the demonstration that WAK1, known to bind OGs, mediates the perception of OGs. Likewise, the EFR design developed in our study might greatly facilitate the characterization of the many plant receptors that still have no known function.

Materials and Methods

Cloning of the Receptors.

Standard protocols were used for plasmid DNA isolation, purification and restriction enzyme digestions. The genes to be cloned were generated using the primers listed in Table S1 and the overlap extension method. A scheme of the amplifications is presented in Table S2.


For ethylene measurements, tobacco agroinfiltrated leaves were cut in small slices and extensively washed with dH2O. Explants (approximately 150 mg, corresponding to an average of 15 explants from different infiltrated leaf sectors) were placed in sealed 10-mL flasks containing 2 mL distilled H2O (dH2O) or elicitor solution (100 μg/mL OGs, or 1 μM flg22, or 10 μM elf18). Headspace samples (450 μL) were withdrawn from the vial 2 h after treatment and analyzed by GC-MS using an Agilent 6850A gas chromatograph coupled to a 5973N quadrupole mass selective detector (Agilent Technologies). Chromatographic separations were carried out on an HP Plot-Q fused-silica capillary column (30 m × 0.32 mm i.d.) coated with polystyrene-divinylbenzene (film thickness 0.20 μm) as stationary phase. Injection mode: splitless at a temperature of 220 °C. The initial temperature of the oven was held at 50 °C for 8 min then ramped to 220 °C at a rate of 15 °C/min and held for 5 min. Helium was used as carrier gas at a constant flow of 1.0 mL/min. Mass spectra were collected both in full scan and in SIM mode monitoring the ions m/z m/z 26, m/z 27 and m/z 28 (ionization energy 70 eV; ion source 280 °C; ion source vacuum 10−5 Torr).

Oxidative burst after elicitor treatment was measured in tobacco agroinfiltrated leaves using a modified luminol-dependent assay (44) using approximately 30 mg of tissue slices.

DAB (3,3’-diaminobenzidine tetrahydrochloride) staining, analysis of callose deposition, and expression of marker genes were performed as previously described (9).

All bioassays were performed with a minimum of three replicates per treatment in least three independent experiments.

Infection of Leaves with A. tumefaciens, P. syringae DC3000, and B. cinerea.

Infections with P. syringae pv. tomato DC3000 were performed by spray-inoculations according to Zipfel et al. (33). B. cinerea growth assays were performed on detached leaves as previously described (11).

Confocal Microscopy Analyses.

GFP-dependent fluorescence was monitored from 2 to 4 days postinfection in lower epidermal cells.

Statistical Analysis.

Data are represented as means ± SE. Unpaired t test with equal variance was used to calculate two-tailed P value to estimate statistical significance of differences between two treatment groups in the whole study. Assays were run in duplicate or triplicate and repeated in a minimum of three independent trials.

Further information is provided in SI Materials and Methods.

Supplementary Material

Supporting Information:


We thank E. Marchetti for technical assistance with the confocal microscope. This work was supported by the Ministero dell'Università e della Ricerca (PRIN2007), by ERA-NET Plant Genomics (Grant RBER063SN4), the European Reasearch Council (ERC Advanced Grant 233083), Ministero delle Politiche Agricole e Forestali (PROTEO-STRESS 2007–2009), Institute Pasteur-Fondazione Cenci Bolognetti (2008–2010) and Università di Roma “La Sapienza” (ATENEO, 2006–2009).


The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1000675107/-/DCSupplemental.


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