A method for functional testing constitutive and ligand-induced interactions of lysin motif receptor proteins

Background Plant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized. Results Interactions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4ED–LYK4ED, LYK4ED–LYK5ED) and chitin-induced interactions (LYK1ED–LYK1ED, LYK1ED–LYK5ED) of the examined ectodomains. Conclusion We present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.


Background
Plants possess specific receptors and signal cascades to alter gene expression in response to invading microbes. PAMPs (pathogen-associated molecular patterns) are conserved elicitor molecules such as fungal chitin and chitooligosaccharides, bacterial peptidoglycan, and flagellin [1][2][3]. PAMPs are typically perceived by corresponding PRRs (pattern recognition receptors) in plant plasma membranes. Many PRRs are receptor kinases consisting of an extracellular, ligand-binding ectodomain, a transmembrane domain, and an intracellular kinase domain. Some PRRs lack an intracellular kinase domain or possess an inactive kinase domain. This implies that these receptors require cooperation with another PRR or an adaptor kinase. PRR-mediated signaling results in activation of PAMP-triggered defense reactions (plant immunity) [1,2,4]. To recognize symbiotic microbes, host plants perceive specific microbial signals to activate expression of symbiosis-related plant genes. Nitrogen-fixing bacteria, commonly referred to as rhizobia, induce nodules on roots of legumes while nutrient-acquiring (mainly phosphorus) mycorrhizal fungi establish a root symbiosis with most land plants. Rhizobial lipo-chitooligosaccharides, commonly known as Nod factors, and chitinous mycorrhizal molecules (chitin, chitooligosaccharides and lipo-chitooligosaccharides) are key signals to induce plant responses required for nodulation and mycorrhization [5,6].

Plant Methods
Numerous PRRs of plants possess LysM domains in their ectodomains. The LysM domain (Pfam PF01476) has been originally discovered in lysins of bacteriophages and bacteria [7]. LysM receptors of plants are implicated in perception of microbial signals containing N-acetylglucosamine such as fungal chitin (or chitooligosaccharides), Nod factors and bacterial peptidoglycan [8][9][10]. Moreover, specific LysM-type proteins may function as receptors for the oligosaccharidic repeating subunit of rhizobial exopolysaccharide [11] or fungal β-1,3-glucans [12]. The Nod factor receptors NFR1 and NFR5 of the model legume Lotus japonicus represent the first characterized plant LysM receptors [13,14]. NFR1 and NFR5 bind Nod factors with high affinity (K d values in the nanomolar range) [15]. The protein kinase activity of NFR1 (NFR5 lacks kinase activity) was found to be required for receptor autophosphorylation and subsequent activation of Nod factor signaling [16]. Ligand-induced activation of the Nod factor receptor pair mediates induction of Nod factor signaling that includes various downstream components such as symbiosis receptor kinase (SymRK), calcium and calmodulin dependent protein kinase (CCaMK), its phosphorylation target (named CYCLOPS) and the key transcription regulator (nodule inception protein) NIN required for expression of Nod factor-induced genes [5,17]. NIN expression is strongly up-regulated by Nod factor signaling in L. japonicus roots [17,18].
Here, we present a method for analysis of interactions between LysM-type receptor proteins. We used the ectodomains of LYK1, LYK4, LYK5 as examples to functionally analyze constitutive and ligand-induced receptor interactions in planta. The method is based on expression of chimeric receptors in L. japonicus roots and expression analysis of the symbiotic marker gene NIN [33]. In this system, each chimeric receptor construct consist of an ectodomain from a given LysM-type protein and the intracellular domain from a L. japonicus Nod factor receptor protein (NFR1 ID and NFR5 ID , respectively). Constitutive or ligand-induced dimerization of functional receptor pairs results in activation of NIN expression as estimated by a co-expressed NIN promoterβ-glucuronidase fusion (NINp-GUS construct).

Plant material
Lotus japonicus (ecotype Miyakojima MG-20) and the Nod factor receptor mutants nfr1-1 and nfr5-2 (ecotype Gifu) were used for expression of chimeric receptors. The nfr1-1 and nfr5-2 mutants (deficient in functional NFR1 and NFR5 proteins) were kindly provided by Simona Radutoiu and Jens Stougaard (Aarhus University, Aarhus, Denmark) [13,14]. The nfr1-1 possesses a stop codon in the kinase domain VIII [13] and the nfr5-2 mutant contains a retrotransposon insertion generating a truncated protein lacking the C-terminal kinase domain [14]. Seeds were treated with concentrated sulfuric acid (10 min), washed with sterile distilled water, 70% (v/v) ethanol (1 min) and then with sterile water again. The seeds were surface-sterilized with sodium hypochlorite (0.05% active chlorine; 5 min), extensively washed with sterile water and then used for hairy root transformation.

DNA constructs
To estimate NIN expression in L. japonicus roots, a 2722bp NIN promoter sequence from Lotus japonicus MG-20 was inserted into the pCAMBIA1305.1 vector (https :// www.cambi a.org) lacking the original CaMV 35S promoter sequence. The β-glucuronidase (GUS) gene in this vector (also known as GUSPlus) contains an intron of a catalase gene from castor bean to prevent expression by A. rhizogenes and ensure detection of GUS activity expressed by L. japonicus. The resulting plasmid (pCAM-BIA-NINp) containing a NIN promoter-β-glucuronidase fusion (NINp-GUS) was then used as an acceptor for chimeric receptor constructs. The hybrid protein constructs consisting of an A. thaliana LYK ectodomain (LYK1 ED , 693-bp fragment of LYK1; LYK4 ED , 813-bp fragment of LYK4; LYK5 ED , 831-bp fragment of LYK5) and an intracellular domain of a L. japonicus Nod factor receptor of (NFR1 ID , 1203-bp fragment of NFR1; NFR5 ID , 1086-bp fragment of NFR5) were obtained by overlap extension PCR. The constructs (namely LYK1 ED -NFR1 ID , LYK4 ED -NFR1 ID , LYK5 ED -NFR1 ID , LYK1 ED -NFR5 ID , LYK4 ED -NFR5 ID , LYK5 ED -NFR5 ID ) were inserted into pRT104 [36] to yield expression cassettes containing a CaMV 35S promoter and a poly-A tail. The chimeric receptor pair constructs were then introduced into the pCAMBIA-NINp vector by using the Hieff Clone ™ Plus Multi One Step Cloning Kit (Yeasen, Shanghai, China). The resulting binary vectors contained expression cassettes encoding the following chimeric receptor pairs: LYK4 ED -NFR1 ID / LYK4 ED -NFR5 ID and LYK5 ED -NFR1 ID /LYK4 ED -NFR5 ID . Moreover, pCAMBIA-NINp with the expression cassette encoding LYK4 ED -NFR5 ID was constructed. Finally, all generated binary vectors were used for hairy root transformation of L. japonicus. Primers and plasmids used are listed in Additional file 1: Tables S1 and S2. Schematic views of expressed chimeric proteins are shown in Fig. 1.

Hairy root transformation
The pCAMBIA-NINp derivatives containing chimeric receptor constructs were mobilized into Agrobacterium rhizogenes strain LBA9402 [37] by electroporation. Bacteria were grown in YMB medium (2 g/L mannitol, 0.4 g/L yeast extract, 0.1 g/L NaCl, 0.66 g/L K 2 HPO 4 ·3H 2 O, 0.2 g/L MgSO 4 ·7H 2 O and 15 g/L agar; pH 7) supplemented with corresponding antibiotics. Transgenic hairy roots of L. japonicus plants (L. japonicus MG-20, Nod factor receptor gene mutants nfr1-1 and nfr5-2) were obtained according to procedures described previously [33,38]. Briefly, germinated seedlings (with a root length of about 1 cm) were placed on 0.8% (w/v) agar (HKM, Guangzhou, China) plates containing Gamborg's 1/2 B 5 Salts and Vitamins medium (Sigma-Aldrich). The plates were kept in a temperature-controlled growth room (~ 24 °C; 16-h photoperiod; ~ 2000 lx light intensity, Philips Lifemax TL-D 36W/54-765 and TL-D 36W/29-530 daylight fluorescent tubes at a ratio 3:1). The plates were partially covered with aluminum foil to protect the roots from light. After incubation for 24 h, roots of seedlings were diagonally cut off by a sterile scalpel and the hypocotyls of the wounded seedlings were immersed in a given A. rhizogenes suspension at 24 °C for 30 min. The transformed seedlings were then transferred to new agar plates. The plates were placed at an angle of approximately 75° and kept in the temperature-controlled growth room. Seedlings were transferred to freshly prepared plates every week. Three weeks after transformation, plants with formed hairy roots were used for elicitor treatments, GUS staining and gene expression analysis.

Elicitor treatment and GUS staining
Transformed L. japonicus roots were treated with a suspension containing 1 μM chitoheptaose (hepta-N-acetylchitoheptaose; Elicityl, Crolles, France) or 10 μg/ mL chitin (Sigma-Aldrich) for 4 h. Control plants were treated with corresponding amounts of sterile water. Finally, harvested roots were subjected to histochemical GUS staining using 5-bromo-4-chloro-3-indol-glucuronide cyclohexylamine salt [39]. Briefly, L. japonicus roots were transferred to 10-mL tubes, then soaked with GUS staining solution and exposed to a vacuum for 20 min. Samples were incubated at 37 °C for 10 h. Finally, roots were rinsed three times with 10% (v/v) commercial bleach and then washed with sterile water. Roots were observed by a stereo microscope and photographed (Lumar.V12, Zeiss, Oberkochen, Germany). Hairy roots with clearly visible blue coloration in at least some root regions were considered as GUS-positive. Transformation efficiency was estimated based on the ratio of GUSpositive hairy roots to the total number of formed hairy roots.

Reverse transcription PCR
Expression of chimeric receptor genes in hairy roots of L. japonicus was confirmed by reverse transcription PCR. Non-transformed roots were used as a negative control. Root RNA was isolated using a HiPure Plant RNA Mini   Figure S1.

qRT-PCR analysis
Expression of chimeric receptor genes in L. japonicus was also analyzed by real-time quantitative reverse transcription PCR (qRT-PCR). Non-transformed roots were used as a negative control. The RNA extractions were performed in triplicate (three independent biological replicates). Isolation of root RNA and synthesis of the first-strand cDNA were conducted as mentioned above. qRT-PCR analysis was performed with obtained cDNAs and primers are listed in Additional file 1: Table S1,

The chimeric receptor method can provide clues to constitutive homodimerization of LysM-type proteins
Hairy roots of L. japonicus expressing a given chimeric receptor pair and a NINp-GUS construct (NIN promoter fused to the GUS reporter gene) were obtain by A. rhizogenes mediated transformation. In addition to MG-20 wild-type plants, we also used the Nod factor receptor gene mutants nfr1-1 and nfr5-2 (ecotype Gifu) for expression of chimeric receptors in hairy roots. In this system, the function of constitutive and ligand-induced receptor pairs can be analyzed (Fig. 1). Three weeks after A. rhizogenes transformation, hairy roots were treated with sterilized water, 1 μM chitoheptaose or 10 μg/mL chitin for 4 h and then stained for GUS activity to monitor NIN gene expression. Three independent experiments were performed with the constructed A. rhizogenes strains. In total, 377 (experiment 1), 350 (experiment 2) and 351 (experiment 3) hairy roots were formed by 216 MG-20 plants. Furthermore, 402 hairy roots from 90 nfr1-1 plants and 375 hairy roots from 90 nfr5-2 plants were analyzed. Details on the number of GUS-positive roots for each test combination are shown in Additional file 1: Tables S3 and S4. The average transformation efficiency of MG-20 plants (as estimated by the ratio of GUSpositive hairy roots to the total number of formed hairy roots) was 48.6 ± 3.6 (Additional file 1: Table S3). Similar values were obtained for the nfr1-1 (48.3 ± 5.1) and nfr5-2 (52.6 ± 7.0) mutants (Additional file 1: Table S4). Analysis by reverse transcription PCR and qRT-PCR confirmed expression of the chimeric receptor genes in the hairy roots (Additional file 2: Figures S1, S2 and S3).
We first asked whether chimeric receptor analysis can be used to obtain information on constitutive homodimerization, i.e., the ectodomain dimerization of a given LysM-type protein in a ligand-independent manner. The chitin receptor complex protein LYK4 of A. thaliana was used as a known example of a LysM-type protein that forms such constitutive homodimers [35]. We therefore examined the ability of LYK4 ED -NFR1 ID and LYK4 ED -NFR5 ID to trigger GUS activity in L. japonicus roots. These hybrid receptor proteins consisted of the ectodomain of LYK4 (LYK4 ED ) fused to the intracellular domains of the Nod factor receptors NFR1 (NFR1 ID ) and NFR5 (NFR5 ID ), respectively (Fig. 1b). Roots treated with water, chitoheptaose or chitin were subjected to GUS staining. Control roots transformed with the single hybrid protein LYK4 ED -NFR5 ID (and also carrying the NINp-GUS construct) did not show GUS activity (Fig. 2a). In contrast, roots co-expressing LYK4 ED -NFR1 ID and LYK4 ED -NFR5 ID showed blue coloration indicating that NIN gene expression was induced (Fig. 2b). Hence, the LYK4 ED -NFR1 ID /LYK4 ED -NFR5 ID proteins formed a functional protein complex independently of whether the roots were treated with water or elicitor. These results are consistent with our previous finding that the ectodomain of LYK4 can form constitutive homodimers [35].

The method also can give information on constitutive heterodimerization of LysM-type proteins
LysM-type proteins can also form constitutive (ligandindependent) heterodimers. We previously found that the ectodomain of LYK4 assembles with the ectodomain of the chitin receptor LYK5 of A. thaliana [35]. We therefore asked whether our chimeric receptor approach can provide information on possible constitutive heterodimerization of LysM-type protein ectodomains. We made a DNA construct encoding LYK5 ED -NFR1 ID , i.e., the ectodomain of LYK5 was fused to the intracellular domain of NFR1 (Fig. 1b). In contrast to LYK4 ED -NFR5 ID alone (Fig. 2a), co-expression of LYK5 ED -NFR1 ID with LYK4 ED -NFR5 ID in either MG-20, nfr1-1 or nfr5-2 plants resulted in blue coloration of GUS-stained roots indicating NIN expression. Roots treated with chitoheptaose or chitin showed similar blue coloration (Fig. 2c). Hence, coexpression of the chimeras resulted in a functional receptor pair, confirming the ligand-independent LYK4-LYK5 heterodimerization reported previously [35].

Analysis of ligand-induced formation of LysM receptor complexes
Ligand-induced dimerization of LysM receptor proteins is believed to play a primary role in triggering activation of downstream signaling pathways. Previous work showed that chitin signaling in A. thaliana is activated by ligand-induced heterodimerization of the chitin receptor complex proteins LYK1 and LYK5 [32,35]. Here, we investigated whether we could obtain similar results with our chimeric receptor method. A. rhizogenes carrying plasmids encoding LYK1 ED -NFR1 ID (ectodomain of LYK1 fused to the intracellular domain of NFR1) and LYK5 ED -NFR5 ID (ectodomain of LYK5 fused to the intracellular domain of NFR5) (Fig. 1b) were used for root transformation of the different L. japonicus genotypes. The formed roots were exposed to water, chitoheptaose or chitin and finally stained for GUS activity reflecting NIN expression. As shown in Fig. 3a, the water treatment did not result in blue coloration of the roots whereas application of either chitoheptaose or chitin caused strong blue coloration. These results indicate a ligandinduced interaction between LYK1 ED and LYK5 ED and thus confirm previous findings on the LYK1-LYK5 protein complex formation [32,35].
Previous work suggested that chitin-induced homodimerization of LYK1 also can activate chitin signaling in A. thaliana [27]. We therefore asked whether a chimeric receptor pair with ectodomains of LYK1 alone is functional in the chimeric receptor system. The combination LYK1 ED -NFR1 ID with LYK1 ED -NFR5 ID (ectodomain of LYK1 fused to the intracellular domain of NFR1 and NFR5, respectively; Fig. 1b) was investigated. Transformed roots treated with water or chitoheptaose showed no visible blue coloration, indicating that NIN expression was not activated. However, blue coloration, often relatively faint, was observed when roots were A co-transformed NINp-GUS construct allowed visualization of receptor-dependent activation of NIN expression. Three weeks after transformation, roots were treated with sterile water, 1 μM chitoheptaose [(GluNAc) 7 ] or 10 μg/mL chitin for 4 h and finally stained for GUS activity. a Roots expressing the single hybrid protein LYK4 ED -NFR5 ID did not show GUS activity. b Blue coloration was observed when the proteins LYK4 ED -NFR1 ID and LYK4 ED -NFR5 ID were co-expressed. c Analysis of LYK5 ED -NFR1 ID combined with LYK4 ED -NFR5 ID also resulted in blue-colored roots. Bars = 0.8 cm treated with chitin (Fig. 3b). These findings suggest that chitin-induced LYK1 homodimerization was possible as reported previously [27].

Analysis of non-functional receptor pairs
In contrast to LYK5, LYK4 did not directly interact with LYK1 in our previous immunoprecipitation and BiFC experiments [35]. The chimeric receptor method provided similar results. The transformed L. japonicus roots co-expressing LYK1 ED -NFR1 ID and LYK4 ED -NFR5 ID were analyzed as stated above. As expected, the examined receptor combination was non-functional. Roots co-expressing LYK1 ED -NFR1 ID and LYK4 ED -NFR5 ID showed no visible GUS staining reflecting NIN expression. Likewise, roots treated with chitoheptaose or chitin failed to induce GUS activity (Fig. 3c). Hence, the chimeric receptor method also could provide information on the lack of direct LysM-type protein interactions.

Discussion
In this article, we describe a method that tests functionality of chimeric LysM receptor protein pairs expressed in L. japonicus roots. This method was originally developed to analyze the chitin-induced interaction between the ectodomains of the receptor proteins OsCERK1 and OsCEBiP in rice [33]. Here we show that the method also can provide information on ligand-independent homodimerization and heterodimerization of LysM receptor proteins. Hence, the method allows discrimination between constitutive and ligand-induced proteinprotein interactions. Furthermore, negative results can be obtained, indicating that the examined ectodomains could not form a functional receptor complex.
The described method is based on expression of chimeric receptors that activate Nod factor signaling in L. japonicus [40]. Nod factor signaling in legumes results in up-regulation of symbiosis-related genes such as NIN, a key transcriptional regulator required for expression of other symbiotic genes [17]. Here we used a NINp-GUS construct to estimate NIN expression in L. japonicus. In addition, transcript levels of other symbiotic genes, such as the GRAS transcriptional regulator genes NSP1 and NSP2, can be determined by qRT-PCR as reported previously [33]. We recommend analyzing NIN expression as a prime response marker because constitutive GUS activity reflecting NIN expression was not detected in any control plants in our current and previous [33] experiments.
The chimeric constructs were expressed from the CaMV 35S promoter to allow sufficient expression of the protein pairs in equal amounts. This promoter has been successfully used to analyze ligand-induced heterodimerization of rice chitin receptor proteins in our previous study [33]. The CaMV 35S promoter is often used Co-transformation of the NINp-GUS construct provided an estimate for NIN gene expression. Prior GUS staining, roots were treated with sterile water, 1 μM chitoheptaose [(GluNAc) 7 ] or 10 μg/mL chitin for 4 h. a Roots co-expressing LYK1 ED -NFR1 ID with LYK5 ED -NFR5 ID showed blue coloration upon treatment with chitoheptaose or chitin. b Co-expression of LYK1 ED -NFR1 ID with LYK1 ED -NFR5 ID resulted in weak blue coloration when roots were treated with chitin. c No blue coloration was observed for the combination LYK1 ED -NFR1 ID with LYK4 ED -NFR5 ID . Bars = 0.8 cm to study protein-protein interactions in planta, including ligand-induced heterodimerization of LYK1 with LYK5 in A. thaliana [32,35]. Furthermore, it is recommended analyzing additional L. japonicus control roots that express a single hybrid protein as performed with LYK4 ED -NFR5 ID in this study and with rice chitin receptor constructs in our previous work [33]. In all examined cases, the single constructs did not cause GUS activation, indicating that the roots showed no endogenous (receptor-independent) NIN expression. Hence, the described method is robust and suitable to analyze functionality of specific LysM-type receptor pairs.
The described method requires co-expression of two chimeric receptor genes and a NINp-GUS construct in L. japonicus. GUS staining reflecting NIN promoter activity can be considered as a measure for successful co-transformation of the expressed constructs. It is also recommended to confirm expression of the chimeric receptor genes by qRT-PCR. The transformation efficiency (as estimated by the ratio of GUS-positive hairy roots to the total number of formed hairy roots) was nearly 50% under the described test conditions. Co-expression of fluorescence proteins and removing of non-fluorescent hairy roots may further increase the transformation efficiency. However, we do not recommend the use of selection media containing antibiotics or herbicides, because Nod factor signaling in L. japonicus may be impaired under such conditions.
In our previous study on chitin receptors from rice, L. japonicus Gifu lines were used that have been stably transformed with a NINp-GUS construct [33]. However, ecotype Gifu plants were difficult to propagate under our growth room and greenhouse conditions. We therefore simplified the system by cloning the chimeric receptor gene constructs into a binary vector containing NINp-GUS (pCAMBIA-NINp). Thus, any L. japonicus genotype with a functional Nod factor signaling pathway, including the early-flowering ecotype MG-20 suitable for indoor handling [41], can now be used.
The current study shows that the GUS staining results with MG-20 wild-type plants and the Gifu Nod factor receptor gene mutants (nfr1-1 and nfr5-2) were similar, indicating that the presence of the full-length NFR1 and NFR5 proteins in MG-20 did not influence the interactions of the examined LYK ectodomains of A. thaliana. These findings indicate that induction of non-specific (NFR1 and NFR5 mediated) downstream signaling was absent or below the detection limit. This is not surprising because NFR1 and NFR5 have been reported to specifically perceive Nod factors to induce downstream signaling [15,16]. Hence, we suggest that MG-20 plants can be used for testing any LysM-type receptor pair with the described method provided the ligand is not a lipo-chitooligosaccharide. In case of characterization of Nod factor receptors and the use of Nod factors, however, the chimeric receptor analysis should be performed with Nod factor receptor gene mutants of L. japonicus.
Whole roots were treated with chitoheptaose and chitin in our experiments. Alternatively, roots can be spottreated with a given elicitor as reported previously [33]. For elicitor treatment of hairy L. japonicus roots, we recommend using an incubation time of 4 h. We have chosen this value because a previous time course experiment with chimeric receptors containing ectodomains of OsCERK1 and OsCEBiP showed increased NIN promoter activity as early as 3 h when roots were treated with chitooctaose. Blue coloration was first observed in the rhizodermis and later in cortical and vascular cells of the roots [33]. In the present study, blue coloration was seen in epidermal, cortical and vascular cells to different extents and was often strongest in the vascular tissue. Differences in GUS activity could be due to insertion of the NINp-GUS construct into different genomic regions. Moreover, different levels of phytohormones in the hairy roots could eventually have modulated the strength of GUS activity, particularly in the central vascular tissue. We therefore recommend analyzing hairy roots from several plants. Furthermore, clearing of GUSstained roots with commercial bleach made the blue coloration within the root more visible but eventually had an attenuating effect on GUS staining at the root surface. Observed tissue-specific variations and quantitative differences in GUS staining intensities should therefore not be over-interpreted.
Immunoprecipitation and BiFC analysis in previous studies revealed constitutive and ligand-induced interactions between LYK proteins of A. thaliana [27,32,35]. The results of the described chimeric receptor method were entirely consistent with these previous findings and provide additional evidence for constitutive homodimerization of LYK4 ED -LYK4 ED [35], constitutive heterodimerization of LYK4 ED -LYK5 ED [35] and ligand-induced heterodimerization of LYK1 ED -LYK5 ED [32,35]. Taken these findings together, we conclude that chitin signaling in A. thaliana is triggered by formation of a tripartite chitin receptor complex consisting of LYK1, LYK4 and LYK5. Upon ligand binding, LYK1 ED interacts with LYK5 ED from the LYK4 ED -LYK5 ED heterodimer as illustrated in Additional file 2: Figure S4. Furthermore, our results support the possibility that a homodimeric LYK1 ED -LYK1 ED complex can be formed in response to chitin treatment [27]. However, chitin-induced LYK1 ED -LYK1 ED dimerization is perhaps rather weak and chitoheptaose was obviously too short for receptor dimerization. Moreover, in agreement with our previous analysis [35], no indications for a functional LYK1 ED -LYK4 ED interaction were found with the chimeric receptor method.

Conclusion
The chimeric receptor approach described in this article provides information on interactions between ectodomains of LysM-type receptor proteins. Taking LYK1, LYK4, LYK5 as examples, we used the method to verify constitutive and ligand-induced interactions between chitin receptor proteins of A. thaliana. The method can be applied to obtain clues for homodimerization of a given LysM-type protein or to test specific candidate receptor pairs for functionality. In future, the method could serve as a powerful tool to identify and characterize co-receptors or scaffold proteins in receptor complexes. In case of ligand-induced dimerization, the method also could be used to identify novel microbial signals perceived by a chimeric receptor pair of hitherto unknown function. Future work will be required to examine the possibility to modify the described method for testing receptor protein-protein interactions in general.