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First published online November 20, 2003; 10.1104/pp.103.027680 Plant Physiology 133:1893-1910 (2003) © 2003 American Society of Plant Biologists Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti1Laboratoire des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique, Boite Postale 27, 31326 Castanet-Tolosan cedex, France (M.-T.N.G., S.C., A.C.J.T., A.N., C.H., E.B., J.V.C.); Signaux et Messages Cellulaires chez les Végétaux, Centre National de la Recherche Scientifique-Université Paul Sabatier, Pôle de Biotechnologie Végétale, Boite Postale 17, 31326 Castanet-Tolosan cedex, France (J.-J.B.); and Centre de Recherches sur les Macromolécules Végétales-Centre National de la Recherche Scientifique (affiliated with Université Joseph Fourier), Boite Postale 53, 38041 Grenoble cedex 09, France (A.I.)
To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.
The lectin-like receptor kinases (LecRKs) are a class of proteins originally described from Arabidopsis (Hervé et al., 1996  ). They have a structure similar to other plant receptor-like kinases (RLKs; Shiu and Bleecker, 2001; Cock et al., 2002) with an N-terminal targeting signal, a presumably extracellular domain, a single transmembrane (TM)-spanning helix, and a cytosolic kinase domain. The Arabidopsis genome contains over 610 RLKs that have been shown to be monophylogenetic with respect to the kinase domain and most closely related to the fruitfly (Drosophila melanogaster) Pelle and related animal cytoplasmic kinases (Shiu and Bleecker, 2001). In cases in which they have been tested, these kinases have been shown to have specificity for phosphorylation on Ser/Thr residues (Shiu and Bleecker, 2001).
The plant RLKs can be grouped into more than 21 structural classes based on their extracellular domains (Shiu and Bleecker, 2001
The legume lectins are a class of proteins that are most abundant in the seeds of legumes (Brewin and Kardailsky, 1997
Because of their homology to legume lectins, it seems reasonable to suppose that LecRKs could be involved in the recognition and transduction of saccharidic signals. However, sequence analysis and molecular modeling of Arabidopsis LecRKs has revealed a poor conservation of the residues involved in monosaccharide binding, whereas the hydrophobic-binding site appears to be better conserved (Hervé et al., 1999
The structure of LecRKs has led to suggestions that in legumes they could be involved in the legumerhizobia symbiosis (Hervé et al., 1996
The other evidence that suggests that LecRKs could be involved in the symbiosis relies on the numerous studies on soluble legume lectins. In the 1970s, these studies culminated in the "lectin recognition hypothesis," which proposed that plant lectins mediate specificity in the legume-rhizobia symbiosis (for review, see Brewin and Kardailsky, 1997
In this article, we describe the first study, to our knowledge, of LecRK genes in a legume, and we specifically address the question of their potential role in relation to the symbiosis with rhizobia. For this work, we have used the model legume Medicago truncatula Gaernt., in which a variety of genomic tools have been developed (Barker et al., 1990
Identification of Four M. truncatula LecRK (MtLecRK) Genes Expressed in Roots
To isolate clones related to LecRK genes potentially playing a role in the legume-rhizobia symbiosis, the following three root or nodule cDNA libraries were screened with a probe consisting of the kinase domain of the Arabidopsis Ath.lecRK-a1 gene: nitrogen-starved roots; roots following inoculation for 6, 24, and 48 h with S. meliloti; and 4-d-old nodules. Of the 35 kinase-containing clones isolated, six were related to either soluble or other receptor kinases. The rest of the clones were related to LecRKs, thus showing that the Ath.lecRK-a1 kinase probe is quite specific for LecRKs despite the large number of kinase genes in higher plants. Two, 25, and two clones were isolated from the three libraries, respectively (corresponding to an abundance of 1:165,000, 1:16,500, and 1:120,000). Restriction enzyme digestion and partial sequence analysis of the clones identified sequences related to four different genes. These genes were mapped on the molecular genetic linkage map of M. truncatula (Thoquet et al., 2002
The longest clones related to each of the four genes were sequenced in their entirety, and for genes MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, the sequences were extended by 5'-RACE. Complete open reading frames (ORFs) were obtained for MtLecRK7;1, MtLecRK7;2, and MtLecRK1;1 (Fig. 1). For MtLecRK7;3, the initiating Met indicated in Figure 1 corresponds to the initiating Met of MtLecRK7;1 and 7;2, but it should be noted that we cannot exclude the possibility that the MtLecRK7;3 ORF starts further upstream. The MtLecRK7;1, MtLecRK7;2, MtLecRK7;3, and MtLecRK1;1 ORFs code for proteins of 659 (73.9 kD), 669 (74.9 kD), 682 (76.0 kD), and 678 (75.8 kD) amino acids, respectively, and these proteins are shown aligned with Ath.LecRK-a1 (At3g59700) in Figure 1. The M. truncatula proteins are more similar in their kinase domains (53%-87% similarity) than they are in their lectin-like domains (39%-83% similarity). MtLecRK7;1 and MtLecRK7;2 are the most closely related, followed by MtLecRK7;3, with MtLecRK1;1 being the most divergent. Structural predictions using the program THMHH suggest that all four proteins contain an N-terminal signal sequence and that they possess a TM-spanning helix of 23 residues situated between the lectin-like and kinase domains. Although there are some variation between the structural prediction programs, these proteins are generally predicted to be Type Ia PM proteins, with cytosolic kinase domains. The kinase domains of all four proteins, by alignment to the kinase domain of Ath.LecRK-a1, contain the 12 sub-domains and the two conserved sequence motifs in the catalytic regions of Ser/Thr kinases (Fig. 1). A region of high variability in these proteins is the carboxy-terminal region, which in protein Tyr kinases is implicated in regulatory functions (Hubbard and Till, 2000
In the lectin-like domain, motif searches using ScanProsite revealed that MtLecRK1;1 contains the lectin_legume alpha and beta chain signatures that are not as highly conserved in the other three proteins. The lectin-like extracellular domains of MtLecRK7;1, MtLecRK7;2, MtLecRK7;3, and MtLecRK1;1 are predicted to be glycosylated with four, eight, seven, and eight N-glycosylation sites, respectively.
BLASTP comparisons with the National Center for Biotechnology Information nonredundant database (Altschul et al., 1997
When compared with the sequences of soluble legume lectins of known structures, the four MtLecRK sequences appear to contain all conserved amino acids necessary for binding Ca2+ and Mn2+ (Fig. 1; Loris et al., 1998
Because MtLecRK1;1 is the only one of the four MtLecRK proteins that displays the required amino acids for binding carbohydrate, a three-dimensional model of the lectin-like domain of this protein was built. From the alignment with selected known three-dimensional structures (Fig. 3), a model of the region between amino acids 37 and 283 could be constructed, with the exception of the loop between Gly-221 and Gly-228 that corresponds to an insertion that is not present in known crystal structures. Because this loop is located at the opposite side to the monosaccharide-binding site, it should have no effect on the docking study, and no attempts to build it from ab initio were made. Because the lectin-like domain of MtLecRK1;1 is glycosylated in planta (see later), the seven putative N-glycosylated sites of the 37 to 283 domain were compared with the ones that have been proven to be occupied in related legume lectins (Fig. 3). N-glycosylation in legume lectins is common but, with the exception of one site, does not display a conserved scheme. The N-glycans are known to affect the oligomerization mode of some legume lectins, but not their carbohydrate binding. In the known structures, only the region close to amino acids 100 to 120 (Asn-113 in Maackia amurensis lectin) contains a partially conserved N-glycosylation site, which corresponds to Asn-144 in the modeled lectin-like domain of MtLecRK1;1. This loop is close to the monosaccharide-binding site, but, in comparison with known structures displaying glycosylation in this position, the N-glycan is predicted to be orientated such that it does not interact at this site. At present, it is difficult to predict if other sites are glycosylated and whether this would affect ligand binding.
The major Nod factor of S. meliloti, NodSm-IV (Ac, S, C16:2), was docked into the protein as follows. A 6-O-acetyl GlcNAc residue was docked into the binding site of MtLecRK1;1 by comparison with the binding of GlcNAc by Ulex europaeus isolectin II (UEA-2; Loris et al., 2000
Three additional GlcNAc residues were added to form a tetrameric Nod factor backbone while performing a systematic conformational search. Several conformations could be generated, but the one retained in the model is the one that presents most interactions between the oligosaccharide and the protein surface (Fig. 4, C and D). No additional hydrogen bonds are observed because all of the three residues interact through their hydrophobic face with a hydrophobic patch of amino acids at the protein surface (Leu-75, Phe-76, and Leu-259). The sulfate group does not make any direct contact with the protein, but it lies not far from a basic residue, Arg-77, that is specific to MtLecRK1;1 and, thus, can participate in electrostatic stabilization. The C16:2 lipid moiety of S. meliloti Nod factors was built into the model from the N-acetyl group of the first docked GlcNAc residue. Different conformations were tested, and the one that results in the best fit between the chain and the protein surface consists simply of an all trans-chain, with expected kinks at the two cis-linkages. In fact, the presence of these two cis-bridges is the factor that brings the chain exactly into a deep hydrophobic crevasse at the surface of the protein (Fig. 4D). The lipid makes most interaction with a strand of hydrophobic amino acids (Ile-131-Pro-Pro-133) that does not exist in the other lectins. Taken together, the model suggests that the lectin-like domain of MtLecRK1;1 may adopt a structure similar to soluble legume lectins and that the major S. meliloti Nod factor could be a possible ligand with its terminal reducing sugar inserted into the position of the putative monosaccharide-binding site.
As an initial approach to study the physiological role of the MtLecRKs, the expression patterns of the four MtLecRK genes were analyzed. This was done by northern analysis using probes to their lectin domains. In Southern analyses, the probes of MtLecRK1;1 and MtLecRK7;3 did not cross-hybridize with the other genes, but a low degree of cross-hybridization was observed between MtLecRK7;1 and 7;2. The abundance of mRNA related to the MtLecRK7;2, MtLecRK7;3, and MtLecRK1;1 probes was found to be higher in roots than in the other plant organs tested (nodules at different stages, leaves, stems, flowers, and shoot apices). Moreover, the mRNA levels of all three genes showed a clear increase in roots following nitrogen starvation (Fig. 5A). The three genes also appeared to be expressed in a cell culture line of M. varia, in which a high-affinity Nod factor-binding site (NFBS2) has been characterized. The MtLecRK7;3 gene seems to be expressed also in leaves, and mRNA related to both MtLecRK7;2 and MtLecRK7;3 could be detected in stems. MtLecRK1;1 seemed to show the highest organ-specific expression, being highly specific for roots but with detectable mRNA levels also in 8-d-old nodules and in stems. The probe to MtLecRK7;1 hybridized very weakly to the blots in Figure 5, suggesting that this gene is expressed only weakly in the plant. The MtLecRK1;1 and MtLecRK7;2 genes were selected for further detailed study based on their expression levels, their sequence divergence, and their different chromosomal locations.
To study the expression of the MtLecRK7;2 and MtLecRK1;1 proteins in M. truncatula, antisera were raised to the lectin-like domains of the two proteins. These proteins were expressed as glutathione-S-transferase fusions in Escherichia coli. The lectin fusion proteins were found to be largely insoluble when the bacteria were grown at room temperature, but by expressing the proteins at 4°C, enough soluble protein was obtained to immunize rabbits. The resulting antisera were tested in western blots with the E. coli-expressed proteins and were found to recognize their own protein but not to cross-react with the other lectin domain (data not shown). These antisera were used in western blots of crude and membrane extracts of M. truncatula roots, but no specific protein corresponding to the endogenous LecRKs could be identified (Fig. 6B). Because the antisera recognize the lectin-like domains when overexpressed in transgenic roots (see below), it seems that the endogenous proteins are of too low an abundance in the root tissues to be detected by western analysis. Therefore, further studies on the regulation of expression of MtLecRK1;1 and MtLecRK7;2 were carried out by northern analysis of their mRNAs.
Different plant-bacterial couples were used to look further at the regulation of MtLecRK7;2 and MtLecRK1;1 during the establishment of the legumerhizobia symbiosis (Fig. 5B). Gene expression during the essentially wild-type symbiosis between M. truncatula cv Jemalong A17 and S. meliloti was compared with expression during either a non-nodulating interaction (wild-type M. truncatula with S. meliloti nodA) or a hypernodulating interaction (between the M. truncatula ethylene-insensitive skl mutant [Penmetsa and Cook, 1997
To investigate the subcellular localization of the MtLecRK1;1 and 7;2 proteins, constructs of each gene were prepared with the GFP marker. Three constructs were prepared for each gene with the GFP tag fused as a C-terminal domain after either the predicted extracellular domain (constructs EX), the TM-spanning helix (constructs TM), or the kinase domain (constructs KIN). These protein fusions (Fig. 6A) were expressed from the CaMV 35S promoter in roots of M. truncatula after Agrobacterium rhizogenes transformation (Boisson-Dernier et al., 2001 By western analysis of the transgenic roots, the size of the EX and TM fusions seemed to be bigger than predicted from the sequences. To investigate this further, the 1;1-EX and 1;1-TM constructs were expressed in an E. coli in vitro transcription/translation system, and their sizes compared with the same protein constructs expressed in the M. truncatula roots (Fig. 6C). The two 1;1 fusion proteins appear to be about 14 kD bigger when expressed in the plant compared with the in vitro system. The 7;2 proteins seemed to be similar in size to the corresponding 1;1 fusion proteins. These results suggest that the lectin-like domains of both proteins are probably heavily glycosylated. The subcellular locations of the fusion proteins were investigated by confocal microscopy (Fig. 7). The 35S promoter gave a characteristically high expression in the vasculature, but particular attention was paid to the epidermis (Fig. 7A) and cortex (Fig. 7B), where the MtLecRKs are more likely to play a symbiotic role. Plasmolysis was used (Fig. 7C) to distinguish between fluorescence in the cytosol, PM, and wall at the periphery of the cells. The GFP-alone construct showed the typical nuclear and cytosolic localization previously shown for this protein. Both TM fusion proteins (1;1-TM and 7;2-TM) were found to be located primarily at the periphery of the cell. After plasmolysis, the GFP fluorescence was found to be located in the PM and not in the cell wall. With the two EX fusions, the GFP fluorescence showed a complex localization within the cell, with the 1;1-EX protein seeming to be mainly located in the endoplasmic reticulum (ER) and the 7;2-EX fusion in the vacuole and cytosol (Fig. 7).
Because the TM fusions were well expressed with the fusion proteins appearing to be glycosylated and targeted to the PM (Figs. 6 and 7), these transgenic hairy roots lines were used in experiments to address the function of the MtLecRK1;1 and MtLecRK7;2 proteins. First, these lines were used to examine whether Nod factors bind specifically to the EX + TM domains of the proteins. A GFP-expressing line was used as a control for either endogenous or nonspecific binding. Cell-free extracts of the roots were prepared and centrifuged sequentially at 5,000g and then 45,000g to obtain different membrane fractions. As expected for PM proteins, the fusion proteins were observed by western analysis in the 45,000g fraction (Fig. 8A). This fraction, from the three extracts, was used in binding experiments with a radio-labeled ligand corresponding to the major Nod factor of S. meliloti (NodSm-IV, Ac, 35S, C16:2). The 45,000g fraction of a M. varia cell culture line, in which NFBS2 has been characterized previously (Gressent et al., 1999
In the second approach, the effect of overexpression of the TM constructs on nodulation was examined. The GFP construct was again used as a control. Chimeric plants with transgenic roots of the three lines were transferred to growth pouches, inoculated with S. meliloti, and nodule number was counted at various times. A pilot experiment and two larger scale experiments were performed. Figure 9 shows the results of the second larger scale experiments for the GFP and 1;1-TM constructs. Although there was considerable variation between the numbers of nodules per plant, the 1;1-TM construct led to an increase in the mean number of nodules per plant compared with the GFP control, starting by 7 d after inoculation. By 14 d, the increase compared with the control was 2.8- and 1.9-fold in the two larger scale experiments. Statistical analysis using the Student's t test showed that the differences from the control were highly significant with P values of less than 0.005 for all the time points in the first experiment and with P values generally of less than 0.005 for the second experiment (Fig. 9). The 7;2-TM construct led to an increase in nodule number in one of the experiments, but no difference was found in the second experiment using the same statistical criteria. For each construct, nodules from several transgenic lines were stained with methylene blue and observed by microscopy. No differences could be observed in nodule structure or infection of the LecRK-expressing roots compared with those expressing the GFP alone.
In this paper, we have tested the hypothesis (Hervé et al., 1996 ; Hirsch, 1999) that LecRKs may play a role in the legume-rhizobia symbiosis. Screening of cDNA libraries and examination of M. truncatula EST banks has shown that the model legume contains at least nine LecRK genes. The four genes studied here (MtLecRK1;1, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3) are the most highly expressed LecRK genes in roots (as judged by their clone abundance in root and nodule cDNA libraries) and, hence, are the most likely LecRK genes to play a role in the symbiosis. Although their mRNAs could be easily monitored by northern analysis, antisera raised against the lectin-like domains of MtLecRK1;1 and MtLecRK7;2 failed to detect their endogenous proteins by western analysis, suggesting that these receptors are low-abundance proteins.
The four genes encode proteins with a classic LecRK structure, with an N-terminal signal peptide, a lectin-like domain, a single TM-spanning helix, and a kinase domain (Fig. 1). They are most likely PM proteins as fusions in which the kinase domains of MtLecRK7;2 and MtLecRK1;1 were replaced by GFP localized to the PM in transgenic roots (Fig. 7). For this localization, the internal TM helix is required because without it, the MtLecRK1;1 fusion became stuck in the ER, and the MtLecRK7;2 fusion located mainly to the vacuole. These localizations are surprising because the default destination of soluble proteins in the secretory pathway is secretion (Barrieu and Chrispeels, 1999
The above localizations suggest that the TM helices of the two LecRKs are required not only to anchor the LecRK proteins in a membrane but also as a positive sorting signal for the PM. In studies in yeast (see Rayner and Pelham, 1997
The lectin-like domains of MtLecRK1;1 and MtLecRK7;2 appear to be heavily glycosylated because the sizes of the EX and TM GFP fusion proteins were about 14 kD greater than the proteins expressed in an E. coli-based in vitro protein production system (Fig. 6). The size of the Ath.LecRK-a1 protein in PM extracts of Arabidopsis suggests that this protein is also highly glycosylated (Hervé et al., 1999
The four MtLecRKs are expected to be active in protein phosphorylation because their kinase domains show a high conservation of the sub-domains, residues, and motifs required for Ser/Thr protein kinase activity (Fig. 1). To date, the kinase domains of only two LecRKs have been tested for functionality (Ath.LecRK-a1 and PnLPK), and both have been shown to be active in autophosphorylation (Hervé et al., 1996
The lectin-like domains of the four MtLecRKs were found be very divergent; hence, clones of these different genes probably would not have been isolated using probes from the lectin-like domain of Ath.lecRK-a1. Thus, the isolation of such divergent MtLecRK genes relied on the strategy of using the kinase domain as a probe. The success of this strategy in isolating mainly LecRK genes, coupled with subsequent sequence analysis, suggest that the kinase domain attached to the variant lectin-like domains have a similar phylogenetic origin, in agreement with studies by Shiu and Bleecker (2001
An obvious question on LecRKs is whether the lectin-like domains are true lectins and bind carbohydrates, perhaps as ligands, to activate the kinase domain. Careful analysis of this domain revealed that the residues shown to be involved in sugar binding in soluble legume lectins are more highly conserved in MtLecRK1;1 (three of four) than any of the other MtLecRKs so far identified. Moreover, of the 42 AtLecRKs, the conservation of the D, G, F/Y, and N residues are shown in two, six, four, and 11 proteins, respectively, with only two proteins showing conservation of three of these residues (Barre et al., 2002 Due to the conservation of key sugar-binding residues in MtLecRK1;1, molecular modeling was performed on this protein using the known structures of soluble legume lectins (Figs. 3 and 4). Because Nod factors are a key signaling molecule in root symbioses and the principle target of our research, the terminal non-reducing sugar of the major Nod factor of S. meliloti was docked into the monosaccharide-binding site, and the Nod factor was fitted into the protein. The results suggest that the two Nod factor substitutions on the terminal non-reducing sugar (the O-acetate on C6 and the C16:2 fatty acyl chain on C2) could fit nicely into cavities on the MtLecRK1;1 protein with the other sugars extending to the surface of the protein. The number of predicted hydrogen bonds between the protein and the Nod factor ligand is low (only two); however, it should be noted that sugars are amphiphilic molecules, and evidence suggests that contact between the hydrophobic face of the glycan and hydrophobic patches on the protein surface are of primary importance for both the affinity and the specificity of binding.
Such an interaction between Nod factors and the lectin-like domains would not be predicted for the other three MtLecRK proteins or for soluble legume lectins. Thus, the structural model predicts that MtLecRK1;1 could be a specific receptor for Nod factors. However, when this hypothesis was tested, no increase in Nod factor-specific binding was found in root extracts overexpressing the MtLecRK1;1-TM fusion in comparison with roots expressing either a similar construction with MtLecRK7;2 or GFP alone (Fig. 8). Thus, no evidence has been obtained that these proteins do bind Nod factors. However, the technique used to measure binding is designed for high-affinity interactions and, thus, we cannot exclude the possibility that Nod factors do bind to this protein but with a dissociation constant (KD) in the micromolar to millimolar rather than the nanomolar range. Moreover, ligand binding could require the activity of the kinase domain, although this is not a general requirement for ligand binding to RLKs (Cock et al., 2002
The lack of many transformants with the KIN constructs and the poor fusion protein expression observed in the few transgenic roots that grew could suggest that overexpression of the LecRK genes is detrimental to root development. Riou et al. (2002
The high expression of the four MtLecRK genes in roots compared with other organs (Fig. 5) suggests roles for these genes predominantly in this organ. Thus, they are predicted to have a different role than two LecRK genes studied in Arabidopsis that are expressed predominantly in aerial tissues (Hervé et al., 1996 Further evidence for such a role for MtLecRK1;1 was obtained by studies of nodulation in roots expressing the TM constructs. Western analysis suggests that the MtLecRK1;1 and MtLecRK7;2 TM constructs were expressed at least 100-fold higher than the endogenous proteins (Fig. 8), and good expression was seen in the epidermal cells that are in direct contact with symbiotic bacteria (Fig. 7). For both constructs, plants produced normal looking nodules that were infected with rhizobia. Thus, expression of these catalytically defective proteins does not interfere with either nodule development or infection. However, roots expressing the MtLecRK1;1-TM construct developed a greater number of nodules than roots expressing the GFP construct alone (Fig. 9).
One explanation for this result could be that the endogenous MtLecRK1;1 gene plays a negative role in controlling nodule number and that overexpressing a catalytically inactive protein acts as a dominant negative mutation inhibiting the normal functioning of the endogenous gene (perhaps by soaking up the normal ligands). Nodule number is known to be controlled by an autoregulatory response involving a shoot-effective gene that controls the number of root and nodule meristems (Caetana-Anolles and Gresshoff, 1991
A second explanation for the increase in nodule number is that overexpression of the MtLecRK1; 1-TM construct could have a positive effect on the establishment of the symbiosis, which is independent of the kinase domain and is perhaps due to the lectin activity of the protein. MtLecRK1;1 would be predicted to have a greater effect than MtLecRK7;2 because it is predicted to be a better lectin. In a manner similar to that proposed for overexpression of soluble lectins (Hirsch, 1999 Clearly, further experimentation is required to understand the potential and rather enigmatic role of the MtLecRK1;1 gene in the control of nodule number. Although the rapid hairy root system has proven invaluable as an expression system to study the structure and location of these proteins, a more detailed functional study of this gene may require stably transformed plant lines in which the plant-to-plant variability is considerably less than in the independently transformed roots of the hairy root system. Moreover, although ectopic overexpression of functional or nonfunctional proteins can lead to indications of the physiological role of a protein, this strategy suffers from the problem that the overexpressed protein may function in a way different to its endogenous homolog. Thus, such studies need to be complemented by strategies designed to inhibit functioning of the endogenous gene either by isolating specific gene mutants or by using antisense or RNA interference strategies. In conclusion, we have shown that M. truncatula contains a family of at least nine LecRK genes. The four genes studied here are the most highly expressed MtLecRK genes in roots. Three of them encode proteins related to the Class A Arabidopsis LecRKs, whereas the fourth gene (MtLecRK1;1) encodes a proteins that is more closely related to a Class B LecRK. The considerable divergence in the sequence of their lectin-like domains suggests that these putative receptors may perceive different ligands and perform different physiological functions. The structure of the MtLecRK1;1 lectin-like domain suggests that this protein is a good candidate for interacting with saccharidic ligands, and studies with transgenic roots suggest that its expression may influence the regulation of nodulation, although probably not through Nod factor binding. Clearly, the identification of the ligands for these proteins, coupled with studies of stably transformed transgenic plants modified in their expression, may help to determine the physiological role of these intriguing receptors in both symbiotic and non-symbiotic conditions.
Growth Conditions and Plant Material
All plant lines and bacterial strains are listed in Table IV of Navarro-Gochicoa et al. (2003
For the experiments described in Figures 6 and 9, composite plants with transgenic roots (Boisson-Dernier et al., 2001
About 980,000 clones were screened essentially as described by Gamas et al. (1996
MtLecRK ESTs were identified by using the four full-length MtLecRK cDNAs in BLAST analyses with the GenBank EST database (Altschul et al., 1997
For the alignment shown in Figure 1 and for the phylogenetic analysis in Figure 2, ClustalX (Thompson et al. 1997
The N-terminal 300-amino acid sequence of MtLecRK1;1 was aligned with the sequences of 15 legume lectins selected among the ones with known structures that are available in the Protein Data Bank (Berman et al., 2000
A GlcNAc residue was docked in the primary binding site with the same location and orientation as in the crystalline complex between UEA-II and GlcNAc (Loris et al., 2000
The techniques for isolating and determining the abundance of specific RNAs by northern analysis are described by Navarro-Gochicoa et al. (2003
The glutathione-S-transferase gene fusion system was used for MtLecRK1;1 and MtLecRK7;2 lectin-like domain purification (Amersham Biosciences, Buckinghamshire, UK). The lectin-like domains corresponding to amino acids 35 to 293 and 19 to 254 for MtLecRK1;1 and MtLecRK7;2, respectively, were cloned into the pGEX 6P-1 plasmid in Escherichia coli strain BL21 DE3. The bacteria were grown at 20°C and induced at 4°C using 0.1 mM isopropylthio-
Frozen plant material and E. coli samples were crushed in a mortar and pestle and extracted by boiling in a 2x concentrated SDS-PAGE loading buffer. After SDS-PAGE on 10% (w/v) acrylamide gels, the proteins were transferred into Hybond-P membrane (Amersham Biosciences) in Laemmli buffer containing 10% (v/v) methanol and 0.02% (w/v) SDS. The protein marker used was a broad range prestained protein marker (New England Biolabs, Beverly, MA). The membrane was pre-incubated in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TTBS) and 5% (w/v) nonfat dried milk. The incubation steps with the appropriate antibody dilutions were performed in TTBS buffer with 1% (w/v) nonfat dry milk and the washing steps in TTBS buffer. The secondary antibody used was an anti-rabbit IgG:horseradish peroxidase-linked whole antibody from donkey, and the western blotting detection was by chemiluminescence (using the ECL Chemiluminescent kit, Amersham Biosciences). In some experiments, membranes were stripped using 100 mM 2-mercaptoethanol, 2% (w/v) SDS, and 62.5 mM Tris-HCl (pH 6.8) before reuse.
Constructs were prepared in the binary vector pGreen0029 that confers resistance to kanamycin in the transformed plants (Hellens et al., 2000 The MtLecRK1;1 EX- and TM-GFP-fusions were also cloned into vector pIVEX2.3 and expressed from the T7 promoter in the in vitro cell-free protein expression system, termed the Rapid Translation System, of Roche Applied Science (Mannheim, Germany). After expression, the reaction mixture was used directly for western analysis.
Transformed roots growing on agar were covered with a gas-permeable plastic foil (bioFolie 25, Sartorius AG, Vivascience Support Center, Göttingen, Germany) and observed with a TCS SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany) using an HCX Plan Apo 63x 1.2-W objective. Optical sections were made with a separating distance of 1 µm between subsequent sections. Image projections were made with the Leica confocal software, and these were processed with Image-Pro plus (Media Cybernetics, Silver Spring, MD). To distinguish between labeling of the PM and the cell wall, root epidermal cells were plasmolyzed by incubation in a 10% (w/v) KNO3 solution.
Transgenic root material from in vitro hairy root cultures was extracted as described by Gressent et al. (1999
A. rhizogenes-transformed composite plants were transferred to growth pouches for 3 to 4 d using media lacking a nitrogen source before inoculation with S. meliloti, essentially as described by Vernoud et al. (1999
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
Map-based cloning of M. truncatula and Lotus japonicus genes involved in Nod factor responses has revealed RLKs with LysM domains as putative Nod factor receptors (Limpens E., Franken C, Smit P, Willemse J, Bisseling T, Geurts R [2003] LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 302: 630-633; Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Gronlund M, Sato S, Nakamura Y, Tabata S. Sandal N, Stougaard J (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425: 585-592).
We thank Brigitte Mangin (Institut National de la Recherche Agronomique, Toulouse, France) for help with statistical analysis and Prof. Douglas Cook (University of California, Davis) for sending us the M. truncatula skl mutant. Received June 2, 2003; returned for revision July 30, 2003; accepted September 15, 2003.
1 This work was supported by the European Community's Framework 5 Human Potential Program (Marie Curie Fellowship to M.-T.N.G., contract no. HPMF-CT-1999-00073, and Research Training Network on Oligosaccharide Signaling in Plants, contract no. HPRN-CT-2002-00251 and by the Région Midi-Pyrénées, France. 
2 Present address: Departamento Ciencias Ambientales, Área de Fisiología Vegetal, Facultad de Ciencias Experimentales, Universidad Pablo de Olavide, 41013 Sevilla, Spain. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.027680. * Corresponding author; e-mail cullimor{at}toulouse.inra.fr; fax 33-5-61-28-50-61.
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ABSTRACT
RESULTS
-sheets (Loris et al., 1998
5' untranslated region were cloned from pBE113-GUS into the HindIII site of the vector, and the CaMV terminator region was cloned into the StuI site near the right border. In this way, a plasmid was created (termed pGr29-35SOMCaMVT) that contains sites for XbaI, BamHI, SmaI, SacI, and EcoRI for gene expression from the 35S promoter. The enhanced GFP sequence from plasmid pEGFP-N2 (CLONTECH Laboratories, Palo Alto, CA) was cloned into the SmaI and SacI sites to create plasmid pGr29-35SOM-EGFP-CaMVT that contains sites for XbaI, BamHI, SmaI, BamHI, and NcoI upstream of the GFP. Using specific primers and PCR, XbaI and NcoI sites were created at the start of the cDNA of MtLecRK1;1 so that the NcoI site encodes the start codon and an SmaI site was introduced either at amino acid 297 (1;1-EX construct), 342 (1;1-TM construct), and 678 (1;1-KIN construct). A similar strategy was used for MtLecRK7;2 except that a BamHI site was created just before the start codon and the SmaI sites were at positions 278 (7;2-EX construct), 326 (7;2-TM construct), and 669 (7;2-KIN construct). The sequence changes were verified by DNA sequencing, and the LecRK-containing fragments were then cloned just upstream of the GFP in pGr29-35SOM-EGFP-CaMVT to produce in-frame LecRK-GFP fusions. The construct-containing plasmids were cotransformed into Agrobacterium rhizogenes ARqua1 (Quandt et al., 1993
