Biochemical and phylogenetic analysis of CEBiP-like LysM domain-containing extracellular proteins in higher plants

Plant Physiology and Biochemistry 49 (2011) 709e720

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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

Biochemical and phylogenetic analysis of CEBiP-like LysM domain-containing extracellular proteins in higher plants Judith Fliegmann a, b,1, *, Sandra Uhlenbroich a, b,1, Tomonori Shinya c, Yves Martinez d, Benoit Lefebvre e, f, Naoto Shibuya c, Jean-Jacques Bono a, b, e, f a

Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales (LRSV), BP 42617, 31326 Castanet-Tolosan, France CNRS, UMR 5546, BP 42617, 31326 Castanet-Tolosan, France Department of Life Science, Faculty of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan d IFR40 CNRS, Pôle de Biotechnologie Végétale, BP42617, 31326 Castanet-Tolosan, France e INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, 31326 Castanet-Tolosan, France f CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, 31326 Castanet-Tolosan, France b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 February 2011 Accepted 4 April 2011 Available online 9 April 2011

The chitin elicitor-binding protein (CEBiP) from rice was the first plant lysin motif (LysM) protein for which the biological and biochemical function had been established. It belongs to a plant-specific family of extracellular LysM proteins (LYMs) for which we analyzed the phylogeny. LYMs are present in vascular plants only, where an early gene duplication event might have resulted in two types which were retained in present day genomes. LYMs consist of a signal peptide, three consecutive LysMs, separated by cysteine pairs, and a C-terminal region without any known signature, whose length allows the distinction between the two types, and which may be followed by a glycosylphosphatidylinositol (GPI) anchor motif. We analyzed a representative of each type, MtLYM1 and MtLYM2, from Medicago truncatula at the biochemical level and with respect to their expression patterns and observed some similarities but also marked differences. MtLYM1 and MtLYM2 proved to be very different with regard to abundance and apparent molecular mass on SDS-PAGE. Both undergo several post-translational modifications, including N-glycosylation and the addition of a GPI anchor, which would position the proteins at the outer face of the plasma membrane. Only MtLYM2, but not MtLYM1, showed specific binding to biotinylated N-acetylchitooctaose in a manner similar to CEBiP, which belongs to the same type. We postulate that LYM2type proteins likely function in the perception of chitin-related molecules, whereas possible functions of LYM1-type proteins remain to be elucidated. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: CEBiP Chitooligosaccharide GPI LYM LysM Medicago truncatula

1. Introduction

Abbreviations: @ATPase, anti-P-type Hþ-ATPase antiserum; @biotin, anti-biotin antiserum; @LYM1, anti-MtLYM1 antiserum; @LYM2, anti-MtLYM2 antiserum; CEBiP, chitin elicitor-binding protein; EGS, ethylene glycol bis(succinimidylsuccinate); Endo H, endo-b-N-acetylglucosaminidase H; GlcNAc, N-acetylglucosamine; GPI, glycosylphosphatidylinositol; GUS, b-glucuronidase; HRP, horseradish peroxidase; LysM, lysin motif; Nod factor, nodulation factor; PAMP, pathogen-associated molecular pattern; PI-PLC, phosphatidylinositol-specific phospholipase C; RLK, receptor-like kinase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales (LRSV), BP 42617, 31326 Castanet-Tolosan, France. Tel.: þ33 534 323823; fax: þ33 534 323802. E-mail addresses: fl[email protected] (J. Fliegmann), uhlenbroich@lrsv. ups-tlse.fr (S. Uhlenbroich), [email protected] (T. Shinya), yves.martinez@lrsv. ups-tlse.fr (Y. Martinez), [email protected] (B. Lefebvre), shibuya@ isc.meiji.ac.jp (N. Shibuya), [email protected] (J.-J. Bono). 1 These two authors contributed equally to this work. 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.04.004

The lysin motif (LysM) was originally described as a small protein domain, often present in repeats in lytic enzymes from bacteria and bacteriophages, and was suspected to be responsible for the binding of the enzymes to the cell wall substrates [1]. Since then, LysMs were found in a variety of bacterial cell wall-degrading enzymes, as well as in proteins from eukaryotes, but they seem to be absent in Archeae [2e4]. The LysM contains typically 45 to 65 amino acid residues and folds into a symmetrical baab secondary structure, in which the two helices pack onto the same side of the two-stranded antiparallel b-sheet, as shown for a LysM of the Escherichia coli lytic murein transglycosylase D (MltD) [5]. LysM domains from bacteria have been shown to bind to different types of peptidoglycan, which all share a common backbone of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid units [6,7], whereas fungal and plant-derived LysM domains were

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shown to interact with chitin and chitooligomers, which are linear polymers of GlcNAc [8e11]. LysM proteins can contain as many as 12 copies of the motif but proteins with a single motif are most common and present in all kingdoms, except Archae [4,12]. Single or multiple LysMs can be associated to other protein domains, with some architectures being widely spread whereas others are restricted to one kingdom only [12]. Plants contain at least three specific domain architectures, which are not found in other kingdoms: LysM receptor-like kinases (LysM-RLKs), where one to three extracellular LysMs are connected via a transmembrane domain to an intracellular kinase domain, F-box LysM proteins, where an F-box domain precedes a single LysM, and LYMs or LYPs, which are composed of multiple extracellular LysMs [2,12]. F-box LysM proteins are encoded by highly conserved single genes (except for poplar and soybean) but a possible function has not yet been established. In contrast, LysM-RLKs are present in gene families comprising up to 21 members [12]. In legume plants, some of the LysM-RLKs are involved in the perception of bacterial symbionts during the establishment of the nitrogen-fixing root nodule symbiosis [13e16]. Most rhizobial bacteria secrete lipoe chitooligosaccharidic nodulation (Nod) factors, which are necessary for the recognition, nodulation, and controlled infection of compatible host legumes [17]. Direct binding of these Nod factors to LysM-RLKs is not yet proven, but genetic analysis and modeling approaches suggest strongly the interaction between extracellular LysMs and the bacterial signaling molecule [18,19]. In non-legumes, a LysM-RLK (CERK1) is involved in the perception of pathogenic organisms by recognizing chitin, a major fungal-derived pathogenassociated molecular pattern (PAMP), thereby activating the defense machinery [9,20,21]. The archetype of the LYMs, the chitin-elicitorbinding protein CEBiP, has been purified due to its intrinsic ability to bind to chitooligomers and was shown to be necessary for chitininduced defense reactions in rice [21,22]. This LysM protein does not contain an intracellular kinase domain but appears to be attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor [22]. We set out to analyze this family of proteins in more detail by, firstly, better defining the members sharing a similar domain structure and by, secondly, characterizing the corresponding proteins from one plant species, Medicago truncatula.

2. Results 2.1. Phylogenetic analysis CEBiP is a LysM-domain containing glycoprotein which was isolated from rice plasma membranes by affinity chromatography on a N-acetylchitooctaose (GlcNAc8) matrix [22]. To generate an overview of the distribution of CEBiP-like sequences in plants, we retrieved similar sequences by TBLASTN search of 16 fully sequenced plant genomes, and combined these with accessions described as LYPs [12]. We then restricted the phylogenetic analysis to proteins of identical domain architecture, which consists of a signal peptide and multiple LysMs followed by a substantial C-terminal domain, named LYMs [15,23]. A list of 53 LYMs was obtained which were classified and renamed according to their structure (Table S1). According to the designation of LYMs from M. truncatula and Arabidopsis thaliana [15,23], the predicted proteins were named LYM1/LYM3 (C-terminal tail of approximately 200 residues, corresponding to LYPs of clade I in Zhang et al. [12]) or LYM2/LYM4 (C-terminal tail of approximately 150 residues, corresponding to clade III LYPs). LYMs were further subdivided with respect to the prediction of the attachment of a GPI anchor: LYM1 and LYM2 refer to LYM proteins with predicted GPI anchor, while LYM3 and LYM4 refer to proteins without GPI (see Table S1). LYMs were not detected in algae or mosses and appeared first in Selaginella moellendorfii, a primitive vascular plant. Most of the annotated plant genomes from vascular plants contain at least two LYM proteins, with the exception of Mimulus guttatus for which only one could be found in the present release (JGI release v1.0). In all other genomes, at least one LYM1/3-like and one LYM2/4-like protein were found. Alignment of the amino acid sequences enabled the prediction of three putative LysMs for each protein, separated by cysteine pairs (Fig. S1), as in LysM-RLKs. The consensus sequence of each of the three predicted motifs shows the conservation of specific positions, such as Tyr at the first position and Asn in the central region (Fig. 1), which were reported to be indicative for LysMs [3]. Phylogenetic analysis of the full length proteins showed a clear separation of LYM1/3 (clade I in [12]) versus LYM2/4 (clade III) (Fig. 2). Interestingly, LYM1/3-type proteins showed a higher

Fig. 1. Graphical representation of the consensus sequence of each of the three LysMs of LYMs. Numbering of each motif corresponds to the respective positions in the multiple protein alignment of 47 LYMs (see Table S1 for the accessions, and Fig. S1 for the alignment). Each position in the sequence logos is represented by a stack, and the overall height of the stack indicates the sequence conservation. The height of symbols within the stack indicates the relative frequency of each amino acid at that position. (A) first LysM; (B) second LysM; (C) third LysM.

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is present in MtLYM2 within the region encoding the three LysMs, unlike other genes encoding LysM-containing proteins in M. truncatula [15] and unlike all other LYM genes analyzed up to now (data not shown). The gene expression atlas of M. truncatula was used to obtain an overview of the expression of the two LYM genes in different organs [24]. MtLYM1 (probeset ID: Mtr.9585.1.S1_at) was equally expressed in the major organs, except for developing seeds, which showed the highest transcript levels. The expression level for MtLYM2 (probeset ID: Mtr.12383.1.S1_at) was generally higher in comparison to MtLYM1, and more variable between organs or treatments. With respect to the major organs, MtLYM2 transcripts were most abundant in roots. We used the Agrobacterium rhizogenes transformation system to obtain more detailed information about the expression pattern of both genes in M. truncatula roots. The promoter of MtLYM1 and MtLYM2 were cloned upstream of the introncontaining b-glucuronidase reporter gene. Histochemical staining demonstrated promoter activity for proMtLYM1::GUS-int and proMtLYM2::GUS-int mainly in the younger parts of roots. Strongest promoter activity was observed in root tips and in emerging lateral roots (Fig. 4). MtLYM1 and 2 presented opposing expression patterns in the root tip: MtLYM1 was expressed in the apical meristem, whereas MtLYM2 was weak in this region but expressed more strongly in the root cap (compare Fig. 4A/B and D/E). Both genes further differed by the unique activity of proMtLYM2 in the rhizodermis and root hairs (Fig. 4E,F). Both promoters were active in emerging lateral roots (Fig. 4C,F). To further corroborate the expression pattern of both genes we generated specific antisera against both proteins and performed Western blotting experiments (see section 2.3 for details) on crude protein extracts derived from roots, stems, flowers, and leaves. MtLYM2 was most abundant in roots, as reported in the gene expression atlas, followed by stems, and not detectable in flowers and leaves (Fig. 5A). In these crude protein extracts, MtLYM1 was not detectable at all (data not shown, see section 2.3 for details). 2.3. Biochemical characterization of MtLYM1 and MtLYM2

Fig. 2. Phylogenetic analysis of LYM proteins. The tree was generated using the web service Phylogeny.fr and rooted with LYM1 from Selaginella moellendorfii (SmLYM1). Branches are labeled with their respective bootstrap values.

overall sequence conservation in comparison to LYM2/4-type proteins. Just one member for each LYM-type was found for the model legume M. truncatula, which were characterized in detail. 2.2. Characterization and expression of the LYM genes in M. truncatula The genes encoding MtLYM1 and MtLYM2 contain four and three introns respectively, from which only one intron position is conserved between both genes (Fig. 3). Interestingly, a small intron

MtLYM1 and MtLYM2 encode preproteins of 412 and 372 amino acid residues, respectively, including predicted signal peptides of 21 and 30 residues (Fig. 3, Table S1). Both proteins furthermore contain a plant GPI lipid anchor motif, which predicts the attachment of a GPI anchor after proteolytic cleavage near the C-terminus, reducing the amino acid sequence of each protein by a further 29 residues. The molecular mass, deduced for the processed proteins thus amounts to 37.9 kDa (MtLYM1, 362 residues) and 33.6 kDa (MtLYM2, 313 residues). Specific antisera were generated against recombinant versions of MtLYM1 and MtLYM2, produced in E. coli, and used to detect the proteins by Western blotting. In crude extracts of roots, immunolabeling after Western blotting using the anti-MtLYM2 antiserum detected a major protein with an apparent molecular mass of 70 kDa instead of the predicted molecular mass of 34 kDa (Fig. 5A). Such a difference in size was already shown for CEBiP, which exhibited an apparent molecular mass of 75 kDa, instead of 34 kDa, due to glycosylation [22]. To compare the characteristics of MtLYM1 or MtLYM2, both proteins were expressed transiently in leaves of Nicotiana benthamiana, which is a rapid and simple expression system for the study of recombinant proteins. Fractionation of transformed leaves and of roots of M. truncatula was performed by differential centrifugation [25] and the resulting subfractions were analyzed by Western blotting (Fig. 6). In N. benthamiana leaves, the anti-MtLYM1 antiserum detected a major protein species with an apparent molecular mass of approximately 95 kDa in the microsomal fraction

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Fig. 3. Gene and protein structures of MtLYM1 and MtLYM2 from Medicago truncatula. Genes (deduced from the gene model Medtr3g097500.1 for MtLYM1 and from region 6955 to 8575 of the accession AC235671.1 for MtLYM2) are represented by black boxes (protein coding exons), black lines (introns) and gray lines (50 and 30 NTR). The protein structures of LYMs include signal peptides and C-terminal transmembrane domains (yellow boxes), three LysMs (gray boxes), separated by regions containing pairs of conserved cysteine residues (red lines, two other conserved cysteines directly before and after the motifs are indicated by hatched red lines), and a variable C-terminal region. A conserved site for the attachment of a glycosylphosphatidylinositol anchor in MtLYM1 and MtLYM2 is marked by dots. The number of residues for each domain for MtLYM1 and MtLYM2 are given below and above the protein structures, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(45 k) and a minor species of approximately 55 kDa (Fig. 6A). The microsomal fraction of M. truncatula roots showed traces of a polypeptide cross-reacting with the anti-MtLYM1 antiserum in the high molecular mass range, but no signal at 55 or 95 kDa was observed (Fig. 6B). Most probably, MtLYM1 corresponded to the high molecular weight species, migrating as a 210 kDa protein in M. truncatula, as documented by the side-by-side comparison of microsomal protein preparations derived from M. truncatula and N. benthamiana (Fig. 6C). MtLYM2 was detected with an apparent molecular weight of 65 kDa in N. benthamiana (Fig. 6D), in comparison to 70 kDa in M. truncatula (Figs. 5A and 6E). With both antisera, no cross-reacting proteins were detectable in N. benthamiana leaves infiltrated with Agrobacteria transformed with the empty vector (Fig. 6A,D, Nb control). Endogenous, as well as recombinant MtLYM1 and MtLYM2 were highly enriched in the microsomal fractions (45 k) of M. truncatula roots and N. benthamiana leaves, respectively (Fig. 6AeE). Phase partitioning of microsomes from M. truncatula was used to further

specify the subcellular localization of MtLYM2, but not of MtLYM1 due to the low abundance of this protein in its native condition. As shown in Fig. 6G, MtLYM2 proved to be enriched in the upper phase containing plasma membranes, as confirmed by the detection of the plasma membrane marker Hþ-ATPase in this fraction (Fig. 6H). The difference between the predicted molecular mass and the migration behavior of both, MtLYM1 and MtLYM2, on SDS-PAGE suggested post-translational modifications for both proteins. In addition to the possible attachment of a lipid anchor, N-glycosylation sites were predicted, 5 for MtLYM1 and 10 for MtLYM2 (NetNGlyc 1.0). Carbohydrate decoration was already shown for two LysM domain-containing plant proteins; CEBiP from rice and NFP, a LysM-RLK from M. truncatula [18,22]. We took advantage of the heterologous expression of MtLYM1 and MtLYM2 in N. benthamiana and in the yeast Saccharomyces cerevisiae to determine the nature and extent of a possible glycosylation (Fig. 7). The results show that MtLYM1 and MtLYM2, expressed in planta and in yeast, have different apparent molecular weights and show differential

Fig. 4. GUS activity in roots of plants transformed with the MtLYM1 and MtLYM2 promoter-GUS fusions. Roots from composite plants transformed with either proMtLYM1::GUS-int (AeC) or proMtLYM2::GUS-int (DeF) were stained for GUS activity with Magenta-Gluc (in pink; A,D) or X-Gluc (in blue; B,C,E,F). Both promoters were active in young roots, root tips and emerging lateral roots. MtLYM1 promoter activity in root tips was strongest in the apical meristem (A), a zone in which MtLYM2 was poorly expressed in comparison to a stronger expression in the root cap (D). Only proMtLYM2::GUS-int was active in the rhizodermis and root hairs (E,F).

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appeared (Fig. 8C), further confirming the predicted plasma membrane attachment of this protein via a glycolipid anchor. Due to the low abundance in M. truncatula, this experiment was not feasible for MtLYM1. 2.4. Chitooligosaccharide binding

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Fig. 5. Expression of MtLYM2 in different organs of M. truncatula. Total protein extracts (90 mg) from roots, stems, flowers, and leaves from M. truncatula were separated in duplicate by 10% SDS-PAGE, and either transferred onto nitrocellulose and incubated with anti-MtLYM2 antiserum (@LYM2, A), or stained with Coomassie to verify equal loading of the protein samples (B). An MtLYM2 polypeptide of approximately 70 kDa was recognized in protein extracts from roots and stems, but was not detectable in flowers or leaves. The positions of co-migrating standard molecular mass markers are indicated on the left.

sensitivity to endoglycosidase H (Endo H). MtLYM1, expressed in planta, exhibited a molecular weight of around 210 kDa (in M. truncatula roots; Fig. 6B,C) or 95 kDa (in N. benthamiana leaves; Figs. 6A,C and 7A), whereas a doublet of 67 and 62 kDa was detected by Western blotting of crude extracts prepared from yeast cells expressing MtLYM1 (Fig. 7C). MtLYM2 did not show this big disparity in the different systems, with 70 kDa in M. truncatula (Figs. 5A and 6E,G), 65 kDa in N. benthamiana (Figs. 6D and 7B), and 69 kDa in yeast (Fig. 7D). Treatment with Endo H did not decrease the apparent molecular mass of MtLYM1 and MtLYM2, expressed in N. benthamiana (Fig. 7A,B). However, the mobilities of the yeastexpressed proteins shifted after incubation with Endo H: MtLYM1, which migrated as a doublet of approximately 67 and 62 kDa in the mock-treated sample, showed an apparent molecular weight of 54 kDa after deglycosylation with Endo H (Fig. 7C). Yeast-expressed MtLYM2 shifted from 69 kDa to 47/45 kDa after deglycosylation (Fig. 7D). The shift of approximately 10 kDa (Sc:MtLYM1) and 20 kDa (Sc:MtLYM2) by deglycosylation is consistent with the prediction of 5 and 10 N-glycosylation sites in MtLYM1 and MtLYM2, respectively, presuming an average molecular mass of 2 kDa for each glycan chain. Endo H is only active on high-mannose type N-glycans and not on complex ones, which are a-(1,3)-fucosylated on the proximal core residue, a modification which is characteristic for plants. Therefore, both LYMs seem to be preferentially glycosylated with high-mannose type N-glycans when they are expressed in yeast and they seem to carry complex-type glycans resistant to Endo H in planta. However, the treatment of the yeast-expressed proteins with Endo H did not result in the release of protein species showing the calculated molecular weights corresponding to the processed, N-glycan-free polypeptides, suggesting further modifications in both cases. Both M. truncatula LYMs were predicted to be GPI-anchored in the plasma membrane (Table S1). Triton X-114 detergent phase partitioning as well as the release from the GPI anchor by phosphatidylinositol-specific phospholipase C (PI-PLC) can be used to verify the prediction of a GPI anchor motif [23]. MtLYM1 or MtLYM2, expressed in N. benthamiana or yeast, respectively, were enriched in the Triton X-114 detergent-rich phases, consistent with the presence of a GPI anchor, rendering the proteins lipophilic (Fig. 8A,B). Treatment of microsomes from M. truncatula with PI-PLC removed MtLYM2 from the microsomes, and a soluble form

The only LYM, for which a function was shown, is CEBiP from rice, which binds to chitooligomers and is necessary for the chitooligosaccharide-induced activation of the synthesis of reactive oxygen species in rice cells [22]. We tested the physical interaction of both M. truncatula LYMs with chitooligomers by affinity labeling with biotinylated chitooctaose using the same experimental design that was successfully developed for CEBiP [26]. MtLYM1 and MtLYM2 were expressed in tobacco BY-2 cells, and microsomes derived from a number of stable transgenic lines were tested for the expression of the heterologous proteins by Western blotting (Fig. 9). As shown in Fig. 9B, immunoreactive polypeptides corresponding to MtLYM1 and MtLYM2 were detected in the microsomal fraction of the respective overexpressing lines. As observed for N. benthamiana, MtLYM1 exhibited a very high apparent molecular weight in comparison to MtLYM2. Microsomes of selected lines were incubated with biotinylated N-acetylchitooctaose (GlcNAc8Bio) in the presence or absence of an excess of unlabeled ligand and treated with a chemical cross-linker to covalently bind the ligand to the proteins. After SDS-PAGE and Western blotting, the affinitylabeled proteins were detected by an anti-biotin antibody (Fig. 9A). The results reported in Fig. 9 clearly show that MtLYM1, expressed in transgenic BY-2 cells, did not show any specific binding to chitooctaose. In contrast, MtLYM2 bound the biotinylated GlcNAc8 ligand, and the binding was competable with an excess of chitooctaose, in a manner comparable to CEBiP [26]. 3. Discussion We describe the phylogeny of an important family of extracellular proteins containing multiple LysMs, called LYMs, and the biochemical characterization of two representative members of this family from the model plant M. truncatula. CEBiP, the chitin elicitor-binding protein from rice, which was the first plant LysM protein for which a biological and biochemical function had been established [22] was used as the archetype to identify LYMs in fully sequenced plant genomes. At least two LYMs were retrieved from all except one genome. In contrast to the family of LysM-RLKs, which in legumes seems to have expanded [15,27], never more than five LYMs could be predicted in a single genome. Genes encoding LYMs were only found in vascular plants, where an early gene duplication event might have generated two forms, both retained in present day plant genomes, giving rise to a clear distinction of LYM1/3-type versus LYM2/4-type proteins. We were able to discern three LysMs of 44e54 residues each in the N-terminal parts of each LYM. These motifs are separated from each other by two pairs of conserved cysteine residues which potentially could form disulfide bridges. Disulfide bonds could have a crucial role for the three-dimensional structure and stability of the domains as shown for a LysM domain from Pteris ryukyuensis chitinase-A [8], and/or protect them against harsh conditions in the apoplast. The primary structures of the three consecutive LysMs in a given LYM are less similar to each other than to the respective motif in another LYM, as observed for LysM-RLKs [2,12,15]. This sequence conservation suggests distinct functional roles for each single motif, implying the evolution of the extracellular domain as an entity. MtLYM2 is the only LYM gene containing an intron between the first and second LysM, which presumably was gained in M. truncatula rather than lost by all other plants. Gain of introns

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Fig. 6. Comparative analysis of MtLYM1 and MtLYM2 proteins in N. benthamiana leaves and M. truncatula roots. Protein extracts from N. benthamiana, transiently expressing either MtLYM1 or MtLYM2, and from M. truncatula roots were prepared and fractionated prior to Western blotting (AeE). (A) The anti-MtLYM1 antibody (@LYM1) visualized a major polypeptide of 95 kDa in the 10,000  g and 45,000  g fractions of N. benthamiana, transformed with pGreen:35S-MtLYM1, but not in empty vector-transformed leaves (Nb control). (B) The microsomal fraction of M. truncatula roots showed a faint signal of a higher molecular weight (arrowhead). (C) Side by side analysis of microsomal fractions (45 k) derived from M. truncatula (Mt) and N. benthamiana (Nb) showed the same blurred migration behavior for the high molecular weight polypeptides which cross-reacted with the antiMtLYM1 antibody, and which centered at 210 kDa and 95 kDa, respectively (arrows). (D) The anti-MtLYM2 antibody (@LYM2) cross-reacted with a polypeptide of approximately 65 kDa only in the 10,000  g and 45,000  g fractions of N. benthamiana leaves transformed with pGreen:35S-MtLYM2, and not in empty vector-transformed material (Nb control). (E) In M. truncatula root proteins, the majority of MtLYM2 (indicated by an arrowhead, apparent molecular mass 70 kDa) was detected in the microsomal fraction (45 k). (F) The protein fractions which were analyzed in B and E (50 mg per lane) were run in parallel (30 mg per lane) and stained by colloidal Coomassie as control for protein loading between the fractions. (G, H) Phase partitioning of microsomes (mic) from M. truncatula roots showed the enrichment of MtLYM2 in the upper, plasma membrane-containing fraction, when compared to the lower phase. Equal amounts of proteins were separated by 7% SDS-PAGE, transferred to nitrocellulose, and developed with the anti-MtLYM2 antiserum (@LYM2, G, 20 mg protein per lane) or with anti-P-type Hþ-ATPase antiserum (@ATPase, [52]) (H, 5 mg protein per lane) for the plasma membrane marker ATPase (apparent molecular mass 100 kDa). The positions of standard molecular mass markers are indicated on the left. S, soluble proteins; 3 k, 10 k, 45 k, proteins sedimenting at 3,000  g, 10,000  g, or 45, 000  g, respectively.

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Sc:MtLYM2 – +

Endo H

67 62 54

-

28

-

36

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det

(kDa) 130 -

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aq

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-

@LYM2

Fig. 7. Glycosylation status of recombinant MtLYM1 and MtLYM2. Endoglycosidase H treatment of yeast e but not of plant e expressed M. truncatula LYMs resulted in the reduction of the apparent molecular weight on SDS-PAGE. (A,B) Western blotting of MtLYM1 and MtLYM2 (5 mg microsomal proteins per lane), each transiently expressed in N. benthamiana leaves, showed the same mobilities in mock-treated () or endoglycosidase H-treated (þ Endo H) samples: MtLYM1 migrated as a 95 kDa protein, and MtLYM2 as a doublet of 64 and 60 kDa (marked by arrows). (C,D) Crude extracts of the yeast S. cerevisiae, expressing either MtLYM1 or MtLYM2, were likewise treated with or without Endo H before Western blotting. MtLYM1, which runs as a doublet of 67 and 62 kDa in the mock-treated sample, showed a shift to one 54 kDa species, whereas the hydrolytic removal of N-glycans from the 69 kDa MtLYM2 gave rise to a doublet of 47 and 45 kDa (arrows). The signal at approximately 50 kDa, which appeared in all Endo H-treated samples, was presumably due to a cross-reaction with the secondary antibody. The positions of standard molecular mass markers are indicated on the left. @LYM1, anti-MtLYM1 antiserum; @LYM2, anti-MtLYM2 antiserum.

is less frequent than loss of an intron at a conserved position in orthologous genes. At present, we do not have any experimental evidence for a possible function of this intron in MtLYM2 but introns can affect transcription and translation, and tissue specificity of intron-mediated enhancement of gene expression has been reported [28,29]. The absence of introns, except for MtLYM2, and thereby lack of exon shuffling inside of the LysM-encoding regions of plant genes during evolution indicates a functional constraint on the integrity of the extracellular LysM domains. The biochemical characterization of MtLYM1 and MtLYM2 revealed common and distinct features. Both proteins exhibited post-translational modifications resulting in an anomalous behavior on SDS-PAGE. MtLYM1 and MtLYM2 both seemed to harbor high-mannose type N-glycans when expressed in yeast, which can be partially removed by treatment with Endo H. Posttranslational modifications in plants, like complex-type glycans, can vary from plant to plant [30] and are known to be resistant to Endo H [31]. Based on the differences between the theoretical masses of MtLYM1 and MtLYM2 and their apparent molecular weights in planta observed on SDS-PAGE analyses, MtLYM1, with only five predicted N-glycosylation sites, might be either hyperglycosylated or contains other posttranslational modifications.

C

@LYM2

membrane soluble

membrane soluble

(kDa) 130 -

det

55 @LYM1

72 55

Sc:MtLYM2

aq

55 -

-

47 45

36

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95

95

55

D

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(kDa) 130 -

@LYM2

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–

+

–

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95

-

95

72

-

72

-

55

-

55

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–

+

PI-PLC

PonceauS

Fig. 8. MtLYM1 and MtLYM2 are GPI-anchored proteins. Microsomal proteins from N. benthamiana (A) or yeast (B), expressing either MtLYM1 or MtLYM2, were solubilized by Triton X-114. Insoluble proteins were removed by centrifugation (insol), and the solubilized proteins were then partitioned into a detergent-rich phase (det), typically containing GPI-anchored proteins, and an aqueous phase (aq). MtLYM1 (95 kDa in tobacco and 67/62 kDa in yeast) and MtLYM2 (64/60 and 69 kDa, respectively), which partitioned preferentially into the detergent-rich phases, are labeled by arrowheads. (C) Microsomes from M. truncatula were mock-treated or treated with PIspecific phospholipase C (PI-PLC), sedimented again and recovered in SDS-PAGE sample buffer. Solubilized proteins were concentrated by precipitation prior to SDSPAGE. Protein samples were analyzed in totality and the nitrocellulose membrane was stained by Ponceau S to verify equal protein loading prior to incubation with antiMtLYM2 antiserum (@LYM2). Molecular mass markers are indicated to the left.

LYM1/3-type proteins exhibit a proline-rich region immediately N-terminal to the GPI-anchor motif and the C-terminal transmembrane domain, which is absent in LYM2/4-type proteins (Fig. S1, positions 400 to 470 in the alignment). The hydroxylation of proline residues and the subsequent glycosylation is a plantspecific modification of some proteins in the secretory pathway. The proline-rich region of MtLYM1 contains two peptides with two adjacent proline residues, which, after hydroxylation, could be arabinosylated, and seven non-contiguous proline residues which could be modified with arabinogalactan glycan chains, according to the Hyp contiguity hypothesis [32]. The extent and complexity of Oglycosylations were reported to depend on the plant species and to be specific for different cell types and tissues [33,34], which might explain our observation of the differently sized polypeptides crossreacting with the anti-MtLYM1 antiserum in the different systems. The existence and exact nature of a possible O-glycosylation of MtLYM1 and its potential role in the stability or the functioning of this protein remains to be elucidated. One major difference between the two proteins concerns their expression in M. truncatula. MtLYM2 was detected in crude extracts prepared from different organs with the highest abundance in roots. In contrast, attempts to detect MtLYM1 remained

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A

LYM1ox

BY-2 NT

-

+

#7

-

#16

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-

+

-

+

LYM2ox

BY-2 NT

#64

-

GN8

(kDa) 250 150 -

(kDa) 150 -

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+

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-

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-

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+

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75 50 50 -

37 @biotin

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GN8

(kDa) 150 100 -

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37 @LYM1

@LYM2

Fig. 9. Binding of chitooctaose to M. truncatula LYMs. (A) Binding activity of MtLYM1 and MtLYM2 to chitooligosaccharides. The microsomal protein fractions from different lines of MtLYM1 (LYM1ox) and MtLYM2 (LYM2ox)-expressing BY-2 cells (50 mg protein) were incubated with GlcNAc8-Bio (0.4 mM) in the presence (þ) or absence () of unlabeled GlcNAc8 (GN8, 40 mM), and cross-linked with EGS. Biotinylated proteins were detected with anti-biotin antibody (@biotin) after SDS-PAGE and transfer to PVDF membranes. (B) Relative expression of MtLYM1 and MtLYM2 in each transformant. The expression level of each line was evaluated using specific antisera against MtLYM1 (@LYM1) and MtLYM2 (@LYM2), respectively. BY-2 NT, non-transformed control cells. Molecular mass markers are indicated to the left.

unsuccessful for any of the tested organs, suggesting that the protein was present in low abundance. Such a contrasting behavior between MtLYM1 and MtLYM2 was not predictable from the transcriptomic data reported in the gene atlas [24]. One possible explanation could be a differential regulation of mRNA or protein stability. Alternatively, it cannot be excluded that MtLYM1 was hardly detectable because of poor extractability of the protein from M. truncatula, which might be related to its nonprotein content. Promoter-GUS fusion experiments confirmed the expression of both genes in roots of M. truncatula. Interestingly, the expression pattern showed some differences: although the activity of both promoters was high in emerging secondary roots and in root tips, GUS activity was observed in the apical meristem for proMtLYM1, whereas proMtLYM2 showed to be active preferentially in the root cap. Moreover, only MtLYM2 was expressed in the rhizodermis and root hairs. In silico analysis of the LYM family predicted the attachment of a GPI anchor for MtLYM1 and MtLYM2. We affirmed the prediction by analyzing the behavior of both proteins during temperatureinduced detergent phase separation and by observing the release of MtLYM2 from its membrane-localized state due to the cleavage of the GPI anchor with PI-PLC. These data correlate with the earlier detection of MtLYM2 in detergent-insoluble membranes prepared from M. truncatula roots by a proteomic analysis [35], in which GPIanchored proteins are expected to be enriched. Likewise, AtLYM1 and AtLYM2, which both are predicted to be GPI-modified, were indeed identified as such [23]. AtLYM2 was moreover isolated as a component of the core of sterol-dependent plasma membrane microdomains [36]. Only MtLYM2, but not MtLYM1 showed specific binding to chitooctaose, comparable to CEBiP, which is also a LYM2-type protein.

AtLYM2 from A. thaliana was also identified as a potential chitinbinding protein in experiments based on affinity chromatography using chitin magnetic beads [9]. Therefore it would be tempting to speculate that members of the LYM2 family have the ability to interact with chitin or chitooligosaccharides. GPI-anchored proteins are often associated with sterol-rich membrane raft-like domains on plasma membranes, which in turn might represent signaling platforms [37]. The presence of LYMs in these domains could suggest a function in the perception and transduction of environmental signals. It is moreover evident that LYM-type proteins, which are devoid of any intracellular domain, need to interact with other components of the plasma membrane in order to transfer the signal. It was shown that CEBiP forms heterodimers with a LysMRLK, OsCERK1, in rice cells [21]. However, it remains to be elucidated if this is dependent on the localization of these proteins in membrane rafts. In summary, we showed that the family of LYMs in plants consists of two major clades, both of which are presumably present in all vascular plants. Three members of the LYM2/4-type have been proven to interact with chitin or chitooligosaccharides, whereas possible ligands for LYM1/3 proteins are not yet described. Interestingly, LYM1/3-type proteins show a higher conservation in primary structure in comparison to LYM2/4 proteins, suggesting a conserved function. The expression of MtLYM1 in the root apical meristem might hint at a role in root growth and development, which needs further evaluation, as well as the strong expression of both genes in emerging lateral roots. From our biochemical characterization of a member of each clade in M. truncatula, it can be concluded that, although they share common structural features, they are distinct in terms of expression patterns and binding

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properties toward chitooligosaccharides, thus excluding redundant functions. 4. Methods 4.1. Phylogenetic analysis LysM-domain containing proteins similar to CEBiP and the LYMs from M. truncatula were retrieved by TBLASTN search of the genomes in the Phytozome version 5.0 dataset (http://www. phytozome.net/), representing the genomes of Chlamydomonas reinhardtii (JGI assembly v4, incl. u9 update), Physcomitrella patens (JGI from July 20, 2006; cured from contaminants in 2009), Selaginella moellendorfii (spikemoss; JGI v1.0 from Dec 20, 2007), Oryza sativa (rice; MSU release 6.0), Brachypodium distachyon (purple false broom; JGI v1.0 8x assembly & MIPS/JGI v1.0 annotation), Sorghum bicolor (cereal grass; release v1.0), Zea mays (maize; 4a.53 assembly & annotation of “B73” & 454 reads of “Mo17”), Mimulus guttatus (monkey flower; JGI release v1.0), Vitis vinifera (wine grape; release & annotation, Sept 2007 [38]), Carica papaya (papaya; release Dec. 2009), A. thaliana (thale cress; Phytozome release incl. TAIR release 9), A. lyrata (lyrate rockcress; JGI release v1.0), Cucumis sativus (cucumber; JGI), Glycine max (soybean; DOEeJGI assembly Glyma1), M. truncatula (barrel medic; release Mt3.0), Populus trichocarpa (Western poplar; JGI v2 assembly), Manihot esculenta (cassava; gene set Cassava1.1), and Ricinus communis (castor bean plant; Phytozome incl. TIGR/JCVI release v0.1). The cluster with the best score was Tracheophyte gene family 22961622, named “LysM domain GPI-anchored protein 1 precursor, putative”, which contained 63 members. These were combined with accessions described as LYPs [12] which were not represented in this gene family. Then, proteins which were smaller than 300 amino acid residues or which were predicted to be intracellular were excluded to restrict the selection to LYMs only. SignalP 3.0 and TargetP 1.1 (http://www.cbs.dtu.dk/services/) were used to predict the presence of signal peptides and putative extracellular localization of the candidates. The application of these restrictions resulted in a list of 54 proteins from which singletons (M. guttatus), accessions derived from a second species in the same genus (A. lyrata), and wrongly deduced proteins were then deleted, resulting in 48 entries. Proteins were compared using “one click” phylogenetic analysis [39]. SmLYM4a and SmLYM4b, from spike moss, an early tracheophyte, showed less conservation than SmLYM1 to LYMs from angiosperms, and were not used further, whereas SmLYM1 was kept to serve as outgroup. The alignment produced during phylogenetic analysis (Fig. S1) was used to define the regions comprising LysMs according to the published consensus sequence [3]. These were degapped and fed separately into the “one click” web service, ignoring alignment curation by Gblocks. Sequence logos representing consensus sequences were prepared using WebLogo 3.0 (http://weblogo.threeplusone.com/). GPI modification sites were predicted using the big-PI plant predictor (http://mendel.imp.ac.at/gpi/plant_server.html). 4.2. MtLYM1 and MtLYM2 gene identification and subcloning EST clusters MtD01600 (MtLYM1) and MtC30180 (MtLYM2), previously identified as described [15], were used for subcloning purposes. In the recent release of the M. truncatula genome Mt3.0, the gene model for MtLYM1 was localized to chromosome 3 (Medtr3g097500.1). The gene encoding MtLYM2 was localized on the BAC clone mth2-75g11 (GenBank accession AC235671.5) and was not yet assigned to a chromosomal location. GENSCAN (http:// genes.mit.edu/GENSCAN.html) was used to predict the gene-free space upstream of the coding sequences of MtLYM1 (2.7 kb) and

717

MtLYM2 (more than 5 kb), respectively. For both genes, 2.5 kb upstream of the start codon were amplified, isolated, and fused to the GUS (uidA) reporter gene [40]. Genomic DNA of M. truncatula A17 and the BAC clone pBeloBac11:178g2, containing MtLYM22, were used for amplification, respectively, using oligonucleotides described in Supplementary Table S2. The PCR products were first subcloned into pJet1.2/blunt (Fermentas) or pGEM-T (Promega), and then inserted via HindIII and XbaI or HindIII and BamHI 50 into the intron-containing GUS-int gene in the plasmid pPR97 [41], yielding proMtLYM1::GUS-int and proMtLYM2::GUS-int, respectively. For bacterial protein expression, the coding sequences for the extracellular domains of MtLYM1 and MtLYM2, omitting the signal peptides and transmembrane domains, were amplified from the EST clones in three consecutive PCR reactions (see Table S2), thereby adding Gateway recombination sites as well as the coding sequence for the prescission protease cleavage site. The open reading frames were cloned into pDONR221 (Invitrogen), yielding the pEntry constructs, which were verified by sequencing. The coding regions were then transferred by recombination into the destination vector pTH19 [42], yielding pTH19:MtLYM1 and pTH19:MtLYM2, for the expression of the recombinant proteins including an N-terminal hexa histidine-tag. For protein expression in yeast, the complete open reading frames of MtLYM1 and MtLYM2 were amplified from the EST clones, concomitantly introducing restriction sites (BsrGI 50 to the first codon, XbaI spanning the stop codon, see Table S2), digested accordingly, and inserted into the shuttle vector pYes2 (Invitrogen), digested with Acc65I and XbaI. For in planta overexpression, the complete open reading frames were excised from the corresponding pYes2 derivatives by digestion with either HindIII and XbaI (MtLYM1) or by restriction with SspI, followed by filling in the 50 -overhang by Klenow polymerase and the release of the fragment by XbaI (MtLYM2). The fragments were inserted into the p35S promoter cassette present in the plasmid p35S, which is part of the pGreen vector set [43], from which the expression cassettes were released by EcoRV and inserted into pGreen0029. 4.3. Bacterial expression and generation of antisera For MtLYM1, the antibody was raised against the recombinant His(6)-MtLYM1, produced in E. coli BL21 (DE3) transformed with pTH19:MtLYM1 (see 4.2), and purified from solubilized inclusion bodies. Briefly, isopropyl b-D-1-thiogalactopyranoside-induced bacterial cultures were centrifuged and the pellet containing insoluble His(6)-MtLYM1 was sonicated 10  30 s in 20 mM Tris/HCl buffer (pH 8.0). The insoluble material sedimenting at 3000  g was resuspended in 20 mM Tris/HCl (pH 8.0), 2 M urea, 0.5 M NaCl, 2% Triton X-100, sonicated 4  10 s and centrifuged (4500  g, 15 min, 4  C). This treatment was repeated twice and the resulting pellet was incubated with solubilization buffer (20 mM Tris/HCl (pH 8.0), 6 M guanidine hydrochloride, 0.5 M NaCl, 10 mM imidazole, 1 mM b-mercaptoethanol) for 1 h at 4  C under constant stirring. The solubilized material, recovered after centrifugation (20,800  g, 15 min) was diluted 10 times in the solubilization buffer and loaded onto a nickel-charged 1 ml HiTrap Chelating HP column (GE Healthcare), equilibrated in the same buffer and placed in an ÄKTApurifierÔ UPC10. The column was washed with 20 ml of buffer A (20 mM Tris/HCl (pH 8.0), 6 M urea, 0.5 M NaCl, 5 mM imidazole, 1 mM b-mercaptoethanol) and urea was progressively removed using a linear gradient from buffer A to buffer B (20 mM Tris/HCl (pH 8.0), 0.5 M NaCl, 10 mM imidazole, 1 mM b-

2

F. Debellé, personal communication.

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mercaptoethanol) in 60 min at 0.5 ml min1. His(6)-MtLYM1 was eluted by a linear gradient of imidazole ranging from 10 mM to 500 mM in buffer B, during 10 min at 1 ml min1. The His(6)MtLYM1 containing fraction was analyzed by preparative SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and the polyacrylamide slices containing the protein were processed for antibody production in rabbits (MilleGene, Labège, France). His(6)-MtLYM2 was produced likewise in E. coli BL21 (DE3), transformed in this case with pTH19:MtLYM2 (see 4.2), but the insoluble His(6)-MtLYM2 protein was directly isolated by preparative SDS-PAGE. Polyacrylamide slices, each containing 150 mg of protein were homogenized in 700 ml of physiological serum and supplemented with 400 ml of Freund’s incomplete adjuvant (Calbiochem) for each immunization of in total two rabbits, which were boosted every 10 days. The serum was collected after three months by centrifugation of the clotted blood, and was stored at 20  C until use. 4.4. Expression in yeast and preparation of cell lysates Yeast strain INVSc2 (Invitrogen) was transformed and grown for recombinant protein production as described [44]. Crude protein extracts were prepared according to [45]. Cell lysates were obtained by mechanical rupture, using glass beads (0.4e0.6 mm, Sigma) in lysis buffer (50 mM Tris/HCl (pH 7.5), 0.3 M sucrose, 0.1 M NaCl, 30 mM MgCl2, containing protease inhibitors (Roche, EDTA-free)). Microsomal proteins were collected from cleared lysates (3000  g, 5 min) by centrifugation at 45,000  g for 45 min and stored at 80  C in 50 mM Tris/HCl (pH 7.5), 0.1 M NaCl, 1 mM MgCl2, 20% glycerol and protease inhibitors until use. 4.5. Overexpression in planta Leaves of N. benthamiana were infiltrated with Agrobacterium tumefaciens strain LBA4404-pBBR1MCS5::VirGN54D, co-transformed, separately, by electroporation with pSoup and either pGreen0029:p35S-MtLYM1, pGreen0029:p35S-MtLYM2, or pGreen0029 (empty vector). Leaves were harvested after 3 days and protein fractions were prepared as described [25]. Suspension-cultured tobacco BY-2 cells were maintained in LSD medium as described [46] and transformed by 2 days of cocultivation with A. tumefaciens strain C58C1, co-transformed with the same plasmid combinations described above. Transformed cell lines were selected on LSD agar medium containing 200 mg ml1 kanamycin and 12.5 mg ml1 Meropenem for 2e3 weeks. Selected cell lines were transferred into LSD liquid medium and used for the preparation of microsomal fractions for binding assays. 4.6. Preparation of protein extracts and analysis by SDS-PAGE and Western blotting Roots, stems, flowers, and leaves were harvested from M. truncatula A17 plants and treated as described [47] to provide whole protein extracts to be analyzed by SDS-PAGE and Western blotting. Fractionation of roots of M. truncatula, grown in aeroponic culture [48], was carried out as described [25]. Plasma membranes were obtained by phase partitioning as described [35]. Proteins were analyzed by 10% SDS-PAGE, unless otherwise noted, transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk in 0.1% Tween-20 in PBS (40 mM Na2HPO4, 8 mM NaH2PO4, 150 mM NaCl (pH 7.4)), and incubated with 1:10 000 diluted primary antibodies (anti-MtLYM1 or anti-MtLYM2 antiserum) in the same solution. Anti-rabbit secondary antibody (Sigma), coupled to horseradish peroxidase (HRP) was used at 1:5000 in PBS/milk/Tween-20. Immunoreactive polypeptides were

detected using chemiluminescence (Immobilon Western substrate, Millipore) and recorded by a digital camera (SynGene). 4.7. Enzymatic treatments and phase partitioning Recombinant yeast-derived crude protein extracts (equal volumes) or microsomal proteins (5 mg), prepared from transiently transformed N. benthamiana leaves, were used for deglycosylation assays by endoglycosidase H (Endo H, Roche). The samples were denatured at 100  C in 0.5% SDS and 40 mM DTT for 10 min prior to treatment with 0.005 U Endo H for 2 h at 37  C. Controls were treated likewise, omitting the enzyme. Microsomes from M. truncatula were treated with 1.5 U ml1 GPIspecific phospholipase C (PI-PLC, Invitrogen) in 20 mM Tris/HCl (pH 7.5 at 37  C), for 2 h with repeated shaking. Solubilized proteins were separated from membrane-resident proteins by centrifugation at 45,000  g for 30 min and precipitated over night at 20  C by adding four volumes of cold acidic methanol/acetone (1/1, v/v, 0.5 mM HCl). Triton X-114 was precondensed in TBS (10 mM Tris/HCl (pH 7.5), 150 mM NaCl) as described [49]. Microsomal proteins (1 mg ml1) were solubilized in TBS/20% precondensed Triton X-114 for 90 min at 4  C on a rotary shaker. Insoluble proteins were removed (10,000  g, 10 min, 4  C) and phase partitioning was performed by heating the solubilized proteins at 30  C for 10 min and subsequent centrifugation at 3000  g for 5 min at room temperature. The phase partitioning was repeated and lipid-rich and lipid-depleted fractions were precipitated as above and analyzed by Western blotting. 4.8. Affinity labeling with biotinylated GlcNAc8 Affinity labeling with biotinylated GlcNAc8 was performed as described previously [26]. Microsomal membrane fractions were prepared from BY-2 cells harvested 6e7 days after transfer to new medium [46] and mixed with 0.4 mM biotinylated GlcNAc8 in the presence or absence of 40 mM non-labeled GlcNAc8, and adjusted to 30 ml with binding buffer. After incubation for 1 h on ice, 3 ml of 3% EGS solution (ethylene glycol bis[succinimidylsuccinate]; Pierce) was added to the mixture and kept for 30 min. The reaction was stopped by the addition of 1 M Tris/HCl, mixed with SDS-PAGE sample buffer, boiled for 5 min, and used for SDS-PAGE. Western blotting was performed on Immun-Blot PVDF Membrane (Bio-Rad). Detection of biotinylated proteins was performed by using a rabbit antibody against biotin (Bethyl) as primary antibody and HRPconjugated goat anti-rabbit IgG (Chemicon International) as secondary antibody. Biotinylated proteins were detected by chemiluminescence (see section 4.6). Recombinant MtLYM1 and MtLYM2 were visualized using the antisera described above (section 4.3). 4.9. Histochemical staining Agrobacterium rhizogenes ARqua1 was transformed by electroporation with proMtLYM1::GUS-int and proMtLYM2::GUS-int (section 4.2), respectively, and used for the transformation of M. truncatula A17 seedlings [50]. Composite plants were used for histochemical staining as described [51]. Stained tissues were observed with a stereomicroscope (MZ FLIII, Leica, Germany) and digitalized with a Leica camera. Acknowledgments We would like to thank Dr. C. Gough for the EST clones, Dr. H. Berglund for providing the pTH19 cloning vector, Dr. P. Ratet for the plasmid pPR97, Dr. M. Boutry for the anti-Hþ-ATPase antiserum, Dr. G. Borderies for continuous support, C. Arlot for the purification of MtLYM1, Y. Sekiguchi for contributing to BY-2 transformation and

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binding assays, and Drs. J. Cullimore, H. Kaku, A. Jauneau, and T. Kroj for helpful discussion. This work was funded in parts by the Région Midi-Pyrénées and the CNRS (PhD grant to SU), the French Agence Nationale de la Recherche (contracts ANR-08-BLAN-0208-01 “Sympasignal” and ANR-05-BLAN-0243-01 “NodBindsLysM”), and by the European Community’s Sixth Framework Program through a Marie Curie Research Training Network (contract MRTN-CT-2006-035546 “Nodperception”).

[22]

[23]

[24]

Appendix. Supplementary material [25]

Supplementary data related to this article can be found online at doi:10.1016/j.plaphy.2011.04.004. [26]

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