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Keywords:

  • Aphanomyces euteiches ;
  • Colletotrichum trifolii ;
  • legume;
  • LysM protein;
  • Medicago truncatula ;
  • plant immunity;
  • symbiosis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Plant LysM proteins control the perception of microbial-derived N-acetylglucosamine compounds for the establishment of symbiosis or activation of plant immunity. This raises questions about how plants, and notably legumes, can differentiate friends and foes using similar molecular actors and whether any receptors can intervene in both symbiosis and resistance.
  • To study this question, nfp and lyk3 LysM-receptor like kinase mutants of Medicago truncatula that are affected in the early steps of nodulation, were analysed following inoculation with Aphanomyces euteiches, a root oomycete. The role of NFP in this interaction was further analysed by overexpression of NFP and by transcriptome analyses.
  • nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A. euteiches, whereas NFP overexpression increased resistance. Transcriptome analyses on A. euteiches inoculation showed that mutation in the NFP gene led to significant changes in the expression of c. 500 genes, notably involved in cell dynamic processes previously associated with resistance to pathogen penetration. nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii.
  • These results demonstrate that NFP intervenes in M. truncatula immunity, suggesting an unsuspected role for NFP in the perception of pathogenic signals.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant roots are in contact with diverse microbes, including symbionts and pathogens. To cope with this biotic environment, plants have evolved receptors to perceive microbe-derived molecules (Jones & Dangl, 2006). This recognition triggers appropriate plant responses that may either control the entry of a beneficial microbe to establish symbiosis (Bonfante & Genre, 2010; Oldroyd et al., 2011) or contribute to rejecting a pathogen to prevent plant disease (Zipfel, 2009). Both outcomes (symbiosis or activation of immunity) are tightly regulated following the perception of microbe-derived compounds at the plant cell surface either by plant receptor-like proteins (RLPs) or by plant receptor-like kinases (RLKs; Antolin-Llovera et al., 2012). Both types of receptor are bound to membranes and have an extracellular domain that allows specific microbe recognition, but the latter also contain an intracellular kinase domain that might be used for signal transduction (Monaghan & Zipfel, 2012).

Among receptors, plant Lysin motif (LysM) proteins have received special attention in the last 10 yr, as some members of this family are pattern recognition receptors (PRRs), which activate pathogen-triggered immunity (PTI; Jones & Dangl, 2006), while others are key players in initiating symbiosis with rhizobacteria or mycorrhizal fungi in legume plants (Gough & Cullimore, 2011). A common feature of LysM receptors, whether they are involved in immunity or symbiosis, is their ability to recognize and bind structurally related microbial glycans that contain a backbone of several N-acetylglucosamine (GlcNAc) residues (Gust et al., 2012).

In Arabidopsis, the LysM-RLK protein CERK1 binds chitooligosaccharides (COs), a major component of fungal cell walls (Kaku et al., 2006; Petutschnig et al., 2010; Liu et al., 2012ab), to trigger intracellular signalling and activation of resistance to fungal pathogens (Miya et al., 2007). It has also been shown recently that Arabidopsis RLP LysM proteins, namely LYM1 and LYM3, are receptors for peptidoglycan (PGN), a major Glc-NAc-containing component of bacterial cell walls (Willmann et al., 2011). In monocots (rice), similar actors have also been identified. OsCEBiP is a CO-binding LysM RLP (Kaku et al., 2006) that forms heterodimers with OsCERK1 following CO treatment, and also controls resistance to a fungal pathogen (Shimizu et al., 2010). Two other LysM proteins, LYP4 and LYP6, were shown recently to be involved in PGN perception by rice and can also act as additional chitin receptors in this plant (Liu et al., 2012a). In legume plants, COs also induce defence-associated responses, but the LysM receptor(s) involved in their perception have not yet been identified (Day et al., 2001; Nakagawa et al., 2011).

As well as being central actors of PTI driven by GlcNAc-sensing mechanisms, LysM receptors are also key components of the early signalling events of endosymbiotic plant–microbe interactions. In these interactions, LysM receptors control the perception of lipo-chitooligosaccharides (LCOs), composed of three to five GlcNAc residues with an acyl chain on the terminal nonreducing sugar (Gough & Cullimore, 2011). In the Rhizobium–legume symbiosis, these molecules, called Nod factors (NFs), trigger infection and nodule organogenesis (Dénarié & Cullimore, 1993; Oldroyd & Downie, 2008). In the two model legumes, Medicago truncatula and Lotus japonicus, genetic analysis identified a pair of LysM-RLKs that are required for NF-dependent nodulation and rhizobial infection. In L. japonicus, recent results have shown that these proteins, NFR1 and NFR5, can bind NFs (Broghammer et al., 2012). In M. truncatula, likely orthologs of these proteins are LYK3 (Limpens et al., 2003) and NFP (Ben Amor et al., 2003; Arrighi et al., 2006), respectively. Both LysM-RLK genes are required for the nodulation process, but only NFP is required for NF sensitivity, whereas LYK3 mediates rhizobial infection. NFP also partially controls perception of Myc-LCOs produced by Rhizophagus irregularis (formerly Glomus intraradices) that stimulate the establishment of the arbuscular mycorrhizal symbiosis (Maillet et al., 2011). Recently, NFP was also shown to control transcriptomic reprogramming of M. truncatula following treatment with Myc-LCOs (Czaja et al., 2012).

Chitooligosaccharide and pathogen perception have been less studied in legume plants than in nonlegumes. However, as legumes are hosts to various pathogens, they present an ideal opportunity among plants to study crosstalk between defence and symbiosis. The model legume M. truncatula is a host for fungi (Tivoli et al., 2006; Ameline-Torregrosa et al., 2008); bacteria (Turner et al., 2009) and oomycetes (Colditz et al., 2005; Djébali et al., 2009). The pathosystem between M. truncatula and Aphanomyces euteiches, a soilborne biotrophic oomycete responsible for pea root rot disease (Gaulin et al., 2007), has been particularly well studied. On the plant side, among accessions that show contrasting responses to this pathogen (Moussart et al., 2007), the F83005.5 (susceptible) and A17 (partially resistant) lines were selected to characterize quantitative resistance of M. truncatula to A. euteiches. While the parasite is able to colonize the root cortex in both lines, a hallmark of A17 resistance is the protection of the root central cylinder, associated with the induction of reactive oxygen species (ROS) scavenging mechanisms that lead to cell wall strengthening (Djébali et al., 2009, 2011). On the pathogen side, it was shown that the A. euteiches cell wall is enriched in noncrystalline GlcNAc polymers, which were called ‘chitosaccharides’ (Badreddine et al., 2008). Chitinase treatment released GlcNAc from the cell wall, suggesting that these compounds, just like chitin, contain β-1,4-linked GlcNAc residues and are potential sources of COs. In addition, labelling with a fluoresceine isothiocyanate (FITC) conjugate of wheat germ agglutinin (WGA), a CO-specific lectin, showed that significant amounts of chitosaccharides were exposed on the surface of hyphae. These features, and the fact that the A17 M. truncatula-resistant line is the genetic background of numerous symbiotic mutants, makes the M. truncatulaA. euteiches pathosystem very appropriate to investigate the role of symbiotic LysM receptors in the context of a pathogenic interaction. Here, we report on the implication of NFP in M. truncatula resistance to both the oomycete A. euteiches and the fungus Colletotrichum trifolii, which indicates a new function for NFP in M. truncatula immunity.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

A17 and F83005.5 are two natural Medicago truncatula (Gaertn.) lines, which can both be nodulated (Kiss et al., 2004), and which were used as references in all experiments performed to assess the degree of resistance of symbiotic mutants to A. euteiches (Djébali et al., 2009). Symbiotic mutants were nfp-1, nfp-2 and lyk3-1 (=hcl-1; Catoira et al., 2001; Arrighi et al., 2006), with the two nfp mutants being derived from independent mutagenized populations of A17 seeds. All mutations are point mutations affecting the coding sequence. No nonsymbiotic phenotypes (in plant growth or development) have been reported for these mutants. Transgenic M. truncatula plants overexpressing NFP were produced as described in the Supporting Information (Methods S1). Plants were grown in vitro under conditions of 16 h light :  8 h dark, at 22 : 20°C, as previously described (Djébali et al., 2009).

Inoculation procedure and symptom analysis

Zoospores of the Aphanomyces euteiches Drechs. strain ATCC 201684, a pea isolate, were produced as described by Badreddine et al. (2008) and adjusted to 105 spores ml−1. Seedlings were inoculated 1 d after transfer onto M medium with a 5 μl droplet of spore suspension deposited in the middle of the root. To assess mutant resistance, three to six independent in vitro infection assays were performed (as indicated in the legends of figures) with 15–30 inoculated plants per genotype in each repeat. The relative length of tissues displaying symptoms (14 d postinoculation (dpi)), along with percentages of dead plants (21 dpi), which have been shown to be the most significant parameters for assessing the degree of resistance of M. truncatula to A. euteiches, were recorded, as described by Djébali et al. (2009).

Colletotrichum trifolii (race 2) conidia were prepared as described by Ameline-Torregrosa et al. (2008) and adjusted to 106 ml−1. Fifteen wildtype (WT) and mutant plants each were inoculated three times independently with 5 μl of this spore suspension and phenotyped as for A. euteiches experiments.

Sample preparation for microscopy

Root sections (100 µm) were prepared and labelled with WGA-FITC, as described by Djébali et al. (2009), to localize A. euteiches hyphae using epifluorescence illumination (excitation filter, BP 450–490 nm). Images were acquired using a charge-coupled device (CCD) camera (colour Coolview; Photonic Science, Robertsbridge, UK).

RNA extraction and quantitative RT-PCR

Total RNA was isolated from roots with the ‘RNeasy for plant and fungi’ kit (Qiagen), followed by DNAse treatment. RNA integrity was checked using a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and reverse transcriptions were performed with 2 μg of total RNA for each sample and the SuperScript reverse transcriptase III (Invitrogen). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in an ABI Prism SDS 7900HT (Applied Biosystems, Foster City, CA, USA) system as previously described (Djébali et al., 2009). Each qRT-PCR reaction was conducted in triplicate. qRT-PCR primer sequences are given in Table S1. An M. truncatula gene encoding a Histone-3 like (Medtr4 g097170 or Mtr.16911.1.S1_s_at as identifier on the Affymetrix chip) was used to normalize plant gene expression or relative pathogen transcript abundances in each analysed sample. Genes encoding an A. euteiches α-tubulin and a C. trifolii elongation factor alpha were used to detect the amount of each inoculated pathogen at 6 and 8 dpi, respectively. The inline image method (Livak & Schmittgen, 2001) was applied to visualize induction or repression of gene expression or to measure the relative amount of pathogen transcript accumulation.

Affymetrix array hybridization and data analyses

Total RNA was extracted using the RNeasy Kit (Qiagen). One microgram of total RNA was transcribed as described in Methods S1 to produce labelled cRNA which was used to hybridize Affymetrix GeneChip® Medicago genome arrays at INRA-URGV (Evry, France). The raw CEL files were imported in R software for data analysis (http://www.r-project.org/). Raw and normalized data are available through the CATdb database (AFFY_aphanomyces_Medicago, (Brunaud et al., 2008)) and from the Gene Expression Omnibus (GEO) repository at the National Centre for Biotechnology Information (NCBI; Barrett & Edgar, 2006): accession number GSE 20587. Following normalization as described in Methods S1, a gene is declared differentially expressed if its P-value is lower than the Bonferroni-corrected P-value threshold of 0.05/N, where N represents the number of M. truncatula genes (50 900) present on the chip. Our transcriptome data can also be found in the Mt Gene Expression Atlas (http://mtgea.noble.org/v3/).

Elicitation assay

The assay was performed according to Nars et al. (2013). Briefly, after sterilization and germination on water agar, the seedlings were transferred into 35 mm Petri dishes, containing 2.5 ml of liquid M medium without sucrose (Bécard & Fortin, 1988), and incubated for 7 d under the same conditions as for plant infection assays. Plants were then treated for 20 min with 1 ml of 100 μg ml−1 of a mixture of chitin fragments (COs) purified from crab shell chitin and enriched in fragments with a degree of polymerization (DP) between 6 and 8, as described in Methods S1. Measurement of ROS production was adapted from (Ashtamker et al., 2007): medium aliquots were collected for the measurement of extracellular hydrogen peroxide, in the presence of 2 U ml−1 horseradish peroxidase and 100 μM amplex red in 25 mM NaHPO4, pH 7.5. Plants were kept under elicitation for 4 h with COs and then collected to extract RNA and perform qRT-PCR on defence genes with primers described in Table S2. Three independent biological repeats containing nine plants each were performed for ROS and qRT-PCR experiments.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Allelic nfp mutants are more susceptible to A. euteiches than WT plants

NFP and LYK3 are putative NF receptor proteins in M. truncatula (Gough & Cullimore, 2011). To assess their possible involvement in response to a pathogen that exposes chitosaccharides at its cell wall surface, roots of strong nfp and lyk3 mutants in the M. truncatula A17 background (Catoira et al., 2001; Ben Amor et al., 2003; Arrighi et al., 2006; Klaus-Heisen et al., 2011) were inoculated with A. euteiches zoospores. Phenotypes of mutant lines were established using a disease score index (DSI) based on the extent of rot symptoms at 14 dpi, and percentages of dead plants at 21 dpi. In all experiments, A17 and F83005.5 were used as partially resistant and susceptible control lines, respectively (Djébali et al., 2009). Hereafter, the A17 line is referred to as WT. These experiments revealed that a lyk3 mutant (lyk3-1) showed a degree of resistance similar to the WT (Fig. 1). In contrast, two independent nfp mutants (nfp-1 and nfp-2) displayed a more susceptible phenotype when compared with the WT, with highly significant (< 0.001) differences of means (Fig. 1). These observations were also validated by qRT-PCR analyses that can be used at an early time-point of the infection (6 dpi), when visual symptoms cannot yet discriminate degrees of resistance between lines, to assess the development of live and active mycelium of A. euteiches in M. truncatula roots (and thus the degree of plant susceptibility). Quantification of A. euteiches α-tubulin transcripts normalized by the transcript of a M. truncatula housekeeping gene indicate that the means of A. euteiches transcript abundances in nfp-1 and nfp-2 plants were five to 10 times higher than those in WT plants (Fig. 1b). The abundances of A. euteiches tubulin transcripts were similar in lyk3-1 mutant and WT plants. Finally, cytological analyses of root sections using WGA-FITC labelling of the cell surface chitosaccharides of A. euteiches (Badreddine et al., 2008) were performed to observe mycelium development within roots (Djébali et al., 2009). Significantly higher frequencies (< 0.001) of stele invasion were observed for both nfp mutants compared with WT, but not compared with F83005.5 (Fig. 1c). Taken together, these results show that the two nfp mutants, but not the lyk3 mutant, are more susceptible than WT to A. euteiches.

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Figure 1. nfp mutants are more susceptible to Aphanomyces euteiches than Medicago truncatula wildtype (WT) plants. (a) Symptoms (14 d postinoculation (dpi)) and percentages of dead plants (21 dpi) on WT (= 151), lyk3 (n = 112), nfp-1 (= 131), nfp-2 (= 118) and the susceptible F83005.5 line, noted F83 (= 122). (b) Quantification of A. euteiches α-tubulin transcripts in planta (6 dpi), normalized by M. truncatula transcripts of the constitutively expressed H3 l (MtGI9-TC118424) gene. (c) Assessment of stele invasion by A. euteiches in WT, nfp and lyk3 mutants, and the F83005.5 line, noted F83. A. euteiches mycelium and oospores are labelled in green with wheat germ agglutinin-fluoresceine isothiocyanate (WGA-FITC) in thin root sections of WT, nfp and lyk3, at 21 dpi. White dotted circles indicate the limits of central cylinders. Bars, 100 μm. On the right, the graph represents the recorded frequencies of stele invasion in the different lines (= 30 plants per line). In all assays, error bars represent ± SE. Means of each line were compared by the t-test for symptom notation; by the χ2 test for proportion of dead plants and frequency of stele invasion; and by the Mann–Whitney U-test for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) results. In all graphs, asterisks indicate significant differences compared with WT results (***, < 0.001; *, < 0.05).

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NFP overexpression increases resistance to A. euteiches

The effect of NFP overexpression on A. euteiches development was assessed in plants transformed with a 35S::NFP cassette (see the 'Materials and Methods' section). The line transformed was 2HA, an easily transformable genotype derived, like A17, from Jemalong (Chabaud et al., 1996). Upon inoculation with A. euteiches, the disease score index and the percentage of dead plants (< 0.001) were significantly higher in control than in transgenic plants (Fig. 2a). The percentages of root steles invaded by A. euteiches were nevertheless similar (10% for 2HA and 12% for 35S::NFP). Quantification of A. euteiches by qRT-PCR indicated that the abundance of the oomycete α-tubulin transcript was significantly (< 0.05) higher (about four times) in control plants than in 35S::NFP plants (Fig. 2a), therefore confirming the greater resistance of these latter plants.

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Figure 2. NFP overexpression increases Medicago truncatula resistance to Aphanomyces euteiches. (a) Phenotypes (left) observed at 14 d postinoculation (dpi; symptoms) or 21 dpi (dead plants and cytological analyses) on 2HA control plants and on transgenic plants transformed with the 35S::NFP cassette and molecular detection of A. euteiches (right). Three independent repeats were performed with a total of 88 plants. Significant differences were detected for symptoms (t-test), dead plants and stele invasion (χ2 test), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (Mann–Whitney U-test) (***, < 0.001; *, < 0.05). (b) Kinetics of NFP expression from 0 to 6 dpi by A. euteiches. NFP transcript abundances were calculated by the inline image method, using H3 l gene for normalization. Results are means of three independent experiments. Error bars represent ± SE.

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As these results suggested a link between NFP expression levels and the degree of resistance to A. euteiches, NFP expression was measured by qRT-PCR at 0, 1, 3 and 6 dpi in WT, nfp-1, nfp-2, F83005.5, 2HA and 35S::NFP transgenic plants (Fig. 2b). These time-points correspond to penetration of the rhizodermis (1 dpi) and root cortex colonization (3 dpi) in both A17 and F83005.5, and invasion of the central cylinder in susceptible F83005.5 plants (6 dpi; Djébali et al., 2011). Results showed that NFP expression before inoculation was over fourfold higher in 35S::NFP transgenic plants than in all the other lines. Expression in the resistant WT (A17) line was also higher than in the susceptible F83005.5 line. After A. euteiches inoculation, NFP transcripts were strongly down-regulated as early as 1 dpi, even in the 35S::NFP line. However, the NFP transcript abundance in this transgenic line remained significantly above the other lines at 1 and 3 dpi, although this difference was drastically reduced at 6 dpi (Fig. 2b).

Although we cannot exclude that the activity of the 35S promoter decreases during A. euteiches infection, as such an alteration has already been observed, for example during symbiosis (Auriac & Timmers, 2007), taken together our results show that NFP transcripts are down-regulated in an A. euteiches-dependent manner and that high NFP expression in the early stages of infection is correlated with an improved resistance to A. euteiches.

Transcriptomes of the nfp-2 mutant and WT plants are divergent upon A. euteiches inoculation

Gene expression profiles of WT and nfp-2 roots were analysed using Affymetrix Medicago Genome Array Genechips® in noninoculated (ni) plants and upon inoculation by A. euteiches at 1 dpi. This time-point was selected to have a homogenous infection stage on whole inoculated roots corresponding to the very early penetration events of A. euteiches, and therefore to enable NFP-dependent signalling and defence responses to be analysed.

Genes whose regulation showed a significant Bonferroni-corrected P-value (i.e. < 9.8231E–07) were retained for global analyses (Table S3, Fig. 3). Comparisons of ni roots showed that basal levels of only 182 out of the 50 900 (0.35%) of the M. truncatula oligonucleotide probes were significantly different between WT and the nfp mutant (Table S4). Among these 182 probes, 101 correspond to genes with an annotated function, but no functional class was detected as significantly overrepresented by a Mann–Whitney U-test (not shown). However, interestingly, two genes encoding LysM proteins (LYK3 and MtLYK4) were found expressed to a higher level in WT. Other known genes associated with symbiosis were also identified in the set of genes differentially detected in control conditions, suggesting a basal activity level of the symbiotic signalling pathway controlled by NFP. This includes two transcription factors (TFs), the GRAS TF MtNSP2 (Hirsch et al., 2009) and the ethylene-responsive TF MtERN1 (Andriankaja et al., 2007), which are more highly expressed in the WT. By contrast, the nodulin gene MtFLOT4 (Haney & Long, 2010) is more highly expressed in nfp-2.

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Figure 3. Comparisons of nfp-2 and wildtype (WT) transcriptomes. Medicago truncatula transcriptomes were assessed with Affymetrix chips. Genes selected for these graphs have a P-value < 9.8231E–07 following normalization (see the 'Materials and Methods' section). Number of genes*, numbers of Affymetrix probes showing differential hybridizations with a significant P-value are taken as good indicators of numbers of regulated genes (despite a slight overestimation since the same gene is sometimes represented by more than one probe on the chip). (a) Left, numbers of genes significantly more highly expressed in WT (blue) or in nfp-2 (grey) in noninoculated plants. A complete list of genes is given in Table S4. Right, Venn diagram obtained following comparisons of genes up- or down-regulated in WT plants (blue) or in the nfp-2 mutant (grey), at 1 d after Aphanomyces euteiches inoculation (1 dpi) compared with noninoculated plants (ni). Genes in the intersection represent common responses of both lines to the pathogen, while the other genes belong to specific responses of WT or nfp-2. Given the selected P-value threshold, induced genes showed a log2 expression ratio (1 dpi vs ni) > 0.97, while for repressed genes this ratio was < −0.96. (b) Distribution in functional classes as defined by Mapman software, of genes belonging to common responses (see Table S5 for the full list of genes). Only Mapman bins or sub-bins with a significant P-value (< 0.05) following a Mann–Whitney U-test and containing at least 10 genes are represented on this graph (log2 expression ratio (nfp-2 1 dpi vs WT 1 dpi) > 0.97 for more highly expressed genes in nfp-2 and log2 expression ratio (nfp-2 1 dpi vs WT 1 dpi) < −0.97 for more highly expressed genes in WT). (c) Left, genes belonging to specific responses (see legend of Venn diagram in panel a) were selected and their ratios of expression between nfp and WT, at 1 dpi, were analysed to identify which ones were significantly more highly expressed in each line. A detailed list of these genes is given in Table S6. Right, distribution of genes belonging to specific responses in functional classes as defined by Mapman software. (see Table S6 for the full list of genes). Only Mapman bins or sub-bins with a significant P-value (< 0.05) following a Mann–Whitney U-test and containing at least 10 genes are represented on this graph.

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Comparisons between sets of genes which were found induced or repressed after A. euteiches inoculation in each line revealed a common molecular response to the pathogen (758 induced and 358 repressed genes; Fig. 3a,b, Table S5), but also a large set of specific genes (3042) whose expression is induced or repressed in either only WT or nfp-2 plants (Table S6). A set of 29 genes belonging to common (11 genes in the most significant functional classes – see later and Fig. 3b) or specific responses (six genes induced only in WT and 12 genes only repressed or induced in nfp-2) were analysed by qRT-PCR. Expression results showed a very good correlation with the microarray data (Table S7) and therefore validate the transcriptome results.

The common response to A. euteiches 1 dpi (Table S5) observed in WT and nfp plants is summarized in Fig. 3(b) using the Mapman classification. Statistically significant regulated functional categories (with a proportion of regulated genes significantly higher than the mean proportion among all the annotated categories of genes), containing at least 10 members, are indicated. Most of these genes correspond to functional classes that are related to plant defence responses or signalling. Genes encoding > 40 kinases, along with genes of the ethylene pathway, were the most significant classes associated with signal transduction, while a significant number of genes associated with ROS scavenging and signalling (i.e. peroxidases (POX) and glutathione-S-transferase (GST)) were also identified. The defence response common to both genotypes was associated with the induction of several genes encoding pathogenesis-related proteins (e.g. chitinases, thaumatin-like protein, PR-1, PR-10, protease inhibitors) that are among the most induced genes. A very strong activation of secondary metabolism was also observed, with many genes induced at different steps of the medicarpin biosynthesis pathway (isoflavonoid pathway), or in the phenylpropanoid pathway leading to lignin synthesis. The key role of secondary metabolism is also reinforced by the significant number of genes belonging to the large family of cytochrome P450s (CYP450), known to participate in a variety of biochemical pathways to produce plant hormonal or defence compounds (Mizutani & Ohta, 2010). Genes involved in cell wall structure were also significantly regulated, especially genes involved in cellulose metabolism (mainly induced) or genes encoding structural arabinogalactan proteins (AGPs) or pectin modelling enzymes (mainly down-regulated; Table S5). Finally, the 20 significantly induced amino acid metabolism genes (notably associated with aromatic amino acid biosynthesis) show that this pathway is also an important component of the common defence response between WT and nfp plants. More than 70% of these genes were also regulated in a similar way in M. truncatula (Table S5) by the fungal root pathogen Phymatotrichopsis omnivora (Uppalapati et al., 2009).

Specific responses involved a large set of 3042 (343 + 821 + 517 + 1361) genes (Fig. 3a). Among them, a higher number of genes, either induced or (mainly) repressed, were detected specifically in nfp plants. For those genes found to be specifically differentially expressed after inoculation of plants (vs non inoculated plants), we compared expression ratios between nfp-2 and WT, 1 dpi to identify the most differentially regulated genes between the two lines. This revealed 544 genes that showed a significant (< 9.8231E–07) difference of expression between inoculated WT and nfp-2 plants (Fig. 3c, Table S6); 323 were significantly more highly expressed in WT and 221 were significantly more highly expressed in nfp-2 plants. A Mann–Whitney U-test applied to this set of genes significantly (< 0.05) highlighted seven annotated Mapman bins or sub-bins (containing a minimum of 10 genes) that are shown in Fig. 3(c) and Table S6. Among these bins, the most significant is ‘regulation of transcription’, which mainly contains genes encoding transcription factors. Two-thirds of these were more highly expressed in nfp plants. ‘DNA synthesis and chromatin structure’ was surprisingly detected as the second most significant bin. All the 27 genes of this class, which mainly encode enzymes involved in DNA replication or histone protein synthesis, are more highly expressed in WT plants. Genes in the cell wall bin, notably encoding cell wall-remodelling enzymes or polygalacturonase inhibiting proteins (PGIPs), were more highly expressed in WT plants. Many genes encoding transporters involved in membrane-trafficking events, along with genes encoding proteins associated with the cytoskeleton or involved in the cell cycle, were significantly repressed in nfp compared with WT plants. Finally, signalling functions associated with genes encoding kinases and also with G-proteins (see Table S6) were largely down-regulated in nfp plants and are thus found significantly more highly expressed in the WT (Fig. 3c). Only 3.5% of these genes were regulated in a similar way by the fungal root pathogen P. omnivora (Uppalapati et al., 2009; data not shown).

In conclusion, this analysis indicates that 1 d after A. euteiches inoculation, the lack of a functional NFP gene does not prevent the activation of some classical defence-related pathways, but leads to a strong alteration in gene expression predicted to affect several cell dynamic processes.

nfp mutants are more susceptible to Colletotrichum trifolii

To assess the potential role of NFP in resistance against other pathogens, we used C. trifolii, a true fungus, to inoculate WT, lyk3 and nfp mutant plants. This hemibiotrophic fungus has already been shown to infect M. truncatula roots (Genre et al., 2009; Kloppholz et al., 2011), and we established an in vitro test involving root inoculation. First, when the symptoms are still weak, 8 dpi, a molecular quantification of a C. trifolii gene encoding an α elongation factor by qRT-PCR, indicated that there was two times more fungus in nfp mutant plants than in the WT (Fig. 4a). Later, 14 dpi, inoculated roots displayed a brown coloration that spread on both sides from the inoculation zone. Measurements of the relative part of symptomatic tissues confirmed significant differences between WT and nfp plants (Fig. 4b). At this time, the observed phenotypes (symptoms and plant development) were indeed different between the two lines. C. trifolii did not affect WT seedling growth such that WT aerial parts, along with root tips, remained symptomless. By contrast, inoculated nfp mutants were smaller than the WT; both aerial parts and roots were less developed and the entire root of each mutant seedling was brown (Fig. 4c). At all analysed time-points, inoculated lyk-3 mutant plants displayed similar responses to A17 (Fig. 4a,b).

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Figure 4. Medicago truncatula nfp mutants are more susceptible to Colletotrichum trifolii. (a) Relative quantification of cDNA transcripts of a C. trifolii gene encoding an α-elongation factor in inoculated plants at 8 dpi (means of three independent repeats with = 15 plants per line). *, < 0.05 (t-test). (b) Percentages of symptomatic tissues on plants (roots and stems) at 14 dpi. Significant differences were detected for symptoms (t-test): **, < 0.01. (c) Phenotypes of wildtype (WT) and nfp plants at 14 dpi (bars, 1 cm). In all experiments, plants were inoculated with a droplet of C. trifolii conidia on the surface of middle of the primary root. Error bars represent ± SE.

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These results indicate that nfp, but not lyk3, mutants are more susceptible than WT plants to C. trifolii.

CO perception by WT and nfp mutant plants

Since nfp mutants are more susceptible to two filamentous microorganisms, we wondered whether the decrease in resistance might be linked to a defect in perception of COs, which are presumably found as structural units of the cell wall of both A. euteiches and C. trifolii. To answer this question, a preparation containing CO fragments (with a DP mainly between 6 and 8) was applied to the roots of young seedlings. In response to CO treatment, extracellular ROS production, an early signalling marker, and the expression of six genes encoding defence-associated proteins (a Pathogenesis-Related 10.2 protein (PR10.2), a thaumatin (THA), a germin-like protein (GLP), a protease inhibitor (PI), an oxophytodienoic acid reductase (OPR) and a strictosidine synthase (SS), either common to the WT and nfp-2 transcriptomic responses (PR10.2, THA, GLP, PI) or specific to the WT transcriptomic response (OPR, SS)), was assessed by chemiluminescence and qRT-PCR, respectively (Fig. 5). None of the measured parameters was significantly different between WT and nfp mutant plants, suggesting that COs can be correctly perceived by nfp plants. Consistent with this, those genes that were found to be induced by CO treatment (PR10.2, THA, GLP and PI) were commonly induced by A. euteiches in both genotypes (Table S5).

image

Figure 5. Early and later responses in Medicago truncatula wildtype (WT) and nfp-2 plants after CO treatment. (a) Measurements of extracellular reactive oxygen species (ROS) production released by WT (grey bars) and nfp-2 (black bars) roots 20 min after CO treatment. Data are means of three repeats, each with nine plants. Bars represent ± SE of the mean. (b) Gene expression data of several defence-related genes in WT (grey bars) and nfp-2 (black bars) plants following treatment by COs for 4 h. Data are means of log2 values of gene expression ratios comparing CO-treated plants with water-treated plants. Similar conditions as in (a) were used to calculate these means and the error bars. THA, thaumatin like protein; GLP, germin like protein; PI, protease inhibitor; OPR, oxophytodienoate reductase; SS, strictosidine synthase.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In plants, LysM-domain receptor proteins are responsible for recognizing microbe-derived signals containing GlcNAc residues. Some of these receptors are involved in pathogen recognition while others are key players of symbiont perception for establishment of symbiotic interactions. In this work, we showed that M. truncatula NFP, initially described as a putative NF receptor, can also play a role in defence against pathogens. Thus, results of the multiple (phenotypic, molecular and cytological) analyses performed on mutants of two LysM-RLKs clearly demonstrated that both allelic nfp mutants, but not a lyk3 mutant, display increased susceptibility to A. euteiches compared with WT. A previous classical genetic approach performed on a recombinant inbred line population derived from a cross between A17 and the susceptible natural line F83005.5 led to the identification of a single major quantitative trait locus (QTL), called prAe1, on the distal part of the chromosome 3 (Djébali et al., 2009). MtNFP does not belong to prAe1, as it is located on chromosome 5 (Arrighi et al., 2006). These two results are, however, not contradictory as it was shown that prAe1 explained only one-third of the observed resistance, indicating that other loci involved in A. euteiches resistance presumably exist but were not detected by this QTL analysis. The reverse genetic approach developed in this work is therefore complementary and identifies MtNFP as a new component of A17 quantitative resistance to A. euteiches. As nfp mutants were also found to be more susceptible to the true fungus C. trifolii, all these results suggest that NFP can possibly be involved in a nonspecific basal resistance mechanism to multiple pathogens.

To explain which molecular mechanisms involved in plant protection might be controlled by NFP, transcriptome analyses were performed at an early stage of A. euteiches infection to compare WT and nfp mutant plant responses. Analyses of noninoculated plants revealed only a very limited set of genes differentially expressed between WT and nfp, suggesting that the mutation of NFP does not significantly modify the transcriptome of healthy plants. Our results indicate that, upon A. euteiches infection, nfp mutant plants are able to mount classical signalling and defence responses to a pathogen, which notably include the induction of expression of many kinases and of the ethylene synthesis and signalling pathways, associated with induction of secondary metabolism and PR-protein genes. As this response is very similar to that observed against another fungal root pathogen, Phymatotrichopsis omnivora, (Uppalapati et al., 2009), this suggests that an NFP-independent core defence transcriptome is activated upon pathogen infection in the M. truncatula A17 line.

Analysis of significant differences in gene expression levels between WT and nfp-inoculated plants led to the identification of c. 550 genes. This set of genes revealed that upon A. euteiches infection, NFP negatively regulates a significant number of transcription factors, and is also responsible for the induction of genes encoding cell wall-remodelling enzymes, cytoskeleton components, membrane transporters, signalling G-proteins, cell division proteins and proteins controlling DNA synthesis and chromatin structure (Table S6). Hence, it appears that the higher degree of resistance observed in WT could be linked to a positive role of NFP in processes associated with cell dynamics. Most of these different processes could be responsible directly or indirectly for an improvement of plant defences. For example, cell wall remodelling, cytoskeleton reorganization and membrane transporters can strengthen structural cell barriers (Huckelhoven, 2007). Moreover, pathogenic fungi and oomycetes often induce cytoplasmic reorganizations in the form of cytoplasmic aggregation at the site of penetration. This cytoskeleton-driven accumulation of organelles and cytoskeleton-dependent transport of secretory products to the infection site contribute to both the formation of cell wall appositions and the localized release of defence-related compounds, and thus to penetration resistance. In Arabidopsis, such a resistance to powdery mildew requires the syntaxin PENETRATION (PEN)1 (Nielsen et al., 2012). Interestingly, a syntaxin gene is found among the genes significantly more highly expressed in the WT than in the nfp mutant after A. euteiches inoculation (Table S6). Identification of several genes encoding G-signalling proteins more highly expressed in the WT reinforced a putative role of NFP in controlling processes associated with secretion at the membrane level and are coherent with the recent finding that a member of this protein family is involved in resistance signalling to A. euteiches (Kiirika et al., 2012).

The higher number of more highly expressed genes involved in cell divisions and DNA synthesis in the WT might only be associated with a more active state of the infected WT root cells. However, the surprisingly significant number of genes encoding histones, which are all repressed in inoculated nfp but not in WT plants, might also reflect the roles of such proteins in the regulation of gene expression, including some that are directly linked to activation of plant defence, as recently demonstrated, via acetylation/deacetylation mechanisms (Ding et al., 2012).

Consistent with the expression of NFP in M. truncatula root hairs (Arrighi et al., 2006), the large differences in gene expression profiles between nfp and WT plants at 1 dpi, a stage where germinated zoospores just start entering the rhizodermis, indicate an early role of NFP in improving resistance to A. euteiches. Because NFP is also involved in resistance to C. trifolii and both microorganisms have in common the presence of chitin or chitin-like compounds in their cell wall, we assessed a putative role of NFP in perception of chitooligosaccharidic PAMPs by comparing WT and nfp responses with a CO preparation containing fragments with an average DP of 7, as larger COs were reported to be most active in defence induction in Arabidopsis and rice (Kombrink et al., 2011). This revealed that early (ROS production) and later (defence gene induction) responses were similar in both lines, indicating that nfp mutants are probably not impaired in chitin perception. Similarly, CO-induced expression of defence-related genes was not affected in L. japonicus nfr5 mutants (LjNFR5 is the likely ortholog of NFP; Nakagawa et al., 2011). In addition, a recent study in M. truncatula, which showed that CO fragments present in mycorrhizal spore exudates can also be perceived as symbiotic signals, inducing DMI1- and DMI2-dependent nuclear calcium spiking in root cells, ruled out the involvement of NFP in this CO-induced response (Genre et al., 2013). Taken together, these data suggest that NFP, in addition to being involved in the perception of symbiotic signals only in the form of COs modified by acylation (LCOs), might also be involved in the perception of GlcNAc-containing PAMPs with distinctive structural features when compared with COs. Studies to analyse M. truncatula responses to PAMPs derived from A. euteiches and C. trifolii will require the purification of such molecules, and these might be different between the two organisms, given the finding that the chitosaccharides present in the cell wall of A. euteiches are noncrystalline and associated with glucans (Badreddine et al., 2008).

Among the observed phenotypes, there was also a striking difference between A. euteiches-inoculated nfp and WT plants in the amount of stele protection. The invasion of the central cylinder seen in about half of the inoculated nfp roots, compared with only 5% in inoculated WT roots, might indicate an additional role for NFP, when hyphae have colonized the root cortex. Given the low levels of NFP expression detected at 6 dpi in all genotypes, it is likely that this late phenotype in nfp plants results solely from a default in pathogen perception at the early stage of infection. However, we cannot rule out a significant presence of the NFP protein at 6 dpi and in this case another hypothesis would predict that an NFP-dependent mechanism is also implicated at later stages of pathogen infection. This situation would then be similar to that occurring during rhizobial symbiosis in which NFP controls both initial NF perception and the infection process (Arrighi et al., 2006; Bensmihen et al., 2011). In addition, NFP controls cortical cell division for nodule organogenesis and both NF- and Myc-LCO-induced stimulation of lateral root formation (Olah et al., 2005). Thus, NFP could control different pathways in both pathogenic and symbiotic interactions. LYK3, in contrast to NFP, is implicated in the specific recognition of NF structure (Limpens et al., 2003; Smit et al., 2007), which supports a symbiosis-specific role for LYK3.

In addition to the well established symbiotic role of NFP, our work extends the role of NFP to pathogenic interactions, raising questions about how specific responses are controlled by NFP in each of these situations. By its dual role in plant adaptation to biotic interactions and in some aspects of plant development, NFP is reminiscent of the BAK1 receptor of Arabidopsis, involved in both PTI and in plant growth regulation (as an enhancer of brassinosteroid signalling; Chinchilla et al., 2009). By analogy to the functioning of BAK1, a likely hypothesis to explain the multiple functions of NFP is its involvement in different heteromeric complexes with other molecular partners that would participate in triggering the appropriate downstream plant response. This is supported by the dead kinase domain of NFP (Arrighi et al., 2006), and the recent suggestion that NFP is not solely responsible for the perception of cognate NFs (Bensmihen et al., 2011). Candidate receptors that would associate with NFP to perceive PAMPs remain to be identified to further understand how this LysM receptor participates in the discrimination of microbial signals for the control of opposite plant responses.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank Drs E-P. Journet and F. de Carvalho-Niebel for providing nfp-2 seeds; C. Remblière and S. Camut for production of the 35S::NFP transgenic plants and S. Danoun for MALDI-TOF mass spectrometry analysis of the chitooligosaccharide samples generated from crab shell chitin. This work was funded by the Région Midi-Pyrénées, the CNRS (PhD grant INEE 36 to A.N.), the Université Paul Sabatier, the Ministère de l'Enseignement Supérieur et de la Recherche (PhD grant to T.R.) and the French Agence Nationale de la Recherche (ANR-08-BLAN-0208-01 ‘Sympasignal’) and was performed in the LRSV, part of the ‘Laboratoire d'Excellence’ (LABEX) entitled TULIP (ANR-10-LABX-41).

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

FilenameFormatSizeDescription
nph12198-sup-0001-MethodS1.docxWord document24KMethods S1 Information regarding the production of transgenic plants overexpressing NFP, microarray experiments and chitin oligomer preparation.
nph12198-sup-0002-TableS1-S7.xlsxapplication/msexcel3579K

Table S1 List of primers used for qRT-PCR validation of transcriptome experiments and pathogen quantification

Table S2 List of primers used for qRT-PCR to analyse defence-related gene expression following CO treatment

Table S3 Complete list of genes whose P-value is under the threshold given by the Bonferroni-corrected P-value (i.e. < 9.8231E-07), in at least one of the analysed conditions

Table S4 List of genes whose expression is significantly different between nfp-2 and WT in noninoculated plants

Table S5 List of genes induced or repressed in WT and nfp-2, 1 d after A. euteiches inoculation

Table S6 List of genes extracted from the Venn diagram in Fig. 3(a) that belong to specific responses of each line and which are significantly more highly expressed in WT or in nfp-2 inoculated plants

Table S7 qRT-PCR validation of the transcriptome data