A Novel Positive Regulator of the Early Stages of Root Nodule Symbiosis Identified by Phosphoproteomics

Abstract

Signals and signaling pathways underlying the symbiosis between legumes and rhizobia have been studied extensively over the past decades. In a previous phosphoproteomic study on the Medicago truncatula–Sinorhizobium meliloti symbiosis, we identified plant proteins that are differentially phosphorylated upon the perception of rhizobial signals, called Nod factors. In this study, we provide experimental evidence that one of these proteins, Early Phosphorylated Protein 1 (EPP1), is required for the initiation of this symbiosis. Upon inoculation with rhizobia, MtEPP1 expression was induced in curled root hairs. Down-regulation of MtEPP1 in M. truncatula roots almost abolished calcium spiking, reduced the expression of essential symbiosis-related genes (MtNIN, MtNF-YB1, MtERN1 and MtENOD40) and strongly decreased nodule development. Phylogenetic analyses revealed that orthologs of MtEPP1 are present in legumes and specifically in plant species able to host arbuscular mycorrhizal fungi, suggesting a possible role in this association too. Short chitin oligomers induced the phosphorylation of MtEPP1 like Nod factors. However, the down-regulation of MtEPP1 affected the colonization of M. truncatula roots by arbuscular mycorrhizal fungi only moderately. Altogether, these findings indicate that MtEPP1 is essential for the establishment of the legume–rhizobia symbiosis but might plays a limited role in the arbuscular mycorrhizal symbiosis.

Introduction

Legumes can fulfill a significant part of their nutrient needs through symbiotic associations with nitrogen-fixing rhizobia and arbuscular mycorrhizal (AM) fungi (Venkateshwaran et al. 2013, Castro-Guerrero et al. 2016). These symbioses are initiated by a signal exchange between the beneficial microbes and their legume partners. In the symbiosis between legumes and rhizobia (hereafter referred to as root nodule symbiosis), the interaction begins with the detection by the rhizobia of flavonoids and isoflavonoids produced by legume roots (Peters et al. 1986, Liu and Murray 2016). In turn, rhizobia produce diffusible ‘Nod factors’ (NF) that are lipo-chitooligosaccharides with specific chemical decorations (Dénarié et al. 1996). The legume host perceives NF via the LysM-domain receptor kinases NF Perception (NFP), LysM domain Receptor-like Kinase 3 (LYK3) and probably by an additional epidermal LysM receptor (Amor et al. 2003, Limpens et al. 2003, Arrighi et al. 2006, Murakami et al. 2018). A similar dialog between plants and AM fungi also involves the detection of a mixture of chitooligosaccharides, including lipo-chitooligosaccharides, which are produced by AM fungi (Genre et al. 2013, Sun et al. 2015, Luginbuehl and Oldroyd 2017). The mixture of chitooligosaccharides produced by AM fungi constitute the so-called ‘mycorrhization factors’ (MF) (Maillet et al. 2011). Upon perception of NF or MF, the transcription and phosphorylation of several symbiosis-related genes and proteins are activated, respectively. These molecular responses are crucial for triggering cellular rearrangements allowing the accommodation of either rhizobia or AM fungi in legume roots (Venkateshwaran et al. 2013, Luginbuehl and Oldroyd 2017).

Despite differences in host range and cellular processes, these two endosymbioses share striking similarities. Genetic studies in the model legumes Medicago truncatula and Lotus japonicus have allowed the characterization of a ‘common symbiosis pathway’ (CSP) that is required for the establishment of symbiotic associations with both rhizobia and AM fungi (Lace and Ott 2018, Wang et al. 2018). Among the components of the CSP in M. truncatula, the leucine-rich repeat (LRR) receptor-like kinase, Does not Make Infections 2 (DMI2), which is localized at the plasma membrane, is assumed to participate in a receptor complex to perceive both NF and MF (Singh and Parniske 2012, Venkateshwaran et al. 2013, Luginbuehl and Oldroyd 2017). Another subset of proteins participating in the CSP includes different types of ion channels, such as the cation channel DMI1 localized at the nuclear envelope, calcium channels of the CNGC15 family, the calcium pump MCA8 and nucleoporins (NUP85, NUP133 and NENA). This set of ion channels is required to generate rapid oscillations in the nuclear and perinuclear calcium concentrations known as calcium spiking (Ané et al. 2004, Kanamori et al. 2006, Peiter et al. 2007, Saito et al. 2007, Groth et al. 2010, Charpentier et al. 2016). We demonstrated that mevalonate production is necessary and sufficient to activate calcium spiking in M. truncatula roots in response to rhizobial and AM fungal signals (Venkateshwaran et al. 2015). These calcium signatures are decoded by a nuclear calcium/calmodulin-dependent protein kinase (CCaMK/DMI3) that further transduces the signal by phosphorylating the transcription factor IPD3/CYCLOPS (Lévy et al. 2004, Miller et al. 2013, Singh et al. 2014). Downstream, different symbiosis-related transcription factors, such as Nodulation Signaling Pathway1 (NSP1)/NSP2, Nodule Inception (NIN), Ethylene Response Factor Required for Nodulation1 (ERN1) and Nuclear Factor YA-1 (NF-YA1)/(NF-YB1), are activated. These transcription factors act in a co-ordinated fashion to regulate the expression of a variety of genes required for the symbiosis with rhizobia and, for some of them, AM fungi (Genre and Russo 2016). Substantial evidence indicates that mutations in any of these core genes impair the signal transduction leading to the abortion of the symbiosis with both rhizobia and AM fungi.

It is widely accepted that CSP is a critical module for initiating both rhizobia and AM symbiosis (Wang et al. 2018). Recent studies have identified a variety of genes that are probably essential components of both rhizobial and AM symbioses. Hence, it has been suggested that those genes might be considered as new members of the CSP. Unfortunately, after detailed analysis, it has been demonstrated that several mutants of the proposed new members of the CSP are still able to establish symbiosis with one or both symbionts (reviewed in Lace and Ott 2018). However, it has also been proposed that genes falling in the first scenario (able still to interact with one of the symbionts) could be classified as members of an auxiliary pathway providing specificity for a particular symbiont (Lace and Ott 2018).

Although over the past two decades, considerable progress has been made to understand the early responses that control the establishment of the symbioses with rhizobia and AM fungi, many components of the signaling cascade controlling these symbioses remain unknown. For instance, the mechanisms of signal transduction from the plasma membrane to the nucleus remain unclear. Given the involvement of several active kinases (e.g. LYK3, DMI2 and DMI3) in this pathway, phosphorylation events are expected to be critical regulators in responses to symbiotic signals. In an attempt to identify new regulators, we previously performed a quantitative phosphoproteomic analysis in M. truncatula roots treated with purified NF from Sinorhizobium meliloti (Rose et al. 2012). Through this analysis, we identified 66 differentially phosphorylated proteins. Rapid phosphorylation of these proteins suggests their involvement in the early steps of this symbiosis. Unfortunately, the majority of these phosphorylated proteins have not yet been functionally characterized.

In this study, we characterized in detail one of these proteins whose phosphorylation levels increased consistently in response to purified NF in M. truncatula (Rose et al. 2012), hereafter termed Early Phosphorylated Protein1 (MtEPP1). An expression profile analysis by quantitative real-time PCR (qRT-PCR) revealed that MtEPP1 is preferentially expressed in roots and nodules. Further expression analyses indicated that upon rhizobial inoculation, MtEPP1 is expressed in curled root hairs. Down-regulation of the MtEPP1 gene in M. truncatula transgenic roots almost abolished the NF-triggered calcium spiking, interfered with the expression of critical symbiosis-related genes (MtNIN, MtNF-YB1, MtERN1 and MtENOD40) and severely decreased nodule development. Phylogenetic and synteny analyses indicated that MtEPP1 is conserved in most land plants, even present in charophyte algae, but absent in the genome of non-host plants for AM fungi (e.g. Arabidopsis thaliana). This evolutionary pattern was similar to that of other genes involved in AM symbiosis (Delaux et al. 2014, Delaux et al. 2015). Despite this characteristic and that CO4 (N,N‘,N’',N'''-tetraacetyl chitotetraose) also triggered the phosphorylation of MtEPP1, the down-regulation of MtEPP1 did not severely affect the root colonization by AM fungi. The data presented here indicate that MtEPP1 is an essential component for the establishment of the root nodule symbiosis, but more dispensable for the symbiosis with AM fungi.

Results

MtEPP1 is a cytoplasmic protein and preferentially expressed in roots and nodules of M. truncatula

We previously reported that 66 proteins were differentially phosphorylated upon a 1 h treatment with purified NF (Rose et al. 2012). Among them, MtEPP1 (Medtr3g099200) was one of the most highly and consistently phosphorylated proteins (2.7-fold change), on Ser77. The MtEPP1 gene contains two introns and three exons (Fig. 1A). It encodes a predicted protein of 41.6 kDa with an α-helix, a β-strand and four disordered regions (Fig. 1B;Supplementary Fig. S1). Additionally, MtEPP1 protein contains: (i) a putative pyridoxal phosphate-dependent transferase (IPR015421) domain that is tentatively located between amino acids 233 and 333 and is highly conserved among legumes (Supplementary Fig. S2); (ii) one serine/threonine-rich region located between amino acids 128 and 150; and (iii) five potential protein-binding sites.

Analysis of the MtEPP1 protein sequence using protein localization prediction programs, such as SignalP 4.1 (Petersen et al. 2011), TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/) and PREDICT PROTEIN server (https://www.predictprotein.org) indicated the absence of signal peptide or potential transmembrane helices, suggesting that MtEPP1 protein is soluble and cytoplasmic. To corroborate this in silico prediction, the MtEPP1 coding region was fused to enhanced green fluorescent protein (eGFP) in the C-terminus under the transcriptional regulation of the 35S promoter and expressed in transgenic M. truncatula roots by infection with Agrobacterium rhizogenes (Fig. 2) or in Nicotiana benthamiana leaves by agroinfiltration with Agrobacterium tumefaciens (Supplementary Fig. S3). Transgenic roots or leaves expressing non-fused eGFP were used as a control. As expected, the non-fused eGFP was observed in both the cytoplasm and nuclei of M. truncatula root cells and N. benthamiana leaf cells (Fig. 2; Supplementary Fig. S3). Green fluorescence (MtEPP1::eGFP) was detected in the cytoplasm of root cells, root hairs and leaf cells, confirming the predictions of the cytoplasmic localization of MtEPP1.

To gain further insight into the expression profile of MtEPP1, we performed a data-mining analysis on transcriptional data from M. truncatula available in the M. truncatula Transcriptome Atlas (https://mtgea.noble.org/v3/; Benedito et al. 2008). Based on available transcription data in M. truncatula, MtEPP1 shows high expression in roots, roots hairs and nodules, and a weak expression in leaves and flowers. To confirm this observation, we evaluated the expression of MtEPP1 in leaves, roots, and nodules from M. truncatula plants by qRT-PCR. This expression analysis by qRT-PCR confirmed the preferential expression of MtEPP1 in roots and nodules (Fig. 1C). Altogether, these data indicate that MtEPP1 is preferentially expressed in roots and nodules and that its protein is located in the cytoplasm.

The expression of MtEPP1 is induced in root hairs upon NF treatment

The fact that MtEPP1 was rapidly phosphorylated upon a 1 h treatment with purified NF, and that its gene is preferentially expressed in roots and nodules, prompted us to evaluate whether these symbiotic signals regulate the expression of MtEPP1. To test this hypothesis, we assessed the expression of MtEPP1 in roots of the wild-type genotype Jemalong A17 treated for 1 h with NF. This expression analysis revealed no significant differences in MtEPP1 expression in response to the NF within this time frame (Fig. 1C).

Given that specific genes can be expressed at particular cell types, assessing their expression in whole organs (e.g. roots) might result in dilution or even loss of signal. Recently, it was reported that Medtr3g099200, which encodes MtEPP1, is one of the M. truncatula genes displaying expression in root hairs (Damiani et al. 2016). Additionally, in the same study, it was reported that the expression of Medtr3g099200 was slightly (fold change of 1.5) induced after 20 h of treatment with purified NF (Damiani et al. 2016). Based on this evidence, we evaluated the spatiotemporal activity of the MtEPP1 promoter in response to purified NF. We cloned a 2 kb fragment of the MtEPP1 promoter (pMtEPP1) that was immediately upstream of the MtEPP1 translation initiation codon and generated transcriptional fusion to the GUS (β-glucuronidase) coding sequence. The pMtEPP1::GUS construct was transfected into M. truncatula through the A. rhizogenes-mediated transformation method, and transgenic roots were treated with NF for 1 h. We observed GUS activity in the entire root (Fig. 3A), with some faint staining in the root hairs of mock-inoculated transgenic roots (Fig. 3C). In contrast, we observed strong staining in the root hairs from NF-inoculated transgenic roots (Fig. 3B, D). This spatiotemporal activity was absent in root hairs from NF-inoculated transgenic roots generated in the M. truncatula nfp-1 mutant (Fig. 3E–H). Altogether, these data indicate that MtEPP1 expression is up-regulated in root hairs upon NF recognition.

The expression of MtEPP1 is induced in rhizobia-induced curled root hairs

Because we observed a definite increase in the activity of the MtEPP1 promoter in the root hairs upon NF treatment, and as rhizobia colonize the legume roots through the infection thread, we investigated whether the activity of the MtEPP1 promoter is located at specific sites of the root hairs in response to rhizobia. Transgenic roots generated in the wild-type genotype Jemalong A17 and expressing the transcriptional fusion pMtEPP1::GUS were inoculated with S. meliloti. At 3 d post-inoculation, we observed a substantial activity of the MtEPP1 promoter in curled root hairs (Fig. 4). These data indicate that MtEPP1 expression is induced in rhizobia-induced curled root hairs.

MtEPP1 is required for root nodulation

Because NF triggered MtEPP1 phosphorylation and NF and rhizobia induce the expression of its gene in the root hair, we thus hypothesized that MtEPP1 might play a role in the establishment of the symbiosis between M. truncatula and S. meliloti. To test this hypothesis genetically, we searched for mutants in the M. truncatula Tnt1-insertion mutant collection of the Noble Research Institute (Tadege et al. 2008) and identified three independent Tnt1-insertion lines, NF20083, NF14597 and NF20274, and heterozygous mutants were obtained (Fig. 1A). Despite many attempts and various experimental conditions tested, we have never been able to recover any homozygous mutant in the progeny of these heterozygous plants. Indeed, heterozygous plants showed similar expression levels of MtEPP1 as well as no significant differences in the number of nodules per plant. In the absence of homozygous mutants, we generated an RNA interference (RNAi) construct targeting MtEPP1 (MtEPP1-RNAi) and used A. rhizogenes-mediated transformation to silence MtEPP1 in the roots of M. truncatula. The expression of MtEPP1 in M. truncatula transgenic roots expressing the RNAi construct was reduced on average by 70% compared with the roots transformed with a control vector (Supplementary Fig. S4). Additionally, the expression of the three homologs of MtEPP1 was not affected in transgenic roots expressing the MtEPP1-RNAi construct, confirming the specificity of this construct (Supplementary Fig. S4). We observed that transgenic roots expressing the MtEPP1-RNAi construct were shorter compared with the transgenic roots expressing the control vector (Supplementary Fig. S5A). In spite of this reduction in the root growth, no apparent difference in the morphology of the MtEPP1-RNAi root hairs was observed (Supplementary Fig. S5B, C). To test whether the reduction in the expression of MtEPP1 affected the establishment of the symbiosis with S. meliloti, we performed nodulation assays on M. truncatula transgenic roots. These nodulation assays indicated that 85% (n = 200) of the control vector transgenics developed on average four nodules per plant. In contrast, 10% (n = 190) of the MtEPP1-RNAi transgenic roots developed one nodule per plant, whereas the other 90% of the MtEPP1-RNAi composite plants did not develop any nodules at all (Fig. 5A, B). These results demonstrate that down-regulation of MtEPP1 negatively and strongly affects nodule development.

MtEPP1 is required for NF-induced root hair branching and the activation of symbiotic gene expression

To test whether MtEPP1 is necessary to activate early responses of the legume–rhizobia symbiosis, transgenic roots expressing either MtEPP1-RNAi or control vector were treated with 10^–8 M purified NF. Sixteen hours after treatment, 90% (n = 100) of the control vector transgenic roots showed characteristic NF-induced root hair branching, whereas only 13% (n = 150) of MtEPP1-RNAi-transformed roots did (Fig. 5C, D;Supplementary Fig. S5D, E).

The fact that MtEPP1 is required to induce NF-induced root hair branching and nodule formation suggests that MtEPP1 is likely to be an essential component participating in the early responses of the legume–rhizobia symbiosis. To investigate further the role played by MtEPP1, we monitored the expression of MtENOD11, in response to NF. We used M. truncatula Jemalong A17 seedlings stably transformed with the promoter of MtENOD11 fused to the coding sequence of GUS, pMtENOD11-gusA (Journet et al. 2001). Agrobacterium rhizogenes-mediated root transformation was performed on these seedlings to silence MtEPP1. GUS activity was determined in roots after treatment with NF. Upon 16 h of NF treatment, we observed a substantial GUS activity in transgenic roots transformed with the control vector (Fig. 6A). In contrast, we saw only a weak GUS activity in MtEPP1-silenced transgenic roots. To validate this MtENOD11 expression data further, we evaluated its expression by qRT-PCR along with four additional symbiosis-related genes: MtNIN, MtERN1, MtNF-YB1 and MtENOD40. As expected, NF did not induce the expression of MtENOD11 in MtEPP1-silenced transgenic roots. In contrast, we observed a 1,000-fold change in control vector transgenic roots (Fig. 6B). Similar results were found when the expression of MtNIN, MtERN1 and MtNF-YB1 was analyzed, which were not induced upon NF treatment in MtEPP1-silenced transgenic roots (Fig. 6C, E). Although we detected an induction (2-fold change) in the expression of ENOD40 in transgenic roots expressing MtEPP1-RNAi, its expression level was much lower than the response observed in the transgenic control roots (Fig. 6F). Together, these results indicate that MtEPP1 is required to activate early events of the legume–rhizobia symbiosis.

MtEPP1 is required for NF-induced calcium spiking

A characteristic response to NF is the induction of nuclear and perinuclear calcium spiking that controls the activation of early nodulin gene expression (Ehrhardt et al. 1996, Wais et al. 2002). Because we observed a reduction in the expression of selected early nodulin genes (Fig. 6), we hypothesized that the down-regulation of MtEPP1 might affect the NF-induced calcium spiking. To test this hypothesis, we analyzed the calcium spiking triggered by NF in transgenic roots of M. truncatula expressing the calcium sensor, Yellow Chameleon 3.6 (YC3.6), along with either the MtEPP1-RNAi construct or control vector. We observed NF-induced calcium spiking in 75% (n = 15) of the analyzed control vector transgenic roots (Fig. 7A, B). In contrast, only 6% (n = 16) of MtEPP1-RNAi transgenic roots showed an irregular calcium spiking (lower intensity and less frequent irregular calcium spikes) (Fig. 7A, B). These results indicate that MtEPP1 is required to induce the calcium spiking triggered by NF.

MtEPP1 is present in legumes and host plants of arbuscular mycorrhizal fungi

Because the expression the MtNIN and MtENOD11 genes as well as the activation of the calcium spiking are required to establish symbiosis with both rhizobia and AM fungi (Kosuta et al. 2003, Boisson-Dernier et al. 2005, Guillotin et al. 2016), and the fact that the down-regulation of the MtEPP1 gene almost abolished these molecular responses, we hypothesized that MtEPP1 might also participate in the establishment of the symbiosis with AM fungi. To test this hypothesis, we first investigated whether MtEPP1 is present in the genome of host plants of AM fungi. To this aim, we constructed a single phylogeny analysis with available genomic and transcriptomic data. This analysis revealed the existence of potential orthologs of MtEPP1 in different legumes. We also found orthologs of MtEPP1 in those plants that can establish symbiosis with AM fungi, including liverworts (Supplementary Fig. S6A). In contrast, we did not find any homolog in the genomes of Arabidopsis thaliana, Arabidopsis lyrata, Aethionema arabicum, Brassica rapa, Capsella rubella, Thellungiella halophila (Brassicaceae), Utricularia gibba (Lentibulariaceae), Beta vulgaris (Amaranthaceae), Nelumbo nucifera (Nelumbonaceae), Pinus taeda, Picea abies (Gymnosperms, Pinaceae) and the moss Physcomitrella patens that are all non-host plants for either rhizobia or AM fungi (Khade et al. 2010, Delaux et al. 2014). To confirm the absence of EPP1 in non-host species, we conducted a synteny analysis on the surrounding region of MtEPP1 loci in M. truncatula, Amborella trichopoda (basal angiosperm), Gossypium raimondii (Malvales), Carica papaya (Brassicales) and the corresponding genomic block in the non-host A. thaliana Col-0, B. rapa, A. arabicum, sugar beet and N. nucifera. Whereas these blocks are well conserved between AM fungi host species, EPP1 is missing in the genome of all five non-host species (Supplementary Fig. S6B). Our data indicate that EPP1 is conserved in legumes and host plants of AM fungi, and might also be essential to interact with this symbiont.

Symbiotic signals of AM fungi induce the phosphorylation of MtEPP1

MtEPP1 was initially selected because of its significant phosphorylation on its Ser77 in response to NF (Rose et al. 2012). However, our phylogenetic analysis indicates the existence of MtEPP1 ortholog genes in the genome of host plants of AM fungi. Additionally, we found that the Ser77 residue is conserved in the EPP1 orthologs of different AM host plants (Supplementary Fig. S6C). With this evidence, we hypothesized that AM fungi symbiotic signals might also trigger the phosphorylation of MtEPP1. To determine whether phosphorylation of Ser77 of MtEPP1 is also induced by CO4, molecules produced by AM fungi, we used a multiple reaction monitoring approach. This targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach allows the quantifiction of a given analyte, a phosphopeptide in this case, using several m/z coming from the specific fragmentation of this analyte. Combined with a synthetic internal standard, this very sensitive approach allows accurate quantification of particular phosphosites. Total proteins were extracted from M. truncatula roots treated with CO4 or mock treated for 24 h. These samples were then used to determine the change in phosphorylation state of Ser77 on MtEPP1. Whereas synthesized standards are detected at a similar level in both cases, the amount of Ser77 phosphorylated peptides was significantly higher (∼30-fold change) in CO4-treated samples compared with mock-treated samples (Supplementary Fig. S7A, B). These results indicate that CO4 also triggers MtEPP1 phosphorylation.

To evaluate whether CO4 induces the expression of MtEPP1, roots from the wild-type Jemalong A17 genotype were treated with this AM fungi-derived molecule, and the expression of MtEPP1 was assessed by qRT-PCR. This expression analysis showed that CO4, like NF, did not induce the expression of MtEPP1 (Fig. 1C). Since MtEPP1 is expressed in the root hairs in response to purified NF, we evaluated the spatiotemporal activity of the pMtEPP1 promoter in response to CO4 by using the transcriptional fusion pMtEPP1::GUS expressed in M. truncatula transgenic roots (Supplementary Fig. S8). This spatiotemporal expression assay indicated that MtEPP1 is expressed in the root hairs upon treatment with CO4 (Supplementary Fig. S8). In summary, the MtEPP1 expression is up-regulated in root hairs, and its protein is phosphorylated upon CO4 treatment.

MtEPP1 plays a limited role in the establishment of the arbuscular mycorrhizal symbiosis

Finally, since MtEPP1 is essential to activate early symbiotic events and was found only in AM host plants, we hypothesized that MtEPP1 might be required for successful colonization by AM fungi. To test the potential role of MtEPP1 in AM symbiosis, we conducted mycorrhization assays on roots transformed either with the MtEPP1-RNAi construct or with the control vector. Six weeks post-inoculation, transgenic roots were screened for AM colonization. Silencing of MtEPP1 barely affected the symbiosis with AM fungi, which was reflected in a reduction by 10% of the density of arbuscules and other fungal structures (e.g. hyphopodia and vesicles) (Fig. 8). This result indicates that MtEPP1 plays a limited role in the AM symbiosis.

Discussion

Over the past two decades, remarkable progress has been made in understanding the genetic mechanisms underlying the establishment of the root nodule symbiosis. For instance, a CSP controlling the molecular dialog with both rhizobia and AM fungi has been identified as a result of this intensive effort (Venkateshwaran et al. 2013, Genre and Russo 2016). Recent large-scale analyses on the early events occurring during the establishment of the root nodule symbiosis have uncovered the existence of potential new regulators. For instance, phosphoproteomic and transcriptomic analyses on M. truncatula roots treated for 1 h with purified NF enabled us to identify a significant number of differentially regulated genes and phosphorylated proteins (Rose et al. 2012). Similarly, Damiani et al. (2016) identified a set of genes that were differentially regulated at the root hairs upon 4 h treatment with purified NF. Nevertheless, few of these identified genes or phosphorylated proteins have been functionally characterized yet.

In this study, we provide evidence supporting the role of MtEPP1, one of the phosphorylated proteins identified in our previous phosphoproteomic analysis (Rose et al. 2012), in the molecular dialogue between M. truncatula and S. meliloti. In this study, we demonstrated that the expression of MtEPP1 is induced in curled root hairs in response to S. meliloti inoculation. Further experimentation on MtEPP1-silenced M. truncatula roots revealed that the activation of the NF-triggered calcium spiking, the expression of critical early nodulin genes (MtNIN, MtNF-YB, MtERN1, MtENOD11 and MtENOD40) and the NF-induced root hair branching were almost abolished. Accordingly, we showed that only 10% of the analyzed MtEPP1-silenced transgenic roots were able to develop just one nodule. Additionally, we showed that orthologous genes of MtEPP1 are only present in legumes and the host plants of AM fungi. Likewise, we demonstrated that CO4 also triggered the MtEPP1 phosphorylation. Nevertheless, the down-regulation of MtEPP1 did not lead to a dramatic reduction of the root colonization by AM fungi. These results led us to propose that MtEPP1 is an essential component for M. truncatula to communicate with rhizobia efficiently, but more dispensable to establish symbiosis with AM fungi. Alternatively, the presence of two paralogs of MtEPP1 in M. truncatula might result in genetic redundancy masking a potentially more pronounced AM phenotype.

Growing evidence indicates that the expression of genes controlling early events of the root nodule symbiosis is induced at the root hairs upon NF detection (Haney et al. 2011, Breakspear et al. 2014, Damiani et al. 2016, Arthikala et al. 2017). For instance, very recently it was reported that the MtNFH1 gene, encoding an NF hydrolase participating in the regulation of the NF levels, is expressed in curled root hairs in response to rhizobia (Cai et al. 2018). Similarly, Arthikala et al. (2017) reported that the PvRbohA gene, which is involved in rhizobia-triggered reactive oxygen species production, is expressed in the root hairs and in the growing infection thread. In this study, we found a similar spatiotemporal expression pattern for MtEPP1, which was detected in rhizobia-induced curled root hairs. These expression data indicate that MtEPP1 is a novel component required to activate early responses in the root nodule symbiosis.

The activation of the nuclear/perinuclear calcium spiking and its subsequent decoding by DMI3 is crucial for activating the transcriptional activator IPD3/CYCLOPS, which, in turn, enables the expression of the transcription factor genes NSP1/NSP2, NIN, NF-YA/B and ERN1 (Venkateshwaran et al. 2013, Genre and Russo 2016). These transcription factor genes are required to activate the expression of different symbiosis-related genes participating in root hair curling, infection chamber formation and entrapping of rhizobia (Cerri et al. 2012, Soyano et al. 2013, Singh et al. 2014, Lace and Ott 2018). It has been demonstrated that dmi1, dmi2, cngc15s, nena, nup85 and nup133 mutant plants are not able to activate calcium spiking in response to NF (Wais et al. 2000, Kanamori et al. 2006, Saito et al. 2007, Charpentier et al. 2016). Additionally, we have reported that mevalonate can trigger calcium spiking, probably through the activation of DMI1 (Venkateshwaran et al. 2015). Here, we demonstrated that the down-regulation of MtEPP1 almost abolished the NF-induced calcium spiking. Accordingly, we showed that the expression of MtNIN, MtNF-YA, MtERN1, MtENOD11 and MtENOD40, as well as the NF-triggered root hair branching, was dramatically reduced in MtEPP1-silenced roots. These data indicate that MtEPP1 is an essential element required to activate calcium spiking in response to rhizobia. Likewise, with these data it is also tempting to speculate that MtEPP1 might indirectly participate in the activation of genes coding for enzymes involved in mevalonate biosynthesis, for instance 3-hydroxy-3-methylglutaryl-CoA reductase1 (HMGR1).

It is widely demonstrated that NF and MF can activate the same core of genes and induce similar calcium signatures (Wang et al. 2018). However, it is still unclear how similar early molecular responses lead to the establishment of two different symbioses. Along this line, it has been proposed that CSP, along with the calcium spiking, is an essential module required to initiate symbiotic responses, but NF and MF might activate an independent auxiliary pathway providing specificity (Lace and Ott 2018). Evidence supporting this hypothesis has been presented through the overexpression of the NF receptors, which led to the activation of specific responses of the root nodule symbiosis (Reid et al. 2014). Here, we provide evidence indicating that MtEPP1 is conserved in legumes and AM fungi host plants, a hallmark of genes participating in the establishment of the symbiosis with AM fungi. Likewise, we demonstrated that CO4 also triggered the phosphorylation of MtEPP1. Despite this evidence, the down-regulation of MtEPP1 reduced the root colonization by AM fungi by 10%. Altogether, these data suggest that MtEPP1 plays a limited role in the symbiosis with AM fungi. Likewise, these data also provide evidence supporting the existence of an auxiliary pathway that provides specificity to rhizobia; however, further research is needed.

Conclusions

In conclusion, the data presented in this study led us to conclude that MtEPP1 is a novel component essential to trigger calcium spiking leading to the activation of early nodulin genes. The fact that the down-regulation of MtEPP1 did not compromise the symbiosis with AM prompted the suggestion that this gene might be dispensable to establish this endosymbiosis; however, further investigation is required to prove this hypothesis.

Materials and Methods

Plant material

Medicago truncatula line Jemalong A17, the R108 line, the transgenic line Jemalong A17 pMtENOD1-gusA (Journet et al. 2001), three Tnt1-insertion mutant lines of MtEPP1 (NF20083, NF14597 and NF20274) derived from R108, as well as nfp-1 (Amor et al. 2003), mutants derived from Jemalong A17, were used in this study. Seeds were scarified and surface-sterilized. Seeds were plated on 1% deionized water agar plates supplemented with 1 μM GA₃. Seeds were subsequently vernalized for precisely 48 h at 4°C and were then germinated by incubating them at room temperature overnight. On the next day, seedlings with equal root length were transferred into plates containing modified Fahräeus medium (Catoira et al. 2000) supplemented with 0.1 μM aminoethoxyvinylglycine (AVG) (Sigma-Aldrich). Fahräeus medium plates were incubated in a growth chamber at 21°C and 16 h of light.

Bacterial strains and culture conditions

The control vector pH7GWIWG2(II)-YC3.6 was propagated in Escherichia coli DB 3.1 cells, whereas the hairpin RNAi construct against MtEPP1 (see below for details), the transcriptional fusion MtEPP1::GUS and the translational fusion MtEPP1::eGFP were propagated in E. coli DH5α cells. Escherichia coli cells were handled using standard procedures.

The A. rhizogenes MSU440 strain was used to induce transgenic roots in M. truncatula plants (see below for details), whereas the A. tumefaciens GV3101 strain was used to transfect N. benthamiana leaves. Agrobacterium rhizogenes and A. tumefaciens cells were grown on 5 mg l^–1 tryptone/3 mg l^–1 yeast extract, 6 mM CaCl₂ (TY) plates for 2 d at 30°C; 50 μg ml^–1 spectinomycin or 50 μg ml^–1 kanamycin was added to select for the presence of plasmid vectors.

Sinorhizobium meliloti strain 1021 was used to inoculate M. truncatula plants. The S. meliloti cells were grown on TY plates supplemented with 50 μg ml^–1 streptomycin for 2 d at 30°C.

Mutant screening

To identify homozygous mutant lines of MtEPP1, we obtained the mutant lines NF20083, NF14597 and NF20274 from the Samuel Roberts Noble Foundation Tnt1-insertion mutant collection that carried a Tnt1 insertion in the coding sequence of MtEPP1. Genomic DNA from 200 individual plants (from each insertion line) was isolated by using the GeneCatch Plant Genomic DNA Purification Kit (Epoch Life Sciences). We used the gene-specific primer (5'-GCTTCAGCTATGATGTGAGCTGG-3') and the Tnt1-specific primer (5'-AGTTGGCTACCAATCCAACAAGGA-3') to identify homozygous Tnt1-insertion mutants by PCR.

Plasmid construction

To analyze the activity of the MtEPP1 promoter, a 2,073 bp DNA fragment upstream of the start codon was PCR-amplified from genomic DNA of M. truncatula A17 Jemalong by using specific primers. To generate the pMtEPP1::GUS construct, we used the Golden Gate cloning strategy as described by Servin-Pujol et al. (2017). The resulting pMtEPP1::GUS cassette was then cloned into the binary vector pCAMBIA_CR1 containing the constitutively expressed DsRED protein (Servin-Pujol et al. 2017).

To analyze the subcellular localization of MtEPP1, the full MtEPP1 CDS (coding sequence) without a stop codon was amplified. The resulting PCR product was then cloned into the pENTR-D-TOPO (Thermo Fisher Scientific) vector. The resulting pENTR-MtEPP1 plasmid was recombined into the pK7FWG2-RR binary vector containing the open CDS of eGFP, yielding the C-terminal MtEPP1::eGFP fusion.

To generate an RNAi construct against MtEPP1, a specific fragment of 350 bp from the C-terminal end of MtEPP1 was amplified using gene-specific primers. The amplified fragment was then cloned into the pENTR-D-TOPO (Thermo Fisher Scientific) vector. The resulting pENTR-MtEPP1-RNAi plasmid was recombined into the pH7GWIWG2(II)-YC3.6 binary vector containing the constitutively expressed fluorescent YC3.6 protein (Riely et al. 2011). The correct orientation was verified by PCR using the primers Forward-Intron (5'-GCACACCAGAGCATATATATTGGTGG-3') and Reverse-35SPromoter (5'-CCACTATCCTTCGCAAGACCCTTCC-3').

All constructs were verified by DNA sequencing. Primer sequences for plasmid constructions are shown in Supplementary Table S1.

Agrobacterium rhizogenes-mediated transformation

Binary vectors with pMtEPP1::GUS, MtEPP1::eGFP or MtEPP1-RNAi constructs were mobilized into A. rhizogenes MSU440 by electroporation. The empty vectors pH7GWIWG2(II)-YC3.6 or pK7FWG2-RR were used as controls. Agrobacterium rhizogenes-mediated transformation was performed according to Boisson-Dernier et al. (2001). Composite plants (plants with the transformed root system and untransformed shoot system) were grown in nitrogen-free Fahräeus medium plates under the environmental conditions described above. YC3.6 or DsRED fluorescence in the transgenic roots was observed with a fluorescence stereomicroscope.

Transfection of Nicotiana benthamiana leaves

Transient expression of the MtEPP1::eGFP fusion protein was conducted in N. benthamiana for subcellular localization of MtEPP1. Briefly, the abaxial epidermis of young leaves of 4-week-old N. benthamiana plants was infiltrated with A. tumefaciens GV3101 suspension harboring either MtEPP1::eGFP or the control vector construct. At 3 d post-infiltration, the infiltrated leaf areas were cut and analyzed under an LSM 510 Meta confocal microscope. The eGFP was excited at 489 nm, and the fluorescence emission was collected at 509 nm.

MtEPP1 promoter activity in response to NF and S. meliloti

To evaluate the MtEPP1 promoter activity, A. rhizogenes-mediated pMtEPP1::GUS transgenic roots were generated in the M. truncatula wild-type line Jemalong A17 as well as in the nfp-1 mutant plant. Composite plants were grown in Fahräeus medium plates under the environmental conditions described above. Roots were treated with 10 nM NF or inoculated with S. meliloti 1021. Upon 1 h NF treatment or 3 d post-rhizobia inoculation, transgenic roots showing DsRED fluorescence were immersed in GUS staining solution [0.05% 5-bromo-4-chloro-3-indolxyl-β-d-glucuronic acid, 100 mM sodium phosphate buffer (pH 7), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM Na₂EDTA and 0.1% Triton X-100] and incubated for 1 h at 37 °C. After being cleared in diluted NaClO and rinsing in phosphate buffer, roots were examined under bright-field microscopy. For these experiments, five biological replicates, each one with 10 composite plants, were included.

Histochemical assays for pMtENOD11::GUS

To analyze the expression of the symbiotic gene MtENOD11 by histochemical staining, A. rhizogenes-mediated MtEPP1-RNAi transgenic roots were generated in M. truncatula plants stably transformed with a pMtENOD11::GUS construct. Composite plants were treated with 10 nM NF for 16 h. Transgenic roots showing YC3.6 fluorescence were immersed in the GUS staining solution and incubated for 2 h at 37°C. After clearing in diluted NaClO and rinsing in phosphate buffer, roots were analyzed under a stereomicroscope and imaged using an EPSON V370 Photo scanner. For this experiment, five biological replicates, each one with 10 composite plants, were included.

Gene expression analysis

To analyze the expression of MtENOD11, MtENOD40 and MtNIN genes, composite plants were treated with 10 nM NF or mock treated (control) for 16 h as described above. For MtEPP1, MtERN1 and MtNF-YB1, composite plants were treated with 10 nM NF or mock treated for 1 h. NF-treated transgenic roots showing YC3.6 fluorescence were harvested, immediately frozen in liquid nitrogen and stored at –80°C until used. Total RNA was isolated from three NF-treated transgenic roots using the GeneCatch Plant RNA Purification kit (Epoch Life Sciences) following the manufacturer’s instructions. cDNA was synthesized from 1 μg of genomic DNA-free total RNA. This cDNA was used to analyze the above-mentioned symbiosis-related genes by qRT-PCR in a Step-One qPCR thermocycler (Applied Biosystems). The housekeeping gene MtActin was used to normalize gene expression levels. The expression level of different genes was calculated according to the equation E = P_eff^(–ΔCt). P_eff is the primer set efficiency computed using the LinRegPCR program (Ramakers et al. 2003), and ΔCt (cycle threshold) was calculated by subtracting the Ct value of the housekeeping gene from the Ct values of a given gene. The nucleotide sequences of the qRT-PCR primers used in this study are provided in Supplementary Table S1. For this experiment six biological replicates, each one with three technical replicates, were included.

Calcium spiking imaging and data analysis

To analyze the NF-induced calcium spiking, M. truncatula composite plants expressing the control vector pH7GWIWG2(II)-RNAi-YC3.6 (control) or the construct pH7GWIWG2(II)-MtEPP1-RNAi-YC3.6 were used. Composite plants with transgenic roots expressing the calcium sensor YC3.6 were selected based on strong yellow fluorescence under a fluorescence stereomicroscope. These composite plants (RNAi or control) were mounted on custom-made plastic slides using a modified Fahräeus medium to which 10 nM NF was added before calcium imaging. Calcium measurements were performed under a Zeiss LSM 510 Meta confocal microscope as reported by Riely et al. (2011). Briefly, young and emerging root hairs from the apical region of the root elongation zone were chosen for calcium measurements. The calcium sensor was excited at 458 nm using an argon laser. The cyan fluorescent protein (CFP; 473–505 nm) and Förster resonance energy transfer FRET; 536–546 nm) emissions were collected. For time-lapse analyses, images were obtained every 3.5 s. Background CFP and FRET signal intensities were subtracted. The FRET/CFP ratio was calculated and was plotted against time.

Root hair deformation analysis

Medicago truncatula composite plants expressing the construct MtEPP1-RNAi or control vector and growing in Fahräeus plates were treated with 10 nM purified NF from S. meliloti. Upon 16 h of treatment, transgenic roots showing YC3.6 fluorescence were observed with a bright-field microscope. For this experiment, 15 biological replicates, each one with 10 plants, were included.

Nodulation assay

Medicago truncatula composite plants expressing the construct MtEPP1-RNAi or control vector and growing in nitrogen-free Fahräeus medium plates were inoculated with 1 ml of S. meliloti 1021 with an OD₆₀₀ value of 0.01. The lower part of the dishes was wrapped in aluminum foil to keep the roots in the dark. Plates were then placed vertically in a growth chamber at 24°C in 16 h light/8 h dark cycle conditions. At 14 d post-inoculation, the total number of nodules was counted in roots showing YC3.6 fluorescence exclusively. For this experiment, 10 biological replicates, each one containing 20 plants, were used.

In vivo phosphorylation of EPP1 by mass spectrometry

To test the effect of hitooligosaccharides on MtEPP1 phosphorylation, plants were grown as previously described and treated with 100 nM CO4 or mock treated (0.5% ethanol in water) for 1 h. Plants were then quickly harvested and flash-frozen in liquid nitrogen, and ground with a mortar and pestle for phosphoprotein extraction. Samples were further homogenized in grinding buffer supplemented with phosphatase inhibitors using sonication (1 cm probe, 4×30 s and 50% duty cycle) while kept on ice as described by Minkoff et al. (2014). The resulting supernatant was filtered through four layers of Miracloth (Calbiochem) and underwent centrifugation (16,000×g, 20 min and 4°C) to remove debris. Proteins were precipitated from the sample supernatant with acetone overnight at –20°C by adding 4 vols. of ice-cold acetone to 1 vol. of plant extract. The resulting precipitate was resuspended in 2% SDS, 1 mM dithiothreitol, Tris–HCl buffer before methanol/chloroform/water protein extraction. Methanol/chloroform extraction was performed by adding 4 vols. of methanol to 1 vol. of resuspended plant extract. The mixture was vortexed, and 1 vol. of chloroform was added before additional vortexing. Three volumes of water were added, and the solution was vortexed and subsequently centrifuged for 15 min (2,500×g, 20 °C). The top layer was removed and discarded via a serological pipette. Then, 3 vols. of methanol were added, and the solution was vortexed before centrifugation. Pellets were washed once with 80% acetone. Extracted protein was resuspended in 6 M urea, 1× phosSTOP (Roche) and quantified using a bicinchoninic acid assay kit. For each sample, 5 mg of protein was spiked with isotopically labeled phosphopeptide standard (WpSGPITP[+6]K), synthesized by the University of Wisconsin-Madison Biotechnology Center’s peptide synthesis core facility. Samples were reduced with 5 mM dithiothreitol (30 min at 55°C) and alkylated using 15 mM iodoacetamide (30 min at room temperature). Samples were digested for 2 h at 37°C using LysC (Wako) at a 1:100 enzyme:protein ratio then diluted with 1.5 M urea using 20 mM Tris pH 8 and further digested with trypsin (Promega) at a 1:250 enzyme:protein ratio overnight at 37°C. Samples were acidified with 0.5% (v/v) trifluoroacertic acid (TFA) to stop enzymatic digestion and desalted using C-18 solid-phase extraction columns (Waters). Phosphopeptide enrichment was performed using homemade TiO₂ columns containing 4 mg of TiO₂ particles (10 μm; GLSciences), as described by Minkoff et al. (2014). LC-MS analysis was performed using the Eksigent NanoLCUltra 2 D system with the cHiPLC nanoflex microfluidic C18 column (75 mm, 120 Å) coupled to the AB SCIEX 5500 QTRAP mass spectrometer. Analytical separation was performed using a linear gradient of 0–30% acetonitrile with 0.1% formic acid over 70 min at a flow rate of 300 nl min^–1. Masses monitored are indicated in Supplementary Table S2. Peak areas were integrated using Skyline version 2.5.

Sequence collection and phylogenetic analysis

To collect sequences corresponding to potential MtEPP1 orthologs, the MtEPP1 sequence was searched in genome and transcriptome data sets using BLASTp and tBLASTn, respectively. Hits with an E-value <10^–50 were selected for the phylogenetic analysis. Sequences used are indicated in Supplementary Table S3. Also, for non-host species, the best hits were reciprocally blasted on the M. truncatula genome to confirm the absence of orthologs. Collected sequences were aligned using MAFFT (http://mafft.cbrc.jp/alignment/server/) and the alignment was manually curated with BioEdit. Phylogenetic trees were generated by both Neighbor–Joining and the maximum likelihood algorithm in MEGA6. Partial gap deletion (95%) was used together with the JTT substitution model (Gamma 2 parameters) and the ‘subtree pruning and regrafting’ heuristic algorithm (SPR3). Bootstrap was calculated using 500 replicates.

Synteny analysis

Synteny analysis of the MtEPP1 locus in M. truncatula, A. trichopoda, G. raimondii, C. papaya, A. thaliana Col-0, B. rapa, A. arabicum, B. vulgaris and N. nucifera genomes was performed as described previously (Delaux et al. 2014). Briefly, 100 kb upstream and downstream of MtEPP1 and the ortholog of Carica papaya were compared using COGE GEvo (https://genomevolution.org/CoGe/GEvo.pl). Syntenic genomic blocks in A. thaliana Col-0, B. rapa, A. arabicum, B. vulgaris and N.nucifera were identified by GoGe-BLAST and added to the analysis.

Arbuscular mycorrhization assays

For AM assays, 2-week-old M. truncatula composite plants were transferred to pots filled with clay-based soil (Turface, Profile^®). Each container was inoculated with 400 spores of Rhizophagus irregularis 197198 (PremierTech^®). Plants were watered three times per week with a Long Ashton solution (Hewitt 1966) with a low phosphate concentration (10 μM) and with water when needed. After 6 weeks, transgenic roots showing YC3.6 fluorescence were harvested, stained with wheat germ agglutinin and conjugated to Alexa Fluor 488 (Invitrogen). Fungal structures (e.g. hyphopodia and arbuscules) were visualized under a Zeiss LSM 510 Meta confocal microscope as reported by Pumplin et al. (2010). For each root system, the numbers of hyphopodia and arbuscules were counted. The percentage of root length colonization was also calculated. Four independent replicates with at least seven plants each were used for this study.

Statistical analyses

All the statistical analyses were conducted using R software 3.0.1. The specific statistical tests performed are indicated in the legend of the corresponding figures.

Acknowledgments

Confocal microscopy analysis was performed at the Newcomb Imaging Center, Department of Botany, UW-Madison, and at the Unidad de Biomedicina, FES-Iztacala UNAM, México. We thank Dr. Jose L. Reyes (Instituto de Biotecnología-UNAM, México), Dr. Georgina Hernandez (Centro de Ciencias Genómicas-UNAM), Dr. Kiwamu Tanaka (Washington State University, Pullman, USA) and Dr. Marc Libault (University of Nebraska-Lincoln) for constructive discussion. We also thank Dr. Roberto Velasco-García (Facultad de Estudios Superiores Iztacala, UNAM, México) for his help in the in silico prediction of the different structures of the MtEPP1 protein.

Funding

This research was supported by the Consejo Nacional de Ciencia y Tecnología [CONACyT grant No. CB2013-219759, a ‘Fortalecimiento a la Infraestructura Cientifica’ grant (Infra#268769) to N.D.B., a fellowship (CVU: 919676) to M.C.I.-A. who is a doctoral student from Programa de Doctorado en Ciencias Biológicas, Universidad National Autónoma de México and a fellowship (347027/239879) to M.R.R.-S. who is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México]; the Programa de Apoyo a Proyectos de Investigación e Inovacción Tecnológica [PAPIIT grant No. IN213017]; Programa de Apoyo a Profesores de Carrera (PAPCA)-UNAM [grant FESI-DIP-PAPCA-2014-3 to O.V.L.]; and from the National Science Foundation [NSF grant Nos. 1237936 and 1546742 to J.M.A. and M.R.S.]

Disclosures

The authors have no conflicts of interest to declare.

References