Nodulation and arbuscular mycorrhization require the activation of plant host symbiotic programs by Nod factors, and Myc-LCOs and COs, respectively. The pathways involved in the perception and downstream signaling of these signals include common and distinct components. Among the distinct components, NSP1, a GRAS transcription factor, has been considered for years to be specifically involved in nodulation.
Here, we analyzed the degree of conservation of the NSP1 sequence in arbuscular mycorrhizal (AM) host and non-AM host plants and carefully examined the ability of Medicago truncatula nsp1 mutants to respond to Myc-LCOs and to be colonized by an arbuscular mycorrhizal fungus.
In AM-host plants, the selection pressure on NSP1 is stronger than in non-AM host ones. The response to Myc-LCOs and the frequency of mycorrhizal colonization are significantly reduced in the nsp1 mutants.
Our results reveal that NSP1, previously described for its involvement in the Nod factor signaling pathway, is also involved in the Myc-LCO signaling pathway. They bring additional evidence on the evolutionary relatedness between nodulation and mycorrhization.
The arbuscular mycorrhizal (AM) symbiosis plays a key role in the nutrient uptake of a majority of land plants and could potentially be an important component of low input sustainable agriculture. The establishment of this symbiosis involves signal molecules released by both partners. The plant produces strigolactones, which are an ancient class of plant hormones that are also required for AM symbiosis (Gomez-Roldan et al., 2008; Umehara et al., 2008; Delaux et al., 2012). At extremely low concentrations strigolactones stimulate AM fungal metabolism, germination and pre-symbiotic growth (Akiyama et al., 2005; Besserer et al., 2006, 2008). The fungus releases lipochitooligosaccharides (Myc-LCOs) and short oligosaccharides (COs), which prepare the plant for symbiosis by activating a symbiotic (Sym) signaling pathway and promoting lateral root formation (Maillet et al., 2011; Genre et al., 2013). The Sym pathway is also involved in the activation of legume nodulation by Nod factors (Oldroyd et al., 2009). Following these early signaling events, the fungus contacts the plant root epidermis, develops hyphopodia and colonizes the root inter- and intra-cellularly. Finally, it forms highly branched structures, called arbuscules, in cortical cells where the nutrient exchanges between the two partners take place.
To date, only two transcription factors of the Sym pathway, RAM1 and NSP2, have been identified as being involved in the Myc signaling pathway (Maillet et al., 2011; Gobbato et al., 2012; Lauressergues et al., 2012). NSP2 is a GRAS transcription factor also involved in the Nod signaling pathway. It interacts with NSP1 and promotes the activation of symbiotic marker genes, such as ENOD11 (Hirsch et al., 2009). NSP1 is another GRAS transcription factor which has been described as a specific component of the Nod signaling pathway (Catoira et al., 2000; Smit et al., 2005). It has been proposed that RAM1 could play a role in mycorrhization similar to that of NSP1 during nodulation by interacting with NSP2 and activating symbiotic genes (Gobbato et al., 2012). However, the fact that nsp1 mutant is affected in the synthesis of strigolactones (Liu et al., 2011) strongly suggests that NSP1 could also be involved in the early steps of the establishment of the AM symbiosis.
To test this hypothesis, we performed a phylogenetic study of NSP1 in plants and carefully examined the mycorrhizal phenotype of the Medicago truncatula nsp1 mutant. We found that NSP1 is well-conserved in angiosperms across the AM host plants and is more divergent in the non-AM host plants of the Brassicaceae family. Also, NSP1 is required for optimum mycorrhization and is involved in the Myc signaling pathway. Taken together, our results show that NSP1 is not specific to the Nod signaling pathway. They bring additional evidence on the evolutionary relatedness between nodulation and mycorrhization.
Materials and Methods
The NSP1 sequences of angiosperms, of the moss Physcomitrella patens and of the lycophyte Selaginella moellendorffii were collected by BLASTp against the GenBank nr database (Supporting Information Table S1). The gymnosperm sequences were obtained by tBLASTn on the available ESTs. The Marchantia polymorpha sequence was identified using the http://www.one-KP.com dataset. In addition, the charophyte databases available on GenBank were also screened. The collected sequences were aligned using MAFFT (http://mafft.cbrc.jp/alignment/server/). The phylogenetic tree was generated by maximum-likelihood using MEGA5 as previously described (Delaux et al., 2012). AtSHR and AtSCL32 and their orthologs in Gymnosperms, S. moellendorffii and P. patens were used as outgroups (Engstrom, 2011). The average pairwise distances of the LHR domains were calculated between the Brassicaceae, AM host dicots and Carica papaya using MEGA5 after gap removal (Tamura et al., 2011).
Estimation of ω parameter
The codon sequences were aligned using MUSCLE (codon) via MEGA5. The gaps were automatically removed. The ratio of non-synonymous substitutions per non-synonymous site (dN) to synonymous substitutions per synonymous site (dS), the ω parameter, was calculated using MEGA5. To determine the ω parameter in each branch of the NSP1 gene tree, a branch-specific approach was conducted using codeml (Yang, 1998). The free-ratio and one-ratio models were generated as proposed by Yang (1998). To determine the most likely model, twice the difference between the log likelihood of each model was compared by Chi-square. For the Chi-square test, df = number of branches in the free-ratio model – 1.
Rhizophagus irregularis sterile spores were purchased from Agronutrition (Carbonne, France). Medicago truncatula A17 seeds were surface-sterilized and germinated on agar plates in the dark for 5 d at 4°C. The nsp1 B85 and C54 (Smit et al., 2005), nsp2-1 (Kaló et al., 2005), ram1-1 (Gobbato et al., 2012) and ipd3-1 (Horváth et al., 2011) alleles were used. The plants were then cultivated in 200 ml pots on Oil-Dri US-Special Substrate (Damolin, Denmark) for 6–8 wk in a growth chamber and watered every 2 d with Long Ashton medium containing 7.5 μM phosphate (Lauressergues et al., 2012). For inoculation with R. irregularis, we used either 400 or 1200 spores per liter of substrate.
Myc-LCOs used in this study are an equimolar mix of the four (Syn)Myc-LCOs: LCO-IV(C16 : 0), LCO-IV(C16 : 0,S), LCO-IV(C18 : 1Δ9Z) and LCO-IV(C18 : 1Δ9Z,S) described in Maillet et al. (2011) at a final concentration of 10−8 M. The plants treated with Myc-LCOs (12 h) were cultivated on Fahraeus medium as described in Combier et al. (2008), containing a low concentration of phosphate (7.5 μM) and a high concentration of nitrogen (10 mM NH4NO3). Some plants were grown in the same conditions but treated with Nod factors of Sinorhizobium meliloti (Lerouge et al., 1990) at a final concentration of 10−8 M. COs with four residues (CO4) (Genre et al., 2013) were used at a final concentration of 10−8 M in the same conditions as Myc-LCOs.
RNAs of Medicago truncatula roots were extracted using the RNeasy Plant Mini Kit (Qiagen). The reverse transcription (RT) was performed using the SuperScript II Reverse Transcriptase (Invitrogen) on 500 ng of total RNA. For the experiment comparing the mycorrhizal and non-mycorrhizal roots, six independent plants were analyzed per condition. For the experiments with molecule treatments, ten independent plants were pooled per replicate. Three replicates (n = 3) were performed with two technical replicates each. Each experiment has been repeated two to three times. The quantitative polymerase chain reaction (qPCR) amplifications were conducted on a Roche LightCycler 480 System (Roche Diagnostics) (see Lauressergues et al., 2012).
The mean values of relative gene expression or mycorrhization rates were compared by using the Kruskal–Wallis test and, when significant, a pairwise comparison was made using the non-parametric Mann–Whitney test. The error bars represent the standard error of the mean (SEM). The stars indicate significant differences (P <0.05).
Results and Discussion
NSP1 is divergent in non-AM host species
To address the question of the involvement of NSP1 in AM symbiosis, we first looked at its conservation in the green lineage by in silico analysis. Indeed, most of the AM symbiosis-related genes are well conserved in land plants and poorly conserved or absent in the Brassicaceae, a non-AM host family (Delaux et al., 2013). We screened the available genomic data of land plants and chlorophyte green algae, the transcriptomic data of Gymnosperms and the recently released transcriptomic sequences of charophyte green algae (Wodniok et al., 2011; Timme et al., 2012) for the presence of NSP1. We identified GRAS domain-containing proteins in land plants and charophyte green algae datasets but not in the chlorophyte sequences. To assess their identity, the collected sequences were aligned and a maximum-likelihood tree was resolved. As previously determined by Engstrom (2011), we found clear orthologs of NSP1 in all the available angiosperm genomes and in the genomes of the Lycophyte Selaginella moellendorffii and of the moss Physcomitrella patens (Fig. 1a). In addition, we found orthologs of NSP1 in the liverwort Marchantia polymorpha and in the Gymnosperm Pinus taeda (Fig. 1a). By contrast, the GRAS domain-containing proteins found in the charophyte green algae belonged to other clades of the GRAS family (data not shown).
To determine whether NSP1 is under different evolutionary constraints in AM host and non-AM host species, we calculated several ω values. The ω value reflects the selection constraints (Yang, 1998). We applied the pairwise comparison approach and calculated the ω values within the symbiotic dicots and the Brassicaceae for NSP1 and four other GRAS transcription factors: SCARECROW (Di Laurenzio et al., 1996), SHORTROOT (Benfey et al., 1993), PAT1 (Bolle et al., 2000) and NSP2 (Kaló et al., 2005). We found that, for NSP1 and NSP2, the ω value of the Brassicaceae is significantly (P <0.001) higher than that of the AM host dicots (Fig. 1b). By contrast, the ω parameters of SCARECROW and PAT1 are higher (P <0.001) in the symbiotic dicots than in the Brassicaceae (Fig. 1b). For SHORTROOT, the ω parameters are similar in both groups (Fig. 1b). Then, to take into account the evolutionary rate of the most common ancestor, we performed a branch-specific analysis on NSP1. In this approach, two models are compared: one which assumes the same ω for all the branches (one-ratio) and the other which assumes a different ω parameter for each branch in the tree (free-ratio). We found that the free-ratio model fits the data significantly better than the one-ratio model (P <0.001). In this model, the branch of the tree at the base of the Brassicaceae lineage displays a ω value (ω = 0.5165) higher than the ω value of the symbiotic dicot branch (ω = 0.1750). These results obtained with the pairwise and the branch-specific approaches suggest that a relaxed selection constraint occurred on NSP1 in the non-AM host Brassicaceae family.
Proteins of the GRAS family contain five domains (LHRI, VHIID, LHRII, PFYRE and SAW) which are well-conserved in each sub-clade of the family (Engstrom, 2011). The two LHR domains are required for the binding of NSP1 to the promoter of MtENOD11, a gene upregulated during the early steps of AM and root nodule symbioses (Boisson-Dernier et al., 2005; Hirsch et al., 2009). To determine the impact of the relaxed selection constraint on NSP1 in the Brassicaceae, the LHR domains of angiosperms were aligned. Then, we calculated the pairwise distance of the LHR domains of the Brassicaceae and of the symbiotic dicots to the LHR domains of Carica papaya. We used C. papaya as reference because it is a non-Brassicaceae member of the Brassicales order and can form AM symbioses (Khade et al., 2010; Franzke et al., 2011). While no significant differences were found for the LHRII domain, the LHRI domain seems to be highly divergent in the Brassicaceae compared to the other, AM host, dicots (Fig. 1c).
Most of the genes involved in the AM symbiosis have been lost in the Brassicaceae (Delaux et al., 2013). However, some of them are still present (DMI1 or NSP2 for instance) suggesting the occurrence of alternative functions that led to their conservation. The purifying selection acting on NSP1 and NSP2 (ω values < 1) argue for this hypothesis. The involvement of these two genes in the regulation of the biosynthesis of the plant hormones strigolactones has been recently demonstrated (Liu et al., 2011) and could explain their conservation. By contrast, some domains, LHRI in NSP1 or the mir171 h-binding domain in NSP2 (Lauressergues et al., 2012), which are important for the symbiotic function, are more divergent, likely due to a locally relaxed selection pressure.
Taken together, these analyses suggest that NSP1 appeared early in the Embryophytes lineage and diverged in the Brassicaceae, an evolutionary pattern shared with NSP2, another GRAS transcription factor of the Sym pathway.
Mycorrhization and Myc-LCOs induce NSP1 expression
NSP1 is known to play a role during nodulation (Smit et al., 2005) and the Medicago gene atlas (http://mtgea.noble.org/v2/) reveals that NSP1 expression is induced during this symbiosis. Interestingly, the Medicago gene atlas also reveals that NSP1 expression is slightly induced in mycorrhizal roots. We first confirmed by qRT-PCR that the expression of NSP1 is slightly (two-fold compared to the control) but significantly induced during mycorrhization (Fig. 2a). In parallel, by analogy with the role of NSP1 in the Nod signaling pathway, we investigated whether the expression of NSP1 could be induced by a mixture of sulfated and non-sulfated Myc-LCOs. We found, under conditions of high nitrogen fertilization (10 mM NH4NO3), which inhibits the Nod signaling pathway (Fig. S1), that NSP1 expression is induced by the Myc-LCO treatment (Fig. 2b). Moreover, this induction is still present in the ram1 and nsp2 mutants but not in the ipd3 mutant (Fig. 2b), suggesting that the induction of NSP1 expression by Myc-LCOs requires IPD3, which acts upstream of NSP1 (Horváth et al., 2011) but is independent of RAM1 and NSP2. The high expression of NSP1 in the non-treated ipd3 mutant was not expected but may suggest the occurrence in the wild-type plant of some negative feedback regulation by IPD3 of the expression of downstream genes. Interestingly, we found no induction of NSP1 expression by treatment with CO4 (Fig. S2), confirming that the symbiotic signaling pathways induced by Myc-LCOs and COs are distinct (Genre et al., 2013).
Mycorrhization is affected in the Mtnsp1 mutant
The screening for mutants affected in the AM symbiosis was initially performed with strong fungal inoculants: a high number of spores or fragments of mycorrhized roots. By using a smaller inoculum (fewer spores), NSP2, which was initially identified as exclusively involved in nodulation (Oldroyd & Long, 2003), was recently found to be also involved in the AM symbiosis and to be a member of the common Sym pathway (Maillet et al., 2011). We first inoculated the Mtnsp1 B85 allele and wild-type plants with a high number of spores (1200 spores per liter). In this condition, the mutant and wild-type plants were similarly colonized (Fig. 3a). However, with a smaller fungal inoculum (400 spores per liter), the mycorrhization of Mtnsp1 mutant plants was significantly reduced compared to the wild-type after either 6, 8 or 12 wk post-inoculation (Fig. 3a). The colonization rate in the mutant plants was two- to three-fold lower than in the wild-type plants (Fig. 3a–c), while in the colonized root sections the frequency and the structure of the arbuscules were the same (Fig. 3b). In addition to the B85 Mtnsp1 allele we tested the Mtnsp1 C54 allele (Smit et al., 2005) for its ability to get colonized. As for B85 we found a reduced colonization rate in Mtnsp1 C54 (Fig. 3c), confirming the requirement of NSP1 for a proper AM symbiosis. These results suggest that NSP1 has a role in mycorrhization, but the protein is probably non-essential, suggesting an overlapping function, perhaps with other GRAS proteins.
Nsp1 participates in the Myc-LCO signaling pathway
To further investigate the mycorrhizal phenotype of the Mtnsp1 mutant, we looked at whether NSP1 is involved in the Myc signaling pathway. For this purpose we tested the effect of Myc-LCOs on the expression of four symbiotic marker genes: ENOD11 (Boisson-Dernier et al., 2005) and three genes known to be up-regulated in Medicago truncatula roots treated with Myc-LCOs (Mtr.52092.1.S1_s_at, Mtr.37912.1.S1_at and Mtr.35524.1.S1_at, which encode for a putative pectinesterase, a NADP-dependent oxidoreductase, and a member of the subtilase family, respectively; Maillet et al., 2011; Lauressergues et al., 2012). To make sure that the Myc rather than the Nod signaling pathway was activated, Mtnsp1 and wild-type plants were treated for 24 h with non-sulfated Myc-LCOs. These LCOs are the most remote to the cognitive Nod factors of Medicago whose sulfate group is essential to activate the nodulation process (Lerouge et al., 1990). In wild-type plants expression of these genes was up-regulated by non-sulfated Myc-LCOs, except for ENOD11, which was previously known to be non-inducible by these molecules (Maillet et al., 2011) (Fig. 4). By contrast, treatment with non-sulfated Myc-LCOs did not affect the expression level of these genes in Mtnsp1 mutant plants (Fig. 4). To strengthen this result, wild-type and Mtnsp1 plants were grown on a medium supplemented with 10 mM NH4NO3. This concentration is sufficient to inhibit plant response to Nod factors (Fig. S1). The plants were then treated for 12 h with a mixture of sulfated and non-sulfated Myc-LCOs. The four genes were up-regulated in wild type plants after treatment with Myc-LCOs (Fig. S3). By contrast, their induction was abolished in the Mtnsp1 mutant plants, confirming that NSP1 is required for the normal response of the plant to Myc-LCOs. Once gene expression and plant responses activated by tetrameric and pentameric chitin oligomers (CO4/CO5, Genre et al., 2013) will be described, similar experiments can be performed to investigate the role of NSP1 in CO-induced signaling pathway.
While RAM1 seems to be required for the penetration of the fungus (Gobbato et al., 2012), NSP1, like NSP2 (Lauressergues et al., 2012), seems to control the fungal colonization in the root. As NSP1 and NSP2 activate genes involved in the biosynthesis of strigolactones (Liu et al., 2011), this control might require local adjustments of strigolactone content.
When examining the lateral root formation of Medicago truncatula in response to Myc-LCOs, Maillet et al. (2011) found that this response was not dependent on NSP1. This result suggested that NSP1 was not involved in the Myc signaling pathway. In light of our results here, showing that the nsp1 mutants are affected in mycorrhizal colonization and in the Myc-LCO induction of mycorrhization marker genes, we hypothesize that the positive induction by Myc-LCOs observed by Maillet et al. (2011) of both mycorrhization and lateral root formation actually involves distinct signaling pathways. Together, these results suggest that NSP1 would be necessary to transduce the Myc-LCO promotion of fungal colonization but not the Myc-LCO stimulation of root development. Finally, as we know that both RAM1 and NSP1 interact with NSP2 (Hirsch et al., 2009; Gobbato et al., 2012), future work will have to further investigate the respective role of each interaction during the fungal penetration of the root and, later, during the fungal colonization and autoregulation of this colonization.
This work was carried out in the Laboratoire de Recherche en Sciences Végétales in Toulouse, which belongs to the Laboratoire d'Excellence entitled TULIP (ANR -10-LABX-41). The authors thank Fabienne Maillet (LIPM, Toulouse, France) for providing mutant seeds and Nod factors, Eric Samain, Sébastien Fort and Sylvain Cottaz (CERMAV, Grenoble, France) for providing the Myc-LCOs and Kari L. Forshey for critical reading of the manuscript. The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper.