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).
Figure 1. The evolution of NSP1 in land plants. (a) Maximum-likelihood tree of NSP1. (b) Pairwise estimation of ω values in the Brassicaceae family (Bra) and in the arbuscular mycorrhizal (AM) host dicots (AM host) for NSP1, NSP2, SHORTROOT (SHR), PAT1 and SCARECROW (SCR). Asterisks indicate a significant difference according to the Mann–Whitney test (P < 0.001). (c) Pairwise distance comparison of the LHR domains (I and II) of Carica papaya to the same LHR domains of the Brassicaceae family (Bra) and of the AM host dicots (AM host). ***, Significant difference according to Student's t-test (P < 0.001); ns, not significant. Accession numbers and species names are available in Supporting Information Table S1.
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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).
Figure 2. NSP1 expression during arbuscular mycorrhizal (AM) symbiosis. (a) Quantification of NSP1 expression by qRT-PCR in non-inoculated (MYC–) and inoculated roots (MYC+) of Medicago truncatula with Rhizophagus irregularis and cultivated for 9 wk (n = 6 independent plants). (b) Quantification of NSP1 expression by qRT-PCR in wild-type (A17) and mutant lines treated or not with 10−8 M of a mixture of sulfated and non-sulfated Myc-LCOs (n = 3 independent pools of 10 plants). Error bars represent standard error of mean (SEM); *, significant difference between the two treatments according to the Mann–Whitney test (P < 0.05).
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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.
Figure 3. Mycorrhizal phenotype of the Mtnsp1 mutant. (a) Percentage of colonization in the roots of wild-type Medicago truncatula A17 and nsp1 B85 allele inoculated with 400 or 1200 spores per liter of Rhizophagus irregularis and cultivated for 6, 8 and 12 wk (wpi, week post-inoculation). (b) Quantification of mycorrhization in wild-type A17 and nsp1 B85 allele 12 wk after inoculation according to Trouvelot et al. (1986). ‘F’, the frequency of colonization in the root system; ‘a’, the arbuscule abundance (in percentage) in the colonized root sections. (c) Percentage of colonization in the roots of wild type (A17) and two alleles of nsp1 mutants (B85 and C54), 8 wk after inoculation. Error bars represent standard error of mean (SEM). *, Significant difference when compared with control according to the Kruskal–Wallis test (n = 6, P < 0.05).
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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.
Figure 4. The response of the nsp1 mutant to non-sulfated Myc-LCOs (NS-LCOs). qRT-PCR analysis of the relative expression levels of mycorrhization marker genes in response to NS-LCOs in wild-type Medicago truncatula A17 and nsp1 mutant roots. (a) ENOD11, (b) Mtr.52092.1.S1_s_at, (c) Mtr.37912.1.S1_at and (d) Mtr.35524.1.S1_at. Error bars represent standard error of mean (SEM). *, Significant difference between the two treatments according to the Mann–Whitney test (n = 3 independent pools of 10 plants, P < 0.05).
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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.