In the previous study, transcript abundance of 201-tag was significantly enhanced at the early appressorium stage of AM symbiosis (Breuninger and Requena, 2004). The amino acid sequence of 201-tag exhibits a high similarity (∼97%) with the Ri14-3-3 protein from R. irregularis (Breuninger and Requena, 2004; Tisserant et al., 2012). With the aim to confirm if this fungal 14-3-3 protein is involved in AM symbiosis, a DNA clone of 1.5 kb in length was obtained by inverse PCR from the cloning procedures on F. mosseae genomic DNA based on the 201-tag. The isolated fragment with 5′ end and upstream region was highly similar to the sequence of Ri14-3-3 gene from R. irregularis (Porcel et al., 2006). Since the 5′ and 3′ end sequences of this gene are not available, 5′ and 3′ RACE experiments on RNA pools of F. mosseae germinating spores were subsequently performed to obtain the full-length CDS sequence. A 1,401 bp full-length cDNA sequence of Fm201, covering the 5′UTR (188 bp) and 3′UTR (411 bp), was thus identified (Accession number: KM258580). The corresponding genomic sequence of Fm201 gene is 1,685 bp in length, containing seven exons and seven introns (Supplementary Figure S1). Interestingly, Fm201 gene contains a 100 nt intron in the 5′ UTR and two transcriptional variants of 3′UTRs (61 and 411 nt in length, respectively). These unusual features of AM fungal 14-3-3 gene firstly reported in the present study may suggest important roles in the regulation of Fm201 expression during AM symbiosis.

To further investigate the phylogenetic and structural features of the Fm201 protein from AM fungi, we exploited the phylogenetic placement and 3D structure of Fm201 protein using bioinformatics strategy. The in silico analysis revealed that the open reading frame (ORF) of Fm201 gene consists of 804 bp corresponding to 267 amino acids with a predicted molecular weight of approximate 30 kDa. A phylogeny of basal fungi and 14-3-3 proteins from Homo sapiens clearly supports Fm201 as a sister clade to Ri14-3-3 (Figure 1A), indicative of the conserved evolutionary origin of the 14-3-3 genes in AM fungi, whereas the RiBMH2 protein from R. irregularis belongs to the closer relative of the yeast BMH2 (Figure 1A). Compared to the 14-3-3 proteins from H. sapiens, Fm201 protein still shares a very high homology. This also demonstrates that 14-3-3 proteins are highly conserved in eukaryotes. As a conserved protein, Fm201 protein shares 97% similarities with Ri14-3-3 protein from AM fungi. The amino acid sequence of Fm201 was compared with BMHs from S. cerevisiae, 14-3-3s from R. irregularis and H. sapiens 14-3-3 epsilon and a high homology with over 72% identity at the amino acid level was observed (Supplementary Figure S2).

We further carried out the Homology modeling using Homo sapiens 14-3-3 epsilon (80.52% identity in amino acid sequences) as a model. The predicted three-dimensional conformation of Fm201 indicates that Fm201 is a typical 14-3-3 protein with 9 alpha helices and 8 loops, with the highly homologous α3, α5, α7, and α9 putatively forming its amphipathic ligand-binding grooves (Figure 1C). Fm201 homologous dimers could form a typical C-shape cup, which provides a basic structure of 14-3-3 dimers for implementing its function (Figure 1D).

To gain further insights into the function of 14-3-3, a S. cerevisiae heterologous expression system was exploited. Since Fm201 shares 82.3% identity at the amino acid sequence with both BMH1 and BMH2 in S. cerevisiae (see Supplementary Figure S2), the ORF of Fm201 was cloned into pMR-12 under the control of the Gal7 promoter and replaced S. cerevisiae BMH1. To test if Fm201 can restore the metabolic activity of S. cerevisiae Δbmhs mutant, as referred in Materials and Methods (also see Supplementary Table S1). S. cerevisiae Δbmhs mutant with pMR-12-Fm201 cannot grow on YPD with 2% glucose as the sole carbon source. However, cell growth was recovered when using 2% galactose as the sole carbon source (Figure 2). Similar results were observed when replacing Fm201 with Ri14-3-3 or RiBMH2 (Figure 2). These data confirmed that Fm201 has similar function as BMH1 in S. cerevisiae.

14-3-3 proteins have been reported to participate in pseudohyphal growth and resistance in yeast (Roberts et al., 1997; Hurtado and Rachubinski, 2002). However, the roles of these 14-3-3 proteins in mycorrhizal fungi are largely unknown. It has been observed that expression specificity of plant 14-3-3 genes in response to various stresses is largely promoter dependent (Aksamit et al., 2005). To analyze the putative CREs (Cis-Regulatory Elements) located in the promoter region of Fm201, a 1.5 kb length promoter sequence upstream of Fm201 coding region (pFm201) was analyzed via Yeastract database¹. The CREs present in pFm201 were compared with pBMH1, pBMH2 from S. cerevisiae and pRiBMH2 in R. irregularis (van Heusden, 2009) (Table 1). Many common CREs in corresponding sites shared by pFm201 and pBMHs have been shown to be recognized by many transcriptional factors (Bruckmann et al., 2004; van Heusden, 2009). In the present study, two CREs possiblely recognized by Msn2 and STE12 were chosen for further investigations. Msn2 is an STRE element (AGGGG/CCCCT) binding transcription factor, which is supposed to be related to fungal infection and resistance to abiotic stress in other filamentous fungi (Schmitt and Mcentee, 1996; Seidl et al., 2004; Elfving et al., 2014; Zhang et al., 2014). STE12, so-called GintSTE, is the transcriptional factor that has been reported in mycorrhizal fungi and is believed to be an indispensable component in the early process of mycorrhizal fungi infection (Tollot et al., 2009; Tang et al., 2016). The common and shared CREs present upstream of Fm201, RiBMH2 and S. cerevisiae BMHs imply that 14-3-3 proteins in AM fungi may be involved in the regulation of resistance to abiotic stress and hyphal growth in AM fungi as BMHs in S. cerevisiae.

Due to the lack of stable genetic transformation approaches in the AM fungi, it is technically challenging to knockout Fm201 gene to confirm the biological function in the early stages during symbiosis (Sanders, 1999; Maldonado-Mendoza et al., 2001). To probe the possible function of Fm201 protein, we employed the site-specific mutagenesis and yeast one-hybrid system to initially explore the essential region of Fm201 promoter. Compared to the site-specific mutagenesis of STRE (CCCCT/AGGGG) located in pFm201(pFm201-ΔSTRE), pFm201 is more sensitive to abiotic stresses, osmotic pressure and drought stress (Figure 3A). This result indicates that the STRE element of pFm201 could be recognized by Msn2 in S. cerevisiae. Although there is no any report, to our knowledge, about the functional properties of RiMsn2 factor mentioned as RiMsn4 in R. irregularis, it may play a major role in eukaryotic abiotic stress response and hyphae differentiation (Tisserant et al., 2013; Zhang et al., 2014).

A yeast one-hybrid system was also performed to confirm the interaction between GintSTE and RiMsn2 with CREs on pFm201 (Figure 3B). As shown in Figure 3B, yeast cells harboring pFm201 with STRE element and RiMsn2 or GintSTE protein grew well, whereas growth of the cells carrying RiMsn2 or GintSTE and Fm201 promoter without STRE element as negative controls were severly inhibited under the same conditions. These data suggest that GintSTE and RiMsn2 proteins interact with the STRE element of Fm201 gene, the similar results were also acquired from the promoter of RiBMH2 (data not shown). It is therefore reasonable to speculate that GintSTE and RiMsn2 proteins positively regulate the expression of 14-3-3 genes in presymbiotic stage through the binding between GintSTE/RiMsn2 and pFm201.

Due to the obligate biotrophic and asexual multinucleate nature of the AM fungi (Sanders, 1999; Maldonado-Mendoza et al., 2001), it is difficult to generate mutants and overexpression strains to analyze the biological functions of Fm201 protein during AM fungal infection. To obtain further insights into the expression profile of Fm201 gene during the colonization process, we performed a time-course analysis of Medicago truncatula roots inoculated with F. mosseae in the pot system, then sampled at 12, 18, 25, and 50 days post-inoculation (dpi) and also collected the quiescent spores and germinated spores. Morphological analyses of mycorrhizal roots showed almost majority of the appressoria and intraradical hyphae at 12–18 dpi. More arbuscules were obviously detected starting from 25 dpi, while the abundance of arbuscules decreased at 50 dpi (Figure 4A), the mycorrhizal colonization of the root samples was also calculated as described by Trouvelot et al. (2015) (data not shown). The transcript abundance of Fm201 gene in F. mosseae at different stages were also analyzed by qRT-PCR. As shown in Figure 4B, the transcript abundance of Fm201 was obviously higher in germinating spores than in quiescent spores, the transcript abundance of Fm201 is ∼40% lower in 50 dpi than in 25 dpi when arbuscules decreased. This expression pattern was similar with fungal colonization in early stages of symbiosis, especially during the hyphopodium formation and root penetration (see Figures 4A,D). The similar results of RiBMH2 and Ri14-3-3 were also obtained from quiescent spores, germinated spores and sampled at 8, 12, 18, 40 days post-inoculation (dpi) by R. irregularis (Figure 4C). The expression levels of both RiBMH2 and Ri14-3-3 obviously increased accompanying the infection process and arbuscules initiation, as demonstrated by the parallel increased transcriptional levels of MtStbM1 (Figure 4E), the host plant subtilase-encoding gene which is considered as a molecular marker of arbuscular mycorrhiza development (Wegel et al., 2007; Takeda et al., 2009).

Because of the obligate property of mycorrhizal fungi, the effect of ΔFm201 mutant on the establishment and maintenance of mycorrhizal symbiosis cannot be confirmed in vivo. RNAi technique has been successfully utilized to inhibit Ri14-3-3 encoding a homologous protein of Fm201 as described above in R. irregularis induced by hairy root lines of M. truncatula during symbiosis (Nowara et al., 2010; Helber et al., 2011). A 262 bp cDNA sequence from R. irregularis Ri14-3-3 gene was cloned into pK7GWIWG2 (II) RR according to the approach mentioned in Materials and Methods. The Host-Induced Gene Silencing (HIGS) results of Ri14-3-3 showed no significant influence on the intraradical structures of R. irregularis within the roots, when compared with the control roots (Figure 5A).

Since the draft of R. irregularis genome was recently released (Tisserant et al., 2013; Lin et al., 2014), we cloned and identified the coding sequence of another 14-3-3 protein subunit termed RiBMH2 (EXX69786.1). The existence of this novel 14-3-3 protein subunit may explain the nice arbuscule observed in HIGS of Ri14-3-3. Thus, the HIGS experiment targeting both Ri14-3-3 and RiBMH2 was designed to address this issue. Mycorrhizal phenotype analysis uncovered that the arbuscules are defective. The almost collapsed arbuscules were present in the hairy root of Ri14-3-3/RiBMH2 RNAi plants, when these two genes were both strongly repressed. Furthermore, the mycorrhizal colonization of each group was also calculated. The data suggests that the abundance of arbuscules in RNAi roots was also significantly lower than that in the control roots (Figures 5A–C). Moreover, the expression levels of symbiotic MtPT4 and RiMST2, which are considered as molecular markers of the functioning of arbuscules (Harrison et al., 2002; Helber et al., 2011), are significantly reduced in the Ri14-3-3/RiBMH2 RNAi roots relative to the control roots (Figures 5D,E), indicating that knock-down of both Ri14-3-3 and RiBMH2 has a significant effect on the symbiotic phenotype of AM symbiosis. These results also suggest that RiBMH2 may be required for arbuscule formation in AM symbiosis. It also provides a direct evidence that AM fungal 14-3-3 proteins play important roles during AM symbiosis.

To further investigate the potential roles of 14-3-3 proteins in response to salinity and drought stresses, the transcript profiles of 14-3-3s in mycorrhizal roots and external hyphae were analyzed by qRT-PCR after 150 mM NaCl treatment for various time (Figures 6A,C). The transcript abundance of 14-3-3 shows slight but significant increase after 1.5 h and relatively stable within 24 h in mycorrhizal roots. In addition, the transcription profile of Fm201 in extraradical hyphae treated with NaCl shows more rapid induction than in intraradical mycelia. To determine whether 14-3-3 genes are responsive to drought stress, the transcript abundance of 14-3-3 genes under 1/2 water holding capacity of drought treatment was also compared (Figures 6B,D). Unlike salinity stress treatment, the transcript abundance of 14-3-3 genes show a ∼4 fold and ∼7 fold up regulation in mycorrhizal roots and extraradical hyphae, respectively. These findings suggest that Fm201 may be responsible for the crosstalk between plant and R. intraradices under salinity and/or drought stresses.

According to bioinformatics analyses, 14-3-3s from AM fungi are typical 14-3-3 proteins with higher similarity to the known 14-3-3 sequences of yeast and human. Additionally, Fm201 protein is conserved across eukaryotes based on the phylogenetic relationships among AM fungi and other basal eukaryotic species as well as the conserved 3D homology structures between F. mosseae and human (Yang et al., 2006). Therefore, it is of interest to find that two similar sequences were found in the recently released genome and transcriptome of another AM fungus, R. irregularis (Tisserant et al., 2012, 2013). Since the sequences of the two additional genes, the so called Ri14-3-3 and RiBMH2, are complete with the full-length of CDSs, and the percentage of identity is relatively high (∼97%), the three AM fungal proteins share the same nine α-helix domain topologies. Among them, Ri14-3-3 gene from R. irregularis has been firstly reported by Porcel et al. (2006). Moreover, RNA-seq data presented a significant induction in planta phase compared to spores (Tisserant et al., 2012). Only the investigation within genomic and transcriptomic data in AM fungi will clarify whether Fm201-related sequences are a general feature among fungi. Consistent with the previous in silico analyses, these three Fm201, Ri14-3-3 and RiBMH2 are able to complement the yeast BMH1 and BMH2 double mutants. This finding is in agreement with those data reported in the earlier studies (van Heusden et al., 1995, 1996), indicating that these genes identified above encode the functional 14-3-3-like proteins in AM fungi. Further studies need to be carried out to confirm whether these 14-3-3-like proteins identified are a conserved feature of Glomeromycota and whether they may have an essential role in the intraradical phase during interaction with the host plants.

The transcription of 14-3-3 genes show a clear increase in the germinating spores as well as the intraradical phase in both R. irregularis and F. mosseae. The data stemming from the time-course experiment presented that the relatively higher transcription levels were achieved in the phases of root penetration and arbuscules formation, while the expression levels of Fm201 and Ri14-3-3 are obviously reduced compared with RiBMH2 in the degenerating mycorrhizal roots. In addition, we also correlated the Fm201 mRNA abundance with the morphological structures of F. mosseae inside the roots (at 12–50 dpi). The results of Fm201 transcription patterns also suggest that it may play an important role in the germination and hyphopodium formation of F. mosseae, which was also proposed by Breuninger and Requena (2004) through SSH of AM symbiosis at the early stage. In addition, transcript levels of Fm201 remain higher during the symbiotic stage (see Figure 4B), suggesting that this 14-3-3 protein may also play important roles during AM symbiosis, especially the formation of arbuscule besides the root penetration stage. It is thus speculated that the expression of 14-3-3s are, to some extent, related to root penetration and arbuscules formation. This hypothesis is supported by the evidence that 14-3-3 transcripts were present in both the laser micro-dissected arbuscule-containing cells and the IRM including intercellular hyphae (Tisserant et al., 2012). Overall these data implicate a relationship between AM fungal 14-3-3 related genes and intraradical hyphal growth and arbuscule differentiation.

The potential involvement of AM fungal 14-3-3 genes Ri14-3-3 and RiBMH2 in the in planta phase of the colonization process was also supported by the HIGS of Ri14-3-3 and/or RiBMH2 during the M. truncatula–R. irregularis mycorrhizal symbiosis. Lacking the stable genetic transformation protocols for AM fungi, HIGS was confined to AM fungi (Helber et al., 2011; Xie et al., 2016).

The data of the knock-down of both Ri14-3-3 and RiBMH2 genes by HIGS resulting in the impaired arbuscule formation of R. irregularis suggest the significance of these AM fungal 14-3-3 proteins for AM symbiosis. Connecting with the transcripts of RiBMH2 during M. truncatula–R. irregularis mycorrhizal symbiosis, RiBMH2 may be required for the development of AM symbioses and the arbuscule differentiation within roots. However, the Ri14-3-3 RNAi roots colonized by R. irregularis exhibited a considerable arbuscule abundance as compared with control mycorrhizal roots. These findings suggest that the AM functionality or arbuscule formation is redundantly regulated by the two 14-3-3-like genes in R. irregularis. Nevertheless, we here propose that RiBMH2 is essential for arbuscule formation, whereas Ri14-3-3 could be involved in the colonization process but not AM functionality. This hypothesis is supported by the evidence that the transcripts of MtPT4 and RiMST2, two symbiotic genes responsible for arbuscule functionality, were strongly reduced in Ri14-3-3/RiBMH2 RNAi roots, while they were not repressed in Ri14-3-3 RNAi roots. Although Ri14-3-3 homologous gene RiBMH2 is identified in the R. irregularis draft genome (Tisserant et al., 2013; Lin et al., 2014) and RiBMH2 was not down-regulated in Ri14-3-3 RNAi roots (see Figure 5B), the normal AM fungal structures observed in this HIGS system indicate a novel but unknown role for Ri14-3-3 in the establishment of AM symbiosis. Based on the above findings and the previous study (Liu et al., 2015), we hypothesize that RiBMH2-mediated signal could be an important signal in the control of arbuscules formation and R. irregularis hyphal growth within roots. This unknown signal relayed by RiBMH2 serves as the essential signal to ensure the metabolic activity of R. irregularis in the hyphal growth and/or arbuscule differentiation during symbiosis. In the absence of this RiBMH2-mediated signal, the arbuscules are impaired, and growth of the fungus is prevented. The R. irregularis itself needs to activate 14-3-3 protein RiBMH2 in response to the environmental clues to meet demands during fungal growth and division. In addition, our functional analysis in yeast cells suggested that Ri14-3-3 and RiBMH2 encode functional signal proteins involved in growth induction (see Figures 2B,C), indicating that these two proteins may play potential roles in signal transduction during the colonization process and arbuscule formation, respectively. Thus, we can speculate the involvement of Ri14-3-3 in fine-tuning fungal growth in the intraradical phase responding to the external stimuli, moreover, RiBMH2 may be indispensable for arbuscules differentiation. This complex mechanism by which arbuscular mycorrhizas are formed in roots requires the elaborate control of the two AM fungal 14-3-3 proteins in the intraradical phase during cross-talk with host plant.

Remarkably, these results from the HIGS experiments revealed that one 14-3-3 protein subunit can adjust its own expression quantity to offset the adverse influence caused by the lack of another 14-3-3 protein subunit. This conclusion is consistent with the previous results derived from yeast system (van Heusden et al., 1995). Based on this point, it is reasonable to hypothesize that the AM fungal 14-3-3 proteins are indispensable for the symbiosis functioning.

The knockdown of both Ri14-3-3 and RiBMH2 by HIGS exhibits somewhat distinct phenotypes, i.e., fewer arbuscule abundance and impaired arbuscules (see Figures 5A,C), repression of the endosymbiosis functioning with regard to transcription of the symbiotic MtPT4 and MST2 genes (see Figures 5D,E). We hypothesize that there exists a positive feedback mechanism in the potential signaling pathway in R. irregularis. It is also proposed that the CREs upstream of a gene always show close relationship with its function, especially for the regulatory proteins (Carey et al., 2012; Petrov et al., 2012), although most CREs are composed of short sequences which may be very abundant in eukaryotic genomes (van Heusden, 2009). As expected, we observed some conserved motifs including STRE elements in the promoters of two AM fungal 14-3-3 genes Fm201 and RiBMH2 (see Table 1), as predicted by YEASTRACT database, in comparison with the promoters of yeast BMH1 and BMH2. Interestingly, the deletion of STRE (CCCCT/AGGGG) element in the promoter of Fm201 showed significantly reduced levels of the reporter gene mRNAs when expressed in yeast cells (see Figure 3A). These hypotheses mentioned above are also supported by the fact that the orthologous Fm201 gene promoter with STRE element (pFm201) directly interacts with transcription factor Msn2 in yeast cells (see Figure 3B). Furthermore, this recognition between RiMsn2 and pFm201 may contribute to the induction of Fm201 in extraradical hyphae in response to the salinity (150 mM NaCl treatment) stress (see Figure 6A). Therefore, based on the site-specific mutagenesis and the yeast one-hybrid analyses, the transcription of AM fungal 14-3-3-like genes during AM symbiosis is Msn2/STRE-element dependent. The zinc finger DNA-binding proteins Msn2 and Msn4 serve as the key factors that controlling fungal growth and stress responses in different fungal species (Martinez-Pastor et al., 1996; Schmitt and Mcentee, 1996; Liu et al., 2013; Zhang et al., 2014). In addition, the Msn2-controlled and STRE-driven gene Fm201 and RiBMH2 from F. mosseae and R. irregularis, respectively, are positively regulated in response to drought stress during AM symbiosis (see Figures 6B,D), reinforcing that AM fungal 14-3-3 genes participate in the Msn2/STRE element-mediated signaling pathway in AM fungal symbiont during AM symbiosis.

Overall these data presented in this study provided new insights into the signaling function of the 14-3-3 proteins in AM fungal cells during crosstalk with host plants. Based on the aforementioned data, we also propose the hypothesis that abiotic stresses such as salinity and drought affect a Msn2/STRE-mediated signaling pathway governing the expression of AM fungal 14-3-3 proteins that promoted fungal colonization and arbuscule formation within roots (see Figure 7). In the first version of the scheme for abiotic stresses induced signaling, it has been proposed that 14-3-3 proteins preferentially expressed in the intraradical phase are involved in AM fungal colonization process and arbuscule functionality by the regulation of Msn2/STRE-mediated signaling pathway that may control the fungal growth and arbuscule lifespan during AM symbiosis.

Further studies, such as characterizing the precise roles of the novel RiMsn2 gene identified in this work, validating the protein-protein interactions in the Msn2-mediated signaling pathway and the biochemical functions of core components of this pathway, and determining the direct evidence of Msn2-dependent mechanisms in R. irregularis, are needed to define the underlying stress response mechanisms in AM symbionts. Furthermore, the RNA-seq data and gene expression analyses show that both R. irregularis and Gigaspora margarita contain multiple distinct MAPK (Mitogen-activated protein kinase)-related proteins (Tisserant et al., 2012; Salvioli et al., 2016; Xie et al., 2016), indicative of the presence of MAPK signaling cascade in AM fungi to respond to external stresses stimuli and adapt to environmental fluctuation. Thus, a major goal in this field will be to uncover a master MAPK protein regulating the AM fungal growth and differentiation during symbiosis under various abiotic stresses.

In summary, we showed that Fm201, Ri14-3-3 and RiBMH2, three genes from two different AM fungi, are preferentially expressed in the intraradical phase and may have impacts on the success of AM colonization and arbuscule formation. Our data also presented that Msn2 protein governs the Fm201 gene transcription, indicating that AM fungal 14-3-3 gene identified is involved in Msn2 factor/STRE element-mediated signaling pathway. Importantly, host-induced gene silencing of both Ri14-3-3 and RiBMH2 impairs the arbuscule differentiation within roots, indicating that the two AM fungal 14-3-3 protein subunits are required for arbuscule formation. Additionally, these AM fungal 14-3-3 genes are up-regulated in response to salinity and drought stresses during AM symbiosis. Based on these new findings, we propose that the AM fungal 14-3-3 genes are essential for the interaction between AM fungi and host plants, and are potentially involved in enhancing plant salinity and drought tolerance by Msn2/STRE element-controlled signaling.

A-grade spores of R. irregularis DAOM197198 were purchased from Agronutrition (Carbonne, France). Spores of F. mosseae BEG12 were kindly provided by the International Bank of Glomeromycota (IBG, Dijon, France) and collected from Medicago truncatula pot cultures by wet sieving to isolate genomic DNA and total RNA. Spores surface sterilized by 2% chloramine T, and then immersed in a solution containing 0.02% streptomycin and 0.02% gentamycin for 10 min (Besserer et al., 2008). Germinated spores of F. mosseae were selected from acetone solution containing 10^-8mol/L GR24 (10^-9mol/L GR24 for R. irregularis in two days) in 25°C dark incubator. Quiescent spores, germination spores and mycorrhizal roots (at 10, 18 25, 50 dpi for F. mosseae; 8, 12, 18, 40 dpi for R. irregularis) were harvested. After washing in sterile water, all materials described above were immediately frozen in liquid nitrogen and stored at -80°C before nucleic acid extraction.

The water-holding capacity of the soil was computated before planting. The soil was weighed before plant, watered uniformly until flowed from the bottom. Keep the pot suspended in midair for 1 day before weighing. The increase weight of soil after waterd is water-holding capacity. M. truncatula mycorrhizal roots inoculated with AM fungi were treated with NaCl (0.5 M) to 150 mM in final concentration (Calculate according to 70% of water holding capacity in soil) (Giovannetti et al., 2001; Li et al., 2011). Drought treatment was measured by 1/2 water-holding capacity treatment which was proved drought stress treatment to M. truncatula in previous experiments. Mycorrhizal roots and extraradical hyphae from sandwich system were harvested at 1.5, 5, 24 h after treatments (Abdel Latef and Chaoxing, 2011; Estrada et al., 2012) to monitor the transcript profiles of 14-3-3 genes under different abiotic stresses by qRT-PCR analysis.

Fm201 promoter-YGFP chimeric gene in S. cerevisiae BY4741 was treated with NaCl (500 mM), CuSO₄ (50 mM), CdCl₂ (0.2 mM), PEG4000 (25%), and 37°C abiotic stresses in YPD medium and then harvested after 1h treatments (Vido et al., 2001).

The total DNA was isolated from AM fungal sporescarps as described by Zézé et al. (1994). Total RNA of different AM fungal tissues was extracted with TRIzol reagent (Invitrogen) according to the protocol. Surface-sterilized spores were placed into 1.5 ml RNase free microtube and then frozen in liquid nitrogen, 0.3ml TRIzol solution was immediately added to the microtube. Electric mill (TIANGEN OSE-Y20, Beijing, China) and Phase Lock Gel (TIANGEN, Beijing, China) were used to make sure the quality of RNA. Total RNA yields and concentrations were measured by the Thermo NanoDrop 2000 spectrophotometer (Thermo). To remove residual genomic DNA, each total RNA sample was treated with RNase-free DNaseI (Thermo) according to the manufacturer’s instructions. The first cDNA strand was synthesized as described in RevertAid First Strand cDNA Synthesis Kit (Thermo).

Transcript profiles of AM fungal genes Fm201, Ri14-3-3, and RiBMH2 as well as host plant genes MtSbtM1 and MtCBF4 in different symbiotic stages and under abiotic stresses were studied by qRT-PCR using ViiA 7 system (Life Technologies, United States), three biological replications were performed. The expression levels were normalized to transcripts of the β-actin gene of F. mosseae or R. irregularis and to transcripts of the MtTEF gene of M. truncatula (Hohnjec et al., 2005). Before real time RT-PCR, gene-specific primers for all target genes were validated on genomic DNA and cDNA. Total RNA was isolated from AM roots comprised plant and fungal materials. The specificity of the primer pairs were also confirmed via PCR method on M. truncatula total DNA. No amplification signals were present on plant DNA. The primers sequences for all genes studied in this work are provided in Supplementary Table S2. qRT-PCR was performed using SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) according to the manufacturer’s instructions. Each 10 μl reaction contained 1 μl of the synthesized cDNA (cDNA pool was diluted to 200 μl), 5 μl SYBR Green Real-time PCR Master Mix, 0.5 μl each primer(10 μM), 3 μl ddH₂O. PCR program consisted of a 30 s incubation at 95°C to active the hot-start recombinant Taq DNA polymerase, followed by 40 cycles of 10 s at 95°C, 15 s at 57°C, and 20 s at 72°C. The relative levels of transcripts were calculated by using the 2^-ΔΔct method (Livak and Schmittgen, 2001).

The Fm201 EST sequence was obtained from NCBI (Accession number: CF803281), (Breuninger and Requena, 2004). Reverse PCR was utilized to get the 5′ flanking sequence of Fm201 gene. The gene-specific primers 201F and 201R were designed to amplify the partial DNA fragment of Fm201 according to the available sequence of Fm201 EST. Genomic DNA of F. mosseae was digested by FastDigest restriction enzyme XhoI (Thermo). DNA fragments were self-ligated by T₄ DNA ligase, and the reaction was carried out in a final volume of 20 μl containing 0.5 μl digested DNA fragments, 2 μl 10× buffer, 0.5 μl T₄ DNA ligase (Thermo), 17 μl ddH₂O, incubated for 12 h at 10°C. Nest-PCR was performed in this experiment, 0.5 μl ligated production was used as PCR template, the specific primers used in the first PCR reaction were 201RF1 and 201RR1, products from first PCR reaction were diluted to 1/1000 as the template for the second PCR reaction, and specific primer 201RF2 and 201RR2 were used.

RACE as a classic method to rapidly obtain the 5′ and 3′ ends of the Fm201 gene (Scotto-Lavino et al., 2006a,b). 3′RACE was carried out on the total RNA from F. mosseae sporecarps by using primer Q_T. Two pairs of primers RACE201F/Q_O and RACE201F2/Q_I were used for the subsequent nest PCR reactions, respectively. Due to the high A/T containing feature of AM fungi genomic DNA, the dGTP and Q_C replaced the dATP and Q_T used in classic 5′RACE, responsively. The first cDNA strand was obtained from F. mosseae sporocarps by using specific primer RACE201R1. Primers Q_C, Q_o and RACE201R2 were used as the first PCR cycle primers, while primers Q_I and RACE201R3 were used as the second PCR cycle. Transfast pfu DNA polymerase (Transgen, Beijing, China) was used in the PCR reactions mentioned above, PCR products were cloned into pEASY-Blunt vector (Transgen, Beijing, China) and sequenced.

Plasmid pMR12 was generated from pMRI-11(Xie et al., 2014), the promoter of Gal7 was amplified from the genome DNA of S. cerevisiae BY4741 by PCR using the specific primers P_Gal7F/P_Gal7R. P_Gal7 and pMR-11 were digested with both SacI and SpeI, respectively, then the digests were cloned into target vector pMR-11. To address the regulation of CREs located in the promoter of Fm201, the expression profiles of YGFP reporter were conducted in S. cerevisiae BY4741. Monoclonal vector pUG35 carrying a YGFP reporter is used in yeast heterologous systems (Cormack et al., 1997). Two restriction sites were SacI and XbaI, which were added to the start codon upstream sequence of Fm201 (pFm201) by using primers 201PFn (n = 1,2,3,4) and 201PR1, then cloned into pUG35 to replace P_MET-25 to produce a series of 5′ truncated promoters-reporter vectors. pFm201 targeted deletion of cis-element was conducted by using SOE-PCR method (Ho et al., 1989). The specific primers used are provided in Supplementary Table S2). The site-directed mutation promoter sequences were also cloned into pUG35 as the promoter truncated verification vectors. qRT-PCR was used for monitoring transcriptional efficiency of Fm201, the Ura gene of pUG35 was chosen as the internal standard, primers UraF, UraR, YGFPF, YGFPR were used in this experiment (Peter et al., 2006).

Yeast one hybrid experiment was carried out using the Matchmaker One-hybrid System (Clontech), the ORF of GintSTE was cloned from cDNA of Rhizophagus irregularis by using primers RiSTE12F and RiSTE12R, and cloned into pGADT7-rec2. Two same 272 bp length promoter fragments contained STE12 targeted cis-element STRE were cloned into pHIS2 in tandem, the same fragment only lacking the cis-element STRE was also inserted into the pHIS2 as a negative control. Yeast one-hybrid experiment of Msn2 was carried out in the same way and the primers RiMsn2F and RiMsn2R were used as mentioned in Supplementary Table S2.

Plasmids for yeast one-hybrid were co-transformed into yeast Y187 strain. Y187 cells carrying the target plasmids were cultivated in SD medium lacking leucine and tryptophan, and were also gradiently inoculated at 1.0 OD₆₀₀ on SD medium lacking leucine, histidine and tryptophan and supplemented with 30 mM 3-AT, which is a competitive inhibitor of the His3 protein.

The RNAi-target sequences of Ri14-3-3 and RiBMH2 were amplified by the specific primers Ri14-3-3F/Ri14-3-3R and RiBMH2ATG/RiBMH2F. The PCR products were cloned to the linearized pDONR221 used CloneExpressII (Vazyme, Nanjing, China), then the LR reaction was done to recombine the target sequences into the pK7GWIWG2(II)RR according to the instructions in Gateway protocol.

Agrobacterium rhizogenes Msu440-mediated root transformation was performed following the method as described in Medicago Truncatula Handbook (International Committee, 2006). In vitro hairy roots were cultured on EM plates containing Benzyl penicillin (200 mg/L) for three times. The root tip (2∼3 cm in length) was used for each subculture. The root was re-cultured in the M medium without antibiotics for half a month. The hairy root lines without bacteria were re-cultured in new M medium for mycorrhization. Mycorrhizal hairy root of P. crispum without DsRed tag was cut into small pieces (∼3 mm) and placed around the hairy root of M. truncatula harboring DsRed marker as described in Supplementary Figure S3. The mycorrhizal roots with red fluorescence were harvested in one month until the external hyphae of R. irregularis beyond the hairy root surface of M. truncatula.

Mycorrhizal roots collected from pot cultures were stained with 0.1% Typan blue, while the mycorrhizal hairy roots expressing red fluorescence grown on plates were stained with WGA488, and the estimation of AM colonization was performed as described by Trouvelot et al. (2015) using MYCOCALC program².

The deduced amino acid sequence of Fm201 was analyzed with the computer program DNAstar. Multiple sequence alignments were performed by DNAMAN8. The unrooted phylogenetic tree constructed by neighbor-joining algorithm was carried out using MEGA6. The computation of physical and chemical parameters was conducted by using ProtParam tool³. Homology modeling of the three-dimensional structure of Fm201 protein was done with the program Swiss Model⁴ using Homo sapiens 14-3-3ε protein (2br9A) as the template (Figure 1B; Yang et al., 2006). The cis-elements of the promoters Fm201 and RiBMH2 were analyzed on YEASTRACT⁵ using S. cerevisiae S288c as the reference.

Yeast mutant strains used in this article are constructed with the methods mentioned by van Heusden (van Heusden et al., 1995, 1996). The detail information for each strain is available at Supplementary Table S1. In the construction of bhms-Fm201(MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; BMH1::KanMX (Gal7[Fm201]); BMH2::ura3), fragments of pMRI-12 which contain a KanMX and a Gal7 promoter and pSH47 which contain a Ura3 marker were used to replace BY4741, BMH1 and BMH2, respectively, by primers PMRI-12F1, PMRI-12R1 and PSH47F, PSH47R. The bmhs-Ri14-3-3 and bmhs-RiBMH2 were also built in the same way.

Statistical analyses were performed through one-way ANOVA. Following ANOVA, Tukey’s test was performed to make comparisons between treatments, using a probability level of p < 0.05(^∗, #), 0.05 ≤p < 0.01 (^∗∗, ##). All statistical analyses were performed using SPSS statistical package (version 23.0, SPSS Inc., United States).