•Arbuscular mycorrhiza (AM) is a mutualistic biotrophic association that requires a complex exchange of signals between plant and fungus to allow accommodation of the mycosymbiont in the root cortex. Signal exchange happens even before physical contact, activating the plant symbiotic program.
•We investigated very early transcriptional responses in Medicago truncatula to inoculation with Glomus intraradices and identified four genes induced by diffusible AM fungal signals before contact. Three of them were previously shown to be mycorrhiza induced at later stages of the symbiosis, while MtMSBP1, encoding a membrane-bound steroid-binding protein, is a novel mycorrhizal marker.
•Expression analyses in plants defective in the symbiotic receptor kinase DMI2 allowed discrimination of two different signaling cascades involved in the perception of the diffusible signals. Thus, while some of the genes are activated in a DMI2-dependent manner, the induction of one of them encoding a proteinase inhibitor is DMI2-independent.
•Downregulation of MtMSBP1 by RNAi led to an aberrant mycorrhizal phenotype with thick and septated appressoria, decrease number of arbuscules and distorted arbuscule morphology. This provides genetic evidence that MtMSBP1 is critical for mycorrhiza development. We hypothesize that MtMSBP1 plays a role in sterol homeostasis in the root.
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Plants have evolved to interact with different microorganisms to establish different types of associations ranging from parasitic to mutualistic. The immune system of the plant has developed to recognize signal/pattern molecules to discriminate between deleterious and beneficial microorganisms (Zipfel & Felix, 2005). Arbuscular mycorrhiza (AM) fungi belong to the latter group and plants forming this association specifically recognize their symbiotic partners and consequently modify their genetic programs to accommodate them (Parniske, 2004). The molecular bases of this recognition process are starting to be understood. They point to common signaling pathways shared with other microbe–plant associations like the nodule symbiosis and to specific signaling pathways commonly elicited by all arbuscular mycorrhizal fungi (Catoira et al., 2000; Stracke et al., 2002; Liu et al., 2003, 2007; Hohnjec et al., 2005; Kistner et al., 2005; Gutjahr et al., 2008; Güther et al., 2009). As only a few plant families are nonmycorrhizal (Smith & Read, 1997), it is clear that most plants have maintained the necessary machinery to associate with these obligate symbionts for at least 450 million yr (Redecker et al., 2000). However, some plants such as the model Arabidopsis thaliana are missing some of the essential plant signaling elements required for mycorrhization (Lévy et al., 2004; Mitra et al., 2004; Kevei et al., 2005). Future research will disclose how many other components are missing or altered in nonmycorrhizal plants.
Similar to the rhizobial symbiosis, where the plant is the first to ‘talk’ to its bacterial partner with a cocktail of flavonoids that induces expression of the nod genes, in the arbuscular mycorrhizal symbiosis the fungus reacts to the production of strigolactones by the roots with hyphal branching (Akiyama et al., 2005). This phenomenon is known to occur upon recognition of compatible host root exudates (Giovannetti et al., 1993; Buee et al., 2000), although it is not sufficient to induce appressoria formation. However, in contrast to the nodule symbiosis, where the identification of the lipochitin–oligosaccharide signals (Nod factors; NF) has led to considerable advances in the dissection of the signal cascade that initiates the symbiotic program in the plant, it is not known how AM fungi ‘talk’ to their host plants. From genetic experiments using legume mutants impaired in nodule symbiosis we know that receptors for NF perception (LYK3, NFP in Medicago truncatula, NFR1 and NFR5 in Lotus japonicus) are not involved in perception of mycorrhizal signals (Wegel et al., 1998; Limpens & Bisseling, 2003;Madsen et al., 2003; Radutoiu et al., 2003). Therefore it is likely that AM fungal signals are of a different chemical nature. Nod factor perception culminates in the activation of at least four specific transcription factors that mediate all NF-induced transcriptional responses (Kalóet al., 2005; Smit et al., 2005; reviewed in Oldroyd & Downie, 2008). Although, these transcription factors are also dispensable for arbuscular mycorrhiza symbiosis (Catoira et al., 2000; Oldroyd & Long, 2003; Hirsch et al., 2009), at least seven of the intermediate signaling components are essential for establishment of the AM symbiosis (Catoira et al., 2000; Kistner et al., 2005). This common pathway has been referred as the SYM (symbiotic pathway) and contains a receptor like kinase, nuclear pore proteins, nuclear ion channels, a calcium/calmodulin-dependent protein kinase and an associated protein (Cyclops) of unknown function (for recent reviews see Parniske, 2008; Oldroyd et al., 2009). In particular, three SYM proteins have been extensively studied and shown to be early transmitters in both symbioses. In M. truncatula they were named DMI proteins (does not make infections). Molecular and genetic analyses of these genes showed that DMI1 (POLLUX in L. japonicus), encoding a cation-permeable channel, and DMI2 (SYMRK in L. japonicus), encoding a receptor like kinase, are hierarchically situated above DMI3, the calcium and calmodulin-dependent protein kinase (Wais et al., 2000). Both proteins are required to induce a calcium oscillation signal that is decoded by DMI3 (Kalóet al., 2005; Smit et al., 2005). While it was long known that NF induce calcium spiking as one of the earliest responses in root hairs, only recently it was shown that AM fungi elicit both, a transient cytosolic calcium elevation (Navazio et al., 2007) and calcium oscillation (Kosuta et al., 2008). In the first case, this happens in response to a fungal diffusible signal constitutively produced (Navazio et al., 2007), while calcium oscillation appears to be dependent on the presence of branched strigolactone-induced hyphae (Kosuta et al., 2008). Although the AM fungal diffusible constitutive signal was able to transcriptionally activate all DMI genes (Navazio et al., 2007), it is not known whether the transmission of this signal travels through this cascade. By contrast, calcium oscillation in response to a diffusible signal released by branched AM hyphae was shown to be dependent on DMI2 and DMI1 (Kosuta et al., 2008).
There are several examples of the divergence in the perception of NF and AM fungal signals, indicating the existence of more than one pathway for the establishment of root symbioses. These pathways are likely to share some components and to be triggered by more than one signal. Thus, while the symbiotic activation of the early nodulin MtENOD11 by NF is DMI dependent, its activation in AM symbiosis is DMI-independent when triggered by a diffusible fungal signal (Kosuta et al., 2003) or DMI-dependent when triggered by appressorium formation (Chabaud et al., 2002). Similarly, the induction of lateral root formation (LRF) induced by both NF and diffusible AM fungal signals requires different elements of the DMI cascade (Oláh et al., 2005). Hence, while NF-induced LRF requires all three components, DMI3 is dispensable for LRF triggered by a diffusible AM fungal signal. This indicates that if calcium oscillations are produced and needed for LRF during mycorrhiza symbiosis, they are not decoded by DMI3. Interestingly, induction of LRF in rice has been recently shown to be independent of the DMI1 and DMI3 rice orthologues (Gutjahr et al., 2009). This could suggest that calcium oscillation might not be necessary for perception of the diffusible AM fungal signal that induces LRF.
In this work we describe a novel M. truncatula gene (MtMSBP1) that is early induced by a diffusible AM fungal signal produced by branched hyphae. The inactivation of this gene leads to changes in the development of the fungus inside the cortex with the appearance of abnormal appressoria and aberrant or collapsing arbuscules. The transcriptional analysis of MtMSBP1 and of the other identified early marker genes clearly shows that at least two different signaling cascades exist for perception of an AM fungal diffusible signal. Even more, it may suggest the possibility that more than one fungal diffusible signal might exist. Thus, while one of these signals would travel independently of the receptor-like kinase DMI2, the perception of the second signal would be DMI2 dependent.
The red fluorescent marker DsRED1 under the control of the constitutive Arabidopsis Ubiquitin10 promoter (PUbq10) (Limpens et al., 2005) was cloned (KpnI) into the binary Gateway vectors pKGWFS7 and pK7FWG2,0 (both Karimi et al., 2002) resulting in the promoter reporter vector pPGFPGUS-RedRoot and the plasmid pCGFP-RedRoot for C-terminal GFP fusion and expression under the 35S promoter of the cauliflower mosaic virus (CMV). Genomic DNA of M. truncatula was isolated with the DNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. A MtMSBP1 promoter fragment of 1518 bp was PCR-amplified from genomic DNA using the primers: PMtMSBP1-forward/reverse. The coding region of MtMSBP1 was amplified from cDNA using the primers MtMSBP1-forward/reverse. The promoter fragment was cloned into pPGFPGUS-RedRoot and the coding region of MtMSBP1 was recombined into pCGFP-RedRoot by using the pENTR/DTOPO and the Gateway system (Invitrogen). The RNAi constructs were created using the binary Gateway vector pK7GWIWG2D(II) for hairpin RNA expression under the control of the CMV 35S promoter (Karimi et al., 2002). MtMSBP1 DNA region −18 bp to +186 bp (with respect to the ATG position, where A is position +1) was amplified from genomic DNA using the primers MTMSBP1-RNAi-forward/reverse and recombined into pK7GWIWG2D(II). Ten hairy root explant lines were obtained and from those five expressing GFP stably after several rounds of subcultivation were further used for real-time polymerase chain reaction (PCR) analyses. All primers used in this manuscript are listed in the Supporting Information, Table S1.
Generation of transgenic M. truncatula hairy root lines
Generation of hairy roots was conducted after the protocol of Boisson-Dernier et al. (2001) with A. rhizogenes ARqua1 (Quandt et al., 1993) using either M. truncatula wild-type or dmi2 mutant plants. Hairy roots emerging from the root section were explanted and propagated stably on M medium (Bécard & Fortin, 1988) supplemented with 25 mg l−1 kanamycin for selection and decreasing concentrations of 400, 200 and 0 mg l−1 augmentin (Amoclav; Hexal, Holzkirchen, Germany) to eliminate A. rhizogenes. For each transformation at least 50 plants were used and between 10 and 20 lines per construct were propagated and investigated for stability of the introduced T-DNA.
In vitro mycorrhizal colonization of M. truncatula
We developed an in vitro system for mycorrhization of M. truncatula roots. Hyphae growing in the distal part of the D. carota–G. intraradices system described by St-Arnaud et al. (1996) were used as a source of mycorrhizal inoculum. Whole M. truncatula plants or hairy root explants were mycorrhized in this system either in liquid or solid M medium, respectively. For whole plant inoculation extraradical mycelium was grown in liquid medium as described before (Helber & Requena, 2008). After 10 d, three to five seedlings were planted into openings on the plate lid close to the border of the bipartite plate. Plants were incubated at 25°C with a photoperiod of 16 h of light.
Hairy root explants were colonized on solid M medium. To achieve this, extraradical hyphae were allowed to grow on a cellophane membrane on top of the M medium. Hyphal passage to the distal compartment was facilitated by a swell (a ramp or step from the distal compartment to the border with the proximal compartment) and a thin layer of M medium poured on top of the cellophane membrane (Fig. 1e). To synchronize the colonization of hairy root explants, extraradical hyphae were grown c. 7 d until the fungal compartment was covered with hyphae. Hairy root explants were then added and covered with some water and a second cellophane membrane was set on top to maximize the contact area between fungus and root.
Appressoria enrichment, time course experiment and sandwich assay of mycorrhizal colonization
Appressoria-enriched root fragments from either whole M. truncatula plants or hairy root explants were isolated, cut and frozen in liquid nitrogen for subsequent RNA extraction and microarray analysis. Growth of extraradical hyphae towards the roots was monitored every day under the stereo microscope and root fragments (2–3 mm long) that came in contact with hyphae were excised and stored in RNAlater solution (Qiagen) at 4°C. Similar root fragments from uncolonized roots were collected as control. Ten different plates for each treatment were used. The time-course of mycorrhizal colonization was carried out in solid medium using M. truncatula hairy root explants. Mycorrhiza formation was monitored periodically by staining with black ink (Vierheilig et al., 1998). Samples for RNA extraction were taken after 3, 6, 8, 13 and 17 d. Roots from at least three plates were pooled at each time-point, frozen in liquid nitrogen and stored at −80°C.
To test the influence of fungal diffusible signals on plant gene expression a sandwich system was employed where M. truncatula hairy root explants were separated from germinated spores (3 d of growth) or extraradical hyphae (7 d of growth) by a permeable cellophane membrane. For inoculation with germinated spores G. intraradices spores were isolated as described earlier (Helber & Requena, 2008). Spores of 40 plates were collected in 2 ml of water and 200 μl of spore suspension were applied directly onto the cellophane. When triggered with extraradical hyphae, mycelium was prepared as described earlier. Plates were incubated for up to 5 d and roots of at least three plates were sampled after 1 d and 5 d. The roots of each time-point were pooled, frozen in liquid nitrogen and stored at −80°C. In all experiments control mock-inoculated roots were also isolated.
Real-time reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR analyses
Total RNA was isolated with RNeasy plant mini kit (Qiagen) according to the manufacturer’s protocol. The integrity of RNA was visualized on denaturing agarose gels and quantification was done spectrophotometrically. A 1-μg sample of total RNA was digested with DNAse I (Amplification grade; Invitrogen) and reverse transcription was performed in a 20-μl reaction using the Superscript II reverse transcriptase and OligodT Primer (Invitrogen).
Transcript levels of genes were determined on a Bio-Rad iCycler MyIQ using MESA Green qPCR MasterMix Plus (Eurogentec, Köln, Germany) and a primer concentration of 0.2 pmol μl−1 in 25-μl reactions. A 1-μl sample of a 1 : 10 dilution of single-strand cDNA was used as a template. For each primer set gradient PCR was performed to determine the optimal annealing temperature. Final PCR conditions were selected as follows: 3 min at 95°C (no cycling); 30 s at 95°C, 30 s at 56°C and 30 s at 72°C (40 cycles). Every real-time PCR run was done at least twice in three biological replicates with three technical replicates per reaction. Melt curve data was collected after each run. Ten-fold dilution standard curves were taken for each primer set to determine the efficiency of amplification. Threshold cycle location as well as further data analysis was performed with the IQ5 optical system software version 2.0 using the ΔΔCt method based on Vandesompele et al. (2002) and taking the primer efficiencies into consideration (Bio-Rad). Transcript levels were normalized to the constitutively expressed translation elongation factor 1-alpha MtTEF1α (TC106470, DFCI M. truncatula gene index) (Hohnjec et al., 2003; Wulf et al., 2003; Nyamsuren et al., 2007).
For RT-PCR, RNA was isolated from M. truncatula roots, shoot, leaves, flowers and pods and cDNA was synthesized as described earlier. The RT-PCR conditions were the same as described for real-time RT-PCR with the exception that only 30 cycles were programmed.
Roots for long observations were prepared for microscopy as described by (Genre et al., 2005). Microscopy was done either with a conventional epifluorescence microscope Zeiss AxioImager Z1 or the Laser Scanning microscopes Zeiss LSM 510 meta and Leica TCS SP5 with water immersion objectives ×60 or ×63.
Identification of M. truncatula early-induced genes during arbuscular mycorrhizal symbiosis
Plants can perceive AM fungi at very early stages of the symbiosis even before physical contact (Kosuta et al., 2003; Weidmann et al., 2004). This perception is manifested in changes in gene expression that presumably prepare the plant for the accommodation of the fungal partner. We attempted here to identify new marker genes of early mycorrhization in M. truncatula. Using a novel experimental set-up under total root sterility seedlings were mycorrhized in vitro in a bicompartmental plate system shown in Fig. 1a. Here AM hyphae are grown in liquid M medium and seedlings are ‘planted’ on the plate. We succeeded with this system to colonize several plants, such as M. truncatula, L. japonicus (Fig. 1b–d) or Solanum lycopersicum. Appressoria formation was monitored under the stereomicroscope after removing the plants from the system and root segments (2–3 mm) harboring contact points or appressoria were harvested and RNA extracted. Transcriptional analysis carried out with the Mt16kOLI1 microarrays (Hohnjec et al., 2005) yielded c. 500 genes regulated vs noncolonized root segments. From those, 237 were upregulated (data not shown), 90 of them at least 2.4 fold (log2-ratio M ≥ 1.25), using a P-value threshold of 0.05 (Table S2). Based on the original data set, several candidate genes induced in appressorium-enriched samples with induction levels between 0.5 and 5 were validated by real-time PCR. From those, four genes were further verified in different biological replicates (Fig. 2a). Three of these genes were previously identified as mycorrhiza specific but their induction was only studied at later stages (TC106351, TC112474, TC107197; Liu et al., 2007; Wulf et al., 2003). The first two encode proteinase inhibitors while the third is a protein of unknown function designated specific tissue protein 2. The fourth gene identified was not previously reported as mycorrhiza induced (TC106972) and showed similarity to membrane steroid-binding proteins. The expression of the early marker gene, MtENOD11 was also included in the analysis as reference (Chabaud et al., 2002) and showed a similar induction to TC106972 in appressoria-enriched samples.
To assess whether the induction of the four genes studied was maintained at later stages of the symbiosis, we carried out a time-course analysis of expression. Mycorrhized M. truncatula hairy root explants were monitored periodically (Fig. 1e,f) and their expression analysed at different time-points. In this system, mycorrhization is very efficient and many appressoria were observed at 3 d post infection (dpi). At 6 dpi intraradical mycelium and some arbuscules are observed, while 8–13 d correspond to high abundance of arbuscules. At 17 dpi vesicles can be observed. The time-course analysis of TC106972 expression showed that the gene was induced at 3 d post inoculation, an appressoria-rich stage, confirming the results shown above (Fig. 2b). Induction was of a similar magnitude albeit a bit lower, indicating a dilution effect owing to the use of whole roots instead of appressoria-enriched samples. Interestingly, the expression of TC106972 returned to basal levels after 6 d and remained low for the rest of the time course. This was in contrast to the other three genes analysed, which remained induced at later stages. However, whereas TC107197 and TC112474 expression was further increased at later stages of mycorrhization, expression of TC106351 was highest at earlier time-points of the symbiosis (Fig. 2c)
MtMSBP1 encodes a membrane steroid-binding protein
blastx analyses showed that TC106972 has homology to a family of proteins called membrane steroid-binding proteins widespread in the plant kingdom but also conserved in animals, insects and fungi. A member of this family in A. thaliana was recently identified as MSBP1, therefore we designated the TC106972 as MtMSBP1 (Yang et al., 2005). Phylogenetic analysis of several eukaryotic members of this protein family showed that MtMSBP1 clusters together with the plant proteins. The closest homology (77% identity) was found to the L. japonicus progesterone-binding protein (PBP) TC20369 followed by a homologue from Glycine max (74% identity) (Fig. 3a). A second M. truncatula membrane steroid-binding protein was found in the TIGR database (corresponding to TC95008 and TC95009), which shares 67% identity with MtMSBP1. We designated it as MtMSBP2. Interestingly, a membrane steroid-binding protein from L. japonicus (Lj TC21243) has been recently found to be induced at early stages of mycorrhiza formation with Gigaspora margarita and repressed in arbuscule-containing cells (M. Güther and P. Bonfante, pers. comm.). Lj TC21243 showed higher similarity to MtMSBP2 than to MtMSBP1. Therefore, we tested whether MtMSBP2 followed a similar trend of early induction. However, MtMSBP2 was not found to be mycorrhiza-induced at any stage of the symbiosis (data not shown). This could indicate that perhaps the L. japonicus protein encoded by Lj TC21243 is the functional orthologue of MtMSBP1.
MtMSBP1 is located in chromosome 6 and it is part of the artificial chromosome AC157779. The corresponding gene consists of two exons (a 410 bp exon containing the start codon, and 660 bp exon containing the stop codon) separated by an intron of 4132 bp. The intron position is conserved in A. thaliana, between the amino acids Ser104 and Arg105. MtMSBP1 cDNA was deduced to encode a polypeptide of 235 amino acids with a calculated molecular mass of 25.8 kDa and a theoretical isoelectric point of 4.55. MtMSBP1 contains a noncleavable signal peptide (amino acids 1–40), a single N-terminal transmembrane region (amino acids 15–37) and a conserved cytochrome b5-like heme/steroid binding domain (72–169 amino acids) Pfam motif PF00173 (Fig. 3b). A conserved tryptophan residue (Trp155), a putative binding site of progesterone, is also present in MtMSBP1. The protein structure indicates an N-terminal anchoring to a membrane and a catalytic domain for steroid binding, typical of membrane-associated steroid receptors (Falkenstein et al., 1996; Meyer et al., 1996; Cenedella et al., 1999).
MtMSBP1 localizes to the ER
The presence of a noncleavable signal peptide and a N-terminal transmembrane region suggests the localization of the protein at a membrane. The wolf psort program (http://wolfpsort.org, Horton et al., 2006) predicts in addition a C-terminal endoplasmic reticulum (ER) membrane retention signal, ADKE, indicating that this protein could be localized to the ER. While animal and fungal homologues of MSBP have been found to localize to the ER, including perinuclear localization (Min et al., 2005), the A. thaliana MSBP1, surprisingly, was found to localize at the plasma membrane (Yang et al., 2005). In order to determine precisely the subcellular localization of MtMSBP1 in M. truncatula we cloned the coding region of MtMSBP1 into the binary vector pCGFP-RR resulting in a C-terminally GFP-tagged version of the protein under the control of the CMV 35S promoter. The M. truncatula hairy roots were generated and the localization of the protein was analysed by epifluorescence and confocal microscopy. The GFP signal in young root hairs was found at a perinuclear localization as well as in membranous structures projecting from the nucleus towards the root hair tip. These structures most likely correspond to the different ER populations: nuclear, cortical and cytoplasmic connections to cortical ER (Fig. 4a,b). Very often MtMSBP1 accumulated as a bright spot around the nucleus (Fig. 4a). Confocal microscopy in cortical cells clearly showed that the GFP signal was localized in the ER network, including the nuclear envelope, but not inside of the nucleus. By contrast, DsRED signal, constitutively expressed, could be observed in the cytoplasm and within the nucleus (Fig. 4c,d,e). The localization of MtMSBP1-GFP at the ER mirrors the localization of the construct P35S:GFP-HDEL containing the HDEL ER retention signal shown recently by Genre et al. (2005). To test whether the ER localization pattern of MtMSBP1was M. truncatula specific, protoplasts of A. thaliana and epidermal cells of N. benthamiana were transformed with the same construct. In both cases MtMSBP1-GFP showed ER localization, including perinuclear localization (Fig. 4f,g).
MtMSBP1 expression analyses
We analysed the expression of MtMSBP1 in different plant tissues by RT-PCR (Fig. 5a) and in silico using the M. truncatula gene expression atlas (http://bioinfo.noble.org/gene-atlas/). The gene was expressed in all tissues, root, shoot, leaves, flowers and pods. In contrast to the homologue in A. thaliana, which is very low expressed in roots compared with other tissues (Yang et al., 2005), MtMSBP1 is similarly highly expressed in all tissues analysed. According to the in silico data, expression is about twofold higher in roots than in other tissues (Benedito et al., 2008). To analyse MtMSBP1 expression in roots in more detail, a 1518 bp promoter region upstream of the ATG was isolated from genomic DNA and fused to the reporter genes GFP-GUS. Transgenic stable hairy root explants were produced and both in vivo fluorescence as well as histochemical GUS staining were carried out. The β-glucuronidase activity showed that MtMSBP1 was constitutively expressed in the central cylinder as well as in the apical meristem (Fig. 5b,c,h). In some cases expression could be seen in epidermal and cortical cells in regions very close to the apical meristem (Fig. 5c).
The temporal and spatial induction of MtMSBP1 in vivo during mycorrhization was studied in stably transformed hairy root explants expressing the GFP-GUS reporter genes by periodical monitoring using fluorescence microscopy. It could be observed that MtMSBP1 was often induced in root areas in contact with branched hyphae originating from runner hyphae running parallel to the root (Fig. 5d,e), in contrast to control roots (Fig. 5g). The GUS staining confirmed this result and showed that in addition to the MtMSBP1 constitutive expression in the vascular cylinder and in the apical meristem observed in control roots, a mycorrhiza-specific GUS activity was induced in root areas in contact with branched hyphae (Fig. 5f). To determine more precisely in which root cells MtMSBP1 was induced upon hyphal contact, we used Z-stack confocal microscopy. The GFP fluorescence could be seen in epidermal and subepidermal cells in the surrounding area of G. intraradices branched hyphae (Fig. 6a–f). However, we cannot exclude induction in other root areas as the fluorescence in deeper tissue layers is difficult to detect.
A secreted AM fungal signal triggers MtMSBP1 expression before hyphal contact
To analyse the onset of mycorrhiza-induced expression, time-lapse video microscopy was carried out. Transgenic M. truncatula hairy root explants expressing the GFP-GUS reporter cassette under the control of the MtMSBP1 promoter were mycorrhized. Root colonization was monitored with fluorescence microscopy. During a period of c. 3.5 h, it could be observed that a short hypha emerged from a runner hypha extending parallel to the root. This short hypha continued growing and branching towards the rhizodermis to form an appressorium. In that time, the promoter of MtMSBP1 was activated in epidermal/subepidermal cells in the vicinity of the branching hypha. Surprisingly, expression was induced before hyphal contact to the root, pointing to a secreted diffusible signal. GFP could be after 2 h and 10 min (Fig. 7 and Video S1) indicating quick diffusion, perception and transduction of the fungal signal. This fact prompted us to investigate whether the expression of MtMSBP1 could also be induced if there was no direct contact between fungus and plant, and signals could pass through a diffusible membrane. A ‘sandwich system’ was used placing a cellophane membrane between fungus and plant. Transgenic hairy root explants were triggered either with germinating spores or with extraradical hyphae, and expression was analysed by real-time PCR after 1 d or 5 d of contact. Indeed, results showed that both fungal propagules produce a secreted diffusible signal able to cross through the cellophane membrane and to induce the expression of MtMSBP1 (Fig.8). Interestingly, this induction was, in both cases, transient, coinciding with results from the time-course analysis (Fig. 2b).
Diffusible AM fungal signal/s travel through two different signaling pathways
It is known that mycorrhiza formation requires an intact symbiotic pathway involving the receptor-like kinase DMI2 and the ion channel protein DMI1 to induce a calcium oscillation signal. Recently, it has been shown that a diffusible signal produced by branched hyphae of AM fungi is able to induce this calcium oscillation and that DMI2 and DMI1 are required for the perception of this signal (Kosuta et al., 2008). Therefore, we addressed the question whether the induction of MtMSBP1 and the other mycorrhiza early-induced genes was caused by a diffusible signal and whether this signal was travelling through the SYM pathway. As shown in Fig. 9, all genes analysed were induced in M. truncatula hairy roots explants with wild-type background when triggered by extraradical hyphae separated by a cellophane membrane. However, with the exception of TC106351, this induction was abolished (TC107197 and TC112474) or reduced (MtMSBP1) when dmi2-1 mutant TR25 hairy roots explants were used. This result shows unambiguously that two signaling cascades are functioning to perceive AM fungal signals.
Inactivation of MtMSBP1 results in a mycorrhiza-defective phenotype
Our work indicated that MtMSBP1 was transcriptionally induced by a diffusible AM fungal signal. This result indicates that its induction might be significant for the development of the symbiosis. To genetically test this hypothesis we used RNA interference and analysed the phenotype of hairy root explants for mycorrhiza formation. We obtained several stable transgenic hairy root explant lines with different levels of gene downregulation. All lines grew similar to the control lines and showed no developmental defects under non-symbiotic conditions (data not shown). Five hairy root lines were analysed by PCR, and four were selected for mycorrhizal analyses based on the highest degree of RNA inactivation. The expression levels of MtMSBP1 in the four RNAi hairy root lines analysed were strongly reduced with respect to several control hairy root lines (transformed only with A. rhizogenes or with other control plasmids) (Fig. 10a). The expression of MtMSBP2 coding for the second isozyme was checked in all lines and it was not significantly affected (data not shown). Transgenic RNAi hairy root lines were mycorrhized in vitro and analysed at 12 dpi by ink staining. Results showed that the frequency of mycorrhizal colonization in RNAi lines was similar to that from control lines (Fig. 10b). However, several important phenotypic differences were observed. Appressoria from RNAi lines often developed clumps of highly septated hyphae that ended in aborted infections (Fig. 10c). Nevertheless, infection succeeded in other areas of RNAi lines and then the fungus developed intracellular hyphae, arbuscules and vesicles. However, the number of arbuscules was strongly reduced in RNAi lines (80% reduction, Fig. 10b). Even more significant was that the abundance of collapsing arbuscules and septated hyphae was strongly increased with respect to the control lines (Fig. 10c).
The establishment and maintenance of a mutualistic symbiosis between plants and microbes is a complex process that requires constant signal exchange between both partners to avoid defense reactions that would jeopardize the association. In the arbuscular mycorrhizal symbiosis this signal exchange occurs before physical contact in both directions. Strigolactones released from host roots have been shown to induce hyphal branching in AM fungi (Akiyama et al., 2005) and one or more diffusible signal molecules from AM fungi elicit transient cytosolic calcium elevation in host plant cells, induction of symbiosis related genes and calcium oscillation (Kosuta et al., 2003, 2008; Weidmann et al., 2004; Navazio et al., 2007). However, the nature of the fungal signal(s) is still unknown. The perception of AM fungal signals travels at least partly through the SYM pathway, but evidence of a second signal cascade has been recently shown (reviewed in Oldroyd et al., 2009). Here we present further evidence of the existence of at least two different signal cascades in M. truncatula for perception of AM fungal diffusible signals.
In this work we identified MtMSBP1 in a screening for early mycorrhiza induced genes together with other early markers (TC107197, TC106351 and TC112474). MtMSBP1 encodes a protein of the recently identified membrane-bound steroid-binding protein family that in plants is associated with the regulation of cell elongation (Yang et al., 2005). Although this gene is highly expressed in roots during asymbiosis in the central cylinder and in the meristem of the root apex, its expression is transiently induced two- to three-fold at very early stages of the AM symbiosis by a diffusible signal. The induced expression was observed in epidermal and subepidermal cells in the vicinity of approaching hyphae and in appressorium-enriched root areas. The induction in appressorium-enriched samples coincides with an induction of the early marker gene MtENOD11 (Journet et al., 2001). MtENOD11, a repetitive hydroxyproline-rich protein, has been widely used as space and time marker of mycorrhization (Chabaud et al., 2002; Kosuta et al., 2003). Similarly to the mycorrhizal-induced expression of MtMSBP1 described in this work, MtENOD11 is induced in root areas underneath branched hyphae (Kosuta et al., 2003). This might indicate that not all hyphae from AM fungi are able to produce (enough) signal(s) inducing expression of these two genes. Therefore it is likely, that the prestimulation of branching by plant strigolactones not only facilitates docking of the fungus to the root but also makes AM fungal hyphae competent for symbiosis.
In our work here, time-lapse video microscopy and cellophane experiments clearly showed that MtMSBP1 expression was induced before fungal contact. This shows that the branching hypha produces a secreted fungal compound, able to pass through a cellophane membrane, that triggers expression within 3 h. This rapid transcriptional induction suggests that the signal perception is transmitted through a pre-established signal cascade, perhaps involving phosphorylation. To date, only the SYM signal cascade mentioned above has been genetically defined for the perception of AM fungal signals. However, evidence that a second cascade might exist is accumulating. Thus, while MtENOD11 is activated upon perception of a diffusible signal in mutant plants defective in three genes of the SYM pathway (DMI2, DMI1 and DMI3) (Kosuta et al., 2003), calcium oscillation induced by a diffusible signal from AM fungi requires DMI2 and DMI1 (Kosuta et al., 2008). Additional indication of an alternative signaling cascade to the SYM route has been recently observed in rice (Gutjahr et al., 2009). The work presented here provides further indication of two different signal cascades operating in this symbiosis. The evidence is based on the differential activation of these target genes among wild-type and mutant plants defective in the symbiotic receptor kinase DMI2. In contrast to wild-type plants, that are able to perceive the AM-diffusible fungal signal/s and activate the four marker genes studied here, in DMI2 defective plants only one of the marker genes (TC106351) is clearly activated, indicating the existence of a DMI2-independent pathway. In addition to two signaling cascades, it is possible that more than one fungal signal might exist. Thus, one of these signals would travel through the SYM pathway and the second through a yet unidentified route. Biochemical analyses will help to discern about the existence of more than one AM fungal signal.
In order to ascertain the role that MtMSBP1 plays in the mycorrhiza symbiosis we localized the protein subcellularly and inactivated it using RNA interference. Surprisingly, although the A. thaliana protein was described to locate to the plasma membrane, we clearly showed here that the M. truncatula protein localizes to the ER as it has been shown for yeast and animal homologues (Rohe et al., 2009). Furthermore, heterologous localization of MtMSBP1 in A. thaliana protoplasts and in N. benthamiana leaves showed ER localization. This is consistent with steroids being molecules able to travel through biological membranes and to reach receptors within the cell lumen. Inactivation of MtMSBP1 by RNA interference showed a surprising but interesting phenotype. Thus, while we did not observe any morphological alterations in root development in the absence of mycorrhiza, the phenotype of the symbiotic structures within the root was affected. Hyphal penetration from appressoria was often aborted by septation of penetrating hyphae. Septation also occurred in intercellular hyphae but, most significantly, the morphology of arbuscules was distorted. Arbuscules developed much less frequently and with a crippled morphology in the RNAi hairy root lines, resembling the phenotype observed in the pea late mutant (RisNod24) or in the M. truncatula phosphate transporter mutant, mtpt4-1, when mycorrhized (Gianinazzi-Pearson, 1996; Lapopin et al., 1999; Javot et al., 2007). These results provide genetic evidence of the relevance of MtMSBP1 for the mycorrhiza symbiosis.
Membrane-bound steroid-binding proteins are a family of proteins first described in animals and later found in plants and fungi (Falkenstein et al., 1996; Hand et al., 2003; Iino et al., 2007). In contrast to plasma steroid-binding proteins that locate to the cytoplasm and shuttle to the nucleus after steroid binding where they act as transcription factors, membrane-bound steroid-binding proteins contain a single transmembrane domain and are presumed to act through second messengers (Lösel et al., 2003). In plants their role has been linked to the control of cell expansion/elongation as it was observed that hypocotyl length changes correlated to induction or repression of AtMSBP1 (Yang et al., 2005). However, recent work with the mammalian and yeast/fission yeast homologues have shown that this family of proteins might have a conserved role in the control of the sterol biosynthesis by binding and regulating ER-located cytochrome P450 enzymes (Mallory et al., 2005; Hughes et al., 2007). In this respect, Yang et al. (2005) also noted that in A. thaliana, from the 115 induced genes in transgenic lines overexpressing AtMSBP1, 13% were related to the steroid/sterol metabolism and signaling. The homeostasis of sterols appears to be of major importance for the establishment of root symbioses. Thus, experiments using sterol biosynthesis inhibitor fungicides have shown that the pattern of root sterols changes dramatically with application of fenpropimorph and this, in turn, impedes arbuscular mycorrhizal symbiosis (Campagnac et al., 2008). Fenhexamide, another sterol biosynthesis inhibitor fungicide does not induce major changes in the sterol profile of the root, or modify the total colonization by G. intraradices, but arbuscule frequency is significantly reduced, phenocopying the effect of inactivation of MtMSBP1. During symbiosis with rhizobia, the DMI2 receptor kinase has been shown to interact with 3-hydroxy-3-methylglutaryl CoA reductase (MtHGMR1), an enzyme of the mevalonate biosynthetic pathway required for the synthesis of isoprenoids (Kevei et al., 2007). Accordingly, application of lovastatin,which inhibits HGMR activity, or inactivation of HGMR by RNAi leads to a decrease in nodulation. We have evidence that DMI2 also interacts with HGMR during mycorrhiza formation (N. Rieger & N. Requena, unpublished results). MtHMGR1 has also been described to be induced at early stages of mycorrhiza symbiosis (Liu et al., 2003). Therefore, it appears that the control of sterol homeostasis is essential to accommodate mutualistic symbionts in the root. We propose that MtMSBP1 could be involved in this regulation by interaction with P450 ER enzymes during mycorrhiza formation.
In summary, we show here that AM fungi are able to produce diffusible signal(s) that are transmitted through two signaling pathways to induce an array of downstream effectors. We propose that one of these signals travels through the well-established SYM pathway or at least enters through this pathway requiring a functional DMI2. This diffusible signal activates TC107197, TC112474 and possibly MtMSBP1, novel markers for early mycorrhization. The second signal travels through a yet unknown signaling pathway to activate the specific mycorrhiza marker TC106351. At present, we cannot rule out whether these are all constitutive or induced signals. However, induction of MtMSBP1 correlated with the presence of branched hyphae. Our results for the inactivation of MtMSBP1 show that AM fungal induction of MtMSBP1 might be related to the need of altering the sterol metabolism to allow plasma membrane invagination and intracellular accommodation of the symbiont in the cortex.
We thank the German Science Foundation (DFG) for financial support to N.R. (DFG Re 1556/4-2 and DFG 1556/3-1) and the Landesgraduiertenförderung Baden-Württemberg, University of Karlsruhe (TH) for the Ph.D. fellowship to H.K. We are thankful to M. Dietmann for her initial help with the in vitro assay of AM and M. Krebs at the ZMBP, University of Tübingen, Germany, for the A. thaliana and N. benthamiana transformation experiments. We thank D. G. Barker for providing the TR25-ENOD11::GUS line.