Introduction

The below-ground association between arbuscular mycorrhizal fungi (AMF) and plant roots is probably the most ancient symbiosis on earth (Redecker et al. 2000) that nowadays involves 80% of terrestrial plants (Smith and Read 2008). In this association, AMF receive their carbon (C) from the host plants, in return for which they compensate the plants by the provision of mineral nutrients (subsequently called ‘nutrients’), in particular the poorly mobile phosphate (P) ions (Smith and Read 2008; Karasawa et al. 2012). Due to their strict dependence on plants for C acquisition, AMF have lost most of the gene-encoding proteins involved in saprotrophic function (Tisserant et al. 2013). Thus, they cannot access soil organic nutrients by themselves. However, other soil microorganisms, e.g., bacteria, can develop near or on the surface of extraradical hyphae where they perform multiple functions (Artursson et al. 2006; Agnolucci et al. 2015; Iffis et al. 2016). Under nutrient-limited conditions, these bacteria may compete for the break-down products solubilized by their exo-enzymes (Leigh et al. 2011; Zhang et al. 2014). Conversely, when the nutrient level is appropriate, these bacteria may cooperate with AMF and enhance the access of the fungi to the organic nutrient (Zhang et al. 2018). These bacteria receive C-rich compounds (e.g., sugars, carboxylates, amino acids, etc.) exuded by the hyphae (Kaiser et al. 2015; Wang et al. 2016), and some of them are able to mineralize soil organic N and P that become available to the AMF (Leigh et al. 2011; Nuccio et al. 2013; Zhang et al. 2016, 2018). In a previous study, Zhang et al. (2018) demonstrated cooperation between the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 43194 and the phosphate-solubilizing bacterium (PSB) Rahnella aquatilis HX2. However, the mechanisms involved in this relationship remain poorly understood.

At the cellular level, the activity of bacteria depends on the high-energy phosphate compound adenosine triphosphate (ATP), which is primarily produced in the tricarboxylic acid (TCA) cycle (Tortora et al. 2016). This cycle comprises eight steps which are catalyzed by nine enzymes (or enzyme complexes). The first step, which is the most limiting step in the cycle, is the synthesis of citric acid catalyzed by citrate synthase (Krebs 1970). Isocitrate dehydrogenase and α-oxoglutarate dehydrogenase are the other pace-making enzymes involved in the cycle (Tortora et al. 2016). Any increase in the amounts or activity of these enzymes will enhance the production of ATP and thus the activity of bacteria that live in close vicinity to AMF hyphae.

In the present study, we investigated at the transcriptional level, how the arbuscular mycorrhizal fungus R. irregularis MUCL 43194 influences the three pace-making enzymes of the TCA cycle in the PSB R. aquatilis HX2. We postulated that the presence of R. irregularis hyphae would increase the expression of genes encoding the three pace-making enzymes in R. aquatilis because hyphal exudates provide C-rich compounds (e.g., sugars, carboxylates, amino acids, etc.) to the bacterium which can be transformed into substrates for the TCA cycle.

Materials and methods

The arbuscular mycorrhizal fungus strain used was R. irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler as “irregulare” MUCL 43194. This strain was provided by the Glomeromycota in vitro collection (GINCO—http://www.mycorrhiza.be/ginco-bel) associated with Ri T-DNA transformed carrot (Daucus carota L.) roots clone DC2 on Petri plates (90 × 15 mm) containing modified Strullu-Romand (MSR) medium (Declerck et al. 1998) solidified with 3 g L−1 Phytagel (Sigma-Aldrich, St. Louis, USA). The bacterial strain used was R. aquatilis HX2, isolated from a vineyard soil in Beijing, China (Guo et al. 2012). This strain has the ability to mineralize organic P, solubilize mineral P, fix nitrogen, and produce pyrroloquinoline quinine, indole-3-acetic acid, and antibacterial substances (Chen et al. 2007; Guo et al. 2009). It colonizes the root of maize and the hyphae of AMF (Chen et al. 2007; Zhang et al. 2016). This strain has been whole-genome sequenced (Guo et al. 2012) and was shown effective in the mineralization and utilization of phytate (Zhang et al. 2016, 2018).

The whole genome sequence of R. aquatilis is available at the National Center for Biotechnology Information (NCBI) (GenBank assembly accession: GCA_000255535). We investigated three genes, i.e., citrate synthase (gltA), isocitrate dehydrogenase (icd), and α-oxoglutarate dehydrogenase E1 component (kgdhc), encoding three pace-making enzymes in the TCA cycle of R. aquatilis. The primers for the genes were designed with the software Primer 5. The sequences of primers and other information related to the genes are described in Supplementary Materials Table S1.

Experimental set-up

Bi-compartmented Petri plates (90 × 15 mm) were used to grow the roots and AMF as detailed in Declerck et al. (2003). Briefly, in one compartment (i.e., the root compartment—RC), an excised transformed root of carrot (Daucus carota L.) clone DC2 was associated with the arbuscular mycorrhizal fungus on 25-ml MSR medium solidified with 3 g L−1 Phytagel. After 4 weeks, the extraradical mycelium (ERM) developed profusely in the RC and started to cross the plastic barrier separating the RC from the other compartment (i.e., the hyphal compartment—HC) in which only the ERM was allowed to develop. In the HC, 4-ml MSR medium solidified with 3 g L−1 Phytagel but without sucrose and vitamins was added in a slope from the top of the plastic barrier to the bottom of the HC (Toljander et al. 2007). The bi-compartmented Petri plates were incubated in a growth chamber at 27 °C in the dark. The cultures were checked on a regular basis, and any roots in the RC close to the plastic barrier were removed before they started to grow into the HC. Two additional control treatments were included under strictly identical conditions. The first control consisted of Petri plates without roots and AMF in the RC. The second control comprised carrot roots associated with the arbuscular mycorrhizal fungus in the RC without proliferation of ERM in the HC (the MSR medium was cut 1 cm away from the plastic barrier in the RC to prevent the hyphae from crossing the partition wall separating the RC from the HC). Three treatments were thus considered: absence of mycorrhizal roots in the RC and R. irregularis in the HC (RC−MR/HC−RI); presence of mycorrhizal roots in the RC but without proliferation of R. irregularis in the HC (RC+MR/HC−RI); presence of mycorrhizal roots in the RC; and proliferation of R. irregularis in the HC (RC+MR/HC+RI) (see Fig. 1). Each treatment consisted of four replicates.

Fig. 1
figure 1

Schematic representation of the experimental set-up. Red lines, extraradical hyphae of R. irregularis; green dots, bacterial cells of R. aquatilis. Treatment codes are as follows: RC−MR/HC−RI, absence of mycorrhizal roots in the RC and R. irregularis in the HC; RC+MR/HC−RI, presence of mycorrhizal roots in the RC but without proliferation of R. irregularis in the HC; RC+MR/HC+RI, presence of mycorrhizal roots in the RC and proliferation of R. irregularis in the HC. RC, root compartment; HC, hyphal compartment; RI, R. irregularis

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After an additional 2 weeks, the ERM of the RC+MR/HC+RI treatment developed extensively on the slope. Ten milliliters of liquid MSR medium without Ca(NO3)2·4H2O, sucrose, and vitamins, but containing 280 μM phytate (Na-phytate, Sigma-Aldrich, Saint Louis, USA), was added to the HC. Phytate was added as unique source of P to investigate the cooperation between R. irregularis and R. aquatilis. The ERM extended from the slope into the whole HC. There were two reasons to remove Ca(NO3)2·4H2O from the liquid MSR medium. First, the uptake of NO3− by the ERM in the HC of the RC+MR/HC+Ri treatment would decrease its concentration as compared with the two other treatments, and at the same time would increase the pH of the MSR medium in this compartment which could then decrease the solubility of phytate. Second, Ca2+ could precipitate the added phytate and decrease its concentration in the MSR medium.

After another 4 weeks, the surface of the HC was covered by actively growing hyphae. The remaining liquid MSR medium (approximately 9 ml) in the HC was transferred to a 15-ml tube with a micropipette and adjusted to 10 ml using the same liquid MSR medium as above but without phytate. Then, 2 ml of bacterial supernatant with a concentration of approximately 108 CFUs ml−1 was added and mixed uniformly. The 12-ml MSR medium containing the bacteria was added back to the HC of each experimental system with a micropipette. The liquid medium was removed carefully and the Petri plates refilled carefully as well. The medium was removed along the border of the Petri plate, where no hyphae were present (at the furthest distance from the hyphae). So, there was no physical contact between the micropipette and the hyphae. Therefore, although we cannot entirely exclude that some hyphae may have been injured, the risk of that was extremely low. The R. aquatilis inoculum was prepared as follows: bacteria were cultured in liquid Luria–Bertani medium with shaking at 180 rpm at 28 °C until OD600 reached 0.4–0.6 (logarithmic phase) and then centrifuged at 5878g for 6 min. The supernatant was discarded, and the pellet was re-suspended and washed with sterilized 0.85% (w/v) NaCl solution three times. The supernatant was then adjusted to OD600 = 0.8 with the autoclaved 0.85% NaCl solution and stored for 4 h before use.

Harvest and gene expression analysis

The liquid MSR medium containing R. aquatilis was added to the HC of each Petri plate. At 1, 6, 12, 24, 48, and 72 h, 0.5-ml medium was sampled with a micropipette and added to 1-ml RNAprotect® Bacteria Reagent (Qiagen) according to the handbook, to stabilize the bacterial RNA. The samples were then stored at − 80 °C until RNA extraction.

Total RNA was extracted from the frozen bacteria using the RNeasy® Mini Kit (Qiagen), according to the manufacturer’s instructions and treated with a TURBO DNA-free™ Kit (Ambion) to remove possible DNA contamination. For single-strand cDNA synthesis, 300 ng of total RNA was reverse-transcribed at 65 °C for 10 min, 55 °C for 20 min, and 85 °C for 5 min in a final volume of 20 μl containing 1 μl random primer using Transcriptor High Fidelity cDNA Synthesis Kit (Roche). The products were then diluted to 100 μl.

Quantitative RT-PCR (qRT-PCR) was performed using a LightCycler® 96 Real-Time PCR System (Roche). Each PCR reaction was carried out in a total volume of 10 μl containing 2.5 μl cDNA, 5 μl 2 × FastStart Essential DNA Green Master (Roche), and 0.5 μl of each primer (5 μM). The following PCR program was used: 95 °C for 600 s, 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. A melting curve was recorded at the end of each run to exclude that the primers had generated non-specific PCR products (Ririe et al. 1997). All reactions were performed on three technical replicates and their mean was used. Baseline range and threshold cycle (Ct) values were automatically calculated using LightCycler® 96 software. The ΔCt was calculated by subtracting the Ct value of a reference gene from the Ct value of each target gene. Relative fold-change of each target gene was normalized by the 2−ΔΔCt method, with reference to the ΔCt value in the RC−MR/HC−RI treatment harvested at the 1-h sampling time. The constitutively expressed RNA polymerase sigma factor RpoD was used as the house-keeping gene.

Data analysis

Statistical analyses were performed using SPSS v. 16.0 (SPSS Inc., Chicago, IL, USA). One-way repeated measures ANOVAs were performed to compare the between-subject effect of arbuscular mycorrhizal fungus, the within-subject effects of harvest time, and the time × arbuscular mycorrhizal fungus interaction on expression of three key genes, i.e., gltA, icd, and kgdhc in TCA cycle. Significant differences among the arbuscular mycorrhizal fungus treatments at a specific time and among the harvest times in a specific arbuscular mycorrhizal fungus treatment were evaluated by a Tukey’s honest significant difference (HSD) test at P ≤ 0.05.

Results

The one-way repeated measures ANOVA analysis showed that the between-subject effect of arbuscular mycorrhizal fungus and the within-subject effect of harvest time significantly affected the expression of all three genes (i.e., gltA, icd, and kgdhc), while the within-subject effect of time × arbuscular mycorrhizal fungus interaction significantly affected the expression of gltA and icd but not kgdhc (see Supplementary Materials Table S2).

No significant difference was found in the expression of gltA between the two control treatments (i.e., RC−MR/HC−RI and RC+MR/HC−RI) at any time, except 1 h. In the RC−MR/HC−RI treatment, the expression of gltA did not significantly differ among 1, 6, 12, and 24 h, but was significantly greater at 48 and 72 h than that at the other times. In the RC−MR/HC+RI and RC+MR/HC+RI treatments, the expression of gltA remained stable from 1 to 72 h. In the presence of the extraradical hyphae in the HC (i.e., the RC+MR/HC+RI treatment), the expression of gltA was significantly greater than either control treatment whatever the harvest time point (Fig. 2a).

Fig. 2
figure 2

Expression of a–c key genes (gltA, icd, and kgdhc) in the TCA cycle of R. aquatilis harvested from the HC of the Petri plates at different times. RC−MR/HC−RI, absence of mycorrhizal roots in the RC and R. irregularis in the HC; RC+MR/HC−RI, presence of mycorrhizal roots in the RC but without proliferation of R. irregularis in the HC; RC+MR/HC+RI, presence of mycorrhizal roots in the RC and proliferation of R. irregularis in the HC. RC, root compartment; HC, hyphal compartment; RI, Rhizophagus irregularis. The error bars represent the SE. The same capital letter means there is no significant difference among the arbuscular mycorrhizal fungus treatments at specific time points at P ≤ 0.05 by Tukey’s HSD test. The same lowercase letter means that there is no significant difference among the harvest times in specific arbuscular mycorrhizal fungus treatments at P ≤ 0.05 by Tukey’s HSD test

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No significant difference was noticed in the expression of icd between the two control treatments at any time point of harvest, except 6 h. In the RC−MR/HC−RI treatment, the expression of icd was the greatest at 6 h, was significantly smaller at 12 h and the following times than that at 6 h, and did not differ significantly between 48 and 72 h. In the RC+MR/HC−RI treatment, the expression of icd increased from 6 to 12 h and was the greatest at 12 h. It was significantly smaller at 24 h and the following harvest times than that at 12 h and did not differ significantly between 48 and 72 h. In the RC+MR/HC+RI treatment, the expression of icd was significantly greater at 1 and 6 h than that at 12, 24, 48, and 72 h. In the presence of the extraradical hyphae in the HC, the expression of icd was significantly smaller than either control treatment at all time points of harvest, except 1 h (Fig. 2b).

Whatever the harvest time, no significant difference was noticed in the expression of kgdhc between the two control treatments. In the RC−MR/HC−RI and RC+MR/HC−RI treatments, the expression of kgdhc remained stable from 1 to 72 h. In the RC+MR/HC+RI treatment, the expression of kgdhc was significantly greater at 1 and 6 h than that at 12, 24, 48, and 72 h. In the presence of the extraradical hyphae in the HC, the expression of kgdhc was significantly greater than either control treatment at 1, 6, and 48 h (Fig. 2c).

Discussion

The surface of extraradical hyphae is the place of important exchanges between AMF, exuding C and other compounds, and bacteria, involved in organic N and P mobilization (Leigh et al. 2011; Zhang et al. 2014). Recently, Zhang et al. (2018) demonstrated that fructose exuded by the arbuscular mycorrhizal fungus R. irregularis MUCL 43194 induces the expression of phosphatase genes of the PSB R. aquatilis HX2, thus supporting cooperation between the two microorganisms. The stimulation of bacterial metabolism, via the provision of sugars or other compounds by the fungus, most probably depends on the cellular ATP level, which is primarily controlled by the TCA cycle (Tortora et al. 2016). In the present study, we demonstrated that the three pace-making genes were influenced differently by the AMF: gltA and kgdhc were increased and icd was decreased, making it difficult to determine the overall effect of the fungus on the TCA rate. We anticipated that the overall effect was positive because the expression of gltA, which has the greatest influence on the TCA rate, was increased, even though this has to be demonstrated conclusively.

In the present study, we selected rpoD as the reference gene and used the SYBR Green method in quantitative gene expression analysis for the following reasons: first, rpoD was selected as the reference gene to calculate the relative expression of phosphatase genes of R. aquatilis HX2 in a previous study (Zhang et al. 2018). In the present study, four reference genes, i.e., 16S rDNA, lpx, recA, and rpoD, were compared and rpoD was again selected as the adequate reference gene. Secondly, SYBR green and TaqMan probes are two methods used in quantitative gene expression analysis. SYBR Green is relatively inexpensive and easy to use and technically based on binding the fluorescent dye to double-stranded deoxyribonucleic acid (dsDNA). Conversely, the TaqMan method is expensive and is based on dual-labeled oligonucleotide and exonuclease activity of Taq polymerase enzyme. Normally, one method is sufficient in qPCR. SYBR is popular, and by optimization of the SYBR Green method, its performance and quality can be comparable to the TaqMan method (Tajadini et al. 2014).

The pace of the TCA cycle is more determined by the citrate synthase reaction than by the other two reactions (i.e., isocitrate dehydrogenase and α-oxoglutarate dehydrogenase) (Krebs 1970). In the present study, the expression of gltA was significantly increased by the presence of the arbuscular mycorrhizal fungus at all observation times. This stimulation was most probably related to an increase in the concentration of substrates needed for the citrate synthase because the production of this enzyme can be induced by its substrates (Holmes et al. 2005). Basically, two substrates could be used by citrate synthase: oxaloacetate and acetyl-CoA. Oxaloacetate can be produced from pyruvate, malate, and aspartate (Utter and Keech 1960), while acetyl-CoA is mainly synthesized from pyruvate (Korkes et al. 1951). Interestingly, the hyphae exudates contain sugars and carboxylates, e.g., fructose, glucose, acetate (Zhang et al. 2018), which are involved in glycolysis and thus in the production of pyruvate. This may then lead to an increasing concentration of oxaloacetate and acetyl-CoA. For example, gltA transcripts in the environment are related to the availability of acetate (Holmes et al. 2005). Hyphal exudates also contain amino acids, e.g., aspartate (Bharadwaj et al. 2012), which may lead to an increasing concentration of oxaloacetate.

Curiously, the expression of icd in the bacteria was decreased at all times in the presence of the ERM. The expression of this gene is influenced by various environmental factors, e.g., the concentration of oxygen and nutrients (Chao et al. 1997; Domínguez-Martín et al. 2018). For instance, it has been shown that under anaerobic condition, the expression of icd in Escherichia coli was fivefold lower than under aerobic conditions (Chao et al. 1997). It is thus not excluded that the ERM of the arbuscular mycorrhizal fungus may compete with the bacterial cells for oxygen, decreasing its concentration near the bacteria (Storer et al. 2017). For instance, in soil conditions, previous studies have proposed that AMF may compete for oxygen with ammonia-oxidizing bacteria and decrease the conversion of NH4+ into NO2− and then NO3−, and ultimately to a decrease in N2O emissions from soil (Bender et al. 2014; Storer et al. 2017). Therefore, it is not excluded that competition for oxygen within the liquid MSR medium may have influenced the expression of icd in R. aquatilis.

Several factors (e.g., Acyl-CoA, metallic ions, and α-oxoglutarate) have been reported to affect the activity of the α-oxoglutarate dehydrogenase protein (Webb 1964; Erfle and Sauer 1969; Peng et al. 2015), but how they influence the gene expression of the enzyme remains mostly unknown. Interestingly, we noticed an increase in expression of kgdhc at the early times (i.e., at 1 and 6 h) of contact with the ERM. The decrease of this gene at the later times seemed to be related to the depletion of some compounds in the hyphae exudates. In the same in vitro culture experiment (Zhang et al. 2018), we demonstrated that fructose depleted at 6 h. Interestingly, fructose-1-phosphate is the preferred effector of the catabolite repressor/activator, which can induce the expression of other genes in the soil bacterium Pseudomonas putida (Chavarría et al. 2014). However, if fructose-1-phosphate can regulate the expression of kgdhc needs further investigation.

From this study, we concluded that the ERM of R. irregularis induces the expression of the gene-encoding citrate synthase, the most pace-making enzyme in the TCA cycle, which may then increase the activity of R. aquatilis. This completed the recent study of Zhang et al. (2018) who demonstrated that the hyphae exudates of R. irregularis induce the expression of phosphatase genes in this bacterium, thus improving the cooperation between the two partners. The interactions between the arbuscular mycorrhizal fungus R. irregularis 43194 and the PSB R. aquatilis HX2 respond to a fine-tuned dialog favoring cooperation within a wider community of microorganisms in which cooperation but also antagonism with AMF may take place. These findings bring new insights on the impacts of AMF at the transcriptional level on the metabolism of phosphate-solubilizing bacteria (i.e., R. aquatilis) developing in the hyphosphere.