• fungal–bacterial interactions;
  • Laccaria bicolor S238N;
  • mycorrhiza helper bacteria;
  • rhizobacteria;
  • transcriptome


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • • 
    The mycorrhiza helper Pseudomonas fluorescens BBc6R8 promotes the presymbiotic survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N in the soil.
  • • 
    An in vitro fungal–bacterial confrontation bioassay mimicking the promoting effects of the bacteria on fungal growth was set up to analyse the fungal morphological and transcriptional changes induced by the helper bacteria at three successive stages of the interaction. The specificity of the P. fluorescens BBc6R8 effect was assessed in comparison with six other rhizobacterial strains possessing mycorrhiza helper or pathogen antagonistic abilities.
  • • 
    The helper BBc6R8 strain was the only strain to induce increases in the radial growth of the colony, hyphal apex density and branching angle. These morphological modifications were coupled with pleiotropic alterations of the fungal transcriptome, which varied throughout the interaction. Early stage-responsive genes were presumably involved in recognition processes and transcription regulation, while late stage-responsive genes encoded proteins of primary metabolism. Some of the responsive genes were partly specific to the interaction with P. fluorescens BBc6R8, whereas others were mutually regulated by different rhizobacteria.
  • • 
    The results highlight the fact that the helper BBc6R8 strain has a specific priming effect on growth, morphology and gene expression of its fungal associate L. bicolor S238N.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The soil is probably one of the most complex ecosystems in which fungal–bacterial interactions operate. Complex deleterious, beneficial and commensal communities of fungi and bacteria surround plant roots. Among those, ectomycorrhizal fungi form symbiotic associations with tree roots and participate in tree nutrition (Smith & Read, 1997). They interact physically and functionally with soil bacterial communities, thus establishing the so-called ‘multitrophic ectomycorrhizal complex’ which impacts gross production and nutrient (Frey-Klett & Garbaye, 2005). The formation of ectomycorrhizal symbiosis can be significantly improved by selected soil and mycorrhizosphere bacteria. These ‘mycorrhiza helper bacteria’ (MHB, Garbaye, 1994) belong to a wide range of genera (Burkholderia, Paenibacillus, Poole et al., 2001; Pseudomonas, Bacillus, Duponnois & Garbaye, 1991, Streptomyces, Maier et al., 2004). They have been described not only in temperate ecosystems, but also in tropical ones (Founoune et al., 2002). Moreover, the ‘mycorrhiza helper bacteria’ concept is not restricted to ectomycorrhizal symbiosis, as some helper bacteria also promote the endomycorrhizal symbiosis (Duponnois & Plenchette, 2003).

The mechanisms by which helper bacteria stimulate mycorrhiza formation remain unclear. Five hypotheses have been proposed by Garbaye (1994): increase in the receptivity of the root towards mycorrhizal infection; improvement of the root–fungus recognition processes; stimulation of fungal growth during the presymbiotic stage; modifications of the physicochemical properties of the rhizosphere soil that improve mycorrhizal infection; and induction of fungal spore germination. So far, only the direct effects of helper bacteria on presymbiotic survival and growth of the mycorrhizal fungi in the soil have been well documented (Bruléet al., 2001; Founoune et al., 2002; Schrey et al., 2005). However, little is known about the molecular mechanisms induced by helper bacteria concerning the fungal growth-promoting effect. Only changes in Amanita muscaria gene expression upon interaction with the helper bacteria Streptomyces sp. have been reported so far (Schrey et al., 2005; Riedlinger et al., 2006). Further studies involving other interaction models of mycorrhiza helper bacteria–ectomycorrhizal fungi are required to assess the specificity of the fungal gene regulations described to date (Bending, 2007).

The mycorrhiza helper Pseudomonas fluorescens strain BBc6R8 significantly promotes the establishment of ectomycorrhizal symbiosis between Douglas fir and the ectomycorrhizal fungus Laccaria bicolor S238N (Frey-Klett et al., 1997). This promoting effect is related to the enhanced survival and growth of L. bicolor S238N mycelium during its presymbiotic phase in the soil, under unfavourable growth conditions (Bruléet al., 2001). The aim of the present work was to elucidate the molecular mechanisms by which P. fluorescens BBc6R8 promotes L. bicolor S238N growth. For this purpose, an in vitro confrontation assay was first set up in which helper bacteria reproducibly improved mycelium growth. Hyphal morphology and growth were monitored along the interaction with P. fluorescens BBc6R8 and six other rhizospheric bacterial strains with mycorrhiza helper or pathogen antagonistic abilities. Using cDNA arrays, the impact of the helper P. fluorescens BBc6R8 on the fungal transcriptome was analysed at three key stages of the fungal–bacterial interaction: before, during and after physical contact. The specificity of the P. fluorescens BBc6R8 effect on gene expression was assessed by comparing the expression level of several target genes with those of the six other rhizobacterial strains.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Microorganisms and confrontation bioassays

The ectomycorrhizal basidiomycete Laccaria bicolor S238N (Maire P. D. Orton) was maintained on Pachlewski agar medium P5 (Di Battista et al., 1996) at 25°C for 3 wk. The MHB Pseudomonas fluorescens strain BBc6R8 is a spontaneous rifampicin-resistant mutant. It phenotypically conforms to the parental strain BBc6 that was isolated from a L. bicolor fruiting body collected in a Douglas fir plantation (Duponnois & Garbaye, 1991; Frey-Klett et al., 1997). The relevant characteristics of the six bacterial strains Collimonas fungivorans Ter331, Paenibacillus sp. EJP73, Paenibacillus sp. F2001L, Bacillus subtilis MB3, Burkholderia sp. EJP67 and Pseudomonas fluorescens Pf29A are listed in Table 1. All strains were maintained at –80°C in Luria-Bertani medium (Sambrook et al., 1989) with 20% glycerol. In the present work, the bacterial strains were first grown on 10% TSA plates (3 g l−1 tryptic soy broth from Difco and 15 g l−1 of agar) at 25°C for 65 h to prepare the bacterial inoculum for the in vitro bioassay. Then, three to four colonies were picked and suspended in 2 ml of sterile deionized water before spreading on to 10% TSA medium. After 48 h of growth at 25°C, the bacteria were harvested and centrifuged at 3300 g for 10 min. The pellet was washed once, then resuspended in deionized water in order to obtain a suspension with A600 nm ~ 0.7 (c. 109 cfu ml−1).

Table 1.  Bacterial strains used in this study
StrainsRelevant characteristicsReference and origin
Pseudomonas fluorescens BBc6R8Helper effect on the Douglas fir-Laccaria bicolor symbiosisFrey-Klett et al. (1997) INRA-Nancy, France
Collimonas fungivorans Ter331Chitinolytic and antagonistic effect on fungi in microcosms and on L. bicolor S238N-P. sylvestris symbiosis in glasshousede Boer et al. (2004) W. de Boer, NIOO-KNAW, the Netherlands
Paenibacillus sp. EJP73Helper effect on the Pinus sylvestris-Lactarius rufus symbiosisPoole et al. (2001); Aspray et al. (2006) G. Bending, Warwick HRI, England
Pseudomonas fluorescens Pf29ABiocontrol agent against the wheat pathogen Gaeumannomyces graminis var triticiChapon et al. (2002) A. Sarniguet, INRA-Rennes, France
Bacillus subtilis MB3Bacillus subtilis; helper bacteria of Douglas fir-L. bicolor mycorrhiza formationDuponnois et al. (1991) INRA-Nancy, France
Burkholderia sp. EJP67Helper effect on the Pinus sylvestris-Lactarius rufus symbiosis in microcosmsPoole et al. (2001) G. Bending, Warwick HRI, UK
Paenibacillus sp. F2001LPresumed intracellular bacterial strain isolated from a liquid culture of L. bicolor S238NBertaux et al. (2003) INRA-Nancy, France

An in vitro confrontation bioassay was developed using 9-cm-diameter Petri dishes containing 20 ml per plate of a Pachlewski medium (Paschlewski & Pachlewska, 1974), which was modified as follows: 0.5 g inline image tartrate, 1 g KH2PO4, 0.5 g MgSO4, 1 g glucose, 1 ml 1/10 diluted Kanieltra microelement solution and 20 g agar l−1 at pH 5.5 (P20Th-). A plug of L. bicolor S238N was cut out from the edge of a colony grown on P5 medium and transferred into the centre of a P20Th plate (Fig. 1). Four 10 µl droplets of sterile deionized water (control treatment) or bacterial suspension (bacterial treatment) were distributed at 1.75 cm from the centre of the fungal plug. Plates were sealed with plastic tape and incubated at 10°C in the dark.


Figure 1. Photograph of the in vitro bioassay after 21 d of dual growth.

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Mycelium growth and morphology measurements

The diameter of the fungal colony was measured every fourth day from day 12 to 40 after the addition of water or bacterial suspensions, with seven replicates per treatment. For the P. fluorescens BBc6R8-inoculated treatment only, the morphology of the fungal mycelium was analysed at three key steps of the interaction: before contact between the mycelium and the bacteria (14 d), at contact time (16 d) and after an extended contact period (21 d). For the other six bacterial strains, observations were made only at the precontact stage. Three biological replicates per treatment were performed. For each replicate, two photographs were taken using an Olympus BX41 microscope (×40 magnification) equipped with a ColorView System camera. The number of apices per microscopic field (3.5 mm2), branching angles, branching densities (number of ramifications divided by the number of apices) and curvature of hyphae were measured on each photograph using AnalySIS software (Soft Imaging System, Olympus, Münster, Germany). The effect of the bacterial treatment on the growth and morphology of the fungal mycelium was determined using analysis of variance (anova) at the threshold value of 0.05 and the Fisher test. The Superanova 1.11 software (Abacus) was used for these statistical analyses.

cDNA array analysis  cDNA libraries from pure culture of L. bicolor S238N mycelium and from three stages of L. bicolor S238N sporocarp development (Lb2 library: stipes and caps of 5–10 mm growing sporocarps; Lb3 library: caps of 30–40 mm mature sporocarps), collected under Douglas fir seedlings grown in a glasshouse, were constructed in the λTriplEx2 vector as previously described (Peter et al., 2003). The cDNA inserts from bacterial clones were PCR-amplified and 4992 cDNAs were arrayed from 384-well microtitre plates on Nylon membranes, as described previously (Peter et al., 2003).

RNA isolation for target preparation L. bicolor S238N mycelium from 50 plates was collected, frozen in liquid nitrogen and pooled. Mycelium was sampled in triplicate at three stages of the interaction: before contact between L. bicolor S238N and P. fluorescens BBc6R8 (14 d), at contact (16 d) and after an extended contact period (21 d). Mycelium was ground in a mortar with liquid nitrogen, and total RNA was extracted using Trizol as recommended by the manufacturer for small quantities of material (Invitrogen AB, Stockholm, Sweden). RNA was further purified using the RNA/DNA mini kit (Qiagen, Hilden, Germany). The quality of the RNA was checked via RNAse-free 1% agarose electrophoresis and by PCR amplification of four full-length cDNAs (mitochondrial fission-related protein, 60S ribosomal protein, cipC and profiline). To analyse the expression of target genes from L. bicolor S238N mycelium in interaction with the other rhizobacterial strains, the in vitro system described earlier was used, replacing drops of P. fluorescens BBc6R8 inoculum with suspensions of the other bacterial strains. Mycelium was collected and RNA was extracted using the RNeasy Plant mini-kit (Qiagen) following the manufacturer's recommendations. The quality of the RNA was checked by RNAse-free 1% agarose electrophoresis.

cDNA array hybridization  Synthesis of complex cDNA probes, cDNA array hybridization and data analysis were performed as described by Peter et al. (2003) and Duplessis et al. (2005).

DNA sequencing  cDNA clones encoding for up- and down-regulated transcripts were amplified by PCR and sequenced as previously described (Kohler et al., 2003). Edited nucleotide sequences were compared with the gene models predicted by the genome sequence assembly v.1.0 of L. bicolor S238N-H82 strain using the BlastN server on the JGI Laccaria Portal ( Conserved domains of the predicted protein sequences were searched in Pfam, Smart and KOG databases (

Quantitative PCR analysis  To validate the cDNA array data, real-time quantitative PCR analyses were performed on seven up- or down-regulated genes selected for their potential biological relevance (Cipc, tectonin II, tra1, glutathione-S-transferase, hypothetical protein with yip1 domain, fumarate reductase and polyadenylate binding protein). Eight nonregulated genes were also used as controls and two of them (Lb17E10 and trehalose phosphorylase) were chosen for data normalization. The expression of genes encoding Tectonin II, Concanamycine-induced protein C (Cipc1), Transcription associated protein (Tra1), Gcn5, Spt3, Ada 3, peroxisomal hydratase dehydrogenase epimerase multiprotein (LbFOX2), Acetoacyl coenzyme A synthetase (LbAaCS) and cyclophiline 40 (LbCyp 40) was monitored by real-time PCR. The primer pairs (Supplementary material, Table S1) were designed using Primer 3 ( and Amplify 3.1 ( The following criteria were used: product size between 100 and 400 bp, melting temperature 60°C ± 1°C and GC% > 50%.

RNA samples were used for cDNA array analysis and for the real-time PCR measurements. For the analysis of BBc6R8-responsive genes during the interaction with other rhizobacteria, cDNA were synthesized from 0.5 mg of total RNA (iScript, Bio-Rad, Hercules, CA, USA). Each qPCR reaction (15 µl total volume) contained 2 µl of cDNA template, 300 nm of each primer and 1X SYBR green PCR master mix (Bio-Rad). Reactions were run using a MJ-opticon2 DNA real-time PCR system (Bio-Rad). The following cycling parameters were applied: 95°C for 3 min and then 40 cycles of 95°C for 30 s, 60°C for 1 min and 72°C for 30 s. A negative control was run for each primer pair. For data analysis, the geometric mean of the three biological replicates for each condition was calculated. The PCR efficiency was checked to be 100% and fold differences were calculated using the ΔΔCt method (Livak & Schmittgen, 2001).


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Mycelium growth and morphology

Pseudomonas fluorescens BBc6R8 significantly stimulated the extension of L. bicolor S238N mycelium (expressed as the diameter of the colony) as early as 14 d of dual cultivation (Fig. 2, Table 2). It also enhanced the hyphal branching angle and the hyphal branching density during the entire interaction (Fig. 3). Whereas the hyphal apex density was significantly enhanced before contact, it was significantly decreased at and after the time of contact. Laccaria bicolor S238N reacted differently to the presence of the six other helper and nonhelper rhizobacterial strains. Its growth was significantly enhanced by the MHB Burkholderia sp. EJP67, the presumed intracellular fungal Paenibacillus sp. F2001L, and later by the biocontrol P. fluorescens Pf29A. The MHB B. subtilis MB3 had no effect on fungal growth. By contrast, it was significantly decreased by the MHB strain Paenibacillus sp. EJP 73 and the chitinolytic C. fungivorans Ter331 (Fig. 2). Pseudomonas fluorescens BBc6R8 and Burkholderia sp. EJP67 were the only strains that had enhanced growth by the time mycelium was collected for transcript analysis and morphology measurements were performed (i.e. after 14 d of confrontation). At this time, the apex number was significantly reduced by Burkholderia sp. EJP67 (Table 2), while the branching angle was significantly enhanced by both Burkholderia sp. EJP67 and C. fungivorans Ter331. The branching density was affected by the four strains EJP67, Pf29A, MB3 and Ter 331. Finally, only Burkholderia sp. EJP67 induced a high amount of curvature in the hyphae (data not shown).


Figure 2. Effect of bacterial strains Pseudomonas fluorescens BBc6R8 (black circles, dash points), P. fuorescens Pf29A (open triangles), Collimonas fungivorans Ter331 (black squares, dash points), Paenibacillus sp. EJP73 (black crosses, dot points), Bacillus subtilis MB3 (open circles), Burkholderia sp. EJP67 (black triangles, dash points) and Paenibacillus sp. F2001L (black diamonds, dot points) on the radial growth of Laccaria bicolor S238N (open squares) in the in vitro bioassay. Each point is the mean (± SD) of seven replicates. At the end of the kinetic, points with the same letters are not significantly different according to a one-factor (bacterial treatment) anova (P > 0.05). The arrow indicates the precontact time at which mycelium was collected for L. bicolor S238N gene expression analysis.

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Table 2.  Effect of six rhizobacterial strains on Laccaria bicolor S238N hyphal extension and morphology after 14 d of dual culture
Bacterial treatmentColony diameter (cm)Apex numberBranching angle (°)Branching density
  1. Hyphal extension, mean value of two perpendicular diameters of the fungal colony for seven biological replicates; apex number, the number of apices per microscopic field; branching density, the number of ramification divided by the number of apices. For the three variables (apex number, branching angle and branching density), each value corresponds to the mean value (± SE) of three biological replicates and two microscopic fields by replicate. In each column, mean values with the same letter are not significantly different according to a one-way anova and the Fisher test (P > 0.05).

Control1.25 ± 0.02 a38.5 ± 3.6 a30.1 ± 1.3 a22.5 ± 2.8 a
P. fluorescens BBc6R81.42 ± 0.02 b52.3 ± 3.6 b37.1 ± 1.8 b31.3 ± 1.1 b
Burkholderia sp. EJP 671.40 ± 0.02 b22.5 ± 2.3 c58.1 ± 4.7 c39.8 ± 2.7 bc
Paenibacillus sp. F2001L1.26 ± 0.01 a35.8 ± 2.4 a30.3 ± 2.1 a28.0 ± 1.4 a
P. fluorescens Pf29A1.26 ± 0.02 a33.7 ± 4.3 a30.2 ± 1.6 a34.7 ± 3.6 b
B. subtilis sp. MB31.26 ± 0.02 a27.3 ± 4.2 a30.1 ± 1.7 a32.6 ± 3.2 b
Paenibacillus sp. EJP 730.99 ± 0.01 c33.0 ± 5.8 a34.4 ± 2.1 a24.0 ± 5.4 a
C. fungivorans Ter3310.91 ± 0.01 d26.0 ± 7.7 a41.1 ± 3.7 bc43.5 ± 6.6 c

Figure 3. Effect of the helper bacterial strain Pseudomonas fluorescens BBc6R8 on the morphology of Laccaria bicolor S238N before contact (14 d of common growth), at contact time (16 d) and after contact (21 d). Open bars, control treatment with water; closed bars, P. fluorescens BBc6R8 treatment. Error bars denote standard error; *, significant differences with the control according to a t-test (P < 0.05) performed at each confrontation stage.

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Transcriptional response of L. bicolor S238N during the interaction with the helper P. fluorescens BBc6R8

Transcripts of L. bicolor S238N showing an altered abundance in the presence of P. fluorescens BBc6R8 were identified by the following criteria: (i) a significant modification (Bayesian t-test) of gene expression was detected in at least two of the three replicates; and (ii) the average of fold changes (P. fluorescens BBc6R8 treatment vs control) in the expression level was > +2 or < –2 (Duplessis et al., 2005). According to these criteria, we found 164 transcripts (3.2% of the total 4992 cDNAs) significantly regulated in least at one stage of interaction with P. fluorescens BBc6R8. The cDNA of 144 of these regulated transcripts were successfully sequenced; they encoded 104 different genes (Table S2). Among the regulated transcripts, 27 were up-regulated and none were significantly down-regulated during the early stage of interaction. At the contact stage, 18 transcripts were up-regulated and 11 were down-regulated. Finally, after a long contact period, 22 and 86 transcripts were up- and down-regulated, respectively. cDNA array results were confirmed by quantitative PCR measurements on six target genes (Fig. S1). MHB-responsive genes are involved in multiple cellular functions as showed by their distribution in various gene ontology (GO) categories (Fig. 4).


Figure 4. Gene ontology to which up-regulated (white bar) or down-regulated (black bar) transcripts belonged. Grey bars, transcript categories that showed variable expression along the interaction. Data are expressed as a percentage of the total number of transcripts up-regulated, down-regulated or variably regulated at all stages of the interaction.

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Some sets of transcripts belonging to the same GO showed concentration variation at only one step of the interaction. For instance, genes encoding protein involved in cell interaction (tectonin II), efflux processes (efflux protein of MFS family) and detoxification processes (two glutathione-S-transferases) were regulated at only the earliest stages (i.e. before or at contact time). Similarly, genes involved in protein synthesis, including 18 different ribosomal proteins, as well as those involved in the translation process (translation initiation factor SU1) and protein degradation (ubiquitin, ubiquitin extension protein, ubiquitin fusion protein) were only repressed after extended contact between mycelium and the bacterial cells. This repression of protein synthesis machinery was associated with a reduction in transcription of several genes involved in energy metabolism (i.e. ATP synthase subunits, cytochrome oxidase, ATP/ADP carrier protein, malic enzyme, PEP carboxylase and fumarate dehydrogenase).

By contrast, other sets of transcripts were affected at different times of the interaction (i.e. some genes before, and others at or after contact time). For example, the expression of genes encoding the histone H4-2, the tra1 subunit of the transcription regulatory SAGA complex (transcription regulation), and the splicing factor 3b (mRNA splicing) were enhanced before contact, while a gene encoding a transcription factor and tra1 showed an increased expression at contact time. Finally, after extended contact, three genes involved in translation regulation (polyadenylate binding protein and U6, a small nuclear RNA-associated RNA binding protein) and the tra1 gene showed decreased expression. Similarly, the expression of genes involved in growth and morphology was modified at different steps of the interaction: the gene encoding the concanamycin-induced protein C (CipC1), which could be involved in hyphal branching, was up-regulated twofold before contact, while transcription of the gene encoding actin-1 was repressed after extended contact.

Analysis of transcription regulatory complex expression

Among the genes found regulated at all stages of the L. bicolor S238N–P. fluorescens BBc6R8 interaction, a gene encoding the Tra1 protein was identified. This protein is involved in four transcriptional regulatory complexes (SAGA, SALSA, SLIK, NuA4) in the yeast S. cerevisiae (Allard et al., 1999; Sterner et al., 1999; Wu et al., 2004), all known for their histone acetyl transferase activity (HAT). Acetylation of N-terminal histone tails reduces the affinity of nucleosomes to DNA and leads to an enhanced binding of transcription factors to their cis-regulatory DNA sites. The concentration of transcripts encoding for the histone H4, one target of HAT, was enhanced before contact (Table 3), suggesting that chromatin structure modifications take place in L. bicolor S238N in response to P. fluorescens BBc6R8. To know if the expression of other genes corresponding to key proteins of the precited transcriptional complexes was also regulated during the fungal–bacterial interaction, we searched for these genes in the annotated L. bicolor S238N-H82 genome and then measured their transcript concentrations by quantitative RT-PCR at the three stages of interaction. The SAGA complex (Spt-Ada-Gcn5–acetyltransferase, Fig. 5) of S. cerevisiae is composed of 20 subunits; among those, 11 are shared with the SALSA complex (SAGA alterated complex, Sterner et al., 2002) and 16 with the SLIK complex (SAGA like, Pray-Grant et al., 2002). We identified the genes encoding 13 of these proteins in the L. bicolor S238N-H82 genome (Table 4). The Spt 20/Ada 5 and HFI1/ada 1 subunits, which are shared by the three complexes and are involved in the interaction with TATA box binding protein (TBP), were not found in the current assembly of the L. bicolor S238N-H82 genome. Similarly, the genes corresponding to the Sgf11, Sgf 29, Sus1 and Rtg2 subunits were not identified in the current genome assembly. By contrast, two components (Esa1 and Tra1) of NuA4 (Nucleosome acetyltransferase of histone H4; Allard et al., 1999) were found. The concentration of the gcn5 and esa1 transcripts did not vary at any stage of the interaction, whereas the expression of ada3 and spt3 was significantly regulated before and at contact time (Table 4).

Table 3.  Transcripts of Laccaria bicolor S238N regulated (t-test, ≥ 2.0, ≤ 2.0, in bold) upon the interaction with the helper strain Pseudomonas fluorescens BBc6R8
Accession numberIdentityAccession number of the best hitScoreBefore contactContactAfter contact
  1. ns, not significant.

  2. cDNA clone ID, identity of the best BlastX, the GenBank accession number corresponding to the best hit and the E-value are given. The transcript ratio (P. fluorescens BBc6R8 treatment: L. bicolor S238N control) is given at the three times of the interaction: before contact, at the contact time and after contact.

Up-regulated before contact
EL740045, EL740122Cipc protein (Emericella nidulans)CAC872724.00E–313.21.60.9
EL739265Efflux protein, putative-MFS family (Cryptococcus neoformans var. neoformans JEC21)CNB020401.00E–1626.41.11.4
EL739689Histone H4,2 (Phanerochaete chrysosporium)P627924.00E–352.11.50.6
EL739275Panthotenate kinase (Cryptococcus neoformans var. neoformans JEC21)AAW424540.
JGI_LbEX5106Splicing factor 3b, subunit 4 (Danio rerio)1923183.00E–818.71.11.8
Up-regulated at the contact time
EL739371Ankyrin repeat protein (Neosartorya fischeri NRRL 181)XP_0012574499.00E–540.92.21.4
EL739436Heat shock protein (Schizosaccharomyces pombe)O143686.00E–731.93.01.8
Down-regulated at the contact time
EL739383Glutathione S-transferase (Paxillus involutus)AAT912502.00E–780.90.41.5
JGI_LbEX8144Glutathione S-transferase PM239 × 14 (GST class-phi) (Arabidopsis thaliana)P427697.00E–811.00.31.0
Up-regulated after contact
EL739467Polyadenylate binding protein (Cryptococcus neoformans JEC21)CNI011600.0nsns5.8
EL739886Citrate synthase 2 (Phanerochaete chrysosporium)jgi|Phchr1|38126|g ww2.–1630.90.62.7
Down-regulated after contact
EL740070Putative ubiquitin extension protein (Oriza sativa)AAT93912e-351.00.60.4
JGI_LbEX1896Ubiquitin fusion protein (Magnaporthe grisea)AAC136895.00E–651.00.80.4
JGI_LbEX876Actin-1 (Beta-actin) (Schizophyllum commune)Q9Y7020.
JGI_LbEX2079Histone H2b (Agaricus bisporus)P785673.00E–
EL739344PEP carboxylase (Cryptococcus neoformans var. neoformans JEC21)CNI035900.
EL738956LSM3 homologue, U6 small nuclear RNA-associated (Saccharomyces cerevisiae)KEGG-272582.00E–401.20.70.4
EL739202Vacuolar ATP synthase 16 kDa proteolipid subunit (Neurospora crassa)P314133.00E–611.40.60.4
JGI_LbEX5683Small heat shock protein (Laccaria bicolor)AAM785956.00E–731.00.60.4
EL739415Ras-related protein (Laccaria bicolor)AAD01986
EL739129NADP-dependent malic enzyme (Flaveria pringlei)P364440.01.1NS0.4
EL739368, EL739199Ubiquitin (Phanerochaete chrysosporium)CAA808510.
JGI_LbEX303Dihydrolipoamide dehydrogenase precursor (Aspergillus fumigatus Af293)EAL873078.00E–311.30.70.3
EL739358Large subunit ribosomal protein L40e (Eremothecium gossypii) with ubiquitin domain IPR000626KEGG-AFR285C5.00E–330.80.80.3
EL739366Eukaryotic translation initiation factor eIF-1 (protein translation factor SUI1) (Saccharomyces cerevisiae)P329111.00E–501.11.20.3
EL739045Probable RNA-binding protein C839.10 (Homo sapiens)Q8WZK05.00E–
JGI_LbEX2263ADP, ATP carrier protein (ADP/ATP translocase) (Neurospora crassa)P027230.
JGI_LbEX5121Integral membrane Yip1 family protein (Arabidopsis thaliana)KEGG-At3g052801.00E–971.8ns0.2
EL740097Fumarate reductase (NADH) (Cryptococcus neoformans var. neoformans JEC21)AAW457540.
JGI_LbEX4993Glyceraldehyde-3-phosphate dehydrogenase (Agaricus bisporus)AAA326341.00E–1501.5ns0.2
Regulated all the time
EL739455Tra1 (Phanerochaete chrysosporium)Phchr1449670.
Up-regulated before contact and at the contact time
EL739445Tectonin II (Physarum polycephalum)AAC062016.00E–
Up-regulated at the contact time and after contact
EL739395Heat shock proteinCNA028300.
EL739391Protein kinase (Glycine max)AAA340026.00E–561.82.92.8
Down-regulated at the contact time and after contact
EL739038Mismatched base pair and cruciformDNA recognition protein (Agaricus bisporus)CAB856902.00E–391.40.50.4
EL739050ATP synthase alpha chain, mitochondrial precursor (Cryptococcus neoformans var. neoformans JEC21)AAW440190.
EL738997F-type H+-transporting ATPase f chain (Eremothecium gossypii)KEGG-ACR203W7.00E–431.10.50.4

Figure 5. Scheme of the hypothetical SAGA structure and function in transcription (adapted from Sterner et al., 1999). Depicted is a hypothetical gene with an upstream activation sequence (UAS) and TATA box; the DNA is bound around nucleosomes (cylinders). The SAGA complex would interact with activators (A) while the histone acetyl transferase activity of the gcn5 subunit would acetylate (Ac = acyl groups) the amino-terminal tails of nucleosomal histone, providing more effective TATA binding of TATA binding protein (TBP). Further regulation would be provided by SAGA–TBP interactions through Spt3 and other factors.

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Table 4.  Expression level of genes encoding key proteins of the SAGA and NuA4 regulatory complexes and of the marker genes tectonin 2, Cipc and fox2 along the interaction between Laccaria bicolor and the helper strain Pseudomonas fluorescens BBc6R8, and only before the contact with the six other rhizobacterial strains
Transcript functionsP. fluorescens BBc6R8P. fluorescens Pf29APaenibacillus sp. F2001LB. subtilis MB3C. fungivorans 331
Before contactContact timeAfter contact
  1. nd, nonmeasured value.

  2. Measurements were performed by real-time PCR on L. bicolor S238N cDNAs from control (without bacteria) and bacterial treatments. Level of expression was calculated by the ΔΔCt method. Each value is the mean of three replicates. Data in bold and italic highlight expression rates up to 2.0 and down to 2.0, respectively.

SAGA complex
 Tra14.5 ± 2.40.5 ± 0.32.4 ± 0.42.6 ± 0.31.5 ± 0.11.0 ± 0.10.2 ± 0.1
 Gcn51.1 ± 0.21.1 ± 0.50.8 ± 0.23.1 ± 1.31.0 ± 0.10.8 ± 0.12.6 ± 0.8
 Ada30.2 ± 0.020.3 ± 0.20.1 ± 0.10.8 ± 0.21.1 ± 0.10.6 ± 0.10.5 ± 0.4
 Spt30.7 ± 0.21.4 ± 0.50.7 ± 0.11.6 ± 0.41.5 ± 0.11.0 ± 0.12.2 ± 0.3
NuA4 complex
 Esa10.8 ± 0.10.4 ± 0.10.5 ± 0.0ndndndnd
 tectonin 23.7 ± 0.63.4 ± 0.7nd9.2 ± 6.01.1 ± 0.11.0 ± 0.26.5 ± 1.2
 cipc22.1 ± 0.1ndnd3.4 ± 1.31.1 ± 0.11.0 ± 0.11.4 ± 0.6
G. mosseae MHB responsive gene
 fox20.6 ± 0.10.4 ± 0.11.2 ± 0.21.5 ± 0.41.1 ± 0.11.0 ± 0.1nd
A. muscaria MHB responsive genes
 AaCS1.3 ± 0.7nd0.6 ± 0.1ndndndnd
 Cyp 400.5 ± 0.10.3 ± 0.20.9 ± 0.2ndndndnd

Expression analysis of BBc6R8-responsive genes during the interaction with other rhizobacteria

The expression of the genes Cipc, tectonin2, fox2 and of four genes encoding proteins of the SAGA complex (tra1, gcn5, spt3 and ada3) was investigated during the early stage of the interaction with the bacterial strains P. fluorescens Pf29A, Paenibacillus sp. F2001L, B. subtilis MB3 and C. fungivorans Ter331. No changes in the expression of the seven target genes was observed with the MHB B. subtilis strain MB3, nor with the presumed intrafungal Paenibacillus sp. F2001L (Table 4). By contrast, the expression of various BBc6R8-responsive genes was modified by the four other bacterial strains. The chitinolytic strain C. fungivorans Ter331 enhanced the expression of tectonin2 and ada3, and decreased the expression of Tra1. The biocontrol strain P. fluorescens Pf29A induced an up-regulation of tectonin2, as well as Tra1, gcn5 and Cipc. None of the tested strains induced modulation of fox2 gene expression. Concerning the two MHB, Burkholderia sp. EJP67 and Paenibacillus sp. EJP73, the three replicates gave contradictory results (data not shown). Therefore we cannot conclude on their impact on the expression of the targeted genes.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Growth and morphology modifications

Some previous studies showed that the effect of helper bacteria on mycorrhiza formation can be correlated with an increase in mycelium growth in vitro (Garbaye, 1994; Becker et al., 1999; Maier et al., 2004; Hildebrandt et al., 2006). It has been suggested from these observations that the helper effect could result, in this case, from an increase in the growth and survival of the mycorrhizal mycelium in the soil, which would therefore increase the probability of root–fungus encounter and consequently, the number of mycorrhizas (Bruléet al., 2001). Here, we showed that the MHB P. fluorescens BBc6R8 not only enhanced the growth of L. bicolor mycelium, but also induced morphological changes of the mycelium in vitro (i.e. hyphal branching, apex density). The impact of bacteria on fungal morphology was previously reported in both antagonistic and mutualistic fungal–bacterial interactions (Bolwerk et al., 2003; Schrey et al., 2005; Ström et al., 2005). Interestingly, not all bacterial strains can modify fungal growth and morphology. The strain B. subtilis MB3, which promotes ectomycorrhizal symbiosis between L. bicolor and Douglas fir in glasshouses and nurseries (Duponnois & Garbaye, 1991), modified neither the growth nor the morphology of the L. bicolor colonies in our in vitro bioassay. On the contrary, the strain Paenibacillus sp. EJP73, which was isolated from a Lactarius rufus mycorrhiza and promoted the L. rufus–Pinus sylvestris symbiosis in vitro (Poole et al., 2001), as well as the L. bicolor–P. sylvestris symbiosis in the glasshouse (Aspray et al., 2006), significantly reduced the growth of L. bicolor colonies in our bioassay. This suggests that additional mechanisms not limited to growth increase are involved in the promotion of mycorrhiza formation by these two strains. The close presence of the plant could be required by EJP73 to exert its MHB activity (Aspray et al., 2006). In opposition, the MHB effect of MB3 likely involves detoxification of the rhizospheric environment from autotoxic compounds produced by ectomycorrhizal fungi (Duponnois et al., 1991).

Nonhelper rhizospheric bacteria, such as the biocontrol P. fluorescens Pf29A, were also able to induce alterations in growth and morphology of L. bicolor S238N. Nevertheless, P. fluorescens BBc6R8 was the only strain that enhanced both diametral growth of the colony, apex density and branching angle at the precontact stage. Interestingly, dramatic alterations in branching of hyphae occur in the earliest stages of root colonization by ectomycorrhizal fungi (Peterson & Bonfante, 1994; Martin et al., 2001). In addition to its effect on the survival and growth of L. bicolor, the helper strain P. fluorescens BBc6R8 also induces hyphal morphological changes that could be beneficial to mycorrhizal infection of the host roots. Similar processes have been described in endomycorrhizal symbiosis where strigolactones from host plant root exudates induced growth and branching of germinating hyphae before root infection (Akiyama et al., 2005; Besserer et al., 2006).

Alteration in L. bicolor S238N transcriptome

Mycorrhiza helper bacteria and nonMHB specific alterations in the transcriptome have been poorly documented thus far (Schrey et al., 2005; Hildebrandt et al., 2006). Here, we analysed for the first time regulation of the fungal transcriptome at several stages of interaction with a helper bacterial strain (i.e. before contact, at contact time and after a prolonged contact, during which bacteria colonized the surface of fungal hyphae). Transcript concentration of L. bicolor S238N was significantly altered in response to bacterial stimuli. As previously observed by Schrey et al. (2005) on the A. muscaria–Streptomyces interaction, the presence of the MHB P. fluorescens BBc6R8 led to a moderate response of L. bicolor S238N at the transcriptome level, as only 3% of the analysed transcripts showed an altered concentration. We distinguished two separated phases in the fungal response. Before contact and after a few hours of direct interaction, BBc6R8-responsive fungal genes were mainly up-regulated; these genes code for proteins involved in recognition processes and transcriptional regulation. After prolonged contact with the MHB BBc6R8, the expression of > 100 genes was down-regulated; 50% of them encoded for proteins involved in protein synthesis and energy metabolism. Yeast orthologues of these genes were repressed when Saccharomyces cerevisiae was grown in starvation or stressful environmental growth conditions (Gasch & Werner-Washburne, 2002; Wu et al., 2004). It was hypothesized that down-regulation of gene expression could help fungal cells to preserve energy while adapting to their growth environment. In our study, we also observed a down-regulation in the transcription of malic enzyme, PEP carboxylase and fumarate dehydrogenase, while citrate synthase (the enzyme involved in first step of the TCA cycle) was up-regulated. This suggests a shift in carbon use in the mycelium of L. bicolor after prolonged contact with P. fluorescens BBc6R8. Similarly, in Neurospora crassa, Xie et al. (2004) showed that glucose starvation induced genes of the TCA and glyoxalate cycle, while genes involved in glycolysis were down-regulated. Therefore, transcriptional modifications observed at this stage probably result from trophic competition occurring between L. bicolor S238N and P. fluorescens BBc6R8 after a long period of common growth in the in vitro assay. As a consequence, we will restrict discussion to the precontact stage, as it accurately mimicked the promoting effect of bacteria on the fungal growth as previously observed in soil (Bruléet al., 2001).

As in most fungal transcriptome analyses, about half of the BBc6R8-responsive genes coded for hypothetical proteins. Interestingly, orthologues were not found in other sequenced genomes for most of these genes (72%), suggesting that they are unique to L. bicolor. Annotation of the L. bicolor S238N-H82 genome has shown that these expressed hypothetical proteins are abundant in L. bicolor (F. Martin et al., unpublished). These orphan sequences should have rapidly evolved as they have no orthologues in the genome of Coprinopsis cinerea, an Agaricale saprotroph phylogenetically close to Laccaria; they may be related to specific adaptation of the fungus to its symbiotic lifestyle (Le Quéréet al., 2006). Further studies will be necessary in order to clarify the role of the BBc6R8-responsive orphan sequences.

BBc6R8-responsive genes involved in bacterial recognition

We have identified a BBc6R8-responsive fungal gene (fourfold up-regulation) encoding for the tectonin II protein. This gene shares 54% similarity with the tectonin II of the amoebae Physarum polycepharum (Huh et al., 1998). This protein participates in bacterial aggregation by the amoebae cells during the phagocytosis process. In L. bicolor, its expression levelled off to its constitutive amount after bacterial colonization of the mycelium. This gene appears to be specific to L. bicolor as no orthologue was found in the sequenced genome of Coprinopsis cinerea, Phanerochaete chrysosporium, Cryptococcus neoformans or Neurospora crassa. Further analysis on mycorrhizal fungi will be needed to confirm this hypothesis. This suggests that the tectonin orthologue of L. bicolor could play a role in cell recognition and/or fungal cell interaction with P. fluorescens BBc6R8, during the earliest phase of the interaction. Its expression was up-regulated not only by the helper P. fluorescens BBc6R8 strain and the biocontrol P. fluorescens Pf29A strain that promoted L. bicolor growth, but also by the antagonistic bacterial strain C. fungivorans Ter331. On the contrary, the helper strain Burkholderia sp. EJP67 that enhanced L. bicolor growth did not significantly induce expression of the tectonin gene. In consequence, transcription of the tectonin gene is not specifically modulated by helper bacteria. However, up-regulation of this gene could be related to the ability of BBc6R8 to attach to L. bicolor mycelium. The parental strain P. fluorescens BBc6 (Sen et al., 1994) and the rifampin mutant BBc6R8 (data not shown) are able to attach to L. bicolor mycelium. Further studies will be necessary to confirm this hypothesis with P. fluorescens Pf29A and C. fungivorans Ter 331.

Role of BBc6R8-responsive genes in fungal metabolism, growth, and morphology

Lipid metabolism was previously shown to be modulated in both endomycorrhizal– and ectomycorrhizal–MHB interactions (Requena et al., 1999; Schrey et al., 2005), as well as the early steps of mycorrhiza formation on pine roots by L. bicolor (Podila et al., 2002). Here, we observed that the mRNA concentrations of pantothenate kinase, which catalyses the limiting step of coenzyme-A synthesis, and of FOX2, which encodes a multifunctional enzyme of the β-oxidation pathway, were both decreased during the precontact phase and at contact time, respectively. In the same way, its orthologue in Glomus mosseae was repressed at contact time with the MHB B. subtilis NR1 (Requena et al., 1999). By contrast, no modification of the expression of the A. muscaria/Streptomyces AcH505 acetoacyl-CoA synthase regulated gene was found during the interaction between L. bicolor S238N and P. fluorescens BBc6R8. All these data strongly suggest that the response of mycorrhizal fungi to helper bacteria involves a modification of both fungal lipid anabolism and catabolism, which could result in an increase of lipid synthesis required for the enhanced fungal growth rate. However, the genes differentially regulated would depend on the fungal–bacterial systems.

The cipc gene was previously reported to correspond to expressed sequence tags from the two ectomycorrhizal fungi, L. bicolor S238N and P. tinctorius (Peter et al., 2003). The early up-regulation of this gene could be linked to the modification of L. bicolor growth and morphology. In fact, this gene encodes for a protein of unknown function which was first identified when analysing changes in Aspergillus nidulans morphology in response to the antibiotic concanamycin A, a specific inhibitor of V-ATPase produced by Streptomyces bacterial species (Melin et al., 2002). The concentration of this protein was enhanced in the fungal mycelium when the bacterial antibiotic was added to the culture medium. An orthologue of this gene was also suggested to be linked to changes in the growth and morphology of the ectomycorrhizal fungus Paxillus involutus in the early step of ectomycorrhiza formation with Betula pendula (Morel et al., 2005). This gene could therefore be considered as a marker of the presymbiotic status of the fungus. Interestingly, the strain BBc6R8 was also proved to up-regulate expression of the cipc gene at the early stage of coculture with L. bicolor. Our results therefore suggest that P. fluorescens BBc6R8 could induce a shift in the mycelial physiology from a saprotrophic to a presymbiotic status, in addition to its effect on the presymbiotic survival and growth of the fungus (Bruléet al., 2001). Further investigations will be needed to confirm this hypothesis.

Alteration of transcription machinery

Regulation of gene transcription involves recruitment of the RNA transcription polymerase II complex, the fixation of transcription factors on promoter sequences, and histone acetylation or methylation (for a review, see Cosma, 2002). In the yeast S. cerevisiae, the SAGA complex is required for the recruitment of the basal transcription machinery and regulates the expression of 10% of the genes (Wu et al., 2004). Here, we demonstrate that expression of the largest subunit of the SAGA complex, the so-called Tra1, was regulated at all stages of the L. bicolor S238N–P. fluorescens BBc6R8 interaction. This protein plays a fundamental role in the functioning of the SAGA complex by interacting with transcriptional activators (Brown et al., 2001). Several genes encoding for other SAGA subunits showed transcription changes in the presence of P. fluorescens BBc6R8 and C. fungivorans Ter331. Complex patterns of expression were observed as some genes were up-regulated, whereas others were down-regulated. But these proteins must play a major role in the response of L. bicolor S238N as the Tra1 gene is one of the largest genes of the genome, with a size of 11 526 kb. Therefore, its increased transcription imposes a high energy cost for the cell. Moreover, some of the fungal transcripts regulated during the interaction with the helper P. fluorescens BBc6R8, such as FOX2, citrate synthase, PEP carboxylase and acetyl-CoA synthase, are homologous to Saccharomyces genes known to be under the control of two transcription factors, Adr1 (FOX2, citrate synthase) and Cat8 (PEP carboxylase, acetylCoA synthase), which themselves need to interact with histone acetyl transferase (Tachibana et al., 2005). Interestingly, expression of the gene encoding histone H4 in A. muscaria, one target of HAT activities, was also stimulated by the MHB Streptomyces sp. AcH505 (Schrey et al., 2005). As a consequence, these data suggest that at least part of the transcriptional response of ectomycorrhizal fungus to MHB involves epigenetic changes in the histone code.

Specificity of mycorrhizal fungus–MHB interactions

A key question arising from our study is whether the gene regulations involved are specific to our ectomycorrhizal fungus–MHB model. Interestingly, we identified several molecular determinants that had never been previously identified and thus appear to be specific to the L. bicolor S238N–P. fluorescens BBc6R8 interaction. Others were mutually regulated at the transcriptome level in the two distinguished ectomycorrhizal fungus–MHB pairs, L. bicolor–P. fluorescens and A. muscaria–Streptomyces sp. In addition, some responsive genes identified in the A. muscaria-Streptomyces sp. and Glomus mosseae-Bacillus subtilis models were not regulated during the L. bicolorP. fluorescens interaction (data not shown). We also observed that some genes overexpressed in the presence of the MHB P. fluorescens BBc6R8 were also regulated by other rhizobacteria. Consequently, our results highlight the fact that some fungal pathways are mutually regulated by different rhizobacteria, whereas others are specific to some MHB.

Our work emphasizes the importance of studying MHBs as models for genomic analysis of fungal–bacterial interactions. This should also benefit other research areas where fungal–bacterial interactions play a major role, such as plant protection and medicine (Frey-Klett & Garbaye, 2005; Bending, 2007).


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by grants from INRA and Lorraine Region. We also greatly thank G. Bending (HRI, University of Warwick, UK) and W. de Boer (NIOO-KNAW, the Netherlands) for providing the EJP67, EJP73 and Ter 331 bacterial strains. The research used the DNA Sequencing Facilities at INRA-Nancy supported by INRA, Lorraine Region and the European Commission. Finally we thank the American Journal Expert for revising the manuscript.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 List of the primers used in this study and the gene model corresponding to each gene in L. bicolor H82 genome

Table S2 Composition of the transcription regulatory complexes SAGA, SALSA and SLIK in the yeast S. cerevisiae and gene model homologues in the genome of L. bicolor H82

Table S3 Transcripts of L. bicolor S238N regulated (t-test, ≤ ≥ 2.0, underlined in grey) upon the interaction with the helper strain P. fluorescens BBc6R8

Fig. S1 Validation of the cDNA-array data by real-time PCR analyses

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