• Beneficial rhizosphere microorganisms may share similar molecular steps during root colonization. To test this hypothesis, we compared Medicago truncatula Gaertn. gene expression in roots colonized, or not colonized, by Glomus mosseae BEG12, Pseudomonas fluorescens C7R12 or Sinorhizobium meliloti 2011.
• Pseudomonas fluorescens C7R12 formed colonies on the surface of M. truncatula roots and colonized root tissues intercellularly and intracellularly in a way similar to that previously described for other plants.
• Semiquantitative reverse transcriptase polymerase chain reaction of a set of 12 mycorrhiza upregulated M. truncatula genes revealed different expression profiles in roots 3 weeks after inoculation with P. fluorescens or S. meliloti. Pseudomonas fluorescens colonization activated seven of the plant genes while nodulated root systems showed increased expression in only three genes and five appeared to be downregulated.
• This first report of similar gene induction by a fluorescent pseudomonad and a mycorrhizal fungus in roots supports the hypothesis that some plant cell programmes may be shared during root colonization by these beneficial microorganisms. Less similarity existed in expression of the gene set with nodulation by S. meliloti.
Plants have developed diverse strategies to combat environmental stresses since their colonization of terrestrial ecosystems. One of the most successful is the ability of root systems to establish beneficial relationships with rhizosphere microflora, creating an environment that favours essential exchanges between roots and the soil (Whipps, 2001). Rhizobacteria and arbuscular mycorrhizal fungi are among the most frequent rhizosphere microorganisms which stimulate plant development, improve mineral nutrition and/or increase tolerance against pathogenic microorganisms (Stacey et al., 1992; Handelsman & Stabb, 1996; Smith & Read, 1997). Arbuscular mycorrhizal fungi, which belong to the Glomeromycota (Schüssler et al., 2001), develop symbiotic associations with the roots of most terrestrial plants (Walker & Trappe, 1993). Rhizobacteria mainly consist of Rhizobiaceae which nodulate with legume species (Franssen et al., 1992) and rhizospheric plant growth-promoting rhizobacteria (PGPR) including Pseudomonas and Bacillus species, which colonize the roots of a wide range of plants (Kloepper, 1994). Plant factors exuded into the rhizosphere activate these different groups of microorganisms and enhance their proliferation, leading to root colonization (Franssen et al., 1992; Giovannetti et al., 1994; Duijff et al., 1997). Rhizobial and arbuscular mycorrhizal associations are the two main plant root endosymbioses (Gianinazzi-Pearson & Dénarié, 1997), and studies have clearly shown that their establishment is characterized by several common features (Marsh & Schultze, 2001), while root colonization by some Pseudomonas fluorescens strains or arbuscular mycorrhizal fungi such as Glomus mosseae can both activate induced systemic resistance responses to pathogen attack (van Loon, 1997; Cordier et al., 1998). Although in morphological terms the outcome of the root interactions differ, the common ability of rhizobacteria and arbuscular mycorrhizal fungi to establish beneficial, mutualistic associations with plant root tissues may imply shared molecular events and/or cell programmes.
Much is known about plant gene regulation in the development of nodule symbioses (Schultze & Kondorosi, 1998). Far less information is available about symbiotic gene expression patterns in arbuscular mycorrhiza (Harrison, 1999; Lapopin et al., 1999), and investigations of genes expressed during root interactions with P. fluorescens have so far been essentially limited to those which that be involved in bioprotection induced by the bacteria (van Loon, 1997; Pieterse et al., 1998). However, there are reports that arbuscular mycorrhizal fungi and nodulating bacteria may activate some common pathways of gene expression in legumes (Gianinazzi-Pearson & Dénarié, 1997; Frühling et al., 1997; Albrecht et al., 1999; Journet et al., 2001). In order to obtain greater insight into plant genes which may be common to beneficial rhizosphere interactions in legume species, the expression of a set of Medicago truncatula genes activated in arbuscular mycorrhiza with G. mosseae isolate BEG12 has been compared in roots colonized by P. fluorescens strain C7R12 or developing nodules with Sinorhizobium meliloti strain RCR 2011.
Materials and Methods
Seeds of M. truncatula Gaertn. cv. Jemalong line J5 (provided by G. Duc INRA Dijon, France) were surface-sterilized for 6 min in 98% sulphuric acid, 5 min in 96% ethanol, 10 min in 3% calcium hypochlorite and rinsed in sterile distilled water. They were germinated on 0.7% Bactoagar (Difco Laboratories, Detroit, MI, USA) at 25°C in the dark and transplanted after 48 h into 75 mL sterilized (autoclaved twice at 120°C) Terragreen (OilDri-US special, Mettmann, Germany). Inoculation with G. mosseae BEG 12 was performed as described by Brechenmacher et al. (2003). Rhizobacteria were introduced into the substrate as a suspension in Long Ashton solution (Hewitt, 1966) containing 106 colony-forming units (CFU) ml−1 of P. fluorescens C7R12 or S. meliloti strain RCR 2011. Pseudomonas fluorescens strain C7R12 is a spontaneous mutant of strain C7 resistant to rifampicin (Eparvier et al., 1991). The wild-type strain C7 was previously isolated from the rhizosphere of flax cultivated in the Châteaurenard, France, a soil that suppresses fusarium wilts (Lemanceau et al., 1998). The strain C7R12 was shown to improve the suppression of fusarium wilts achieved by nonpathogenic Fusarium oxysporum (Lemanceau & Alabouvette, 1991) and to be rhizosphere competent (Eparvier et al., 1991). Controls for G. mosseae received filtered washings (Whatman no. 2, Whatman International Ltd., Maidstone, UK) of inoculum to reconstitute associated bacterial microflora. Control plants for P. fluorescens and S. meliloti received only Long Ashton solution. Plants were grown under constant conditions (16-h photoperiod, 19°C night/22°C day, 360 mol m−2 s−1 (LI-189; Li-Cor Radiation sensors, Lincoln, NE, USA), 70% relative humidity (r.h.)) and received twice weekly 5 ml Long Ashton solution without phosphate (Hewitt, 1966) enriched in iron (1 g sequestrene l−1) or without nitrogen for P. fluorescens and S. meliloti studies, respectively. Thirty plants were harvested for each treatment 3 wk after inoculation (wai), shoots and roots weighed and roots immediately stored in liquid nitrogen.
Root colonization, shoot and root fresh weight were evaluated from a sample of five plants. Rhizoplane colonization by P. fluorescens was estimated after vortexing 300 mg roots per plant in 5 ml sterile distilled water with 2 g glass beads (Powder G2458510). Pseudomonas fluorescens strain C7R12 was enumerated as CFU after plating serial dilutions of the rhizoplane suspensions on King B+ agar (Geels & Schippers, 1983) supplemented with 100 mg l−1 rifampicin. Parameters of mycorrhizal colonization by G. mosseae were determined according to the method of Trouvelot et al. (1986) (http://www.dijon.inra.fr/bbceipm/Mychintec/). Nodules were counted on root systems of plants inoculated with S. meliloti.
Because P. fluorescens root colonization had not previously been described in M. truncatula, light microscope investigations were also performed. The M. truncatula seeds were disinfected as described in Plant inoculation and seedlings grown on 1% agar plates for 72 h. Root tips were spot-inoculated with a 1 l suspension of 106 cells ml−1 of P. fluorescens strain C7R12 and Petri dishes were incubated at an angle of 60° in the dark for 4 d at 22°C. Root pieces 3–5-mm long were sampled, incubated in fixation buffer (2% glutaraldehyde, 0.1 m cacodylate, 200 mm CaCl2) overnight at 4°C, and washed five times in 0.1 m cacodylate and once in NH4Cl for 1 h at 4°C. Root tissues were dehydrated through a graded ethanol series and embedded in LR White resin (London Resin Company, London, UK) according to Gianinazzi and Gianinazzi-Pearson (1992). Semithin sections (0.5 m) were cut with an ultramicrotome (Ultracut Reichert, Leica, Rueil Malmaison, France), stained with toluidine blue (1% sodium borate buffer, 1% toluidine blue) and examined in a Leica light microscope.
Total RNA extraction and DNase treatment
Total RNA was isolated from uninoculated M. truncatula roots and roots inoculated with P. fluorescens, G. mosseae or S. meliloti according to the method of Franken and Gnädinger (1994) and treated with DNase for 30 min at 37°C (25 g total RNA, 40 U RNase inhibitor, 3 U RNase-free DNase, 6 l 10× buffer, and diethyl pyrocarbonate (DEPC) water to 60 l). DNase was removed by phenol–chloroform–isoamyl alcohol (25 : 24 : 1), RNA was precipitated overnight at −20°C (0.1 vol. 3 m sodium acetate, 2.5 vol. 95% ethanol) and then resuspended in DEPC water. RNA concentration was evaluated from absorption values at 260 nm and 280 nm.
Reverse Northern analyses
Expressed sequence tags (ESTs) from an suppressure subfracture hybridization (SSH) library obtained previously using nonmycorrhizal (driver) and mycorrhizal (tester) cDNA (Brechenmacher et al., 2003) served as templates for polymerase chain reaction (PCR). The PCR reactions (PTC-200; MJ Research, Watertown, MA, USA) were conducted in a 20 l reaction volume containing 1 l 1 : 100 cDNA clone in water, 0.5 U Taq polymerase (Invitrogen Life Technologies, Cergy-Pontoise, France), 125 m dNTP and 0.5 m each primer (18.1 for GTCACGACGTTGTAAAACG and 18.2 rev AGCTATGACCATGATTACG). Amplifications consisted of an initial denaturation step for 1 min at 95°C, 29 cycles with annealing 1 min at 56°C, 1 min 30 at 72°C, with a final extension for 5 min at 72°C.
The cDNA, synthesized from 2.5 g total RNA, was 32P-labelled by reverse transcriptase polymerase chain reaction (RT-PCR) and purified on ProbeQuant G-50 Micro Columns (Amersham Bioscience, Orsay, France) before denaturing 5 min at 95°C. The PCR products prepared from ESTs were separated on 1.2% agarose gels, transferred to Hybond-XL (Amersham Bioscience) by capillary blotting and fixed under UV light (70 000 J cm−2). Membranes were prehybridized for 1 h at 60°C and hybridized with probes overnight at 60°C in Church buffer (Church & Gilbert, 1984), then washed twice 5 min in 2× standard saline citrate (SSC)−0.1% sodium dodecyl sulphate (SDS) at room temperature, twice 20 min in 0.5× SSC−0.1% SDS at 60°C and twice 20 min in 0.5× SSC−0.1% SDS at 65°C. Hybridization signals were quantified in a Storm 860 phosphorimager with ImageQuant software (Molecular Dynamics, Amersham Pharmacia Biotech, Orsay Cedex, France) and normalized using the Mtgap1 gene (see below).
Semiquantitative RT-PCR was performed according to Taylor and Harrier (2003). cDNA was prepared from 1 g total RNA added to 1.5 g oligodT15, dNTP (2.5 mm each) and made up to a final volume of 11.5 l with sterile distilled water. RNA was denatured 5 min at 70°C, placed on ice and 5 l MMLV 5× reaction buffer, 300 U moloney murine leukemia virus (MMLV) reverse transcriptase and 80 U RNase inhibitor were added. First-strand cDNA was synthesized at 25°C for 15 min followed by 50 min at 42°C and 2 min at 96°C. Gene-specific fragments were amplified by PCR using the primers, annealing temperature and number of cycles described in Table 1. Amplification was 95°C for 5 min, 93°C for 45 s, annealing for 45 s, 72°C for 1 min, and a final extension at 72°C for 5 min. Amplification products were analysed by 1.2% agarose gel electrophoresis, stained with ethidium bromide and quantified in a Storm 860 phosphorimager with ImageQuant software (Molecular Dynamics, Amersham Pharmacia Biotech).
Table 1. Oligonucleotide primer sequences, polymerase chain reaction (PCR) conditions and number of cycles used for analysis by semiquantitative reverse transcriptase PCR
The Mtgap1 gene (encoding a glyceraldehyde phosphate dehydrogenase) was used as an active reference control for equivalent reverse transcription to cDNA and equivalent amplification in the PCR. Constitutive levels of expression were checked by semiquantitative PCR of transcripts on cDNA synthesized from RNA of M. truncatula roots inoculated or not inoculated with P. fluorescens, G. mosseae or S. meliloti as described earlier. Polymerase chain reaction was performed on reverse transcription products diluted to 1 : 4, 1 : 8 and 1 : 16 using specific primers of the Mtgap1 gene: Gapfor (5′-TGA GGT TGG AGC TGA TTA CG-3′) and Gaprev (5′-AGC CTT GGC AGC TCC AGT GC-3′), designed from the consensus sequence of the Mtgap1 cluster MtC00030_GC (medicago. toulouse.inra.fr/Mt/EST:DOC/MtB.html). Polymerase chain reaction was performed as described above except at a Tm of 55°C. Amplification products were analysed and quantified as described above.
Data from semiquantitative PCR evaluations of gene expression of replicate RNA batches from three independent pools of plants were statistically compared between uninoculated and inoculated (by G. mosseae, P. fluorescens and S. meliloti) treatments, using Student's t-test.
Rhizoplane colonization by P. fluorescens was evaluated as 2 × 108 bacteria per root system at 3 wai. In semithin transverse sections of inoculated M. truncatula, cells of P. fluorescens were observed as colonies on the root surface in the apical region (Fig. 1a), between root cap tissues (Fig. 1b), and more rarely, proliferated within cortical cells (Fig. 1c). For plants inoculated with G. mosseae, the proportion of root systems showing cortex colonization was 48.5 ± 11.9%. Plants grown 3 wk with S. meliloti had an average of 100 ± 8 nodules per root system.
Expression of 12 plant genes activated in mycorrhizal roots of M. truncatula (Brechenmacher et al., 2003) was first compared by reverse Northern hybridization of corresponding ESTs using cDNA probes from M. truncatula roots inoculated or not inoculated with G. mosseae, P. fluorescens or S. meliloti. Hybridization signal intensities were normalized using the Mtgap1 gene, which showed a similar expression in inoculated and uninoculated roots by semiquantitative RT-PCR (Fig. 2). Activation of all 12 genes was confirmed in mycorrhizal roots (Table 2). Genes corresponding to six of the ESTs showed at least 2.5-fold greater expression in P. fluorescens-inoculated roots than in uninoculated roots (Table 2); these encoded a germin-like protein, a putative wound-induced protein, a nodulin 26-like aquaporin, a glutathione-S-transferase and two putative proteins of unknown function. Only two genes were upregulated more than 2.5-fold in nodulated root systems (putative wound-induced protein and nodulin 26-like aquaporin).
Table 2. Reverse Northern analysis of expression patterns of Medicago truncatula genes 3 wk after inoculation with Glomus mosseae and Pseudomonas fluorescens or Sinorhizobium meliloti
MtGmLs EST number
Matching sequence (E-value Blast X, Blast N)
Fold change in gene expression in colonized roots
EST, expressed sequences tag (Brechenmacher et al., 2003). Values in bold type indicate ≥ 2.5 fold increase in gene expression; ns, signal too weak for quantification.
Gene expression was further analysed by semiquantitative RT-PCR. This technique has two advantages over Northern analyses in that it allows (1) specific detection of a gene family member and (2) quantification of plant gene expression if the target gene has a low level of expression or a limited amount of tissue is available for RNA extraction (Burleigh, 2001). Using this approach, differential expression in P. fluorescens-inoculated roots of the six genes detected by reverse Northern analyses was confirmed and one additional gene (glutamine synthetase) was found to be significantly upregulated (Fig. 3). Reverse transcriptase polymerase chain reaction is a more sensitive technique than reverse Northern analysis. Indeed, it allows, using specific primers, to distinguish between the expression of member genes of a multigenic family such as glutamine synthetase. The identity of the PCR fragment was confirmed by the unique band of the expected size, and by sequence analyses. MtGmLs136, MtGmLs164 and the PR10-encoding gene showed no differential expression, and MtGmLs281 and MtGmLs311 gave no quantifiable amplification products.
To determine common gene expression in root interactions with rhizobacteria, alterations in expression patterns in P. fluorescens-colonized roots were compared with S. meliloti-nodulated roots by semiquantitative RT-PCR (Fig. 4). Five patterns of differential gene regulation were observed for the 12 plant genes upregulated in mycorrhiza. Expression was increased (P = 0.05) in both P. fluorescens and S. meliloti-inoculated roots for genes encoding the putative wound-induced protein, nodulin 26-like aquaporin and glutathione-S-transferase, while genes encoding the germin-like protein, MtGmLs11, MtGmLs291 and glutamine synthetase, which were activated in P. fluorescens-inoculated roots, were downregulated (P = 0.05) in nodulated roots. The gene corresponding to MtGmLs164 showed no significant change in expression in either P. fluorescens-inoculated or nodulated roots. The remaining genes, MtGmLs136 and the PR10-encoding gene, which showed no significant differential expression in P. fluorescens-inoculated roots, were downregulated in nodulated roots. The MtGmLs281 and MtGmLs311 genes, again, gave no quantifiable amplification products in P. fluorescens-inoculated or nodulated roots.
Beneficial rhizosphere microorganisms, such as fluorescent pseudomonads, rhizobia and arbuscular mycorrhizal fungi, may use similar molecular pathways when interacting with root tissues. To test this hypothesis, we compared M. truncatula gene expression in roots colonized, or not, by the mycorrhizal fungus G. mosseae BEG12 and the rhizobacteria P. fluorescens C7R12 and S. meliloti RCR 2011. Because P. fluorescens–root interactions have not previously been studied in M. truncatula, a prerequisite to this study was to establish and define root colonization of this model legume. Population estimates and microscope observations showed that P. fluorescens strain C7R12 proliferated over the root surface and colonized root tissues both inter and, more rarely, intracellularly in a way similar to that described in tomato in association with another bioprotective P. fluorescens strain, WCS417r (Duijff et al., 1997).
Expression analyses of 12 mycorrhiza upregulated M. truncatula genes revealed different expression profiles in roots 3 wk after inoculation with P. fluorescens or S. meliloti. Pseudomonas fluorescens colonization activated seven of the plant genes, while nodulated root systems showed increased expression in only three genes and six appeared to be downregulated. Interestingly, the nodulin 26-like aquaporin encoding gene was among the plant genes activated by all three microorganisms, suggesting a broad role of this multifunctional aquaporin in beneficial root–microbe interactions. A number of Nod 26 homologues are expressed in different tissues of plants including root elongation and tip zones and, although nodulin 26 has been postulated to be a channel protein that facilitates transport of small molecules across the peribacteroid membrane in nodules, the presence of distinct homologues of Nod 26 in a same plant suggests a wider role of these proteins in cell function (Miao & Verma, 1993).
Two genes associated with defence or stress responses (glutathione-S-transferase and putative wound-induced protein) were also activated in root interactions with G. mosseae, P. fluorescens and S. meliloti. Glutathione-S-transferases are multifunctional, detoxifying enzymes encoded by a highly divergent, ancient gene family and are involved in the metabolism of xenobiotic and reactive endogenous compounds (Edwards et al., 2000). While mycorrhizal fungi have previously been reported to induce glutathione-S-transferase transcription and translation in different plants (Strittmatter et al., 1996; Bestel-Corre et al., 2002; Wulf et al., 2003), these are novel observations for the rhizobacteria. The gene encoding a probable PR10 protein was not differentially expressed in P. fluorescens-colonized roots and appeared to be downregulated in nodulated roots. The lack of modification in PR10 gene expression with P. fluorescens is in accord with reports that pathogenesis-related gene activation is not associated with induced systemic resistance to pathogens by this group of rhizobacteria (Pieterse et al., 1998). The result with S. meliloti contrasts with reports that the two M. truncatula genes MtN1 and MtN13, homologs of PR10, are expressed during nodulation (Gamas et al., 1998) and that Rhizobium leguminosarum induces accumulation of PR10 transcripts in pea (Ruiz-Lozano et al., 1999). However, the PR10 gene family is large and ubiquitous (Sikorski et al., 1999), and responses therefore probably depend on the gene member studied. Similarly, it is surprising that the gene encoding glutamine synthetase, which plays a central role in the nitrogen metabolism of higher plants, was not activated by S. meliloti because different members of this family have been found to be associated with nodule formation (Carvalho et al., 2000). The M. truncatula germin-like protein gene also responded differently to S. meliloti compared with G. mosseae or P. fluorescens. Germin-like proteins constitute another large and highly diverse family of ubiquitous plant proteins (Bernier & Berna, 2001) and their precise functions have not yet been elucidated. Some are enzymes (oxalate oxidase and superoxide dismutase) while others appear to be receptors or structural proteins (Ohmiya, 2002) and their proposed roles include restructuring of cells walls, salt and heavy metal responses, and plant defence (Bernier & Berna, 2001).
A number of similarities between rhizobial and arbuscular mycorrhiza symbioses have been reported (Frühling et al., 1997; Gianinazzi-Pearson & Dénarié, 1997; Albrecht et al., 1999; Journet et al., 2001) and the isolation of isogenic mutants of legume species, unable to form either mycorrhiza or nodules, has provided evidence that some steps of the two symbioses are controlled by the same plant genes (Marsh & Schultze, 2001). A receptor-like kinase gene has been isolated from L. japonicus (Stracke et al., 2002) and M. sativa (Endre et al., 2002) where it is considered to be essential to a signal transduction pathway involved in the establishment of both mycorrhiza and nodule symbioses. However, the present work represents the first comparison to be made of both these root symbioses with a beneficial P. fluorescens association. The results obtained support the hypothesis that some plant cell programmes may be shared during root colonization by these beneficial microorganisms. In our study, less similarity was shown in root responses of M. truncatula to S. meliloti than to G. mosseae and P. fluorescens at the stage (3 wai) of interactions studied here. This may be partly linked to the fact that root colonization by S. meliloti results in nodule formation while P. fluorescens and G. mosseae colonize tissues endophytically without causing gross changes in root organization. Further research to identify early molecular events and regulation pathways associated with initiation of root colonization of M. truncatula by P. fluorescens C7R12 and G. mosseae BEG12, and comparison with those known for nodulation (Catoira et al., 2000) will give provide clues to the mechanisms whereby roots distinguish friend from foe.
The authors are grateful to Odile Chatagnier, Christine Arnould, Denise Dubois and Thérèse Corberand for technical assistance, to Valérie Monfort for providing inoculum of G. mosseae and to Gérard Duc for providing M. truncatula seeds. This project was supported by doctoral grants to L. S. and L. B. (INRA/Conseil Régional de Bourgogne) and by the EU project MEDICAGO (QLG2-CT-2000-00676).