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Keywords:

  • auxin-regulated gene;
  • GH3;
  • Hebeloma cylindrosporum;
  • mycorrhiza;
  • Pinus pinaster

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants.
  • • 
    Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia.
  • • 
    Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin.
  • • 
    By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.

Introduction

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

In temperate forests, ectomycorrhiza is a prevalent mutualistic symbiosis between filamentous soilborne fungi and tree roots (Wiemken & Boller, 2002). By supplying water and mineral nutrients, the fungal partner plays a major role in the mineral nutrition of associated trees. As a consequence, the growth and fitness of the plant partner are improved significantly (Smith & Read, 1997). In return, carbohydrates and organic compounds from root exudates are transferred to the fungal partner, allowing growth of the mycelium in the soil and fruit-body production.

Although ectomycorrhizal symbiosis is a key component of forest ecosystems, the molecular mechanisms underlying its establishment and maintenance are poorly understood (Martin & Tagu, 1995; Martin et al., 1997). Ectomycorrhiza is the ultimate stage of complex ontogenic processes that necessitate coordinated expression of both plant and fungus genomes. Although global gene-expression changes have been shown to occur in several ectomycorrhizal systems (Voiblet et al., 2001; Johansson et al., 2004), signalling networks that coordinate the symbiosis-related alteration of gene-expression patterns remain to be identified (Martin et al., 2001). As a diffusible molecule, auxin released by the fungal partner is a good candidate as a signal molecule involved in the early molecular cross-talk between symbiotic partners (Gay et al., 1994; Gea et al., 1994; Tranvan et al., 2000; Laurans et al., 2001). As indole-3-acetic acid (IAA)-overproducing mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum have been shown to modify dramatically the structure of pine ectomycorrhizas, it has been suggested that fungal IAA could be involved in the control of some particular morphogenetic processes controlling mycorrhiza formation (Gay et al., 1994; Gea et al., 1994; Laurans et al., 2001). Noticeably, fungal IAA could facilitate Hartig-net formation, thereby improving the efficiency of reciprocal nutritional exchanges between symbiotic partners. In order to determine whether auxin-regulated plant genes could play a role in ectomycorrhizal symbiosis establishment, Charvet-Candela et al. (2002b) screened a cDNA library from auxin-treated roots of Pinus pinaster and isolated several auxin-inducible cDNA clones. This screen led to the identification of two auxin-inducible P. pinaster genes that were shown to be upregulated on establishment of ectomycorrhizal symbiosis (Charvet-Candela et al., 2002a; Reddy et al., 2003). One of these, Pp-iaa88, encodes a putative transcription factor that may play a role in triggering a cascade of molecular events in the pine roots ultimately leading to the formation of mycorrhizas (Charvet-Candela et al., 2002b).

The aim of the present work was to characterize Pp-GH3.16, a cDNA encoding an auxin-inducible GH3-like protein. The expression of Pp-GH3.16 was investigated in pine roots subjected to different hormonal and environmental conditions. In an attempt to determine whether this gene was expressed differentially during the earliest stages of ectomycorrhiza formation, transcripts were quantified in pine roots inoculated with two auxin-producing ectomycorrhizal fungi, H. cylindrosporum and Rhizopogon roseolus. We also used a nonmycorrhizal mutant of H. cylindrosporum in attempt to determine whether the differential expression pattern detected in mycorrhizal root systems had to be related to the symbiotic process, or was solely caused by the presence of fungal hyphae in the rhizosphere. Results showed that the auxin-upregulated gene Pp-GH3.16 was specifically downregulated on establishment of ectomycorrhizal symbiosis.

Materials and Methods

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

Plant material and fungal strains

Pinus pinaster (Ait.) Sol. seedlings were obtained from seeds (CEMAGREF batch), surface sterilized and germinated according to Debaud & Gay (1987). Mycorrhizal syntheses were performed using three different strains of Hebeloma cylindrosporum Romagnesi and one strain of Rhizopogon roseolus (Corda) Th. M. Fr. The haploid monokaryotic strain h1 of H. cylindrosporum was isolated from single-spore germination (Debaud & Gay, 1987). The IAA-overproducing mutant referred to as 331 in the present work corresponds to the haploid mutant strain h1 FIR4 F1331 isolated by Gay et al. (1994); the nonmycorrhizal mutant 552 was isolated as described previously (Reddy et al., 2003; Combier et al., 2004). The dikaryotic strain of R. roseolus was isolated under P. pinaster in Les Landes forest, south-western France.

Plant culture and induction

For induction studies, 3-wk-old seedlings were transferred to Petri dishes (140 mm in diameter) filled with 80 ml liquid Melin–Norkrans medium (Gea et al., 1994) containing 0.5 g l−1 glucose. The seedlings were first placed for 2 d at 22°C under 16 h photoperiodic illumination (80 mmol photons m−2 s−1). This pretreatment was performed to reduce any stress effects caused by transferring the seedlings. Seedlings were subsequently immersed for 6 h in Melin–Norkrans nutrient solution supplemented or not with either 50 µm IAA, 50 µm 2,4-dichlorophenoxy acetic acid (2,4-D), 50 µm abscisic acid (ABA), 50 µm gibberellic acid (GA3), 50 µm zeatin, 10 µm quercetin or 100 µm 2-chloroethylphosphonic acid (ethephon) for ethylene induction. All chemicals were obtained from Sigma-Aldrich (L’Isle d’Abeau, France). The concentrations used were previously shown to induce auxin-regulated genes in P. pinaster (Charvet-Candela et al., 2002a; Reddy et al., 2003). Cycloheximide (CHX, 50 µm) was used in protein synthesis-inhibition assays. Combined 50 µm CHX + 50 µm IAA treatment was preceded by a 30-min pretreatment consisting of 50 µm CHX to ensure complete protein synthesis inhibition from the onset of the combined treatment. Standard duration of the induction was 6 h. Water-stress treatment was applied for 6 h according to Dubost & Plomion (2003) by lowering the osmotic potential of the nutrient solution to −0.45 MPa using 180 g l−1 polyethylene glycol (PEG 3350, Sigma-Aldrich) as an osmoticum. Anoxia was induced by placing root systems for 6 h in 5-cm-deep Melin–Norkrans medium contained in a test tube (25 mm in diameter). Heat shock was performed by transferring seedlings from the 22°C standard temperature to 35°C. This treatment was applied for 6 h. Except for heat shock, all treatments were performed at 22 ± 1°C in the dark. Control plants consisted of seedlings treated in the same manner, except that the inducer was omitted.

Fungal culture and axenic mycorrhizal synthesis

The fungal cultures were routinely grown in the dark at 22°C on yeast-malt-glucose (YMG) agar (Rao & Niederpruem, 1969). Mycelia for inoculation of pine roots were grown from agar plugs of mycelium placed on cellophane-covered YMG agar medium. Mycelium strips approx. 60 × 5 mm were excised from the edges of the thalli. Strips were then placed on cellophane-covered plates filled with Melin–Norkrans agar medium (Gea et al., 1994) and the mycelia were allowed to grow for 1–2 wk. Mycelium strips obtained from the margins of these thalli were used as inocula for mycorrhizal syntheses.

Mycorrhizal syntheses were performed under axenic conditions in Petri dishes by placing strips of mycelia over the entire length of the tap root of 4-wk-old P. pinaster seedlings, as described previously (Tranvan et al., 2000). Controls consisted of uninoculated seedlings grown in the same conditions as inoculated ones. At selected times, roots were sampled, frozen in liquid nitrogen and stored at −70°C until RNA extraction.

cDNA cloning and sequence analysis

Differential screening of a cDNA library constructed from auxin-treated P. pinaster roots allowed the identification of different auxin-upregulated cDNAs (Charvet-Candela et al., 2002a, 2002b). One of these, Pp-GH3.16, was studied in the present work. For sequencing, plasmid DNA was purified using a Qiagen kit (Qiagen, Crawley, UK) and the insert was analysed by DNA sequencing in both directions using gene-specific primers. Computer-assisted searches for nucleotide and amino acid sequence homology were carried out using tblastx programs through the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST). A search for functional domains was performed using the motif search software (http://motif.genome.jp) and search engines available on the Infobiogen website (http://www.infobiogen.fr/deambulum). Multiple alignments were carried out using the clustalx program (Higgins & Sharp, 1988).

RNA extraction and transcript quantification by competitive RT–PCR

Total RNA was isolated from whole root systems according to the method of Kiefer et al. (1999), including a DNase I (Amersham, Saclay, France) treatment. Reverse transcription was performed as described previously (Reddy et al., 2003). The absence of contaminating genomic DNA was confirmed on each sample by amplifying a control gene with primers flanking an intron sequence (data not shown).

Pp-GH3.16 mRNA accumulation was quantified by competitive RT–PCR. After reverse transcription of all mRNA from root samples using poly dT primers, an internal fragment of the Pp-GH3.16 cDNA and a competitor DNA sequence were coamplified by PCR using the same set of primers specific for the Pp-GH3.16 sequence. The competitor sequence was a plasmid-cloned DNA fragment homologous to the sequence to be quantified, which differs from the latter by a length polymorphism so that both PCR-amplified sequences can be separated by gel electrophoresis and quantified separately. As both sequences are amplified simultaneously with the same primer pair, the final amplification ratio of the two sequences is proportional to the initial (known) amount of competitor sequence and an unknown (to be quantified) amount of target sequence added to the PCR mix.

In the case of RNA extracted from uninoculated root samples, a 384-bp-long fragment of the Pp-GH3.16 cDNA was amplified using primers Pp-GH3.16 U (5′-CCATTCTTTCTGCTCACC-3′) and Pp-GH3.16 L (5′-GAACACAGTATCGCCTCC-3′). In this case, the competitor DNA was the cloned Pp-GH3.16 genomic sequence that contains two introns between primers Pp-GH3.16 U and Pp-GH3.16 L; this competitor is 155 bp longer compared with the corresponding cDNA sequence.

This primers/competitor combination could not be used in the case of RNA extracted from inoculated roots as, for an unknown reason, these RNA samples were most of the time slightly contaminated with genomic DNA, despite extended incubations in the presence of DNase I. In this case, we amplified a 297-bp cDNA fragment using primers Pp-GH3.16 L and Pp-GH3.16INT (5′-TCTCACAAGCTCTGGAAC-3′). The latter primer overlaps the right and left borders of an intron and therefore eliminates unwanted amplification of the genomic copy in the PCR reaction. In this case, the competitor DNA was the cloned cDNA, in which a 116-bp-long NcoI DNA fragment was inserted in an NcoI site located between primers Pp-GH3.16 L and Pp-GH3.16INT.

Standard curves were constructed for each competitor by coamplifying different known amounts of cloned target DNA (between 0.0075 and 0.5 pg) with a constant amount of competitor DNA (0.03 pg). A standard curve is obtained by plotting the log values of the amplification ratios of target DNA/competitor against the log values of the amplified DNA (pg of target DNA) added to the PCR mix before amplification. It was found that 0.03 pg of competitor per 25 µl PCR reaction could be used for an accurate quantification of Pp-GH3.16 present in the different RT reaction mixtures.

The PCR reactions were performed as described previously (Reddy et al., 2003) in a final volume of 25 µl using 1 µl RT cDNA mixture, 0.03 pg competitor, 0.2 mm of each primer, 2 mm MgCl2, 100 mm dNTP, 2 U Taq DNA polymerase and the appropriate buffer (Invitrogen, Cergy-Pontoise, France). Amplification conditions were 3 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 54°C and 1 min at 72°C followed by a final elongation step of 5 min at 72°C. After electrophoresis in 2.5% agarose gels and staining with ethidium bromide, the relative amounts of the two coamplified DNA fragments were quantified using the gelDoc 1000 system (Bio-Rad, Marnes-la-Coquette, France).

RNA was extracted from three independent batches of 10 plants. Two reverse transcriptions were performed from each extract, and three to four competitive PCR reactions were carried out on each cDNA sample. Each experiment was replicated three times, and all plants in a given replicate were treated simultaneously. Hence results obtained in a replicate could be expressed by reference to a control that was either transcript level at zero time (see Figs 2, 6) or in untreated plants (see Figs 3–5), and transcript level was expressed using arbitrary units where 1 corresponds to level at zero time or in untreated plants. In the case of induced roots, Pp-GH3.16 transcript accumulation was calculated per mg total RNA. In the case of inoculated roots, Pp-GH3.16 transcript accumulation was standardized by reference to plant 5.8S RNA (see below). For induction kinetics that showed an unexpected bimodal pattern, the results were confirmed by repeating this procedure twice. The two replicates gave similar results.

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Figure 2. Kinetics of Pp-GH3.16 mRNA accumulation in Pinus pinaster roots treated (triangles) or not (squares) with 50 µm indole-3-acetic acid. Bars represent ± SE (P ≤ 0.05).

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image

Figure 6. Kinetics of Pp-GH3.16 transcript accumulation during the establishment of ectomycorrhizal symbiosis. (a) Pinus pinaster roots were inoculated either with the indole-3-acetic acid-overproducing mutant 331 of Hebeloma cylindrosporum (squares) or with the corresponding wild-type strain h1 (triangles). (b) Roots were inoculated with a wild-type dikaryon of Rhizopogon roseolus (triangles) or with the nonmycorrhizal mutant 552 of H. cylindrosporum (squares). For both (a) and (b) Pp-GH3.16 mRNAs were also quantified in uninoculated control roots (circles). Bars represent ± SE (P ≤ 0.05).

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Figure 3. Dose–response curve of indole-3-acetic acid-induced Pp-GH3.16 mRNA accumulation in Pinus pinaster roots. Roots were treated for 6 h with different auxin concentrations. Bars represent ± SE (P ≤ 0.05).

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image

Figure 4. Specificity of the induction of Pp-GH3.16 transcript accumulation. Pinus pinaster roots were incubated for 6 h in the presence of 50 µm indole-3-acetic acid; 50 µm 2,4-dichlorophenoxy acetic acid (2,4-D); 100 µm ethephon (for ethylene induction); 50 µm abscisic acid; 50 µm 6-benzylaminopurine (cytokinin); 50 µm gibberellic acid (gibberellin); or 10 µm quercetin. Heat shock was performed by transferring seedlings from the basal temperature (22 ± 1°C) to 35°C. For water stress, the osmotic potential of the nutrient solution was lowered to −0.45 MPa. Anoxia was induced by placing roots inside a nutrient agar medium. Control corresponds to untreated roots. Bars represent ± SE (P ≤ 0.05).

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Figure 5. Effect of cycloheximide on the accumulation of Pp-GH3.16 transcripts in Pinus pinaster roots. Roots were treated for 6 h with either 50 µm indole-3-acetic acid (IAA); 50 µm IAA plus 50 µm cycloheximide (IAA + CHX); or 50 µm cycloheximide alone (CHX). Control corresponds to untreated roots. Bars represent ± SE (P ≤ 0.05).

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Estimation of plant RNA in symbiotic tissues

To estimate the amount of plant RNA in samples extracted from inoculated root systems, 10 µg total RNA were dot-blotted onto nylon membranes and hybridized with the P. pinaster-(γ32P)ATP-radiolabelled 5.8S oligonucleotide probe (Pp5.8S 5′-TATAGAGCCGAGAACAATGC-3′). This oligo was shown not to cross-hybridize to the fungal 5.8S rRNA (not shown). Radioactivity was quantified using a gelDoc 1000 Bio-Rad radioimager.

Results

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

Cloning of Pp-GH3.16 and characteristics of the predicted polypeptide

The differential screening of a cDNA library obtained from pine roots treated with 50 µm IAA (Charvet et al., 2002a, 2002b) led to the identification of a 1845-bp open reading frame, designated Pp-GH3.16, predicted to encode a 615-aa residue polypeptide with a molecular mass of 69.9 kDa and an isoelectric point of 5.37. Most amino acid residues were hydrophilic, indicating that it is likely to be a soluble protein. The alignment of the deduced protein sequence to known sequences in databases showed strong homology to the GH3 proteins from Arabidopsis thaliana, Nicotiana tabacum and Glycine max throughout the entire sequence length (Fig. 1). Sequence identities ranged from 61 to 73% and sequence similarities from 75 to 85%. The alignment indicates some regions (e.g. aa residues 106–117, 541–547 or 558–564 in Pp-GH3.16) to be considerably conserved, which eventually suggests regions corresponding to functional domains.

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Figure 1. Alignment of the full-length amino acid sequence deduced from Pp-GH3.16 with those of GH3 homologues from Arabidopsis thaliana (DFL1, accession number AB050596.1; putative GH3, AY096516.1); Nicotiana tabacum (Nt-gh3, AF123503.1); and Glycine max (GH3, X60033.1). Numbers on left indicate position of amino acids in proteins. Gaps introduced to produce the best alignment are indicated by dashes. Identical amino acid residues are boxed. Grey boxes encompass functionally similar amino acid residues.

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Characterization of Pp-GH3-16 expression in pine roots

The kinetics of Pp-GH3.16 induction was studied using roots exposed to 50 µm IAA for 0–24 h (Fig. 2). Transcripts were detectable in pine roots at zero time (before IAA treatment), and their level remained fairly constant over the 24-h period in untreated roots. Pp-GH3.16 mRNAs increased rapidly in abundance during the first 6 h following auxin treatment, with a 3.2-fold accumulation over the control at 6 h. Transcript amount dropped at 9 h and subsequently increased, first rapidly until 12 h and then slowly until 24 h, when it was four times as high as the control. This bimodal kinetics of induction suggests that auxin could induce Pp-GH3.16 mRNA accumulation via two distinct mechanisms: the first being rapid but transient whereas the second is slower but stable over 24 h.

To determine the sensitivity of the Pp-GH3.16 response to auxin, pine roots were treated with various IAA concentrations. Transcript accumulation in roots was induced by auxin concentrations as low as 10−10 m (Fig. 3). Increasing auxin concentration from 10−10 to 10−6 m did not increase mRNA accumulation significantly. A maximal response was recorded for 10−5 m IAA, which increased transcript accumulation by 3.5-fold over the control. A higher concentration (10−4 m) induced mRNA accumulation much less efficiently.

The specificity of Pp-GH3.16 upregulation was first tested by treating roots with different phytohormones. As shown in Fig. 4, the auxin analogue 2,4-D enhanced transcript accumulation as efficiently, as did IAA. Other phytohormones tested (ethylene, ABA, BAP and GA3) did not induce Pp-GH3.16 mRNA accumulation. Different stresses were also tested: heat shock, anoxia, water stress and quercetin, a flavonoid involved in plant–microbe interactions. None of these treatments affected Pp-GH3.16 transcript accumulation.

The rapid Pp-GH3.16 transcript accumulation in response to auxin treatment (Fig. 2) suggests that Pp-GH3.16 upregulation could be a primary (direct) response to auxin. To investigate this hypothesis, we studied the effect of the protein-synthesis inhibitor cycloheximide (CHX). After a 6-h treatment, the mRNA level of Pp-GH3.16 was not significantly different in roots treated by 50 µm IAA alone and in roots treated by 50 µm IAA together with 50 µm CHX (Fig. 5). This indicates that CHX did not inhibit IAA-induced Pp-GH3.16 transcript accumulation, suggesting that Pp-GH3.16 upregulation is a primary (direct) response to auxin which does not require de novo protein synthesis. Cycloheximide alone was observed to induce Pp-GH3.16 transcript accumulation in pine roots, but at a lower level than IAA.

Expression of Pp-GH3-16 in P. pinaster roots on colonization by different ectomycorrhizal fungi

To understand better the potential implication of Pp-GH3.16 in the formation of ectomycorrhiza, we studied its expression during ectomycorrhizal symbiosis establishment using different fungal strains. As this gene was found upregulated by auxin, we compared transcript accumulation in pine roots inoculated by the IAA-overproducing mutant 331 of H. cylindrosporum and by the corresponding wild-type strain h1. This mutant strain was previously shown to be more infective than the wild type, and to form a hypertrophic Hartig net (Gea et al., 1994). Transcript accumulation in root systems was monitored for the first 10 d following inoculation. This corresponds to the early stages of symbiosis establishment that lead to the differentiation of a Hartig net in tap-root cortex (Tranvan et al., 2000). At 10 d, no dichotomously branched ectomycorrhiza could yet be seen on the tap root.

Except for a transient 14–30% increase at day 2, Pp-GH3.16 transcript level in noninoculated pine roots remained fairly constant during the course of the experiment (Fig. 6a,b). At any time, the transcript level was lower in roots inoculated with H. cylindrosporum than in controls (Fig. 6a). A transient accumulation of Pp-GH3.16 transcripts was also observed in roots inoculated with the wild-type strain. It could be detected at day 1, when hyphae reached the plant root, and lasted for 2 d. The level of transcripts subsequently decreased over time. In contrast to noninoculated roots or the presence of the wild-type strain, root inoculation with the mutant did not result in a short-term induction in Pp-GH3.16 expression and the transcript level decreased steadily over time (Fig. 6a). This trend was observed as early as 1 d after inoculation, as soon as fungal hyphae reached plant roots, and before any morphological or anatomical modification of the host root system had taken place (Tranvan et al., 2000). At 4 d after inoculation, at the beginning of the differentiation of the Hartig net, transcript accumulation was similar in roots inoculated with mutant and wild-type strains, representing approx. 60% of the amount of transcripts detectable in uninoculated control roots. At 10 d postinoculation, the amounts of transcript measured in roots inoculated with either fungal strain were 2.5 times less than those measured in uninoculated roots.

In an attempt to determine whether Pp-GH3.16 downregulation was specifically triggered by H. cylindrosporum, or whether it could be induced by any mycorrhizal fungus, we studied transcript accumulation in roots inoculated with R. roseolus, another ectomycorrhizal fungus commonly associated with P. pinaster. The dikaryotic strain of R. roseolus used in this work was shown to be slightly more infective than the monokaryotic h1 strain of H. cylindrosporum, but less so than the IAA-overproducing mutant (data not shown). It induced a decrease of Pp-GH3.16 transcript accumulation similar to that detected with both H. cylindrosporum strains (Fig. 6b). The transient accumulation 1 d after inoculation was not statistically significant and the decrease in transcript accumulation followed the kinetics detected with H. cylindrosporum. By 10 d after inoculation, Pp-GH3.16 mRNA accumulation in roots inoculated with R. roseolus was only 30% of that seen in the control. This indicates that Pp-GH3.16 downregulation on inoculation with ectomycorrhizal fungi is not species-specific and could be a response to mycorrhizal fungi in general.

We investigated whether Pp-GH3.16 downregulation was connected with symbiosis establishment, or could be attributed to the presence of the fungus in the rhizosphere. To address this question we studied transcript accumulation in root systems inoculated by a nonmycorrhizal mutant of H. cylindrosporum that is unable to colonize pine roots and to form a mantle and a Hartig net. Interestingly, Pp-GH3.16 transcript accumulation in P. pinaster roots was not affected by inoculation with the nonmycorrhizal H. cylindrosporum mutant 552 (Fig. 6b). The transcript level remained constant, and not significantly different from the control values, throughout the 10-d culture period. As this mutant shows a mycelial growth rate similar to that of the two other H. cylindrosporum strains used in this work (data not shown), the present results cannot be ascribed to a low proliferation of the mutant strain in contact with the inoculated roots. This suggests that Pp-GH3.16 downregulation is connected with ectomycorrhizal symbiosis establishment.

Discussion

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

Differential screening of a cDNA library from auxin-treated P. pinaster roots allowed the identification of a full-length cDNA, referred to as Pp-GH3.16, representing an auxin-upregulated transcript that is downregulated in P. pinaster roots in response to ectomycorrhizal symbiosis establishment. Deduced protein-sequence alignments showed that the corresponding gene is a GH3 homologue. Members of this family were first identified in soybean (Hagen et al., 1984) and subsequently in several plants, and are known to be auxin-inducible (reviewed by Hagen & Guilfoyle, 2002). The GH3 proteins are cytoplasmic but their function is not well established (Nakazawa et al., 2001; Takase et al., 2004; Staswick et al., 2005). GH3-related genes also exist in mouse, man and other vertebrates, but not in fungi (Hagen & Guilfoyle, 2002). To our knowledge, Pp-GH3.16 is the first GH3 homologue to be characterized in gymnosperms.

Pp-GH3.16 transcripts were detectable in roots in the absence of any hormonal treatment, suggesting that endogenous auxin level is sufficient to induce Pp-GH3.16 expression, or that auxin is not required for basal expression in roots. The former hypothesis is consistent with the fact that Pp-GH3.16 was upregulated by IAA concentrations as low as 10−10 m. The same has been reported in the case of soybean and tobacco (Hagen et al., 1984; 1991; Lund et al., 1997; Roux & Perrot-Rechenmann, 1997).

Transcript accumulation in IAA-treated roots was detectable 3 h after the start of the treatment and followed a bimodal distribution, peaking at 6 h followed by long-term induction for at least 24 h. So far, the kinetics of induction of GH3 homologues has been studied only in shoots. Results show either a transient induction (Hagen & Guilfoyle, 1985; Roux & Perrot-Rechenmann, 1997) or a steady increase in transcript accumulation over time for at least 24 h (Wright et al., 1987) or even 40 h (Hagen et al., 1991). Our results suggest that, in pine roots, IAA induces Pp-GH3.16 mRNA accumulation via two different mechanisms. This hypothesis is consistent with results of Liu et al. (1994), who identified at least three sequence elements within the soybean GH3 promoter that are auxin-inducible and can function independently of one another.

Cycloheximide did not inhibit IAA-induced Pp-GH3.16 transcript accumulation at 6 h, indicating that the cellular components required for induction to occur were present before auxin treatment. Therefore the peak of Pp-GH3.16 transcripts recorded at 6 h can be considered as a primary action of auxin characteristic for GH3 genes (Hagen & Guilfoyle, 1985; Franco et al., 1990; Roux & Perrot-Rechenmann, 1997). The increase in transcript level we detected from 9 h onwards could be a secondary response to auxin treatment. As reported previously by Roux & Perrot-Rechenmann (1997) for Nt-gh3, incubation with CHX, in the absence of IAA, resulted in increased Pp-GH3.16 transcript accumulation. CHX could induce Pp-GH3.16 by preventing the synthesis or altering the functionality of a short-lived repressor protein and/or by stabilizing IAA-regulated mRNAs.

In pine roots, Pp-GH3.16 mRNA accumulation was specifically induced by auxins. It was not induced by other phytohormones or quercetin, a nonhormonal compound thought to act as a molecular signal in plant–microbe symbioses. Heat shock, water stress and anoxia did not induce transcript accumulation either, and none of the treatments repressed Pp-GH3.16. Similar results were recorded by Hagen et al. (1984); Hagen & Guilfoyle (1985); Wright et al. (1987); Hagen et al. (1991); Roux & Perrot-Rechenmann (1997).

In addition to being auxin-inducible, Pp-GH3.16 was downregulated in pine roots following inoculation by the three mycorrhizal strains studied of H. cylindrosporum and R. roseolus. The fungal strains used in this work release IAA when grown in pure culture (Gay et al., 1994; unpublished results). The IAA-overproducing mutant releases approx. 0.9 × 10−7 mol IAA l−1 culture medium, whereas other strains release approx. 0.3 × 10−7 mol IAA l−1. According to Fig. 3, such concentrations do not significantly affect Pp-GH3.16 mRNA accumulation in pine roots. Although the amount of fungal IAA released locally on contact between fungal hyphae and plant roots is not known, we can reasonably speculate that the expected IAA concentration does not exceed 10−4 m. As such a concentration does not inhibit Pp-GH3.16 mRNA accumulation in pine roots, Pp-GH3.16 downregulation following symbiosis establishment may not be a consequence of fungal IAA production. This hypothesis is supported by the fact that the nonmycorrhizal mutant strain 552 of H. cylindrosporum (which releases IAA at the same level as did the symbiotic strains) did not affect Pp-GH3.16 expression in P. pinaster roots. From these results, it can be concluded that Pp-GH3.16 downregulation in mycorrhizal root systems is a component of the molecular cross-talk between symbiotic partners at the origin of symbiosis establishment. In this respect, it can also be considered as a molecular marker of successful differentiation of symbiotic structures. Considering that (i) this gene was not found downregulated by auxin; and (ii) except for auxins, none of the phytohormones we tested affected Pp-GH3.16 expression, we assume that the transduction pathway leading to Pp-GH3.16 downregulation in mycorrhizas does not involve a hormonal component. As neither the stresses tested nor quercetin affected Pp-GH3.16 expression, the signal(s) at the origin of the downregulation in mycorrhizas remain(s) to be elucidated. Besides auxin, the only signal reported to regulate GH3 genes is light (Hagen & Guilfoyle, 2002), which is unlikely to play a role in a root symbiosis.

Our results can be compared with those reported by Mathesius et al. (1998), who showed that clover-nodulating rhizobia induce a rapid, transient and local downregulation of a GH3::gusA chimeric cassette during nodule initiation followed by upregulation of the reporter gene at the site of nodule initiation. In contrast, non-nodulating Rhizobium strains did not affect GH3::gusA expression. Together with the results presented here, this suggests that plant GH3 homologues could play a general role in symbiotic interactions with microorganisms.

More recently, Nakazawa et al. (2001) and Takase et al. (2004) demonstrated experimentally that two Arabidopsis GH3 homologues, DFL1 and YDK1, negatively regulate lateral root formation in this plant. Indeed, plant lines defective in the expression of either of these two genes show altered lateral root formation and elongation. Considering this result, we hypothesize that the Pp-GH3.16 downregulation we observed in mycorrhizas could be, at least in part, at the origin of the typical hyperbranching pattern of P. pinaster ectomycorrhizas. Recently, Staswick et al. (2005) showed that several GH3 genes (including AtGH3-6 (DFL-1), which showed the highest homology to Pp-GH3.16) encode IAA-amido synthetases which conjugate excess IAA to amino acids. The demonstration of such activity in case of Pp-GH3.16 would help as to understand the role of this protein in differentiation of ectomycorrhiza.

Current results, together with those of Charvet-Candela et al. (2002a, 2002b) and Reddy et al. (2003), show that the very early molecular cross-talk between partners in ectomycorrhizal symbiosis involves auxin-regulated genes. The downregulation of Pp-GH3.16 on formation of mycorrhiza indicates that plant genes recruited for differentiation of symbiotic structures can have an unusual regulation in this particular situation. In this respect, studies of their regulation and function in symbiotic structures could assist in understanding their function in plants.

Acknowledgements

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

This work was supported by the Indo-French programme CEFIPRA (project 2003-2).

References

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