Differential gene expression during pre-symbiotic interaction between Tuber borchii Vittad. and Tilia americana L

RESEARCH ARTICLE

M. Menotta Æ A. Amicucci Æ D. Sisti

A. M. Gioacchini Æ V. Stocchi

Differential gene expression during pre-symbiotic interactionbetween Tuber borchii Vittad. and Tilia americana L.

Received: 2 March 2004 / Revised: 8 June 2004 / Accepted: 17 June 2004 / Published online: 16 July 2004� Springer-Verlag 2004

Abstract Ectomycorrhizal formation is a highly regu-lated process involving the molecular reorganization ofboth partners during symbiosis. An analogous molecu-lar process also occurs during the pre-symbiotic phase,when the partners exchange molecular signals in order toposition and prepare both organisms for the establish-ment of symbiosis. To gain insight into genetic reorga-nization in Tuber borchii during its interaction with itssymbiotic partner Tilia americana, we set up a culturesystem in which the mycelium interacts with the plant,even though there is no actual physical contact betweenthe two organisms. The selected strategies, suppressivesubtractive hybridisation and reverse Northern blots,allowed us to identify, for the first time, 58 cDNA clonesdifferentially expressed in the pre-symbiotic phase. Se-quence analysis of the expressed sequence tags showedthat the expressed genes are involved in several bio-chemical pathways: secretion and apical growth, cellulardetoxification, general metabolism and both mutualisticand symbiotic features.

Keywords Tuber borchii Æ Pre-infection phase ÆSuppressive subtractive hybridization Æ Expressedsequence tags

Introduction

Ectomycorrhizae are symbiotic associations of plantroots and filamentous fungi. Nutrients are exchangedbetween the two partners by new functional biochem-ical pathways in the ectomycorrhizal structure. Theplant provides organic carbon compounds to the fun-gus (Salzer and Hager 1991; Chen and Hampp 1993;Nehls et al. 1998), while the fungus, in turn, providesaccess to nitrogen, phosphorus and other nutrientsotherwise unavailable to the plant (Rousseau et al.1994; Brun et al. 1995; Perez-Moreno and Read 2000).Furthermore, there is evidence that mycorrhizal plantsare more likely to survive in unfavourable conditionsbecause the ectomycorrhizal association significantlyimproves resistance to plant pathogens (Duchesne et al.1989).

The development of a functional ectomycorrhiza re-quires a new genetic and biochemical procedure (Taguet al.1993; Martin et al. 2001; Sundaram et al. 2001;Voiblet et al. 2001). First, the fungus needs to grow to-wards the host roots. In a second stage, it must envelopthe radical apparatus of its host plant and, finally, itmust infect the fine roots, allowing development of thesymbiotic structure.

Several studies have been carried out on differentlyexpressed fungal genes during the infection stage and theectomycorrhizal phase (Burgess et al. 1995; Martin et al.1997; Laurent et al. 1999), with a particular focus oncarbon and nitrogen metabolisms (Salzer and Hager1991; Chen and Hampp 1993; Nehls et al. 1998, 1999).In contrast, little is known about fungal differential geneexpression during the pre-infection stages. The aim ofthe present study is to identify differentially regulatedgenes during the early interaction between plant andfungus in order to understand the molecular eventswhich lead to the formation of ectomycorrhizae in Tuberborchii Vittad.

Tub. borchii is an hypogeous ascomycetous fungusbelonging to the Pezizales order. It is capable of forming

Communicated by U. Kuck

M. Menotta (&) Æ A. Amicucci Æ V. StocchiIstituto di Chimica Biologica Giorgio Fornaini,Universita degli Studi di Urbino, Via Saffi 2,61029 PU Urbino, ItalyE-mail: [email protected]

D. SistiIstituto e Orto Botanico, Universita degli Studi di Urbino,Via Bramante 28, 21029 PU Urbino, Italy

A. M. GioacchiniIstituto di Ricerca sull’Attivita Motoria,Via Sasso, 61029 PU Urbino, Italy

Curr Genet (2004) 46: 158–165DOI 10.1007/s00294-004-0518-4

ectomycorrhizae on the fine roots of gymnosperms andangiosperms. Although Tub. borchii ascocarp is not aspopular as Tub. magnatum Pico, it is important com-mercially in Mediterranean areas.

An in vitro system in which the two symbionts (Tiliaamericana L., Tub. borchii) interact without direct con-tact was compared with the same in vitro system con-taining only the fungus; and 58 clones were identified,representing fungal genes differentially expressed in thepre-contact phase.

Most of these genes are involved in secretory andapical growth processes, while others seem to be in-volved in infection processes. There are also some in-volved in mitochondrial metabolism, gene expressionregulation and general metabolism.

Materials and methods

Biological material The biological material for theconstruction of the subtracted library consisted ofvegetative mycelia of Tub. borchii (strain 10 RA) grownon Nylon membranes laid on solid MS/2 medium(Murashige and Skoog 1962) at pH 6.5 and 24�C, with10 g/l of glucose as carbon source, for 30 days under16 h of light provided by cool white fluorescent lamps.These conditions were used for the Driver sample. TheTester sample was prepared as follows: Tub. borchiimycelia were grown under the above-mentioned con-ditions, but in the presence of the host plant Til.americana, from which they were separated by a Nylonmembrane.

Independent samples for reverse Northern blotexperiments were prepared according to the method ofSisti et al. (1998). Plantlets of Til. americana were firstmicropropagated and then inoculated with tissue blocksof Tub. borchii mycelium in tube cultures (4 cm diameter· 30 cm height) filled with 22 ml of peat-moss vermic-ulite (1:30 v/v) and embedded with 10 ml of MS/2medium, at pH 6.5 and 10 g/l of glucose (interactiontubes). All the cultures were maintained for 30 days(ectomycorrhizae take longer) in a culture room at24±1�C under 16 h of light provided by cool whitefluorescent lamps (3,500 lx). Tubes in which the myce-lium was permitted to grow alone were prepared ascontrol tubes.

Isolation of total RNA Total RNA for subtraction andreverse Northern experiments was extracted using aQiagen RNeasy kit according to the manufacturer’sinstructions. A DNase (Ambion) digestion step wasperformed before all subsequent reactions.

Subtracted library construction and clone selec-tion RNA obtained from the Tester and Driver cul-tures was used for the PCR select cDNA subtractionexperiment (Clontech) according to the manufacturer’sinstructions, with the exception of the ligation efficiencytest and the subtraction efficiency test, which were car-

ried out through amplification of the actin gene(AF462034) with specific Tub. borchii primers (forward5¢-GAGATGAGGCCCAATCCAAAC-3¢, reverse5¢-CCAGAATCCAAACGATACCGG-3¢). The result-ing fragments were inserted in pGEM vector II (Pro-mega) and subsequently cloned using Escherichia coliXL1-Blue. A total of 115 recombinant bacterial clones,obtained by blue–white selection on plates containingisopropyl-b-D-thiogalactopyranoside/X-Gal, were se-lected and sequenced by a private laboratory. A total of58 clones of reliable sequence were considered for fur-ther analysis, using the BLASTX program (NationalCenter for Biotechnology Information; NCBI). Cloneswere considered significantly similar to known proteinsfor E-values lower than 1·10�4.

Dot blotting and reverse Northern experiments cDNAinserts of purified plasmids from the subtracted librarywere amplified by PCR with 0.5 lM universal primersSP6 and T7, 200 lM each deoxynucleotide, 0.1 units ofTaq DNA polymerase and buffer supplied by the man-ufacturer (Qiagen). The PCR was performed using theApplied Biosystem GeneAmp 9700 PCR systemaccording to the following parameters: 94�C for 5 minand 30 cycles of 94�C for 0.5 min, 55�C for 0.5 min and72�C for 1 min. All PCR products were checked on1.2% agarose gel stained with ethidium bromide. Iden-tical quantities of the PCR products were blotted onseveral Hybond-N+ Nylon filters using blotting solu-tion (0.4 N NaOH) and the BioRad Bio-Dot apparatus.Further, serial dilutions of an internal standard (actingene) were added in each filter.

The RNA extracted from the tubes (three controltubes, three interaction tubes) was used for the synthesisof labelled cDNA for probes. The cDNA was obtainedusing oligo(dT) primers and Clontech PowerScript re-verse transcriptase according to the manufacturer’sinstructions. One microlitre of each cDNA was em-ployed for labelled probe synthesis by the Amershamrandom prime labelling system Rediprime II kit in thepresence of 30 lCi of (32P)dATP. The six replicatedmembranes were probed at 60�C for 12 h in hybridisa-tion solution (0.3 M sodium phosphate, pH 7.2, 1 mMEDTA, 1% bovine serum albumin, 7% SDS). Thewashing procedure was carried out twice in 2· SSC/0.1% SDS and once in 1· SSC/0.1% SDS.

Data acquisition and analysis Membranes were exposedby the Biorad BI imaging screen cassette and subse-quently analysed using the Biorad GS 250 molecularimager system. The raw images were imported with Gel-Pro Analyzer ver. 3.1 software and the intensity of eachspot was calculated using the ‘‘outline close to the dotoption’’. All values were normalised by constitutive ac-tin gene expression (serial dilution from well F1 to F3)and the results were imported with SPSS statisticalpackage ver. 10.1 software in order to perform theMann–Whitney U-test on all spots of control andinteraction samples.

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Morphological analysis After 30 co-culture days, Tub.borchii hyphae were gently removed from the membranesurface, fixed with a drop of FAA (40% formaldehyde/70% ethanol/acetic acid, 5:90:5), washed and mounted.Microscopic observation was performed with a LeicaDMLB light microscope equipped with a DC300F(charge-coupled device) camera.

Results

A high-quality subtracted library was obtained, asdemonstrated by subtraction efficiency experiments(Fig. 1) in which the subtracted product and a controlwere amplified with Tub. borchii actin specific primers.Up to 115 clones were selected from the subtracted li-brary and 58 genes were analysed.

Each sequence was queried against the NCBI proteindatabank using the BLASTX program. A total of26 clones showed significant similarity to known genes(E-value <1·10�4), 24 clones had no significanthomology with known genes (E-value >1·10�4) andeight clones represented unknown genes (Table 1). Onlytwo sequences had a high homology with the same gene(S31, S56).

The reverse Northern experiments allowed us toconfirm that all the selected clones are differentially ex-pressed during the pre-symbiotic interaction, as reportedby the Mann–Whitney U-test (P<0.05). In fact, all themedians of the calibrated intensities of the interactionblots were statistically different from the medians of thecalibrated intensities of the control blot (Figs. 2, 3).

The clones that showed the highest homology withknown proteins may be involved in: (1) cellular organelle

dynamics and cell wall construction [S102 (GAS-2homologue protein), S32 (centractin-like protein), S53/2(putative secreted protein), S97 (syntaxin binding pro-tein 1), S43 (Rho GDP dissociation inhibitor, GDI), S22(aspartic protease), S100 (putative GDP-mannose py-rophosphorylase)], (2) mitochondrial/microsomalmetabolism and cellular detoxification processes [S38(glyoxal oxidase), S41 (cytochrome P450), S71 (COIintron 9 protein), S67 (related to trichodiene oxygenasecytochrome P450), S76 (COX1-i1 protein)], (3) cellularsignaling [S35 (inorganic pyrophosphatase), S4 (nucleartransport factor 2), S29 (DNA-binding protein amdA)],or (4) cell cycle accomplishment and general metabolism[S87 (asparagine synthase), S103 (ribonucleotide reduc-tase), S93 (26S proteasome regulatory subunit mts4),S59 (histone H4), S56/2 (60S ribosomal protein L6), S28(probable mRNA maturase aI5-alpha), S27 (alpha-L-rhamnosidase A precursor), S42 (histidine-rich protein).Other clones analysed showed less similarity to knowngenes and hence it was not possible to confirm theirpossible role in the cell (S2, S11, S56, S31, S75, S26, S73,S37, S68, S79, S82, S96, S48/2, S44, S63, S23, S53, S24,S112, S3, S17, S74, S35/2, S48, S46, S61).

These results lead us to believe that a complex seriesof molecular mechanisms are activated in the very firststages of ectomycorrhizal formation (pre-infectionphase), before the plant and fungus actually make con-tact. We may gain insight into the interaction betweenTub. borchii and its host plant by looking at threeprincipal events taking place during this phase.

The first event is a more rapid mycelium growth as itgets close to the host roots (Fig. 4). In addition, theapical hyphal cell stops its apical growth in order todifferentiate a subapical hypha (Fig. 5). The expressionof genes involved in the secretory process may be in factcorrelated with the variation in the apical growth ofmycelia which occurs during this phase.

In particular, the S97 clone encodes for the proteinsec1, a molecule responsible for binding syntaxin andinvolved in vesicle membrane fusion (Brummer et al.2001; Peng and Gallwitz 2002). The Candida glabrataGAS-2 protein-like molecule, encoded by clone S102, isa homologue of GAS1p in Saccharomyces cerevisiae andis also a homologue of C. albicans PHR1 and PHR2(Weig et al. 2001). GAS1p, PHR1 and PHR2 are in-volved in cell wall assembly, in that they code a 1,3-b-glucanosil transferase (Mouyana et al. 2000). Moreover,the apical growth events are consistent with the expres-sion of clone S32, which is directly involved in cell nu-clear migration towards the apical tips of hyphae (Xiangand Morris 1999; Hirozumi et al. 1999).

The second metabolic process may be represented bythe enzymes involved in cellular detoxification processes,encoded by clones S41 and S81. In particular, S81encodes for a protein related to the fluconazole resis-tance protein FLU1 in Neurospora crassa. This proteinis an ABC transporter providing azole resistance to theorganisms expressing the gene (Calabrese et al. 2000).Evidence of the activation of detoxification processes

Fig. 1 Subtraction efficiency test with Tub. borchii actin specificprimers. Lane M Marker VIII (Boehringer), lanes 1, 3, 5unsubtracted control PCR product at 18, 23 and 28 cycles,respectively, lanes 2, 4, 6 subtracted PCR product at 18, 23 and28 cycles, respectively

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can also be found in the detection of azole molecules andother volatile organic compounds during the pre-infec-tion stage in the Til. americana–Tub. borchii mycorrhizalsynthesis system (Menotta et al. 2004). Clone S41

encodes for the protein cytochrome P450, which isprobably also involved in cellular detoxification pro-cesses (Lamb et al. 1999; Seo et al. 2000). In fact, thisprotein is strongly induced by benzothiazole, one of the

Table 1 Tub. borchii genes differentially expressed during the pre-symbiotic interaction between Tub. borchii Vittad. and Til. americanaL. The genes are sorted by E-values

Wellposition

cDNAname

Size(bp)

Best BLASTX database match Identity(%)

Similarity(%)

E-values

54 S103 834 Ribonucleotide reductase (Emericella nidulans) 66 74 5.00·10�6643 S32 455 Centractin-like protein (Pneumocystis carinii) 77 87 3.00·10�5953 S102 817 GAS-2 homologue (Candida glabrata) 61 75 9.00·10�4349 S93 589 26 s proteasome regulatory subunit mts4 (Schizosaccharomyces pombe) 51 68 2.00·10�4132 S59 417 Histone H4 (Neurospora crassa) 100 100 1.00·10�3952 S100 758 Putative GDP-mannose pyrophosphorylase (Candida albicans) 61 70 4.00·10�3516 S35 438 Inorganic pyrophosphatase (Pichia pastoris) 70 83 2.00·10�3345 S87 581 Asparagine synthase Asn2p (Saccharomyces cerevisiae) 70 84 2.00·10�3142 S76 395 COX1-i1 protein (Yarrowia lipolytica) 55 78 3.00·10�263 S4 338 Nuclear transport factor 2 (Aspergillus nidulans) 52 76 8.00·10�2531 S56/2 473 60S ribosomal protein L6, YL16-like (Saccharomyces cerevisiae) 69 84 7.00·10�2412 S28 728 Probable mRNA maturase aI5-alpha (Saccharomyces cerevisia) 41 58 9.00·10�2335 S67 519 Related to trichodiene oxygenase cytochrome P450 (Neurospora crassa) 41 62 2.00·10�197 S22 394 Aspartic protease (Aspergillus oryzae) 50 59 5.00·10�1919 S38 327 Glyoxal oxidase (glx1) [Arabidopsis thaliana] 46 66 2.00·10�1738 S71 370 COI intron 9 protein (Podospora anserina) 69 84 6.00·10�1720 S41 561 Cytochrome P450 (Fusarium sporotrichioides) 36 54 3.00·10�1647 S91 581 Hypothetical protein (Schizosaccharomyces pombe) 31 61 1.00·10�1527 S50 453 Hypothetical protein (Schizosaccharomyces pombe) 33 43 2.00·10�1329 S53/2 295 Putative secreted protein (Streptomyces coelicolor A3(2)) 40 54 3.00·10�1322 S43 643 Rho gdp dissociation inhibitor. (Schizosaccharomyces pombe) 39 54 5.00·10�1348 S97 577 Syntaxin binding protein 1, sec1 family (Schizosaccharomyces pombe) 41 63 3.00·10�1244 S81 326 Related to fluconazole resistance protein (FLU1) [Neurospora crassa] 33 51 2.00·10�913 S29 802 DNA-binding protein amdA (Emericella nidulans) 35 51 6.00·10�711 S27 471 Alpha-L-rhamnosidase A precursor (Aspergillus aculeatus) 40 58 1.00·10�621 S42 734 Histidine-rich protein (Plasmodium lophurae) 38 43 1.00·10�44 S11 377 Possible nuclear pore complex associated (Schizosaccharomyces pombe) 40 52 3.00·10�430 S56 418 RNA-dependent RNA polymerase (Ophiostoma mitovirus 3a) 48 65 3.00·10�414 S31 490 RNA-dependent RNA polymerase (Ophiostoma mitovirus 3a) 48 65 6.00·10�41 S2 453 CG9682 gene product (Drosophila melanogaster) 28 43 1.00·10�341 S75 582 Hypothetical protein (Schizosaccharomyces pombe) 30 40 2.00·10�310 S26 586 Putative integral membrane protein (Streptomyces coelicolor A3(2)) 53 66 1.10·10�239 S73 437 Alpha/beta-gliadin precursor (Triticum aestivum) 48 59 1.50·10�218 S37 810 Filament-associated late protein FALPE (Amsacta moorei entomopoxvirus) 44 63 1.60·10�236 S68 453 CG9682-PA (Drosophila melanogaster) 28 34 1.60·10�250 S96 564 Hypothetical protein XP_146970 (Mus musculus) 41 54 4.58·10�226 S48/2 473 Actin cytoskeleton-assiociated (Schizosaccharomyces pombe) 47 58 5.50·10�223 S44 435 Putative Myb-related transcription activator protein (Arabidopsis thaliana) 32 51 1.70·10�134 S63 309 Cold-inducible RNA binding protein (Xenopus laevis) 41 49 2.11·10�18 S23 587 Nar1p (Saccharomyces cerevisiae) 35 50 2.70·10�128 S53 685 Myosin-IA (Acanthamoeba castellanii) 42 44 3.76·10�19 S24 368 Intercellular adhesion molecule 1 (Rattus norvegicus) 38 59 5.90·10�158 S112 546 Envelope glycoprotein (Human immunodeficiency virus type 1) 29 60 8.80·10�12 S3 486 Unnamed protein product (Homo sapiens) 29 42 1.096 S17 279 Hypothetical protein (Cytophaga hutchinsonii) 23 49 1.5040 S74 268 Hypothetical protein XP_119113 (Homo sapiens) 31 46 2.0017 S35/2 283 Hypothetical protein XP_096386 (Homo sapiens) 38 53 2.5025 S48 361 CG15406-PA (Drosophila melanogaster) 24 44 3.0024 S46 425 Hypothetical protein (Chlamydia trachomatis) 35 55 3.5033 S61 412 Hypothetical protein (Plasmodium falciparum 3D7) 31 46 8.0055 S104 347 Unknown 0 056 S106 191 Unknown 0 057 S111 243 Unknown 0 05 S12 317 Unknown 0 015 S34 348 Unknown 0 037 S70 484 Unknown 0 046 S84 223 Unknown 0 051 S94 223 Unknown 0 0

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volatile organic compounds previously detected (Men-otta et al. 2004; Suwanchaichinda and Brattsten 2002).

Finally, a part of the identified clones includes genesinvolved in the general metabolism of the mycelium,

such as S59, S71, S76, S87, S103 and S27. In particular,S76 (which encodes for a protein with similarities to aCOX1 protein) and S71 (which encodes for a COI intron9 protein), may be involved in the protein turnover of

Fig. 2a, b Representativehybridisation results of reverseNorthern experiments.Hybridisations were carried outusing labelled cDNA obtained afrom control tubes RNA and bfrom interaction tubes RNA

Fig. 3 Expression levelscalculated by reverse Northernexperiments. The values areexpressed in arbitrary units. Allcontrols (V driver) arestatistically different from Vtester (U-test, P<0.05). Thesamples are numeratedsequentially from Dot A1 toDot E10. A serial dilution ofactin gene (from F1 to F3) isused as an internal standard forsignal normalisation

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mitochondria, suggesting an increase in mitochondrialactivity in this stage. Their expression or over-expressionduring the early interaction of Tub. borchii with Til.americana strongly suggests that mycelial metabolismincreases in the presence of the plant, probably as aresponse activated by mitogenetic processes.

Discussion

Many studies have been carried out during the past fewyears concerning the molecular changes which take placein Tub. borchii ectomycorrhizae (Polidori et al. 2002) orin other ectomycorrhizal species (Tagu et al. 1993;Martin et al. 2001, Voiblet et al. 2001). In contrast,limited information is available regarding early interac-tions in other ectomycorrhizal species (Podila et al.2002) and very little is known about the biochemicalprocesses that are at work during the pre-infection stagein Tub. borchii just before the establishment of symbio-sis. In the current study, 58 genes differentially expressed

at this stage were detected in Tub. borchii and cultivatedwithout contact with its symbiotic plant partner,Til. americana.

The reverse Northern experiments showed that genesselected by suppressive subtractive hybridization areactually expressed independently from the culture sys-tem tested. In fact, both the mycelia grown in ectomy-corrhizal synthesis tubes and the mycelia grown onNylon (see Materials and methods) suggest that the se-lected genes are not affected by the culture system.Furthermore, the pre-symbiotic interaction model pro-vided to be a useful tool for studies of the pre-symbioticphase of these organisms.

This evidence leads us to believe that a complex seriesof molecular mechanisms switches on during the veryfirst stage of ectomycorrhizal formation, before the plantand fungus make contact. In particular, for the firsttime, we highlight the expression of several genes thatmay be involved in the apical growth of Tub. borchiitowards the roots of its symbiotic plant, as shown bymorphological analysis.

Fig. 4 Culture system used inthe present study. The enlargedimage shows Tub. borchiimycelia growing around Til.roots

Fig. 5 Representativemicroscopic images of a Driversample and b Tester sample. Inb, numerous subapical hyphalgrowth events are visible

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Proteins such as 60S ribosomal protein and cyto-chrome P450 (corresponding to clones S56/2 and S41,respectively) have been shown to be directly or indirectlyinvolved in fungus–plant signaling in arbuscolar sym-bioses (Gianinazzi-Pearson et al. 2002). Furthermore, animproved mitochondrial metabolism was recently dem-onstrated in Gigaspora rosea (Tamasloukht et al. 2003).The results reported in this paper are consistent withthose findings.

This evidence suggests that there may be a commonexpression mechanism in a wide range of species duringthe pre-symbiotic plant–fungus interaction, both inendo- and in ectomycorrhizal species.

In our findings, we have highlighted the expression ofgenes having a high homology with genes involved in thepathogenic and saprophytic interaction of fungi. In fact,clone S38 encodes for a glyoxal oxidase, an enzyme thatis necessary for the oxidation activity of ligninolyticperoxidases in Phanerochaete chysosporium (Kerstenet al. 1995; Cullen 1997). The ability to degrade ligninmight be a necessary step for Tuber infection. Glyoxaloxidase is also expressed by the pathogenic fungi Fusa-rium oxysporum and Trichoderma atroviride. Finally,clone S102 encodes for a C. glabrata GAS-2 protein-likemolecule. This protein has been shown to be necessaryfor virulence during the infection of host tissue (Weiget al. 2001; De Bernardis et al. 1998; Muhlschlegel andFonzi 1997; Saporito-Irwin et al. 1995; Ghannoum et al.1995).

In conclusion, the present study represents a first steptowards gaining a better understanding of the molecularmechanisms at work in the initial phases of symbiosis inTuber; and it attempts to highlight the early signal ex-changes that occur between the two symbionts prior toactual contact. Several genes involved in various bio-chemical mechanisms, not yet cloned in the genus Tuber,were isolated. Even if further studies are required, inlight of the results obtained herein and those reported inliterature cited above, it can be asserted that, in the pre-contact phase, the fungus switches on a series of mech-anisms, such as those to recognise and attack plant rootsand defend the fungus from substances secreted by theplant. All of those mechanisms are accompanied byhighly regulated metabolic modifications.

Studies on the molecular mechanisms at work in thepre-infection phase in Tub. borchii–Til. americana are animportant part of research on symbiosis. Most studieshave mainly focused on endo-mycorrhizal fungi andectomycorrhizal basidiomycete fungi, whereas the pres-ent study concerns an ascomycete fungus.

Moreover, since some of the mechanisms hypothes-ised here are similar to those used by pathogenic ormycorrhizal fungi during the infection of host-planttissue, there may be a common ancestral infection pro-cess that subsequently evolved into different mechanismsof interaction: symbiotic, pathogenic and all the inter-mediate stages. Considering recent advances in the studyof fungal behaviour in plant–fungus interactions (Hallet al. 2003), we could hypothesise a more complicated

relationship. In fact, many ‘‘ectomycorrhizal fungi’’ suchas Tricholoma matsutake can switch from a symbioticstage to a parasitic one, depending on host-plant species,general health of the host plant and pedo-climatic con-ditions.

References

Brummer MH, Kivinen KJ, Jantti J, Toikkanen J, Soderlund H,Keranen S (2001) Characterization of the sec1-1 and sec1-11mutations. Yeast 18:1525–1536

Brun A, Chalot M, Finlay RD, Soderstorm B (1995) Structure andfunction of the ectomycorrhizal association between Paxillusinvolutus (Batsch) Fr. & Betula pendula Roth. I. Dynamics ofmycorrhizae formation. New Phytol 129:487–493

Burgess T, Laurent P, Dell B, Malajczuk N, Martin F (1995) Effectof the fungal isolate aggressivity on the biosynthesis of symbi-osis—related polypeptides in differentiating eucalypt ectomy-corrhiza. Planta 195:408–417

Calabrese D, Bille J, Sanglard D (2000) A novel multidrug effluxtransporter gene of the major facilitator superfamily fromCandida albicans (FLU1) conferring resistance to fluconazole.Microbiology 146:2743–2754

Chen XY, Hampp R (1993) Sugar uptake by protoplast of theectomycorrhizal fungus Amanita muscaria (L Ex Fr) Hooker.New Phytol 125:1883–1887

Cullen D (1997) Recent advances on the molecular genetics ofligninolytic fungi. J Biotechnol 53:273–289

De Bernardis F, Muhlschlegel FA, Cassone A, Fonzi WA (1998)The pH of the host niche controls gene expression and virulenceof Candida albicans. Infect Immun 66:3317–3325

Duchesne LC, Peterson RL, Ellis BE (1989) The time course ofdisease suppression and antibiosis by the ectomycorrhizal fun-gus Paxillus involutus. New Phytol 111:693–698

Ghannoum MA, Spellberg B, Saporito-Irwin SM, Fonzi WA(1995) Reduced virulence of Candida albicans PHR1 mutants.Infect Immun 63:4528–4530

Gianinazzi-Pearson V, Brechenmacher L, Tamasloukht M, BecardG, La Camera S, Gianinazzi S, Franken P (2002) Functionalgenomics of fungal–plant communication in the arbuscolarmycorrhizal symbiosis. Int Mycol Congr 7:11–17

Hall RI, Yun W, Amicucci A (2003) Cultivation of edible ecto-mycorrhizal mushrooms. Trends Biotechnol 21:433–438

Hirozumi K, Nakajima H, Machida M, Yamaguchi M, Takeo K,Kitamoto K (1999) Cloning and characterization of a gene(arpA) from Aspergillus oryzae encoding an actin-related pro-tein required for normal nuclear distribution and morphologyof conidiophores. Mol Gen Genet 262:758–767

Kersten PJ, Witek C, Wymelenberg AV, Cullen D (1995) Phan-erochaete chrysosporium glyoxal oxidase is encoded by twoallelic variants: structure, genomic organization, and heterolo-gous expression of glx1 and glx2. J Bacteriol 177:6106–6110

Kim SJ, Zheng J, Hiremath ST, Podila GK (1998) Cloning andcharacterization of a symbiosis-related gene from an ectomy-corrhizal fungus Laccaria bicolour. Gene 222:203–212

Lamb D, Kelly D, Kelly S (1999) Molecular aspects of azoleantifungal action and resistance. Drug Resist Updates 2:390–402

Laurent P, Voiblet C, Tagu D, de Carvalho D, Nehls U, De BellisR, Balestrini R, Bauw G, Bonfante P, Martin F (1999) A novelclass of ectomycorrhiza-regulated cell wall polypeptides inPisolithus tinctorius. Mol Plant Microbe Interact 12:862–871

Martin F, Lapeyrie F, Tagu D (1997) Altered gene expressionduring ectomycorrhiza development in the mycota. In: LemkeP, Caroll G (eds) Plant Relationship, vol VI. Springer, BerlinHeidelberg New York, pp 223–242

Martin F, Duplessis S, Dintengou F, Lagrange H, Voiblet C, La-peyrie F (2001) Development of cross talking in the ectomy-corrhizal symbiosis: signals and communication genes. NewPhytol 151:145–154

164

Menotta M, Gioacchini AM, Buffalini M, Amicucci A, Sisti D,Stocchi V (2004) Headspace solid-phase microextraction withgas chromatography and mass spectrometry in the investigationof volatile organic compounds in an ectomycorrhizae synthesissystem. Rapid Commun Mass Spectrom 18:206–210

Mouyana I, Fontaine T, Vai M, Monod M, Fonzi WA, DiaquinM, Popolo L, Hartland RP, Latge JP (2000) Glycosylphos-phatidylinoisotl-anchored gluconyltransferases play an activerole in the biosynthesis of the fungal cell wall. J Biol Chem275:14882–14889

Muhlschlegel FA, Fonzi WA (1997) PHR2 of Candida albicansencodes a functional homolog of the pH-regulated gene PHR1with an inverted pattern of pH-dependent expression. Mol CellBiol 17:5960–5967

Murashige T, Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol Plant15:473–497

Nehls U, Weise J, Guttenberger M, Hampp R (1998) Carbonallocation in ectomycorrhizae: identification and expressionanalysis of an Amanita muscaria monosaccharide transporter.Mol Plant Microbe Interact 11:167–176

Nehls U, Ecke M, Hampp R (1999) Sugar and nitrogen-dependentregulation of an Amanita muscaria phenylalanine ammoniumlyase gene. J Bacteriol 181:1931–1933

Peng R, Gallwitz D (2002) Sly1 protein bound to Golgi syntaxinSed5p allows assembly and contributes to specificity of SNAREfusion complexes. J Cell Biol 157:645–655

Perez-Moreno J, Read DJ (2000) Mobilization and transfer ofnutrients from litter to tree seedling via the vegetative myceliumof ectomycorrhizal plants. New Phytol 145:301–309

Podila GK, Zheng S, Balasubramanian S, Sundaram S, HiremathS, Brand JH, Hymes MJ (2002) Fungal gene expression in earlysymbiotic interaction between Laccaria bicolor and red pine.Plant Soil 244:117–128

Polidori E, Agostini D, Zeppa S, Potenza L, Palma F, Sisti D,Stocchi V (2002) Identification of differentially expressed cDNAclones in Tilia Platyphyllos–Tuber borchii ectomycorrhizaeusing differential screening approach. Mol Genet Genomics266:858–864

Rousseau JVD, Sylvia DM, Fox AJ (1994) Contribution of ecto-mycorrhiza to the potential nutrient adsorbing surface of pine.New Phytol 128:639–644

Salzer P, Hager A (1991) Sucrose utilization of ectomycorrhizalfungi Amanita muscaria and Hebeloma crustuliniforme dependson the cell wall-bound invertase activity of their host Piceaabies. Bot Acta 104:439–444

Saporito-Irwin SM, Birse CE, Sypherd PS, Fonzi WA (1995)PHR1, a pH-regulated gene of Candida albicans, is required formorphogenesis. Mol Cell Biol 15:601–613

Seo KW, Park M, Kim JG, Kim TW, Kim HJ (2000) Effects ofbenzothiazole on the xenobiotic metabolizing enzymes andmetabolism of acetaminophen. J Appl Toxicol 20:427–430

Sundaram S, Kim SJ, Suzuki H, Mcquattie CJ, Hiremath ST,Podila GK (2001) Isolation and characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria bicolor.Mol Plant Microbe Interact 14:628

Suwanchaichinda C, Brattsten LB (2002) Induction of microsomalcytochrome P450s by tire-leachate compounds, habitat com-ponents of Aedes albopictus mosquito larvae. Arch Insect Bio-chem Physiol 49:71–79

Tagu D, Phyton M, Cretin C, Martin F (1993) Cloning symbiosisrelated cDNAs from eucalypt ectomycorrhizal by PCR assisteddifferential screening. New Phytol 125:339–343

Voiblet C, Duplessis S, Encelot N, Martin F (2001) Identificationof symbiosis regulated genes in Eucaliptus globus–Pisolithustinctorius ectomycorrhizal by differential hybridization ofarrayed cDNA. Plant J 25:181–191

Weig M, Haynes K, Rogers TR, Kurzai O, Frosch M, Muhl-schlegel FA (2001) A GAS-like gene family in the pathogenicfungus Candida glabrata. Microbiology 147:2007–2019

Xiang X, Morris NR (1999) Hyphal tip growth and nuclearmigration. Curr Opin Microbiol 2:636–640

Sisti D, Zambonelli A, Giomaro G, Rossi I, Ceccaroli P, Citterio B,Benedetti PA, Stocchi V (1998) In vitro mycorrhizal synthesis ofmicropropagated Tilia platyphyllos Scop. plantlets with Tuberborchii Vittad. mycelium in pure culture. Acta Hortic 457:379–387

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