Curr Genet (2002) 42: 161–168 DOI 10.1007/s00294-002-0343-6 R ES E AR C H A RT I C L E Sabrina Zeppa Æ Chiara Guidi Æ Alessandra Zambonelli Lucia Potenza Æ Luciana Vallorani Æ Raffaella Pierleoni Cinzia Sacconi Æ Vilberto Stocchi Identification of putative genes involved in the development of Tuber borchii fruit body by mRNA differential display in agarose gel Received: 26 July 2002 / Revised: 27 September 2002 / Accepted: 3 October 2002 / Published online: 26 November 2002
Springer-Verlag 2002 Abstract In order to analyse gene expression during fruit body development of the ectomychorrizal fungus Tuber borchii Vittad., a modified differential display procedure was set up. The procedure used is easier and faster than the traditional one and generates reproducible cDNA banding patterns that can be resolved on a standard ethidium bromide-agarose gel. From 16 cDNA fingerprints, 25 amplicons with apparent differential expression were identified and cloned without a previous reamplification. Fifteen clones showed significant similarity to known proteins that are involved in dikaryosis and fruiting, cell division, transport across membranes, mitochondrial division, intermediary metabolism, biosynthesis of isoprenoid compounds and putative RNA/DNA binding. Northern blot analyses confirmed that seven cDNAs were indeed differentially expressed during fruit body development. The characterisation of these cDNAs represents a starting point in understanding the molecular mechanisms of cellular differentiation leading to the development of the T. borchii fruit body. Keywords Ectomycorrhizal fungi Æ Fruit body development Æ mRNA differential display Æ Gene expression analysis Sabrina Zeppa and Chiara Guidi contributed equally to this work Communicated by U. Kück S. Zeppa Æ C. Guidi Æ L. Potenza Æ L. Vallorani Æ R. Pierleoni C. Sacconi Æ V. Stocchi (&) Istituto di Chimica Biologica ‘‘Giorgio Fornaini’’, Università degli Studi di Urbino, Via Saffi 2, 61029 Urbino (PU), Italy E-mail: v.stocchi@uniurb.it A. Zambonelli Dipartimento di Protezione e Valorizzazione Agroalimentare, Università di Bologna, Via Filippo Re 8, 40126 Bologna, Italy Introduction Ascomycetes are considered as ‘‘higher fungi’’ along with Basidiomycetes owing to their structural complexity. The Ascomycetes include truffles, well known for their high organoleptic properties and the economic value of their fruit bodies. The truffle life cycle, like that of other symbiotic filamentous fungi, begins with a limited extraradical phase of vegetative growth in which the hyphae proliferate before coming into contact with the roots of the host plant. After this contact the symbiotic phase begins, leading to the development of the ectomycorrhiza, a new organ that is functionally and morphologically distinct from the two individual partners. In the final stage, the mycelium is organised into the fruit body, the role of which is to produce sexual fructifications, later dispersed in the environment. The developing fruit body is a distinct phase of the Tuber life cycle, characterised by the aggregation of different types of hyphae: vegetative hyphal cells and highly specialised reproductive hyphae (asci). The development of ascospores within asci involves features of cell differentiation that are unique within the Kingdom Fungi (Read and Beckett 1996). In the subdivision Ascomycotina, sexual reproduction results in the formation of endogenous ascospores within asci. In Tuber immature fruit bodies the presence of antheridia and oogonia has been demonstrated (Marchisio 1964; Paguey-Leduc et al. 1990), suggesting that sexual reproduction also occurs in Tuberales. Ascospores are typically formed immediately after karyogamy and meiosis and their formation involves cytoplasmic compartmentalisation by double delimiting membranes between which the wall of the ascospores develops (Read and Beckett 1996). Ascospore liberation from mature asci may be either an active or, as in the truffles, a passive process. Recently, biochemical and molecular biological techniques have been applied to the study of Tuber, including the molecular identification of truffle species (Amicucci et al. 1998; Bertini et al. 1998), the 162 characterisation of Tuber borchii mycelial strains and bacteria associated with the life cycle of the fungus (Saltarelli et al. 1998; Rossi et al. 1999; Barbieri et al. 2000), and the isolation of differentially expressed genes (Zeppa et al. 2001; Polidori et al. 2002). Even though developmentally regulated genes have been isolated (De Bellis et al. 1998; Balestrini et al. 2000; Zeppa et al. 2001), aspects of the molecular biology underlying fruit body development remain unclear. In the present study mRNA differential display in agarose gel was used to compare mRNA populations from the unripe and ripe fruit body of the white truffle T. borchii in an attempt to identify genes involved in fruit body development. T. borchii was chosen as the experimental organism because it is one of the few Tuber species that can be grown in vitro for plant mycorrhizal synthesis (Sisti et al. 1998). The characterisation of differentially expressed genes is the main step in understanding the molecular basis of a biological system. Several technical approaches, such as subtractive hybridisation, have been proposed to compare different mRNA populations. Liang and Pardee (1992) proposed the use of mRNA differential display as an alternative to conventional methods for analysing changes in gene expression. In the last decades, this technique has been preferred for the study of different organisms, from plants and fungi to mammals, and in different research areas, such as tumours, diabetes, heart disease and microorganism interactions (Benito et al. 1996; Robinson et al. 1997). Since the birth of the differential RNA display technique, several modifications have been made in order to simplify and optimise this powerful tool. The requirement for radioactive polymerase chain reaction (PCR) products has limited the use of this technique to laboratories with the appropriate equipment. This limitation has been overcome with the use of digoxigenin-conjugated or biotinylated oligo(dT) primers and colorimetric or chemiluminescence-based detection of PCR products (An et al. 1996; Chen and Peck 1996). Despite the use of non-radioactive PCR, the laborious preparation of polyacrylamide gels and band detection cannot be avoided. In a previous study, mRNA differential display in agarose gel has been set up in order to detect differences in mRNA populations appearing after infection of plant leaves (Dioscorea bulbifera) with pathogens (Colletrotrichum gloeosporioides) (Rompf and Kahl 1997). Introducing some modifications to this protocol, we have set up a reliable method for identifying mRNAs differentially expressed during T. borchii fruit body development. Materials and methods Mycelial strains and fruit bodies A mycelial strain designated 1BO (ATCC 96540) was isolated from a T. borchii fruit body harvested in a Pinus pinea and Pinus pinaster forest located near Cervia (Ravenna, Central Italy). A dried voucher specimen and mycelium are preserved in the Dipartimento di Protezione e Valorizzazione Agroalimentare of the University of Bologna, Italy. The isolates were grown in the dark at 24C, with no agitation, in modified Melin-Norkrans nutrient solution (MMN), pH 6.6 (Molina 1979) in 100 ml flasks, each containing 70 ml of medium inoculated with fungus cultured in potato-dextrose-agar plugs. T. borchii fruit bodies were collected in Northern and Central Italy and harvested in an experimental truffle orchard located near Marina di Ravenna, Italy (Zambonelli et al. 2000). Fruit bodies at each stage of maturation were collected at the same time. The degree of maturation of the ascocarps was defined using the following categorised stages on the basis of the percentage of asci containing mature spores: stage 0, 0%; stage 1, 5%; stage 2, 6–25%; stage 3, 26–50%; stage 4, 51–75%; and stage 5, 76–100%. The maturation stage of the spores was defined morphologically: the mature spores were yellow-reddish brown in colour with reticulate ornamentation. The ascocarps were confirmed as belonging to the T. borchii species by morphological and PCR-based techniques (Pegler et al. 1993; Bertini et al. 1998). RNA isolation Total cellular RNA was isolated from 1 month old T. borchii mycelia and unripe and ripe fruit bodies using an RNeasy Plantmini kit from Qiagen, following the manufacturer’s instructions. The purification and yields of RNA (from fruit body samples) were improved by phenol-chloroform extraction before processing of samples. The final concentration and quality of RNA samples were estimated either spectrophotometrically or by agarose gel electrophoresis with ethidium bromide staining. Total RNA was treated using a Dnase I, DNA-free kit (Ambion), according to the manufacturer’s instructions. Differential display Total RNA (500 ng) was reverse-transcribed in a 20 ll reaction volume using 200 U PowerScript Reverse Transcriptase (Clontech) and 10 lM of four different anchored primers T12VG, T12VC, T12VA and TV12T (V=A/G/C). The cDNA preparation was stored in 40 ll of TE buffer (10 mM TRIS, pH 7.6, 1 mM EDTA). Differential display PCR was carried out in 40 ll containing 3 ll of cDNA, 0.25 lM of the appropriate anchored primer, 0.125 lM of one of the random 13-mer oligonucleotides (CZ1, 5¢-GCTCTACCACCTT-3¢; CZ2, 5¢-CCTGAGCCCGCCG3¢; CZ3, 5¢-CATGATGGTCAA-3¢; and CZ4, 5¢-CACCCTCATC GTC-3¢), 100 lM dNTPs, 1.25 mM MgCl2, 1·PCR buffer (Takara Biomedicals) and 2 U Taq polymerase (Takara Biomedicals). The amplification was performed in a PCR System 2400 Thermal Cycler (Perkin Elmer) as follows: an initial denaturation at 94C for 30 s, 40 cycles at 94C for 30 s, 42C for 40 s and 72C for 60 s followed by a 7 min extension at 72C. The total RNA was routinely checked for contamination by chromosomal DNA using parallel PCRs that were programmed with an aliquot of RNA that had not been reverse-transcribed prior to amplification. The PCR samples were electrophoresed on a 1.8% agarose gel and stained with ethidium bromide. Bands of interest were cut from the gels, purified using the QIAquick Gel Extraction Kit (Qiagen) and checked by agarose gel electrophoresis. Cloning and screening for inserts by colony-PCR The differential display fragments were cloned using the pGEM-T Vector System (Promega), according to the manufacturer’s instructions. Colonies containing vectors with cloned inserts were screened using the colony-PCR technique (Zeppa et al. 2000). Amplification products were visualised by electrophoresis in a 1.5% agarose gel stained with ethidium bromide. 163 Restriction analyses of the colony-amplification products To check whether the composition of the amplification products was homogeneous, 10 colonies from each transformation were digested with EcoRI, HindIII and PstI (Promega). The reactions were performed on unpurified PCR products (15 ll) using 4 U of restriction enzyme, according to the manufacturer’s instructions. The restriction fragments were size-fractionated by 2.5% agarose gel electrophoresis. Identical restriction patterns were obtained for 86.4% of the transformations. Northern blotting analyses For Northern blots, 15 lg total RNA from T. borchii mycelium, unripe and ripe fruit bodies were loaded on a formaldehyde 1.2% agarose gel (Sambrook et al. 1989) and electrophoresed. RNA was blotted onto version 2.0 Hybond-N+ positively charged nylon membranes (Amersham Life Science) in accordance with the manufacturer’s instructions. The quantity and quality of the blotted RNA were checked by staining the membranes with 0.02% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.5 for approximately 3 min before destaining with distilled water. The filters were hybridised in phosphate buffer (Sambrook et al. 1989) with the fragments obtained by differential display, 32Plabeled using the RediPrime labelling kit (Amersham Life Science). Final wash conditions were 15 mM NaCl, 1.5 mM trisodium citrate (0.1·SSC) and 0.1% SDS at 65C. As a control for loading, membranes were rehybridised with a fragment of the T. borchii 18S gene, obtained using the 18S1 and 18S2 primers (Balestrini et al. 2000). Each hybridisation signal was detected and quantified using the Quantity One program (BioRad). Sequencing of the isolated clones The cloned inserts were sequenced, in both directions, using the ABI PRISM BigDye terminator cycle-sequencing-ready reaction kit (Perkin-Elmer), according to the manufacturer’s instructions. The sequence was obtained using the ABI PRISM 310 Genetic Analyzer (PE Applied Biosystem). The differential display reverse transcribed (DDRT) cDNA fragments were deposited in GenBank under the accession numbers indicated in Table 1. Results mRNA differential display Total RNA isolated from completely immature fruit bodies with 0% of asci containing mature spores (stage Table 1 Characteristics of the cDNA clones with similarity to known proteins present in the NCBI data bank Clone Accession cDNA number length (bp) Best database matcha Identity Similarity E (%) (%) value M Differentially expressed clones: VC12 AF487329 718 Mating type protein 1 of Alternaria brassicicola 26 (AAK85542) VT16 AF487320 930 3-Hydroxy-3-methylglutaryl-coenzyme A reductase 32 of Gibberella fujikuroi (Q12577) VT3 AF487327 619 Involved in mitochondrial division Fis1p 50 of Saccharomyces cerevisiae (NP_012199) 56 VT19 AY082799 873 Putative protein G6G8.8 of Neurospora crassa belonging to 3-phosphoserine phosphatase family protein (AAK07846) VT15 AF487319 599 Dihydrolipoamide acetyltransferase 42 of N. crassa (P20285) VC13 AF487316 364 SMT3 protein of S. cerevisiae (NP_010798) 56 VT13 AF487318 521 Serine-rich protein of Schizosaccharomyces 28 pombe (CAA16838) Not differentially expressed or not detectable clones VT1 AF487328 609 Hypothetical protein SPBC1709.14 of S. pombeb (T39642) VT4 AF487315 549 Septin of Aspergillus nidulans (AAK21000) VT14 AF487317 633 Putative helicase of Oryza sativa (AAK54302) VT18 AF487321 650 Hypothetical protein B14D6.440 of N. crassac (T49492) VA5 AF487323 592 Putative sodium P-type ATPase of N. crassa (CAB65298) VA6 AF487325 619 Probable transport protein of S. pombe (T40399) VA11 AF487324 552 Putative sulphate transporter of S. cerevisiae (P53394) VC7 AF487322 1099 Putative methylenetetrahydrofolate reductase of S. cerevisiae (NP_011390) a Homology searches were performed using the BLASTX program on the NCBI server b Belonging to transglutaminase/protease-like superfamily c Belonging to Na+/Ca2+ exchanger protein family d The values were determined from Northern analyses; the transcript levels of all cDNAs were normalised against 18S rRNA and Relative mRNA levelsd 42 0.059 50 1e–25 70 - UF RF 1 - 1.5 1 32e 9e–11 1 2 1.3 69 4e –74 2 1 - 58 2e–35 6 3.5 1 72 49 5e–04 3e–04 6.5 16 7.7 10.5 1 1 37 51 1e–05 1 1.1 85 41 30 88 59 49 1e–83 1e–39 5e–08 64 80 2e–64 1 1 1.1 32 28 57 46 3e–04 2e–09 1 1 1.2 1 1.1 1 51 62 5e–93 1.13 1.15 1.2 1 1 1 1 Not detectablef Not detectablef the lowest value obtained was set to 1.00. M: T. borchii 30-day-old mycelium; UF: T. borchii unripe fruit body (stage 0); RF: T. borchii ripe fruit body (stage 5) e These values were referred to the highest hybridization signals f These clones probably represents rare transcripts not detectable in Northern blot experiments 164 0), and fruit bodies with 76–100% of asci containing mature spores (stage 5) was treated with deoxyribonuclease, checked for integrity by denaturing gel electrophoresis and used for DDRT-PCR experiments. These fruit bodies were collected from an experimental truffle orchard (see Materials and methods). Sixteen combinations of primers were utilised; from the resulting differential display patterns in agarose gel, 22 amplicons, showing differential expression, were selected for further analyses (Fig. 1). These cDNA fragments were purified from the gels and directly cloned. This procedure is especially handy because it enables the amplification of differentially expressed products to be detected on agarose gel and directly cloned. By combining the use of longer primers (13 bp in length) and a powerful reverse transcriptase we obtained cDNA fragments larger in size (350–1.200 bp) than the cDNA fragments (100–500 bp) obtained by the traditional differential display technique. Furthermore in an attempt to reduce non-specific primers annealing and to obtain more specific and reproducible patterns, the PCR annealing temperature was increased to 42C. The mRNA differential display protocol utilised revealed reproducible differences in banding patterns with all primer combinations, when using mRNA derived from two samples for each maturation stage (stages 0 and 5). Characterisation of cDNA fragments To ensure that the composition of the amplification products was homogeneous, ten colonies from each transformation were digested with three different restriction enzymes. Identical restriction patterns were obtained for 86.4% of the transformations, suggesting that the corresponding DDRT-PCR bands represent single products, whereas 13.6% of the transformations gave rise to different restriction patterns, revealing DDRT-PCR bands heterogeneous in sequence. Twenty-five clones were obtained and sequenced. These sequences were compared with those present in the NCBI database using the BLASTX and BLASTN algorithms (Altschul et al. 1997). Fifteen clones showed similarity to known proteins, as reported in Table 1, eight clones were either novel or showed only poor matches to known sequences, whereas two clones corresponded to fragments of 28S rRNA. Among the analysed clones, the 873 bp clone VT19 appeared to be full length (Fig. 2A). A database search identified this cDNA as encoding a putative 3-phosphoserine phosphatase protein, exhibiting 69% similarity with Neurospora crassa G6G8.8 protein (AAK07846), belonging to the 3-phosphoserine phosphatase family. The VT19 clone can also be aligned with Saccharomyces cerevisiae YNL010w (S62922), Schyzosaccharomyces pombe SPAC823.14 (CAB90159) (two proteins similar to N. crassa 3-phosphoserine phosphatase) and Arabidopsis thaliana 3phosphoserine phosphatase (BAA33806.1) (Fig. 2B). Expression analysis The expression pattern of all the cDNAs isolated was analysed by at least two independent Northern blot experiments, using RNA from the same two samples as used in differential display experiments and other samples, collected in the same site. Seven cDNA clones were confirmed to be differentially expressed during fruit body development. One month-old-mycelium of T. borchii, showing active metabolism (Saltarelli et al. 1998), was also included in Northern blot analyses in order to detect changes in gene expression levels in the vegetative and fructification phases (Fig. 3). The relative mRNA stages of the cDNAs were expressed relative to the lowest expression stage for each transcript (Table 1). The VC12 clone is expressed only in completely immature ascomata, suggesting that it is a specific unripe fruit body gene (Fig. 3). Fig. 1A, B Examples of mRNA differential display in agarose gel. The fingerprints, showing the differences in mRNA populations between unripe and ripe Tuber borchii fruit body, were obtained using T12VC anchored primer and random primer CZ2 (A) and T12VT anchored primer and random primer CZ3 (B). M: Marker VIII, Boehringer Mannheim; RF: T. borchii ripe fruit body (stage 5); UF: T. borchii unripe fruit body (stage 0). The arrows indicate the selected cDNA bands, corresponding to differentially expressed clones Discussion In this paper the first attempt in understanding the molecular basis of truffle fruit body development is reported. We analysed two different stages of ascomata maturation: the fruit body with 0% of asci with mature spores (completely immature ascomata) and fruit body 165 Fig. 2 A Nucleotide and deduced amino acid sequences of the VT19 clone, a putative fulllength cDNA encoding 3-phosphoserine phosphatase. The coding sequence is in uppercase. The CT-rich sequences are underlined; the A in the –3 position (Kozac consensus sequence) is in bold and underlined. The start and stop codons are in bold. B Alignment of the putative translation product of clone VC19 with Neurospora crassa putative protein G6G8.8 (AAK07846) similar to 3-phosphoserine phosphatase, Schizosaccharomyces pombe SPAC823.14 (CAB90159) similar to N. crassa G6G8.8, Saccharomyces cerevisiae YNL010w (S62922), similar to N. crassa G6G8.8 and 3-phosphoserine phosphatase of Arabidopsis thaliana (BAA33806.1). Residues that are identical to VC19 in at least two of the related proteins are marked by asterisks above the sequences with 76–100% of asci with mature spores (mature ascomata). The truffle fruit body is composed of cytologically different and functionally distinct components: an outer peridium and an inner gleba, which consists of vegetative hyphal cells and reproductive structures (Balestrini et al. 2000). As the fruit body matures, the specialised hyphae are differentiated in the asci and the ascospores are formed. These morphogenetic processes are accompanied by new membranes and cell wall synthesis. Meiosis and mitosis occur during spore formation and maturation. The mRNA differential display in agarose gel method was set up in order to identify genes that were differentially expressed during ascomata development. This 166 Fig. 3 Northern analysis of the seven differentially expressed cDNA clones. M: Tuber borchii 30 day old mycelium; RF: ripe fruit body (stage 5); UF: unripe fruit body (stage 0). RNA sizing: ethidium bromide-stained ribosomal RNA bands showing the integrity of total RNA. 18S: control hybridisation with 18S rRNA as a probe technique has several advantages compared with the classic mRNA differential display in polyacrylamide gels, such as the use of non-radioactive PCR, simplified band detection and direct cloning of bands purified without previous reamplification. Therefore, it is significantly less time consuming and lower in cost. Furthermore, the traditional decamer primers often mismatch and hybridise to distinct regions of the same cDNA and for this reason we used 13 bp random primers (Zhao et al. 1995; Graf et al. 1997). Twenty-five cDNAs were isolated from the 16 fingerprints, cloned and sequenced. Fifteen clones have significant similarity to known proteins and the cDNAs can be grouped into the following functional categories: dikaryosis and fruiting (mating type protein 1), cell division control (septin, SMT3, helicase), transport across membranes (sulphate transporter, Na+–Ca2+ exchanger protein, probable P-type ATPase and probable transport protein), mitochondrial division (Fis1p), intermediary metabolism (methylenetetrahydrofolate reductase, 3-phosphoserine phosphatase, dihydrolipoamide acetyltransferase, transglutaminase/protease-like protein), biosynthesis of isoprenoid compounds (3-hydroxy-3-methylglutarylcoen-zyme A reductase) and putative RNA/DNA binding (serine-rich protein). These similarities allowed us to obtain a contribution to our knowledge of T. borchii genome. The cDNA clones were used in Northern blot experiments in order to quantify their expression level in mycelium, unripe fruit body and ripe fruit body. Seven out of the 25 cDNA fragments corresponded to differentially expressed genes (Fig. 3), while the others were not differentially expressed or not detected, representing false mRNA differential display positives or rarer transcripts more difficult to visualise. The sequence homology of the differentially expressed clones suggests their putative gene function, allowing us to extend our knowledge of the molecular aspects of T. borchii fruit body development. The deduced amino acid sequence of clone VT3 was found to have 70% similarity with Saccharomyces cerevisiae Fis1p (NP_012199), an integral mitochondrial protein required during fission for the correct assembly, membrane distribution and function of Dnm1p, a soluble, dynamin-related GTPase. This protein assembles on the outer mitochondrial membrane at sites where organelle division occurs (Mozdy et al. 2000). Northern blot analysis of VT3 revealed higher expression in the T. borchii unripe fruit body (Fig. 3), suggesting that an increase in mitochondrial division and biogenesis could support the great energy demand of the cells during fruit body development and ascospore formation. Clone VT19 contains the complete sequence of the gene encoding a putative 3-phosphoserine phosphatase, the enzyme responsible for the dephosphorylation of 3-phosphoserine to serine, in the phosphorylated pathway of serine biosynthesis. This amino acid is a key intermediate in a number of important metabolic pathways. Furthermore, the serine is involved in the synthesis of phospholipids, porphyrins, purines and thymidine (Ireland and Hiltz 1995). Northern blot analysis of clone VT19 (Fig. 3) showed high expression in the vegetative phase and lower expression in the unripe fruit body; however, it was not detectable in the ripe fruit body. This revealed the importance of the phosphorylated pathway enzymes in fast proliferating tissues, which require a high serine supply (Ho and Saito 2001). Clone VT15 codes for a part of dihydrolipoamide acetyltransferase (E2p), a dynamic multifunctional multidomain protein of the pyruvate dehydrogenase complex (PDH). The PDH complex catalyses a key reaction that generates acetyl-CoA, linking glycolysis to the tricarboxylic acid cycle and to the biosynthesis of fatty acids. Expression of clone VT15 is increased sixfold in the mycelium, revealing the active metabolism of this stage and 3.5-fold in the unripe fruit body (Table 1), probably in order to meet the energy demand for fruiting. Furthermore, the fatty acids that can be produced by acetyl-CoA, could be utilised for the active synthesis of new membranes during ascospore formation. The deduced amino acid sequence of clone VC13 showed 56% identity with the SMT3 protein of Saccharomyces cerevisiae (NP_010798). SMT3 is a ubiquitin-like protein modifier, known to be conjugated to other proteins and to modulate their functions in various important processes. Modification of cellular proteins by 167 the ubiquitin-like protein SMT3 is essential for nuclear processes, including control of telomere length and chromosome segregation and cell cycle progression in yeast (Tanaka et al. 1999). Clone VC13 is expressed at a higher level in the unripe fruit body (7.7-fold) and in mycelium (6.5-fold) relative to the ripe fruit body (Table 1); this could indicate more active cellular division in the immature stage. The predicted amino acid sequence of clone VT13 showed 49% similarity to a serine-rich protein of Schyzosaccharomyces pombe (CAA16838). Serine-rich proteins, such as transcriptional or splicing factors, are often involved in RNA/DNA binding (Horie et al. 1998). The VT13 cDNA is highly expressed in the vegetative phase; however, its expression decreases in the unripe fruit body and is lowest in the ripe fruit body. Clone VC12, showing homology to the mat1 genes, is expressed only in the completely immature fruit body (Fig. 3). The mating type is a single regulatory locus that controls sexual reproduction in Ascomycetes. The mating type idiomorphs encode proteins with confirmed or putative binding motifs, suggesting that mat genes encode transcriptional regulators and control the expression of some genes required for sexual reproduction (Turgeon et al. 1998). In Saccharomyces cerevisiae, the mating type genes are known to act as master genes (producing trascriptional factors) controlling the expression of multiple genes (downstream regulation), which then impact on the sexual development pathway, such as on fertilisation and sporulation. The T. borchii VC12 clone showed the highest homology with the mating type protein 1 of Alternaria brassicicola (AAK85542), MAT1 of Cochliobolus heterostrophus (1913430A) and FMR1 of Podospora anserina (CAA45519.1). In P. anserina it has been reported that the FMR1 gene, together with the SMR2 and FPR1 genes, acts as an activator and repressor of fertilisation and internuclear recognition (Debuchy et al. 1993). In T. borchii, VC12, a specific unripe fruit body gene (Fig. 3), could be required after fertilisation for fruit body development as in P. anserina (Debuchy et al. 1993). Clone VT16, part of the gene encoding 3-hydroxy 3-methylglutaryl CoA reductase (HMGR), is highly expressed in ripe fruit body whereas it shows lower expression in the 30 day old mycelium and unripe fruit body; furthermore multiple transcripts appear in ripe fruit body RNA (Fig. 3) and this might be due to alternative RNA splicing or to the existence of multiple HMGR genes, like in other fungi and plants (Lum et al. 1996). HMGR is the first specific enzyme of the isoprenoid pathway, which leads to several classes of primary and secondary metabolites such as sterols, quinones, carotenoids and gibberellins. High expression of HMGR in the ripe fruit body could be explained by increasing synthesis of the ergosterol for the biosynthesis of new membranes in ascospores. The mature fruit body may also produce terpenic compounds that could contribute to the flavour of the truffle ascomata and have antimicrobial and antifungal activity. Furthermore, like other ectomychorrizal fungi, T. borchii could produce gibberellin-like compounds, although nothing is known about the role of these hormones in fungi (Hanley and Greene 1987). The mRNA differential display in agarose gel method is a valid strategy for identifying differentially expressed genes in the development of the complex truffle fruit body. The results reported here provide new insight into gene expression during fruiting of the ectomycorrhizal fungus T. borchi. Furthermore, the isolation of differentially expressed genes suggests new directions for further molecular studies and represents an interesting first step towards a further characterisation of the life cycle of this fungus. Acknowledgements This work was supported by P. S.: ‘‘Biotecnologia dei funghi eduli ectomicorrizici: dalle applicazioni agroforestali a quelle agro-alimentari’’ CNR, Italy. 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