The isoprenoid pathway in the ectomycorrhizal fungus Tuber borchii Vittad.: cloning and characterisation of the tbhmgr, tbfpps and tbsqs genes

Curr Genet (2006) 50:393–404 DOI 10.1007/s00294-006-0097-7 R E SEARCH ART I CLE The isoprenoid pathway in the ectomycorrhizal fungus Tuber borchii Vittad.: cloning and characterisation of the tbhmgr, tbfpps and tbsqs genes C. Guidi · S. Zeppa · G. Annibalini · R. Pierleoni · M. Guescini · M. BuValini · A. Zambonelli · V. Stocchi Received: 12 June 2006 / Revised: 25 July 2006 / Accepted: 8 August 2006 / Published online: 8 September 2006  Springer-Verlag 2006 Abstract The isoprenoid pathway of the ectomycorrhizal fungus Tuber borchii Vittad is investigated to better understand the molecular mechanisms at work, in particular during the maturation of the complex ascomata (the so-called “truZes”). Three T. borchii genes coding for the most important regulatory enzymes of the isoprenoid biosynthesis, 3-hydroxy-3methylglutaryl-CoA reductase, farnesyl-diphosphate synthase (FPPS) and squalene synthase (SQS), were cloned and characterised. The analyses of their nucleotide and deduced amino acid sequences led us to identify the typical domains shown in homologous proteins. By using a quantitative real-time PCR the expression pattern of the three genes was analysed in the vegetative phase and during the complex ascoma maturation process, revealing an over-expression in the mature ascomata. The enzymatic activity of the T. borchii 3hydroxy-3-methylglutaril-CoA reductase (HMGR) was investigated with a HPLC method, conWrming that the signiWcant isoprenoid biosynthesis in ripe ascomata proceeds not only via a transcriptional activation, but also via an enzyme activity control. These Wndings imply that isoprenoids play a fundamental role in Tuber ascomata, particularly in the last phases of their maturation, when they could be involved in antifungal or/and antimicrobial processes and contribute to the famous Xavour of the truZe ascomata. Communicated by U. Kües. Introduction Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s00294-006-0097-7 and is accessible for authorized users. C. Guidi · R. Pierleoni · M. BuValini · V. Stocchi (&) Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino “Carlo Bo”, via SaY 2, 61209 Urbino (PU), Italy e-mail: v.stocchi@uniurb.it S. Zeppa · G. Annibalini · M. Guescini Istituto di Ricerca sull’Attività Motoria, Università degli Studi di Urbino “Carlo Bo”, via I Maggetti 26, 61029 Urbino (PU), Italy A. Zambonelli Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi di Bologna, via Fanin, 46, 40127 Bologna, Italy Keywords Tuber borchii ascoma development · Isoprenoid pathway · 3-Hydroxy-3-methylglutaryl CoA reductase · Farnesyl-dyphosphate synthase · Squalene synthase · Mycorrhizal fungi The isoprenoid biosynthesis regulatory enzymes in the ectomycorrhizal fungi belonging to the Tuber genus, ascomycetous symbiotic fungi well known for the appreciated organoleptic properties of the ascomata of some species (e.g. Tuber magnatum and Tuber melanosporum), have not yet been investigated. The production of the appreciated truZes under controlled conditions is not yet possible. Recently, several studies were performed to better understand the molecular bases regulating the complex morphogenetic process of truZes production (Lacourt et al. 2002; Zeppa et al. 2002; Pierleoni et al. 2004; Gabella et al. 2005). Interestingly, several isoprenoids were identiWed among the volatile organic compounds produced by ripe Tuber borchii ascomata (Zeppa et al. 2004); this Wnding 123 394 suggests that mature fruit bodies possess a high activity of the isoprenoid pathway. Similarly, Gabella et al. (2005) described an increased expression of the genes coding for 3-hydroxy-3-methylglutaryl-CoA synthase, acetyl-CoA acetyltransferase and isopentenyl diphosphate isomerase in mature ascomata. Isoprenoids (also known as terpenoids) are a large family of compounds derived from the isoprene building block involved in several physiological, metabolic and structural roles: hormones and intracellular messengers (gibberellins, cytokines, farnesylated mating factors in yeast, juvenile hormones in insects and steroid hormones in mammals); photosynthetic pigments (carotenoids, phytol); electron carriers (ubiquinone, plastoquinone); protein glycosylation (dolichol); sterols (especially, ergosterol in yeast and cholesterol in animals). Other isoprenoids, classiWed as secondary metabolites (monoterpenes, diterpenes, sesquiterpenes, etc.), play less essential roles, but can be found through nature, particularly in plants and fungi, and provide a wide range of commercially useful products, including solvents, Xavouring and fragrances, adhesives, industrially useful polymers and a number of pharmaceuticals and agrochemicals (McGarvey and Croteau 1995; Barkovich and Liao 2001). Because of the multiple roles played by isoprenoids, the level of their synthesis must be strictly controlled. The isoprenoid biosynthesis pathway is also known as the mevalonic acid (MVA) pathway. One of the most challenging aspects of this multibranched complex metabolic process is the identiWcation of the enzymes that catalyse the rate limiting steps in the pathway. Investigations performed during the last three decades established that 3-hydroxy-3-methylglutaril-CoA reductase (HMGR) (EC 1.1.1.34), the enzyme synthesising MVA, is the major regulatory enzyme of the MVA (Enjuto et al. 1994; Bach 1995). HMGR is among the most tightly regulated enzymes in nature: it is subjected to a number of complex metabolic regulatory mechanisms including transcriptional and translational control and modulation of enzyme activity by degradation and phosphorylation. Recently, it has been also accepted that additional key enzymes are involved in the control of the Xux through the pathway to maintain the appropriate cellular balance of isoprenoids under diVerent physiological conditions (Cunillera et al. 1996). In fact, there are increasing data showing that biosynthesis of dolichols, ubiquinones and isoprenilated proteins is regulated by enzymes distal from HMGR; in particular, other important regulatory enzymes are the branch point enzyme farnesyl-diphosphate synthase (FPPS) (EC 2.5.1.1/ EC 2.5.1.0) and the Wrst committed enzyme in the sterol 123 Curr Genet (2006) 50:393–404 biosynthesis, squalene synthase (SQS) (EC 2.5.1.21) (Szkopinska et al. 2000; Karst et al. 2004). FPPS catalyses the production of the 15-carbon compound farnesyl diphosphate, which is the starting point of a variety of important isoprenoid end-products (dolichols, ubiquinone, heme a, prenylated proteins). SQS catalyses the Wrst reaction of the sterol biosynthesis, leading to the main end-product of the MVA pathway, cholesterol in mammals and ergosterol in fungi. These compounds are the essential component of plasma membranes, aVecting Xuidity, permeability and the activity of membrane-bound enzymes. In the present work we have cloned and characterised the T. borchii genes for HMGR (tbhmgr), FPPS (tbfpps) and SQS (tbsqs). Furthermore, the expression of these genes in the mycelium and during the ascoma maturation process has been investigated and discussed. Finally, the T. borchii HMGR enzyme activity has been evaluated by using a novel HPLC method (BuValini et al. 2005), to better understand its involvement and physiological role during the development of the ascomata. This research represents the Wrst step towards a better understanding of the isoprenoids metabolism of the complex Tuber fruit bodies and the molecular mechanisms at work in their formation and maturation. Materials and methods Biological material Vegetative mycelia of T. borchii (strain MYA 1018) were grown in the dark at 24°C for 30 days, without shaking in modiWed Melin Norkrans nutrient solution. T. borchii ascomata were collected in an experimental truZe orchard, located in Northern Italy (Zambonelli et al. 2000), analysed by morphological and molecular methods (Zeppa et al. 2002) and classiWed in three diVerent maturation stages, on the basis of asci-containing mature spores: unripe (5–15%); intermediate (16–60%); mature (more than 60%). The maturation stage of the spores was deWned by a morphological method (Zeppa et al. 2002). DNA and RNA isolation DNA extraction was performed as described by Erland et al. (1993). Total cellular RNA was isolated using the RNeasy Plant-mini kit (Qiagen, Crawley, UK). The puriWcation and yields of ascoma RNA were improved by phenol-chloroform extraction before processing of the samples. The Wnal concentration of the DNA and Curr Genet (2006) 50:393–404 RNA samples was estimated either spectrophotometrically or by gel electrophoresis, staining with ethidium bromide. Degenerated primers selection and PCR reactions The degenerated primers were derived from the consensus nucleotide sequences deduced from two alignments, containing, respectively, fungal FPPS and SQS proteins. Successful oligonucleotides to identify the T. borchii tbfpps gene were: Ffor (5⬘-CGBCGBGGYC ARCCYTG YTG-3⬘), corresponding to the amino acid sequence RRGKPC (amino acids 109–114 in the Saccharomyces cerevisiae FPPS protein, accession number A34441) and Frev (5⬘-GCRACRGGVAGRTARAA VG-3⬘), corresponding to the amino acid sequence SFYLPVA (amino acids 202–208 in the S. cerevisiae FPPS protein). Successful oligonucleotides to identify the T. borchii tbsqs gene were: Sfor (5⬘-CARAARSCYAAYATHA THCG-3⬘), corresponding to the amino acid sequence QKTNIIR (amino acids 219–224 in the S. cerevisiae SQS protein, accession number P29704) and Srev (5⬘-T GRGTRGCDATRGCCATRAC-3⬘), corresponding to the amino acid sequence MAIATL (amino acids 302–308 in the S. cerevisiae SQS protein). The PCR reactions were performed using an automated Thermal cycler (Perkin Elmer, Boston, MA, USA) in a Wnal volume of 25
l, containing 200 ng of mycelial DNA, 1X PCR buVer (Qiagen), 100
M of dNTPs, 20 pmol of each primer and 0.2 U of Taq DNA polymerase (Qiagen). Reaction conditions were as follows: 1 cycle of 4 min at 95°C; 40 cycles of 1 min at 95°C, 1 min at 40°C, 1 min at 72°C, followed by a Wnal elongation cycle (7 min at 72°C). Under these conditions, one PCR fragment of 297 bp and one of 267 bp were generated by using, respectively, the primers Ffor–Frev and the primers Sfor–Srev. The two fragments were automatically sequenced in both directions, using an ABI 373S System, Perkin Elmer. Based on the preliminary sequence data obtained, more speciWc primers were designed: F2for 5⬘-CTCAG GTCAAGGTCTAC-3⬘ and F2rev 5⬘-GGGAGGT AGAAAGAGTAG-3⬘ for tbfpps; S2for 5⬘-CGTGA TTACAGGGAGGATC-3⬘ and S2rev 5⬘-CATGACC TGGGGAATAG-3⬘ for tbsqs. The PCR reactions were as follows: 1 cycle of 4 min at 95°C; 27 cycles of 30 s at 95°C, 30 s at 57°C, 30 s at 72°C, followed by a Wnal elongation cycle (7 min at 72°C). The PCR products obtained were cloned using the pGEM-T vector system (Promega, Madison, WI, USA) and sequenced in both directions, obtaining two fragments, one of the tbfpps gene and one of the tbsqs gene. 395 cDNA library screening A
Zap cDNA library (Stratagene, Cedar Creek, TX, USA) of 30-day-old T. borchii mycelium was screened with three diVerent 32P-probes: the two fragments obtained by PCR (see above), and the clone VT16, corresponding to a part of the tbhmgr gene, obtained in a previous work (Zeppa et al. 2002). Approximately, 3 £ 105 plaques were analysed, following the manufacturers’ instructions. For each probe, Wve positive cDNA phages were selected, re-screened until plaquepure and then converted into pBlueScript II SK-. Final wash conditions were 1X SSC and 0.1% SDS at 60°C. The cDNA clones obtained were automatically sequenced in both directions, using an ABI 373S System (Perkin Elmer). 5⬘-end ampliWcation of the tbhmgr gene A “genome walker” method was used to clone the unknown 5⬘-end of the tbhmgr gene. Four aliquots of T. borchii mycelial DNA (2.5
g each) were digested with the restriction enzymes ScaI, PvuI, SmaI and StuI (Takara Biomedicals, Tokyo, Japan), that have sixbase recognition sites and generate blunt ends. The digested DNA was then puriWed with a phenol-chloroform method and ligated to an adaptor of 44 nucleotides (5⬘-CTAATACGACTCACTATAGGGCTCGA GCGGCCGCCCGGGCAGGT-3⬘). To walk from the known sequence of the tbhmgr gene, we ampliWed the four libraries by PCR, using the “Advantage Genomic Polymerase mix” kit (Clontech, Heidelberg, Germany), speciWcally developed for long-distance PCR. We performed a Wrst PCR using an outer adaptor primer (5⬘-CTAATACGACTCACTATAGGGC-3⬘) and a tbhmgr gene-speciWc primer (5⬘-GCTGCAAGA TGGAGTGTC-3⬘), designed on the known 5⬘-end of the tbhmgr sequence available. The cycling conditions were as follows: 7 cycles of 94°C for 25 s and 72°C for 3 min; 32 cycles of 94°C for 25 s and 67°C for 3 min; a Wnal incubation of 67°C for 4 min. One microlitre of the primary PCR mixture obtained was then diluted into 49
l of deionised water and used as a template for a secondary PCR, with a nested adaptor primer (5⬘-TCGAGCGGCCGCCCG GGCAGGT-3⬘) and a nested tbhmgr gene-speciWc primer (5-GCTATGACTCCAACGATTCC-3⬘). The cycling conditions were as follows: 5 cycles of 94°C for 25 s and 65°C for 3 min; 20 cycles of 94°C for 25 s and 60°C for 3 min; a Wnal incubation of 60°C for 4 min. The PCR products from the secondary PCR were loaded on a 1.5% agarose gel and fractionated by electrophoresis. Four DNA fragments > of 1,000 bp were 123 396 puriWed using the Qiagen gel extraction kit, cloned using the pGEM-T vector system (Promega) and automatically sequenced in both directions, using an ABI 373S System (Perkin Elmer). Database searches using the BLAST programs permitted the identiWcation of a fragment of 2,488 nucleotides, localised upstream the known 5⬘-end of the tbhmgr gene, containing the lacking 5⬘-end and the putative promoter region. Detection of introns Two speciWc primers were selected, respectively at the 5⬘- and 3⬘-UTR region of the cDNA sequences, in order to evaluate the possible presence of introns: Hfor: 5⬘-GCTAAGGTGTGTCGGTAC-3⬘ and Hrev 5⬘–CGCCAACGCCTCTGCTCGC–3⬘ for the tbhmgr gene; F3for 5⬘-GCCTCTATCCCCCTTTC-3⬘ and F3rev 5⬘-GTTTTTCTGTATTTCGACC-3⬘ for the tbfpps gene; S3for 5⬘-CACCTCCTCATTACAC-3⬘ and S3rev 5⬘-CACCTGTTGCCAAACTG-3⬘ for the tbsqs gene. Each pair of primers was used to amplify total T. borchii DNA; the PCR products obtained were cloned using the pGEM-T vector system (Promega) and sequenced in both directions. The sequences obtained were deposited in GenBank under the following accession numbers: DQ408535 (tbhmgr); DQ408536 (tbfpps); DQ408537 (tbsqs). Sequence analysis Database searches and sequence analyses were carried out using the BLAST programs at the National Centre for Biotechnology Information, National Institute of Health http://www.ncbi.nlm.nih.gov. The ExPASy program (http://www.expasy.ch/tools/ protparam.html) was used to analyse the physicalchemical parameters of the amino acid sequences coded by tbhmgr, tbfpps and tbsqs. The CDD (http://www.ncbi.nlm.nih.gov/Structure/ cdd/cdd.shtml) and the SCANPROSITE (http://www. expasy.ch/tools/scanprosite) programs were used to identify amino acid conserved domains. The membrane topology of the amino acid sequences coded by tbhmgr and tbsqs was examined by using six diVerent programs, set to standard parameters and relying on single-sequence predictions: HMMTOP (http:/ /www.enzim.hu/hmmtop/index.html); PRED-TMR2 (http://www.o2.biol.uoa.gr/PRED-TMR2); SOSUI (http:/ /www.sosui.proteome.bio.tuat.ac.jp/cgi-bin/sosui.cgi? /sosui_submit.html); TMHMM (http://www.cbs.dtu.dk/ services/TMHMM); TMpred (http://www.ch.embnet. org/software/TMPRED_form.html); TopPred 2 (http:// www.bioweb.pasteur.fr/seqanal/interfaces/top pred.html). 123 Curr Genet (2006) 50:393–404 Southern blot analysis Fifteen micrograms of T. borchii mycelial DNA were digested with restriction enzymes that presenting no sites in the gene sequences, respectively: BamHI, PstI and NotI for tbhmgr; BamHI, NotI and XhoI for tbfpps; BstZI, HindIII and NotI for tbsqs. Three gels were created and loaded in the same conditions, and successively blotted onto version 2.0 Hybond N+-positively charged nylon membrane (Amersham, Life Science, Freiburg, Germany). Each membrane was then hybridised in phosphate buVer (Sambrook et al. 1989) with a 32 P-labelled probe, using the RediPrime labelling kit (Amersham, Life Science). Final wash conditions were 0.1X SSC and 0.1% SDS. Quantitative real time PCR Five-microgram aliquots of DNase-treated (DNA-free; Ambion, TX, USA) total RNA from immature, intermediate and mature ascomata were denaturated at 70°C for 2 min and then reverse-transcribed in a 10
l reaction mixture containing each dNTP at 250
M, 200 U of reverse transcriptase (PowerScript Reverse Transcriptase, Clontech), 50 nM of random hexamer primer, 1 £ Wrst-strand buVer and 0.5 mM of DTT at 42°C for 1 h cDNAs were then diluted by adding 50
l of DEPC-treated distilled water. SpeciWc primer for tbhmgr (H2for: 5⬘-GCTTGTCAAGAACGCGGAG-3⬘ and H2rev: 5⬘-AACGTCAAGTGCATGGACAA AT-3⬘), tbfpps (F4for: 5⬘-GAGCTTTTCCATGATGT CACCTGG-3⬘ and F4rev: 5⬘-TCAATACTATCTTC GGGGGATG-3⬘) and tbsqs (S4for: 5⬘-AAACCTG CGATCTACTTGTGA-3⬘ and S4rev: 5⬘-GGACTCG ATGAATTGTTCAACCTTG-3⬘) were designed. 18S rRNAs from T. borchii (tb18S) was used as reference (Guescini et al. 2003). The PCR was performed in a Bio-Rad iCycler iQ Multi-Colour Real-Time PCR Detection System using 2 £ Quantitect SYBR Green PCR kit (Qiagen). The quantitative PCR reaction consisted of: 95°C for 10 min to activate HotStart DNA polymerase followed by 50 cycles of the two-step at 95°C for 30 s and at 60°C for 30 s. The speciWcity of the ampliWcation products obtained was conWrmed by examining thermal denaturation plots and by sample separation in a 3% DNA agarose gel. The amount of the target transcript was related to that of the reference gene by the method described by PfaZ (2001). Potential signiWcance of diVerences in RT-PCR signals was determined by one-way ANOVA. Each sample was tested in triplicate by quantitative PCR, and the experimental groups (immature, intermediate and mature ascomata) consisted of at least Curr Genet (2006) 50:393–404 three independent experiments. The signiWcance of the diVerence in expression levels among the groups has been evaluated by a non-parametric approach (Kruskal–Wallis test P > 0.05). 3-hydroxy-3-methylglutaryl-CoA reductase assay The HMG-CoA reductase speciWc activity was evaluated according to the method of BuValini et al. (2005). BrieXy, T. borchii ascomata (0.5 g of dried weight) was homogenised using a Potter homogeniser (Steroglass, Perugia, Italy) with an appropriate amount (at least 500
l) of the extraction buVer consisting of 20 mM of sodium phosphate buVer, pH 7.5, 10
M -mercaptoethanol (-MSH), 0.25% (v/v) Tween-20 and 10
M phenylmethylsulphonyl Xuoride (PMSF). The suspension obtained was centrifuged at 18,000g for 10 min at 4°C and the supernatant was used as a crude extract for the enzyme assay. Protein content was determined by the method of Lowry et al. (1951). For HMG-CoA reductase assays ascomata proteins were pre-incubated for 5 min at 37°C with 200 mM of sodium phosphate buVer, pH 7.5 and 10 mM -MSH and then the reaction was initiated by the addition of 0.24 mM of NADPH and 40
M of HMG-CoA (1 ml Wnal volume of the reaction mixture). The mixture obtained was incubated at 37°C for 20 min and then stopped by adding 10
l of 6 M HCl. Controls were obtained adding together co-factor, substrate and HCl (T = 0 min). After the lactonisation of the reaction with HCl the mixture was incubated for an additional 40 min at 37°C to promote the generation of mevalonolactone. Then, the supernatant was neutralised with 0.1 M of sodium carbonate buVer, pH 10.5. Aliquots (200
l) of each sample were injected onto a Gold HPLC system from Beckman (Beckman Coulter Inc, Fullerton, CA, USA) and elution was carried out at a Xow-rate of 1 ml min¡1 at 37°C, using HPLCgrade water as mobile phase. The detection was performed at 200 nm. Sample activity was determined by calculating the concentrations of mevalonolactone in spiked samples against calibration curve. Results The tbhmgr gene: cloning, sequence analysis and Southern blotting In a previous study, we identiWed a fragment (VT16 clone) of the T. borchii tbhmgr gene (Zeppa et al. 2002). To clone the entire gene, we screened a cDNA 397 library of 30-day-old T. borchii mycelium by using VT16 clone as a probe. Five positive phages were selected from the third screening and converted into pBlueScript II SK- by in vivo rescue. The sequence analysis led us to conWrm that they were the same clone, 2,378 bp in length, containing an ORF of 1,950 nucleotides. Database searches were carried out using the BLASTP programs, revealing a high degree of homology (75%) with HMGR proteins from diVerent fungal species, such as Aspergillus fumigatus (EAL87464.1) and Gibberella fujikuroi (CAA63970.1). Unfortunately, the sequence obtained corresponded to a part of the tbhmgr gene, because the corresponding amino acid sequence was deprived of about 400 amino acids in the N-terminus. To clone the lacking region and to evaluate the presence of introns, we used a “genome walker” strategy, followed by several PCR on total T. borchii DNA (see Materials and Methods). The Wnal sequence is 4,920 bp in length, contains an ORF of 3,294 bp, four exons and three introns; a fourth intron is present in the 5⬘untranslated region (see Fig. S1). In all of the introns, the dinucleotides GT and AG are present at the 5⬘- and at the 3⬘-end, respectively, as in almost all eukaryotic genes (Breathnach et al. 1978). Furthermore, the intron length (99, 73, 63, 54 nucleotides, respectively) and AT content correspond to those of other fungal introns (Bon et al. 2003). Two in-frame methionine codons at positions 1 and 25 of the amino acid sequence are present; based on the “Wrst methionine rule”, it is likely that the translation begins at the Wrst methionine (Kozak 1981). The region upstream the ATG start codon (1,008 nt in length) was analysed with respect to the known consensus sequences typical of a promoter region. A CAAT-box is located at position 198, while a TATAAA motif can be found at position 244 (TATTAC). Several CT-rich sequences are present (-925, -847, -800, -692, -494, -470, -340, -170 and -108): these tracks are generally found in highly expressed genes (Kinghorn 1987). Furthermore, particularly important is the identiWcation of the promoter sequences that may be relevant for the regulation of tbhmgr gene. It is known that sterol-mediated regulation of HMGR promoter is dependent on a short segment, the “sterol regulatory element” consensus sequence (SRE): 5⬘-CACC(C/G)CAC-3⬘. This is a target for positive feedback regulation by sterols, via the SRE-binding proteins (SREBPs). A putative SREmotif was found at position -489 in the tbhmgr promoter region. Putative binding sites (GATA motifs) for a nitrogen regulatory protein (AREA, NIT2; Kudla et al. 1990) are also present at positions -738, -560, -347 and -261. 123 398 Curr Genet (2006) 50:393–404 The 3⬘-untranslated region of tbhmgr gene contains two putative polyadenylation consensus sequences (AAUAA). To evaluate the genomic organisation of the tbhmgr gene, a Southern blot analysis was performed. A fragment localised at the 3⬘ region of the tbhmgr gene (from nt 3,129 to nt 3,396) was ampliWed and then used as a probe on T. borchii genomic DNA. The presence of only one hybridisation signal revealed that tbhmgr is a single-copy gene in the T. borchii genome (see Fig. S2A). Analysis of the tbhmgr deduced amino acid sequence The tbhmgr open reading frame codes for a 1098amino acid polypeptide (TBHMGR) (see Figs. S1 and S3), showing high homology with several fungal HMGCoA reductases. The BLASTX and SCANPROSITE analysis revealed a Sterol Sensing Domain (SSD) located in the N-terminal region (aa209–aa382), and Fig. 1 Alignment and comparison of the deduced amino acid sequence of tbhmgr gene with HMGR from: A.n. Aspergillus nidulans (AA085434.1), N.c. Neurospora crassa (CAA65645.1), T.b. Tuber borchii, S.c. Saccharomyces cerevisiae (A34441), S.p. Schizosaccharomyces pombe (NP_595334.1), H.s. Homo sapiens (AAH33692.1), M.a. Mesocricetus auratus (AAA36989.1). Only the conserved catalytic domains were compared. The two binding sites both for HMG-CoA (aa752–aa762 and aa779– aa788) and NAD(P)H (aa877–aa882 and aa1027– aa1034) are evidenced. Asterisks indicate the conserved residues involved in catalytic activity (glutamate, aspartate and histidine at position 783, 993 and 1090, respectively); Plus indicates the position of the serine involved in phosphorylation processes, absent in TBHGMR 123 the C-terminal catalytic domain (aa683–aa1095). As found for other HMGR, the linker region of TBHMGR connecting the membrane and catalytic domains is highly divergent and enriched in amino acids such as valine, lysine and glutamic acid. The analysis of TBHMGR catalytic domain revealed two binding sites for HMG-CoA and NAD(P)H, respectively (Fig. 1). Within the HMGR Cterminal domains some amino acids are functionally conserved and may play an important role in the structural conformation and/or in the catalytic properties of the enzyme (Woitek et al. 1997): TBHMGR presents the glutamate, aspartate and histidine amino acids at positions 783, 993 and 1090, respectively (Fig. 1). Of particular interest, with respect to the regulation of HMG-CoA reductase activity of several organisms, is a serine residue within the catalytic domain which can be phosphorylated by a cAMP-dependent protein kinase, resulting in the loss of catalytic activity (Sato et al. 1993). Interestingly, TBHMGR, like S. cerevisiae A.n. N.c. T.b. S.p. S.c. H.s. M.a. HGACCENVIGTLPLPLGVAGPLVIDGQSYFIPMATTEGVLVASASRGAKAINAGGGAVTV HGACCENVIGYMPLPVGVAGPLVIDGQSFFVPMATTEGVLVASTSRGCKAINSGGGAVTV LGACCENVIGYMPLPVGIAGPLNIDGEIFYLPMATTEGVLVASTSRGCKAINAGGGAVTV LNACCENVIGYMPLPLGVAGPLIIDGKPFYIPMATTEGALVASTMRGCKAINAGGGAVTV FGACCENVIGYMPIPVGVIGPLIIDGTSYHIPMATTEGCLVASAMRGCKAINAGGGATTV MGACCENVIGYMPIPVGVAGPLCLDEKEFQVPMATTEGCLVASTNRGCRAIGLGGGASSR MGACCENVIGYMPIPVGVAGPLCLDGKEYQVPMATTEGCLVASTNRGCRAIGLGGGASSR * 775 851 806 735 733 582 581 A.n. N.c. T.b. S.p. S.c. H.s. M.a. LTGDGMTRGPCVGFPTLARAAAAKVWLDSEEGKSVMTAAFNSTSRFARLQHLKTALAGTY LTADGMTRGPCVQFETLERAGAAKLWLDSEKGQSIMKKAFNSTSRFARLETMKTAMAGTN LTGDGMTRGPVVEFPSVRRAGAAKNWIDSEEGQRRLKKAFDSTSRFARLQTIKTALAGTY LTRDQMSRGPCVAFPNLTRAGRAKIWLDSPEGQEVMKKAFNSTSRFARLQHIKTALAGTR LTKDGMTRGPVVRFPTLIRSGACKIWLDSEEGQNSIKKAFNSTSRFARLQHIQTCLAGDL VLADGMTRGPVVRLPRACDSAEVKAWLETSEGFAVIKEAFDSTSRFARLQKLHTSIAGRN VLADGMTRGPVVRLPRACDSAEVKAWLETPEGFAVIKDAFDSTSRFARLQKLHVTMAGRN 835 911 866 795 793 642 641 A.n. N.c. T.b. S.p. S.c. H.s. M.a. LYIRFKTTTGDAMGMNMISKGVEKALHVMATECGFDDMATISVSGNFCTDKKAAALNWID LYIRFKTTTGDAMGMNMISKGVEHALSVMYNEG-FEDMNIVSLSGNYCTDKKAAAINWID LFIRFRTTTGDAMGMNMISKGVECALHVMSTECGFDDMFIVSVSGNYCTDKKPAAINWIE LFIRFCTSTGDAMGMNMISKGVEHALVVMSNDAGFDDMQVISVSGNYCTDKKPAAINWID LFMRFRTTTGDAMGMNMISKGVEYSLKQMVEEYGWEDMEVVSVSGNYCTDKKPAAINWIE LYIRFQSRSGDAMGMNMISKGTEKALSKL--HEYFPEMQILAVSGNYCTDKKPAAINWIE LYIRFQSKTGDAMGMNMISKGTEKALLKL--QEFFPEMQILAVSGNYCTDKKPAAINWIE 895 970 926 855 853 700 699 A.n. N.c. T.b. S.p. S.c. H.s. M.a. GRGKSVVAEAIIPGDVVRNVLKSDVDALVELNTSKNLIGSAMAGSLGGFNAHASNIVTAI GRGKSVVAEAIIPADVVKNVLKTDVDTLVELNVNKNLIGSAMAGSMGGFNAHAANIVAAI GRGKSIVAEAIIPAAVVKSVLKSDVDALVELNISKNLIGSAMAGSVGGFNAHAANIVTAV GRGKSVIAEAIIPGDAVKSVLKTTVEDLVKLNVDKNLIGSAMAGSVGGFNAHAANIVTAV GRGKSVVAEATIPGDVVKSVLKSDVSALVELNISKNLVGSAMAGSVGGFNAHAANLVTAL GRGKSVVCEAVIPAKVVREVLKTTTEAMIEVNINKNLVGSAMAGSIGGYNAHAANIVTAI GRGKTVVCEAVIPAKVVREVLKTTTEAMIDVNINKNLVGSAMAGSIGGYNAHAANIVTAI 955 1030 986 915 913 760 759 A.n. N.c. T.b. S.p. S.c. H.s. M.a. FLATGQDPAQNVESSSCITTMKN---TNGNLQIAVSMPSIEVGTIGGGTILEAQGAMLDL FLATGQDPAQVVESANCITLMRN---LRGNLQISVSMPSIEVGTLGGGTILEPQSAMLDM FLATGQDPPQNVESSNCITVMKKT--VEGALQISVSMPSIEVGTIGGGTILEPQGAMLDL YLATGQDPAQNVESSNCITLMDN---VDGNLQLSVSMPSIEVGTIGGGTVLEPQGAMLDL FLALGQDPAQNVESSNCITLMKE---VDGDLRISVSMPSIEVGTIGGGTVLEPQGAMLDL YIACGQDAAQNVGSSNCITLMEASGPTNEDLYISCTMPSIEIGTVGGGTNLLPQQACLQM YIACGQDAAQNVGSSNCITLMEASGPTNEDLYISCTMPSIEIGTVGGGTNLLPQQACLQM * 1012 1087 1044 972 970 820 819 A.n. N.c. T.b. S.p. S.c. H.s. M.a. LGVRGSHPTNPGDNARQLARIVAAAVLAGELSLCSALAAGHLVRAHMAHNRSAAPTRSAT LGVRGPHPTNPGENARRLARIVAAAVLAGELSLCSALAAGHLVKAHMAHNRSAPPTRTST LGVRGAHQTNPGDNARKLARIVAAAVLAGELSLCSALAAGHLVKSHMAHNRKV------LGVRGAHMTSPGDNSRQLARVVAAAVMAGELSLCSALASGHLVKSHIGLNRSALNTPAMD LGVRGPHPTEPGANARQLARIIACAVLAGELSLCSALAAGHLVQSHMTHNRKTNKANELP LGVQGACKDNPGENARQLARIVCGTVMAGELSLMAALAAGHLVKSHMIHNRSKINLQDLQ LGVQGACKDNPGENARQLARIVCGTVMAGELSLMAALAAGHLVRSHMVHNRSKINLQDLQ * + 1072 1147 1097 1032 1030 880 879 Curr Genet (2006) 50:393–404 HMGRs, does not contain a serine residue in the corresponding region (Fig. 1), thus suggesting that no enzyme inhibition by phosphorylation occurs in this fungal species. Since eukaryotic HMGRs are integral membrane glycoproteins of the endoplasmic reticulum with 1–8 transmembrane segments in the N-terminal region, the membrane topology of the amino acid sequence coded by tbhmgr was investigated. Using six diVerent prediction programs (see Materials and Methods), eight individual transmembrane helices were identiWed in the amino acid sequence coded by tbhmgr (see Fig. S1). These six programs produced diVerent results regarding the precise localisation of the borders of the individual membrane-spanning helix, so we followed the “majority-vote” criterion: consensus border residues conWrmed by at least three of the six methods were considered the most likely border residues. The tbfpps and tbsqs genes: cloning, characterisation and deduced amino acid sequences analysis To isolate T. borchii farnesyl diphosphate synthase (tbfpps) and SQS (tbsqs) genes, degenerated primers, corresponding to the amino acid sequences of highly conserved domains known for fungal FPPS and SQS were designed. Following PCR reactions and cDNA library screenings the cDNA sequences of the two genes were identiWed and cloned (see Materials and Methods). The nucleotide sequence of tbfpps, 1,577 nucleotides in length, contains an open reading frame of 1,044 nucleotides and four introns (see Fig. S4). The intron length (118, 57, 57 and 51 bp, respectively), AT content and 5⬘- and 3⬘-end sequences correspond to those of other fungal introns. The 5⬘-UTR region contains typical elements of a promoter region: the A in the -3 position is present, in agreement with the Kozak consensus sequence (Kozak 1981); two putative GATA-motifs (105 and -9), a CT-rich region (from -56 to -25) and a putative CAAT-box (-99) are present. The 3⬘-UTR region contains three putative polyadenylation consensus sequences. The tbsqs gene, 2,288 nucleotides in length, contains an open reading frame of 1,254 nucleotides and two introns; a third intron is present in the 3⬘-UTR region. The introns length (102, 55 and 55 bp, respectively), AT content and 5⬘- and 3⬘-end sequences correspond to those of other fungal introns. The region upstream the ATG start codon contains an A in the -3 position, in agreement with the Kozak consensus sequence, while the 3⬘ untranslated region contains a putative 399 polyadenilation consensus sequence (AATTA) (see Fig. S5). In order to investigate the genomic organisation of tbfpps and tbsqs, Southern blotting analyses were performed. T. borchii genomic DNA was digested and hybridised with 32P-labelled ampliWcation products (see Materials and Methods). Each probe detected only one band, suggesting that tbfpps and tbsqs are single-copy genes (see Figs. S2B and C). The amino acid sequence coded by tbfpps (TBFPPS), 347 in length (Figs. 2, S3 and S4) has been analysed by using the BLASTP program, revealing a 76% homology with the Neurospora crassa farnesyl-pyrophosphate synthetase (CAD21355.1). The alignment of TBFPPS with orthologues available in the databases showed four highly conserved regions: domain I contains lysine and arginin, probably involved in substrate binding; domain III is also involved in binding the allylic substrate; II and IV are aspartate-rich domains present in all polyprenyl synthases, probably involved in substrate binding through the formation of magnesium salt bridges between the pyrophosphate moieties of the isoprenoid substrate and the carboxyl group of the aspartate (Homann et al. 1996). TBFPPS contains also the lysine 192, like the ascomycetes G. fujikuroi and N. crassa, which is thought to Wx the pyrophosphate moiety of geranyl diphosphate. The deduced amino acid sequence coded by tbsqs, 417 aa in length (Figs. 3, S3 and S5) shows a high homology with SQSs from several organisms. Furthermore, the amino acid sequence conserves the squalene and phytoene sequence motifs: Y(C/S/A)XXVA(G/ A)XVG (positions 179–185) and (L/I/V/M)GXXXQ XXNIXRD(L/I/V/M/F/Y)XX(D/E) (positions 215– 232). Other conserved domains contain the motif DX(XX)D, an aspartate-rich domain present in all polyprenyl synthases, like in the amino acid sequence coded by tbfpps; Wnally, also three highly conserved residues of tyrosine (aa180), phenylalanine (aa295) and glutamine (aa302), essential for the catalysis, are present in TBSQS. Squalene synthase is a microsomal membrane-associated enzyme, so we analysed the primary structure of the amino acid sequence coded by tbsqs for possible transmembrane domain, by using the six prediction programs indicated in Materials and Methods. One short C-terminal membrane-spanning domain was identiWed (residues 293–313), while the N-terminal catalytic domain faces the cytosol (Figs. 3 and S5); this orientation would permit TBSQS to accept water-soluble FPP and NADH from the cytosol, and release lipophilic squalene, as previously described in other organisms (Tansey and Shechter 2000). 123 400 Fig. 2 Alignment and comparison of the deduced amino acid sequence of tbfpps gene with FPPS from: N.c. Neurospora crassa (CAA65645.1), G.f. Gibberella fujikuroi (CAA65641.1), T.b. Tuber borchii, S.c. Saccharomyces cerevisiae (A34441), M.m. Mus musculus (AAL09445.1), B.t. Bos Taurus (AAL58886.1), A.t. Arabidopsis thaliana (AAL34286.1), S.p. Schizosaccharromyces pombe (NP_595334.1). The four conserved domains are evidenced; the aspartate-rich domains are in bold; plus indicates the important lysine 192 Curr Genet (2006) 50:393–404 N.c. G.f. T.b. S.c. M.m. B.t. A.t. S.p. -------MAKTTTLKEFESVFP-KLEEALLEYAKAYKLPEQMLSWYKQSLEVNTLGGKCN -------MAQQTTLQEFNSVYP-KLEEALLDHARSYKLPQEQLDWYKRSLEVNPLGGKCN ---------MATTRAQFEEVFTNVIIPELIAEVKKTQISENVIQWIERNFLHNTLGGKYN -----MASEKEIRRERFLNVFP-KLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLN MNGNQKLDAYNQEKQNFIQHFSQIVKVLTEKEL-GHPEIGDAIARLKEVLEYNALGGKYN MNGDQKLDAYAQERQDFIQHFSQIVKVLTEEDI-GHPEIGDAITRLKEVLEYNAIGGKYN ---------MADLKSTFLDVYS-VLKSDLLQDP-SFEFTHESRQWLERMLDYNVRGGKLN --MVNDFNEKNGIKKRLLDFFP-VVLEGIREILESMQYFPEETEKLLYSIKRNTLGGKNN 52 52 51 54 59 59 49 57 N.c. G.f. T.b. S.c. M.m. B.t. A.t. S.p. RGMSVPDSASILLGRP---LTEEEYFQAATLGWMTELLQAFFLVSDDIMDSSITRRGKPC RGMSVPDSVSLLLEKP---LTEEQYFQAATLGWMTELLQAFFLVSDDIMDSSITRRGQPC RGMTVPETYRLLTGKET--LSLAEYKLSAILGWCTELLQAMFLVADDIMDSSKTRRGSPC RGLSVVDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPC RGLTVVQAFQELVEPKK--QDAESLQRALTVGWCVELLQAFFLVSDDIMDSSLTRRGQIC RGLTVVITFRELVEPGK--QDPDSLQRALTVGWCVELLQAFFLVSDDIMDSSLTRRGQTC RGLSVVDSYKLLKQGQD--LTEKETFLSCALGWCIEWLQAYFLVLDDIMDNSVTRRGQPC RGLAVLQSLTSLINRE---LEEAEFRDAALLGWLIEILQGCFLMADDIMDQSIKRRGLDC 109 109 109 114 117 117 107 114 N.c. G.f. T.b. S.c. M.m. B.t. A.t. S.p. WYRQEGVGMVAINDAFMLESAIYTRLKKYFRSHPRYVDFLELFHEVTFQTEMGQLCDLLT WYRQEGVGMIAINDAFMLESAIYTLLKKYFRSHPAYFDLIESFHETTFQTELGQLCDLLT WYLMEGVGNIAINDSFLLESSIYVLLKKYFKGTDYYIDLVELFHDVTWKTELGQLVDLLT WYKVPEVGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLIT WYQKPGIGLDAINDALLLEASIYRLLKFYCREQPYYLNLLELFLQSSYQTEIGQTLDLMT WYQKPGIGLDAINDAFLLESSIYRLLKLYCREQPYYLDLIELFLQSSYQTEIGQTLDLIT WFRKPKVGMIAINDGILLRNHIHRILKKHFREMPYYVDLVDLFNEVEFQTACGQMIDLIT WYLVVGVRR-AINESQLLEACIPLLIRKYFRNMPYYVDLLDTFREVTFLTELGQQEDLLS 169 169 169 174 177 177 167 173 N.c. G.f. T.b. S.c. M.m. B.t. A.t. S.p. + APEDKVDLDNFSMDKYTFIVIYKTAYYSFYLPVALAMYMLDIATPENLKQAEDILIPLGE APEDNVNLDNFSLEKYSFIVIYKTAYYSFYLPVALALHQLNLATPSNLKQAEDILIPLGE SPEDSIDLNRFNYDKYYFIVRYKTAFYSFYLPVALAMHQAGIATPDNLTRARDVLIPLGE APEDKVDLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGE APQGHVDLGRYTEKRYKSIVKYKTAFYSFYLPIAAAMYMAGIDGEKEHANALKILMEMGE APQGNVDLGRFTEKRYKSIVKYKTAFYSFYLPVAAAMYMAGIDGEKEHAHAKKILLEMGE TFDGEKDLSKYSLQIHRRIVEYKTAYYSFYLPVACALLMAGENLEN-HTDVKTVLVDMGI SRDGEASLRSFDLMKYDFIITYKTSFYSFYLPIKCALLLSRNSNQKAYDTTIKLSKLLGY 229 229 229 234 237 237 226 233 N.c. G.f. T.b. S.c. M.m. B.t. A.t. S.p. YFQVQDDYLDNFGLPEHIGKIGTDIQDNKCSWLVNKALSIVTPEQRKTLEENYGRKDKAK YFQIQDDYLDNFGKPEHIGKIGTDIKDNKCSWLVNQALAVATPEQRKILEENYGRKDDEK YFQVQDDYLDCYGNPDYIGKIGTDILDNKCGWLVNKALEIVTPEQRKLLEDNYGQKNAAA YFQIQDDYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVA FFQVQDDYLDLFGDPSVTGKVGTDIQDNKCSWLVVQCLLRASPQQRQILEENYGQKDPEK FFQIQDDYLDLFGDPSMTGKIGTDIQDNKCSWLVVQCLQRASPEQRQILQENYGQKEAEK YFQVQDDYLDCFADPETLGKIGTDIEDFKCSWLVVKALERCSEEQTKILYENYGKAEPSN YFQVQDDYLDCFGDYTVLGKVGMDIQDNKCTWLVCYAEKFASADQLNLLRAHYGKAGSEN 289 289 289 294 297 297 286 293 N.c. G.f. T.b. S.c. M.m. B.t. A.t. S.p. EAVIKQLYDDLKLEDHYKQYEEERVGEIRKMNDAIDESKGLKKQVFEAFLGKIYKRSK EKVVKKLYDDLNLEQRYLDYEEKVVGQIRERIANIDENDGLKKTVFEAFLAKIYKRSK EKKVKDLYLELKLEDHYRRYESESIAMVKGLIDGVDESQGLKRSVLEGFLNKIAGRDK EAKCKKIFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK VARVKALYEALDLQSAFFKYEEDSYNRLKSLIEQCSAP--LPPSIFMELANKIYKRRK VARVKALYEEMNLSAVYMQYEEDSYNHIMGLIEQYAAP--LPPAIFLGLAQKIYKRKK VAKVKALYKELDLEGAFMEYEKESYEKLTKLIEAHQSK--AIQAVLKSFLAKIYKRQK IAVIKQLYHELQIPELYHKFEDDMVDSISKEIDLIDESTGLKKCIFTKFFQLIYKRSR Tbhmgr, tbfpps and tbsqs expression analysis in the vegetative phase and during ascoma ripening: a quantitative real-time assay The expression patterns in the vegetative phase and during the complex process of T. borchii ascoma ripening were analysed for the tbhmgr, tbfpps and tbsqs genes. Total RNAs extracted from 30-day-old mycelia and ascomata of three diVerent maturation degrees (immature, intermediate and mature) were used to synthesise cDNA and to set up quantitative real-time assays. To avoid genomic ampliWcation, speciWc primers were designed to include splice junctions. The per- 123 347 347 347 352 353 353 342 351 formance of tbhmgr, tbfpps and tbsqs assays were characterised in control experiments over a range of template concentrations, from 1:5 to 1:500 dilutions of the starting cDNA. The amount of the target transcript was related to that of the reference gene (see Materials and Methods). Figure 4a–c shows real-time PCR results: all the three genes are up-regulated in mature fruit bodies, respect to the immature ones and to the mycelium. In particular, tbhmgr, tbfpps and tbsqs gene expression was about 4-fold higher, 10-fold higher and 4-fold higher, respectively, in mature ascomata with respect to the immature ones. Curr Genet (2006) 50:393–404 Fig. 3 Alignment and comparison of the deduced amino acid sequence of tbsqs gene with SQS from: S.c. Saccharomyces cerevisiae (A34441), P.j. Pichia jadinii (spO74165), C.a. Candida albicans (EAK95451.1), T.b. Tuber borchii, S.p. Schizosaccharomyces pombe (NP_595363.1), S.t. Solanum tuberosum (BAA82093.1). The squalene and phytoene sequence motifs are evidenced; the aspartate-rich domains are in bold and underlined; plus indicates the important residues of tyrosin, phenilalanin and glutamine; the TBSQS putative membrane-spanning domain (aa293–aa313) is in bold 401 S.c. P.j. C.a. T.b. S.p. S.t. --MGKLLQLALHPVEMKAALKLKFCRTPLFSIYDQSTSPYLLHCFELLNLTSRSFAAVIR --MGKLLQLALHPDELASIVQFKLFRKNENARNPATESAELIRCYELLNLTSRSFAAVIE --MGKFLQLLSHPTELKAVIQLFGFRQPLHPGKRDVNDKELVRCYELLNLTSRSFAAVIE MKTSDILYLLAHPNQLRAIIQWKIWHNPLHERNIEKESPTLRRCFEFLDLTSRSFSAVIQ ------MSLANRIEEIRCLCQYKLWNDLPSYGEDENVPQNIRRCYQLLDMTSRSFAVVIK --MGTLRAILKNPDDLYPLIKLKLAAR--HAEKQIPPEPHWGFCYLMLQKVSRSFALVIQ 58 58 58 60 54 56 S.c. P.j. C.a. T.b. S.p. S.t. ELHPELRNCVTLFYLILRALDTIEDDMSIEHDLKIDLLRHFHEKLLLTKWSFDGNAPDVK ELHPELRNVIMVFYLVLRALDTVEVDMSIENSVKLPVLRQFHEKLDTKDWTFDGNSPNEK ELHPELRDAVMIFYLVLRALDTIEDDMTIKSSIKIPLLREFDTKLNTKNWTFDGNGPNEK ELCPELCVSVALFYLILRGLDTIEDDMTIPLGIKEPLLRSFDEILEKEGWSFDGNGPKEK ELPNGIREAVMIFYLVLRGLDTVEDDMTLPLDKKLPILRDFYKTIEVEGWTFNESGPNEK QLPVELRDAVCIFYLVLRALDTVEDDTSIPTDVKVPILISFHQHVYDREWHFACG--TKE 118 118 118 120 114 114 S.c. P.j. C.a. T.b. S.p. S.t. DRAVLTDFESILIEFHKLKPEYQEVIKEITEKMGNGMADYILDENYN-LNGLQTVHDYDV DRCVLVEFDRILGQYHELKPQYQKVIKEITEKMGNGMADYIENENFN-SNGLLTIEDYDL DRTVLVEFDKILNVYHRLKPQYQDIIKSITFKMGNGMADYILDEEFN-VNGVATVEDYNL DRQLLVEFYTVIKEFALLPESHRVIIKDITKKMGNGMADYANNAEHN-VNGVNTVADYDL DRQLLVEFDVVIKEYLNLSEGYRNVISNITKEMGDGMAYYASLAEKNDGFSVETIEDFNK YKVLMDQFHHVSTAFLELGKLYQQAIEDITMRMGAGMAKFICK-------EVETTDDYDE 177 177 177 179 174 167 S.c. P.j. C.a. T.b. S.p. S.t. + YCHYVAGLVGDGLTRLIVIAKFANESLYSN-EQLYESMGLFLQKTNIIRDYNEDLVDG-YCYYVAGLVGDGLTQLIVLAKFGNSELSVN-KQLFKSMGLFLQKTNIIRDYEEDQVDG-YCHYVAGLVGEGLTNLFVLANFGDKTLTENNFAKADSMGLFLQKTNIIRDYHEDLQDG-YCYYVAGLVGDGLTRLFVDTGKANPALLER-PHLINSMGLFLQKTNIIRDYREDLDDK-YCHYVAGLVGIGLSRLFAQSKLEDPDLAHS-QAISNSLGLFLQKVNIIRDYREDFDDN-YCHYVAGLVGLGLSKLFHAS--GTEDLASD--SLSNSMGLFLQKTNIIRDYLEDINEVPK 234 234 235 236 231 223 S.c. P.j. C.a. T.b. S.p. S.t. -RSFWPKEIWSQYAPQLKDFMK---PENEQLGLDCINHLVLNALSHVIDVLTYLAGIHEQ -RAFWPKEIWGKYANELSDFMK---PENQSQGLWCISELVCNALDHVIDVLQYLALVEEQ -RSFWPREIWSKYTENLQDFHKVKTPAKEFAGVSCINELVLNALGHVTDCLDYLSLVKDP -RRFWPKEIWTKHIEKYEELPL---PENEEAALNCISEMCLNSLQHADECLFYLAGIKDQ -RHFWPREIWSKYTSSFGDLCL---PDNSEKALECLSDMTANALTHATDALVYLSQLKTQ CRMFWPREIWSKYVNKLEDLKY---EENSVKAVQCLNEMVTNALSHVEDCLTYMFNLRDP 290 290 294 292 287 280 S.c. P.j. C.a. T.b. S.p. S.t. + + STFQFCAIPQVMAIATLALVFNNREVLHGNVKIRKGTTCYLILKSRTLRGCVEIFDYYLR TSFNFCAIPQVMAIATLELVFQNPQVLTQHVKIRKGTTVSLILESRTLEGCARIFRRYLR SSFSFCAIPQVMAVATLAEVYNNPKVLHGVVKIRKGTTCRLILESRTLPGVVKIFKEYIQ SVFNFTAIPQVMAIATLALVFRNKDVFQKNVKIPKGEACELMIEVGNLRSTCDIFRKYMK EIFNFCAIPQVMAIATLAAVFRNPDVFQTNVKIRKGQAVQIILHSVNLKNVCDLFLRYTR SIFRFCAIPQVMAIGTLAMCYDNIEVFRGVVKMRRGLTAKVIDRTKTMADVYGAFFDFSC 350 350 354 352 347 340 S.c. P.j. C.a. T.b. S.p. S.t. DIKSKLAVQDPNFLKLNIQISKIEQFMEEMYQDK--------LPPNVKPNETPIFLKVKE KIHHKSHPSDPNYLRLGITIGKIEQFLDGMYPHY--------VPKGITPQTTSIRTQVVK VINHKSSVRDPNYLKIGIKCGEIEQYCEMIYPNKQ------ALPPSMKSLPENKFTKIVA IIHKKNTPKDPNYLNISVTLGKVEQFIESIFPTP------------VEPKAVAARAQMRA DIHYKNTPKDPNFLKISIECGKIEQVSESLFPRRFREMYEKAYVSKLSEQKKGNGTQKAI MLKSKVNNNDPNATKTLKRLDAILKTCR----------------------DSGTLNKRKS 402 402 408 400 407 378 S.c. P.j. C.a. T.b. S.p. S.t. -RSRYDDELVPTQQEEEYKFNMVLSIILSVLLGFYYIYTLHRA----------RLQLDEPMKRDIDEEILKTRILLLSLGVAVFG--VVYGVVRII--------SRESIDLSVQRRIEQENFNCNVVLFGIGALILS--LIYFVLY----------TGETPEQAAAAKKRRCS-----------------------------------LNDEQKELYRKDLQKLGISILFVFFIILVCLAVIFYVFNIRIHWSDFKELNLF YIIRSEPNYSPVLIVVIFIILAIILAQLSGNRS-------------------- TBHMGR activity during Tuber borchii ascoma ripening In a previous work we proposed and validated a novel and simple HPLC method for determination of 3hydroxy-3-methylglutaryl-CoA reductase activity (BuValini et al. 2005). According to this method limited time of analysis and smaller amount of initial material are required because 15–20 min long HPLC runs employing a size-exclusion/ion-exchange column, are used for the detection of mevalonolactone, the 444 443 448 417 460 411 indirect product of HMGR enzyme activity, as well as none microsomal procedure is required. 30-day-old mycelial tissue was previously analysed, revealing that TBHMGR activity is 760 pmol mg¡1 of protein min¡1. Here, the enzymatic activity of TBHMGR was evaluated in the ascomata during their maturation process. The T. borchii HMGR enzyme activity shows the following speciWc values: 960 pmol mg¡1 of protein min¡1 (unripe ascomata); 400 pmol mg¡1 of protein min¡1 (intermediate ascomata); 3,300 pmol mg¡1 of protein min¡1 (ripe ascomata) (Fig. 4d). 123 402 Curr Genet (2006) 50:393–404 6 5 A 4 3 2 1 0 m 14 12 1 2 3 B 10 8 6 4 2 0 6 m 1 2 m 1 2 3 C 5 4 3 2 1 0 4000 3500 3000 2500 2000 1500 1000 500 0 3 D 1 2 3 Fig. 4 a–c Real-time PCR quantiWcation in Tuber borchii mycelia (m) and during ascoma development (1 immature ascomata, 2 intermediate ascomata, 3 mature ascomata) a Expression levels of tbhmgr, b expression levels of tbfpps, c expression levels of tbsqs, d Tuber borchii HMGR enzyme activity during ascoma development Discussion Studies on truZe biology are diYcult because, unlike other Wlamentous fungi such as Pisolithus tinctorius, their mycelia grow very slowly in vitro and it is diYcult (or impossible for some species) to obtain mycorrhizae or ascomata under axenic and controlled conditions. Mycelial T. borchii strains, the species addressed in this study, can be propagated in vitro; furthermore, in vitro T. borchii ectomycorrhizae and a productive truZe orchard where the ascomata can be collected are available (Saltarelli et al. 1998; Zambonelli et al. 2000; Sisti et al. 2003). For these reasons, T. borchii life cycle is started to be analysed by molecular tools in the last 123 years; nevertheless, the molecular mechanisms controlling its complex ontogenetic cycle remain still unknown. In this paper the cloning and characterisation of three T. borchii genes, involved in an important metabolic pathway, allowed to widen the information regarding the genomics of this organism and are necessary for the further advancement of Tuber functional genomics. For instance, the availability of the tbhmgr promoter region will represent the basis for future analyses to identify the transcription regulatory elements which lead to the high expression of the gene in the mature ascomata. In addition, the knowledge of the complete sequences of tbhmgr, tbfpps and tbsqs, allowed us to analyse and compare their putative deduced amino acid sequences with other homologous proteins present in databases. The tbhmgr deduced amino acid sequence, like all the known HMGR, from bacteria to eukaryotes, contains two domains; whereas, the catalytic domain is highly conserved, the hydrophobic one presents a variable number of transmembrane segments: in particular, TBHMGR contains eight transmembrane helices, like other fungi, such as Phycomyces blakesleeanus, Ustilago maydis and Schizosaccharomyces pombe. It is known that HMGR is among the most tightly regulated enzymes in nature (Goldestein and Brown 1990); also the T. borchii HMGR may be regulated at a post-translational level: it contains a SSD, which is conserved across phyla and confers sensitivity to regulation by sterols. The similarity within the catalytic domain of TBHMGR to HMGR from a wide range of organisms is very high (e-value: 5e¡176), indicating the high-sequence conservation of this enzyme during evolution, which may be associated with substrate and/or co-factor binding sites or catalytic activity. Also the tbfpps and tbsqs deduced amino acid sequences show the speciWc domains and structures of the known FPPS and SQS, respectively, conWrming their highly conserved character already shown in other ascomycetes fungi (Homann et al. 1996). Recently, it was found that genes for metabolic pathway are clustered not only in prokaryotic organisms but also in Wlamentous fungi (Keller and Hohn 1997; Tudzynnski and Holter 1998). Nevertheless, Southern blot analyses revealed that the three isoprenoids biosynthesis T. borchii genes do not hybridise to a fragment of the same size when cut with the same restriction enzyme, so probably they are not clustered. Furthermore, the three genes are single copy genes in the T. borchii genome; hmgr is a single copy gene in several other fungi, such as Fusarium graminearum, N. crassa, Aspergillus nidulans and Magnaporte grisea Curr Genet (2006) 50:393–404 (Seong et al. 2006) and all analysed fungi contain a single copy both of the fpps and of the sqs gene, in contrast to animals (Homann et al. 1996). The isoprenoid pathway is involved in the biosynthesis of many primary and secondary metabolites; the cloning of tbhmgr, tbfpps and tbsqs, coding for the three most important regulatory enzymes of isoprenoids biosynthesis, has allowed not only to analyse their genetic organisation and the characteristics of their deduced amino acid sequences, but also to obtain new information regarding this important metabolic pathway in Tuber, in particular during the complex process of ascoma maturation. HMGR and FPPS genes were constitutively expressed in the ascomycetes fungus G. fujikuroi (Homann et al. 1996; Woitek et al. 1997; Tudzynski 2005). Instead, in T. borchii, quantitative Real-time analysis revealed diVerent expression levels, depending on the phase of the life cycle (vegetative or fruiting) and on the ascomata maturation degree. A previous study highlighted the presence of isoprenoids in mature ascomata, but a non-quantitative approach was used (Zeppa et al. 2004); some genes involved in the isoprenoid synthesis are identiWed (Gabella et al. 2005); nevertheless, no information was available regarding the genes coding for the three most important regulatory enzymes of the isoprenoid biosynthesis in Tuber. Here the quantitative Real-time analysis, evaluating not only unripe and ripe fruit body, but also the intermediate ones, highlighted a signiWcative increase in the expression of the three genes during the maturation process, providing a real molecular conWrmation of the isoprenoids production in the T. borchii ascomata. The expression in the mycelium showed values similar to those obtained in the unripe ascomata; accordingly, the TBHMGR activity in the vegetative phase, 760 pmol mg¡1 of protein min¡1 (BuValini et al. 2005) and in the unripe ascomata (960 pmol mg¡1 of protein min¡1, this work) was similar. As reported for the tbhmgr gene expression, HMGR enzyme activity is found to be up-regulated in the ripe ascomata, with respect to the intermediate and the unripe ones. In particular, a lower value is found in the intermediate stage respect to the unripe ones, whereas, a signiWcant increase of the enzymatic activity occurred in the Wnal phase of the maturation process. This trend may be explained as follows: the initial phases of fructiWcation are strictly associated with high-metabolic activity due to the fact that spores form and diVerentiate thus requiring the synthesis of cell walls, membranes and energy supply. Then, in the central phases of the maturation process, the HMGR enzyme activity markedly decreased as the major part of spores are already formed. Finally, in the last phase 403 of fructiWcation the enzyme activity reached a value 3-fold higher respect to that found in the initial phases of the process, conWrming the importance of terpenic compounds, and thus of HMGR enzyme, in mature fruit bodies. These Wndings imply that isoprenoids play a fundamental role in Tuber ascocarps, particularly in the last phases of their maturation, when they could be involved in antifungal or/and antimicrobial processes and contribute to the famous Xavour of the truZe ascomata. In conclusion, the isolation of the T. borchii isoprenoid biosynthetic genes, their sequence and expression analysis represent important steps towards the understanding of the molecular processes of the isoprenoid metabolism in the ectomycorrhizal fungi of the Tuber genus. References Bach TJ (1995) Some new aspects of isoprenoid biosynthesis in plants: a review. Lipids 30:191–202 Barkovich R, Liao CJ (2001) Metabolic engineering of isoprenoids. Metab Eng 3:27–39 Bon E, Casaregola S, Blandin G, Llorente B, Neuveglise C, Munsterkotter M, Guldener U, Mewes HW, Van Helden J, Dujon B, Gaillardin C (2003) Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns. Nucleic Acids Res 31:1121–1135 Breathnach R, Benoist C, O’Hare K, Gannon F, Chambon P (1978) Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci USA 75:4853–4857 BuValini M, Pierleoni R, Guidi C, Ceccaroli P, Saltarelli R, Vallorani L, Zeppa S, Stocchi V (2005) Novel and simple highperformance liquid chromatographic method for determination of 3-hydroxy-3-methylgltaryl-coenzyme a reductase activity. J Chromatogr B Anal Technol Biomed Life Sci 819:307–313 Cunillera N, Arro M, Delourme D, Karst F, Boronat A, Ferrer A (1996) Arabidopsis thaliana contains two diVerentially expressed farnesyl-diphosphate synthase genes. J Biol Chem 271:7774–7780 Enjuto M, Balcells L, Campos N, Caelles C, Arro M, Boronat A (1994) Arabidopsis thaliana contains two diVerentially expressed 3- hydroxy-3-methylglutaril-CoA reductase genes, which encode microsomal forms of the enzyme. Proc Natl Acad Sci USA 91:927–931 Erland S, Henrion B, Martin F, Glover LA, Alexander IJ (1993) IdentiWcation of the ectomycorrhizal basidiomycete Tylospora Wbrillosa by RFLP analysis of the PCR-ampliWed ITS and IGS regions of the ribosomal DNA. New Phytol 126:525–532 Gabella S, Abba S, Duplessis S, Montanini B, Martin F, Bonfante P (2005) Transcript proWling reveals novel marker genes involved in fruiting body formation in Tuber borchii. Eukaryot Cell 4:1599–1602 Goldestein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425–430 Guescini M, Pierleoni R, Palma F, Zeppa S, Vallorani L, Potenza L, Sacconi C, Giomaro G, Stocchi V (2003) Characterization 123 404 of the Tuber borchii nitrate reductase gene and its role in ectomychorrizae. Mol Genet Genomics 269:807–816 Homann V, Mende K, Arntz C, Ilardi V, Macino G, Morelli G, Bose G, Tudzynski B (1996) The isoprenoid pathway: cloning and characterization of fungal FPPS genes. Curr Genet 30:232–239 Karst F, Plochocka D, Meyer S, Szkopinska A (2004) Farnesyl diphosphate synthase activity aVects ergosterol level and proliferation of yeast Saccharomyces cerevisae. Cell Biol Int 28:193–197 Keller NP, Hohn TM (1997) Metabolic pathway gene cluster in Wlamentous fungi. Fungal Genet Biol 21:17–29 Kinghorn JR (1987) Gene structure in eukaryotic microbes. IRL Press, Oxford, pp 93–99 Kozak M (1981) Mechanisms of mRNA recognition by eukaryotic ribosomes during initiation of protein synthesis. Curr Top Microbiol Immunol 93:81–123 Kudla B, Caddick MX, Langdon T, Martinez-Rossi NM, Bennett CF, Sibley S, Davies RW, Arst HN Jr (1990) The regulatory gene are a mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations aVecting speciWcity of gene activation alter a loop residue of a putative zinc Wnger. EMBO J 9:1355–1364 Lacourt I, Duplessis S, Abba S, Bonfante P, Martin F (2002) Isolation and characterization of diVerentially expressed genes in the mycelium and fruit body of Tuber borchii. Appl Environ Microbiol 68:4574–4582 Lowry OH, Rosebrough AL, Farr RJ, Randall J (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275 McGarvey DJ, Croteau R (1995) Terpenoid metabolism. Plant Cell 7:1015–1026 PfaZ MW (2001) A new mathematical model for relative quantiWcation in real-time RT-PCR. Nucleic Acids Res 29:2002– 2007 Pierleoni R, BuValini M, Vallorani L, Guidi C, Zeppa S, Sacconi C, Pucci P, Amoresano A, Casbarra A, Stocchi V (2004) Tuber borchii fruit body: 2-dimensional proWle and protein identiWcation. Phytochemistry 65:813–820 Saltarelli R, Ceccaroli P, Vallorani L, Zambonelli A, Citterio B, Malatesta M, Stocchi V (1998) Biochemical and morphological modiWcations during the growth of Tuber borchii mycelium. Mycol Res 102:403–409 Sambrook J, Fritsch EF, Maniatis TA (1989) Molecular cloning: a laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold Springer Harbor, NY 123 Curr Genet (2006) 50:393–404 Sato R, Goldstein JL, Brown MS (1993) Replacement of serine871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion. Proc Natl Acad Sci USA 90:9261–9265 Seong K, Li L, Hou Z, Tracy M, Kistler HB, Xu JR (2006) Cryptic promoter activity in the coding region of the HMG-CoA reductase gene in Fusarium graminearum. Fungal Genet Biol 43:34–41 Sisti D, Giomaro G, Cecchini M, Faccio A, Novero M, Bonfante P (2003) Two genetically related strains of Tuber borchii produce Tilia mycorrhizas with diVerent morphological traits. Mycorrhiza 13:107–115 Szkopinska A, Swiezewska E, Karst F (2000) The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 267:473–477 Tansey TR, Shechter I (2000) Structure and regulation of mammalian squalene synthase. Biochim Biophys Acta 1529:49–62 Tudzynski B, Holter K (1998) Gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence for a gene cluster. Fungal Genet Biol 25:157–170 Tudzynski B (2005) Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology. Appl Microbiol Biotechnol 66:597–611 Woitek S, Unkles SE, Kinghorn JR, Tudzynski B (1997) 3-hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterisation. Curr Genet 31:38– 47 Zambonelli A, Iotti M, Rossi I, Hall I (2000) Interaction between Tuber borchii and other ectomycorrhizal fungi in a Weld plantation. Mycol Res 104:698–702 Zeppa S, Guidi C, Zambonelli A, Potenza L, Vallorani L, Pierleoni R, Sacconi C, Stocchi V (2002) IdentiWcation of putative genes involved in the development of Tuber borchiii fruit body by mRNA diVerential display in agarose gel. Curr Genet 42:161–168 Zeppa S, Gioacchini AM, Guidi C, Guescini M, Pierleoni R, Zambonelli A, Stocchi V (2004) Determination of speciWc volatile organic compounds synthesised during Tuber borchii fruit body development by solid-phase microextraction and gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 18:199–205