Plant Physiology and Biochemistry 44 (2006) 506–510 www.elsevier.com/locate/plaphy Short communication Molecular characterisation of a Tuber borchii Smt3 gene S. Zeppaa,*, C. Guidib, E. Barbieria, M. Guescinib, E. Polidoria, D. Agostinib, V. Stocchia,b,* b a Istituto di Ricerca sull’Attività Motoria, Università degli Studi di Urbino "Carlo Bo", via I Maggetti, 26, 61029 Urbino (PU), Italy Istituto di Chimica Biologica “Giorgio Fornaini”, Università degli Studi di Urbino “Carlo Bo”, via A. Saffi, 2, 61029 Urbino (PU), Italy Received 11 October 2005; accepted 6 July 2006 Available online 22 August 2006 Abstract Tbsmt3 gene from the ectomychorrizal fungus Tuber borchii was identified and sequenced. The Tbsmt3 gene encodes for a protein sharing significant amino acid homology with the yeast SMT3, a ubiquitin-like protein that is post-translationally attached to several proteins involved in many cellular processes. The comparison between the Tbsmt3 genomic and cDNA sequences established that the encoding sequence is interrupted by an intron of 312 bp. Southern blot analysis revealed only one copy of Tbsmt3 gene in the T. borchii genome. Tbsmt3 is expressed in all phases of T. borchii life cycle: mycelium, ectomycorrhiza and ascoma. However, the Tbsmt3 mRNA decreased during fruit body maturation. © 2006 Elsevier Masson SAS. All rights reserved. Keywords: Tuber borchii; Ectomycorrhizal fungi; Ubiquitin-like proteins 1. Introduction The ubiquitin system plays a central role in many biological regulatory mechanisms, including aspects of signal transduction, cell cycle progression, differentiation and cell response [1,2]. Small ubiquitin-related modifier (SUMO) or SMT3 in yeast is a ubiquitin-like proteins (Ubls) that has important roles in many organisms. As in ubiquitinylation, sumoylation involves the covalent attachment of SUMO to multiple proteins in vivo. However, conjugation of SUMO does not typically lead to degradation of the substrate and instead has a more diverse array of effects on substrate function. While the molecular mechanisms by which sumoylation targets protein localisation are still poorly understood, it is clear that this modification system is an important regulator of intracellular protein localisation, particularly involving nuclear uptake and intranuclear accumulation. SUMO regulates nuclear transport, stress signal transduction in eukaryotes and is essential for cell-cycle progression in yeast [3–5]. In this organism a wide variety of SUMO system substrates have been identified which have been subdivided in dis- tinct functional clusters: stress related proteins, chromatin and genome stability related proteins such as sumoylated topoisomerase II which may contribute to the cohesive properties of the centromere, transcription and translation related proteins, RNA metabolism and metabolic enzymes, among these the majority are glycolytic ones [5–8]. In Schizosaccharomyces pombe the disruption of the pmt3+ gene, homologue of smt3, was not lethal, but mutant cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Furthermore, the loss of Pmt3p function caused a striking increase in telomere length, suggesting that pmt3+ is required for telomere length maintenance [9]. In this paper we reported the cloning of Tbsmt3 cDNA, mRNA expression, and characterisation of genomic DNA from Tuber borchii Vittad., an ascomycetous fungus which produces edible fruit bodies, commonly called truffle, as a result of a mutualistic symbiosis with the roots of some tree species [10]. 2. Material and methods 2.1. Mycelial strain, fruit bodies and ectomycorrhizae * Corresponding author. Tel.: +39 0722 30 5262; fax: +39 0722 32 0188. E-mail addresses: s.zeppa@uniurb.it (S. Zeppa), v.stocchi@uniurb.it (V. Stocchi). 0981-9428/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2006.08.003 A mycelial strain designated 1BO (ATCC 96540) was isolated from a T. borchii fruit body and grown as previously S. Zeppa et al. / Plant Physiology and Biochemistry 44 (2006) 506–510 described in Zeppa et al. [11]. T. borchii fruit bodies were collected in northern and central Italy and harvested in experimental truffle orchards. The ascocarps were identified by morphological and molecular methods [10,12], and subdivided into six different stages of maturation as previously described [11]. Tilia platyphyllos–T. borchii ectomycorrhizae were synthesised using mycelium strain 1BO and host plants micropropagated in vitro using the method set up by Sisti et al. [13]. 2.2. DNA and RNA isolation Genomic DNA of 1-month-old cultures of T. borchii mycelium was isolated following the protocol described by Erland et al. [14]. Total cellular RNA was isolated from 1-month-old mycelium 1BO, fruit bodies at six stages of maturation, T. platyphyllos roots and T. platyphyllos–T. borchii ectomycorrhizae as previously described in Zeppa et al. [11]. 2.3. cDNA library screening A λZap cDNA library (Stratagene) of 30-day-old mycelium was screened using VC13 cDNA obtained in a previous work, as a probe [11]. Approximately, 3 × 105 plaques were analysed following the manufacturer’s instructions. Two positive phages plaques were selected, rescreened until plaque-pure and then converted into pBlueScript II SK- clones by in vivo rescue [15]. Final wash conditions were 1X SSC and 0.1% SDS at 60 °C. These cDNA clones were sequenced in both directions automatically using an ABI 373S System, and both were found to correspond to the same gene. 2.4. Detection of introns In order to characterise the introns, two specific oligonucleotides S1 (5′-CCATCACGTCAACCACTAC-3′) and S2 (5′-GAGGCAGCTTGGAAGTGGTG-3′) were designed, respectively, at 5′ and 3′ untranslated region of the cDNA sequence. The amplification reaction was carried out in a total volume of 25 μl with 200 ng of genomic DNA, reaction buffer 1X, 1.5 mM of MgCl2, 100 μM dNTPs, 10 pmol of each primer and 0.5 Units of AmpliTaq DNA polymerase (Perkin Elmer, CA). After an initial denaturing step at 94 °C for 5 min, 30 cycles of 30 s at 94 °C, 1 min at 55 °C and 2 min and 30 s at 72 °C were performed followed by a final incubation of 7 min at 72 °C. PCR-amplified product was cloned using the TA Cloning Kit (Invitrogen) and sequenced in both directions using the M13 universal reverse and forward primers. We compared the sequences against the nonredundant databases of the National Centre for Biotechnology Information, (Bethesda, MD) with the Internet BLAST server. The sequence was named Tbsmt3 and deposited in GenBank under the accession no. DQ114472. 507 2.5. Virtual Northern, Northern and Southern blots For Northern blots, 15 μg of total RNA were loaded on a formaldehyde 1.2% agarose gel and electrophoresed [16]. 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 dH2O. One micro-gram of total RNA extracted from T. platyphyllos–T. borchii ectomycorrhizae, non-inoculated T. platyphyllos roots and free-living T. borchii mycelium, was utilised for cDNA synthesis and Virtual Northern blotting as reported in Polidori et al. [17]. For Southern blot analysis, 10 μg of genomic DNA from T. borchii mycelia were digested with the enzymes EcoRI and PstI and electrophoresed on a 0.8% agarose gel. Both RNA and DNA were blotted onto version 2.0 Hybond-N+ positively charged nylon membranes (Amersham, Life Science) in accordance with the manufacturer’s instructions and hybridised in phosphate buffer [16] with Tbsmt3, which was 32P-labelled using the RediPrime labelling kit (Amersham, Life Science). Final wash conditions were 0.1X SSC and 0.1% SDS at 65 °C. 3. Results and discussion In a previous study using mRNA differential display in agarose gel we have identified several differentially expressed cDNAs in the profiles of T. borchii unripe and ripe fruit bodies [11]. A cDNA (VC13), encoding for a part of the Tbsmt3 gene, was used as a probe for the screening of a cDNA library of T. borchii 30-day-old mycelium. Two positive cDNA clones were selected from the screening. The sequence analysis led us to confirm that they were the same clone, containing a small ORF of 291 bp in length, which encodes a putative protein of 97 amino acids with a significant homology (54% identity, 73% similarity) with the Saccharomyces cerevisiae SMT3 protein (GenBank Accession no. Q12306). Based on this finding this novel cDNA sequence was termed Tbsmt3 (GenBank Accession no. DQ114472). Two primers, S1 and S2, were designated at 5′- and 3′-end, respectively, to amplify the entire gene. The sequence analysis of the products obtained showed the presence of a single intron, 312 bp in length, at the nucleotide position 226, with respect to the ATG start codon. In this intron the dinucleotides GT and AG occur at the 5′- and 3′-end, respectively, in common with almost all eukaryotic genes [18]. This intron presents a 3′ splice site terminating in TAG (YAG) and a 5′ splice site GTAGGT, resembling the GTANGT which is very common in filamentous fungi. The sequence analysis revealed that the intron is of type III (inserted after the third nucleotide in a codon). In the sequence surrounding the ATG start codon the -3 position is A, in agreement with the Kozak consensus sequence [19]. The 265 nucleotides in the 3′-UTR region of the Tbsmt3 gene was investigated and no polyadenilation consensus 508 S. Zeppa et al. / Plant Physiology and Biochemistry 44 (2006) 506–510 Fig. 1. Alignment of the deduced TBSMT3 amino acid sequence and its orthologues in Ascomycota fungi: Ashbya gossypii AAS54069; Aspergillus fumigatus EAL90412; Aspergillus nidulans EAA65784; Botrytis cinerea AL115316; Candida albicans EAK94742; Candida glabrata CAG61436; Debaryomyces hansenii CAG88153; Giberella zeae EAA70631; Kluyveromyces lactis CAH01119; Magnaporthe grisea EAA549461; Neurospora crassa XP330463; S. cerevisiae AAB01675; S. pombe CAB44758. The putative ubiquitin-like domain and the Gly–Gly dipeptide (^^) are evidenced. sequence (AAUAA) was revealed. The consensus, or a similar sequence, does not appear in many other fungal genes, therefore its functional significance in fungal genes is uncertain at the present time. The TBSMT3 encoded-protein is 97 amino acids in length and was analysed using the ExPASy programme, ProtParam [20], revealing a deduced molecular mass of 10,728 kDa, a theoretical isoelectric point of pI = 4.77 and an instability index which classifies this protein as unstable. Furthermore, TBSMT3, like the yeast SMT3 protein, is a small hydrophilic protein. Ubiquitin and Ubls are synthesised in a precursor form, with one or more amino acids following a Gly–Gly dipeptide that will form the mature C-terminus which is required for efficient conjugation [21]. The comparison with the SMT3 orthologues showed the presence of a putative Gly–Gly dipeptide also in TBSMT3 (Fig. 1). The carboxyl group of the C-terminal glycine residue of the Ub-like proteins covalently binds to the εamino group of an internal lysine residue of receptor proteins, like the ubiquitin conjugation [22]. Furthermore, a putative ubiquitin-like domain is present in TBSMT3 from the 17 to 93 amino acid residue as shown in Fig. 1B. In order to investigate the genomic organisation, Southern blot analysis was performed. T. borchii genomic DNA was digested with restriction enzymes PstI and EcoRI, which did not have any sites in the Tbsmt3 sequence, and hybridised with this gene. Under stringent conditions (SSC 0.1X, SDS 0.1%), the coding sequence probe produces only one hybridisation signal in the PstI- and EcoRI-digested DNA, indicating the presence of a single copy gene of Tbsmt3 in T. borchii genome (Fig. 2). Expression analyses of this gene were carried out during the all phases of T. borchii life cycle. The fruiting of ectomycorrhizal Tuber depends on a complex set of variables, including metabolites and signals produced by the host plant, the nutritional status of the substrates and unknown environmental cues [23]. Since truffle fruit bodies cannot yet be obtained under controlled conditions, our knowledge of the morphogenetic events leading to asco- Fig. 2. Southern blot analysis. T. borchii genomic DNA was digested with PstI and EcoRI and probed with 32P-labelled Tbsmt3 gene. carp development and maturation [24], as well as their underlying molecular bases are still limited [11,24–26]. As the fruit body matures the specialised hyphae are differentiated in asci and meiosis and mitosis occur during spore formation. The molecular bases of such events are largely unknown, with the exception of some model fungi [27–29] in which a perfect cascade of developmentally modulated genes regulates sporulation. The characterisation of these genes during fruit body development is an initial step towards the understanding this complex mechanism. In order to evaluate also the Tbsmt3 transcript during the ascoma maturation Northern blot analysis using 15 μg of total RNA from each of six stages of fruit body maturation, was carried out. The result obtained shows an up-regulation of Tbsmt3 in the early stages of fruit body ripening (stage 1, 2, 3) (Fig. 3A), S. Zeppa et al. / Plant Physiology and Biochemistry 44 (2006) 506–510 509 Furthermore, we evaluated Tbsmt3 expression in the symbiotic phase of T. borchii life cycle. Due to the difficulty to obtain a large amount of RNA from ectomycorrhizae and uninfected roots a Virtually Northern blot experiment was performed [17], revealing that Tbsmt3 mRNA is also present in the mutualistic association (Fig. 3B). The role of the ubiquitin-like proteins as regulator of many cellular processes is confirmed by several and recent researches. How the SMT3 ligation contributes to these different regulatory mechanisms remain an excitant topic. The herein reported data provide a characterisation of T. borchii Tbsmt3 and its expression through the various phases of the truffle life cycle, offering a starting point for further detailed studies on the processes in which TBSMT3 could be involved. Acknowledgements We thank Professor Alessandra Zambonelli from University of Bologna, Italy for providing truffle fruit bodies. Fig. 3A. Tbsmt3 expression during T. borchii fruit body ripening. 0: fruit body with 0% of asci containing mature spores; 1: fruit body with 5% of asci containing mature spores; 2: fruit body with 6–25% of asci containing mature spores; 3: fruit body with 26–50% of asci containing mature spores; 4: fruit body with 51–75% of asci containing mature spores; 5: fruit body with 76– 100% of asci containing mature spores. 18S: control hybridisation performed using T. borchii 18S rRNA as a probe. Fig. 3B. Virtual Northern blotting. 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