TMchs4, a class IV chitin synthase gene from the ectomycorrhizal Tuber magnatum

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Mycol. Res. 104 (6) : 703–707 (June 2000). Printed in the United Kingdom.

TMchs4, a class IV chitin synthase gene from the ectomycorrhizal Tuber magnatum

Lilia GARNERO1, Barbara LAZZARI2, Davide MAINIERI2, Angelo VIOTTI2 and Paola BONFANTE1 " Dipartimento di Biologia Vegetale – UniversitaZ di Torino and Centro di Studio sulla Micologia del Terreno – CNR, Viale P.A. Mattioli 25, 10125 Torino, Italy, and # Istituto Biosintesi Vegetali – CNR, Via Bassini 15, 20133 Milano, Italy. E.-mail : p. bonfante!csmt.to.cnr.it Received 3 June 1999 ; accepted 18 September 1999.

Chitin synthase genes of Tuber magnatum were sought in order to investigate the molecular bases of its growth. Primers designed from highly conserved Chs domains were used to identify a chs gene portion. A genomic library was used to obtain the full gene sequence (TMchs4) with an open reading frame which encodes a predicted protein of 1230 amino acids. The sequence is similar (62 %) to the class IV chitin synthase from Neurospora crassa. The putative protein shows hydrophobicity pattern that suggests the presence of several intramembranous domains and other peculiar features common to the other chs of class IV. Northern experiments demonstrated that the gene is expressed in ascomata sampled at different maturation steps. These data suggest that ascomata growth requires new chitin deposition, which is based on a chs gene activation and not only on an enzymatic activity.

INTRODUCTION Ectomycorrhizal fungi can establish a symbiosis with the roots of many woody plants and may improve their mineral nutrition, phosphorus and nitrogen, in exchange for photosynthate (Smith & Read 1997). Plant carbohydrates are required for the growth of the fungus during all the steps of its life cycle : extraradical mycelium, sporophores, ectomycorrhizas. The molecular mechanisms controlling morphogenesis are largely unknown, though events related to mycorrhizal formation are beginning to emerge (Barker, Tagu & Delp 1998). A number of genes involved in the fungal wall proteins are differentially regulated in the early contacts between plant and fungus (Martin, Lapeyrie & Tagu 1997). Chitin, the polymer of β-1,4 linked N-acetylglucosamine (GlcNAc), is a cell wall component common to both the vegetative and the reproductive structures of fungi. A study into chitin synthesis in T. magnatum could be used to elucidate both the growth and developmental processes occurring in this ectomycorrhizal fungus. The enzymes involved are the chitin synthases (Chs), namely membrane proteins catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into chitin chains (Cabib et al. 1996). To identify chitin synthase genes (chs) in the ectomycorrhizal Tuber magnatum, as genes representative of fungal growth, we used primers designed from highly conserved Chs domains (Bowen et al. 1992, Specht et al. 1996). A full length gene (TMchs4), was isolated from a genomic library. The gene has been demonstrated to belong to the class IV chitin synthase and to be expressed during ascomata development.

MATERIALS AND METHODS Isolation of nucleic acids, gel blot analyses and PCR reactions Thirty ascomata of Tuber magnatum collected near Monta' d’Alba, Piedmont, Italy, were washed, brushed, and peeled of their peridium. Their degree of maturation was evaluated on the ratio of immature and mature ascospores. Immature spores have no ornamentation, mature ones are yellow- to reddishbrown, and have a reticulated ornamentation (Pegler, Spooner & Young 1993). Four classes of maturation were identified (5, 10, 50 and 100 %) looking at hand sections observed under a light microscope at low magnification (objective i10). About 80–90 asci can be simultaneously observed. DNA was extracted by the method of Henrion, Le Tacon & Martin (1992). The precipitated DNA was resuspended in TE buffer (10 m Tris-HCl pH 8, 1 m EDTA). Southern blot experiments were performed by separating digested DNAs on 1 % agarose gels and transferring the fragments onto HybondNj membrane (Amersham). Total RNA was extracted, purified and then analysed on a 1 % formamide-formaldehyde agarose gel (Bernard, Ciceri & Viotti 1994). Probe labelling, hybridisation and post-hybridisation washes were carried out as described by Bernard et al. (1994). The degenerate primers P3–P4 (Specht et al. 1996) were used in PCR reactions carried out in a final vol. of 50 µl containing 10 m Tris-HCl pH 8n3, 50 m KCl, 1n5 m MgCl2, 0n2 m dNTPs, 100 pmol of each primer and 2n5 U of AmpliTaqGOLD DNA polymerase (Perkin–Elmer). Two concentrations (undiluted and 1\10) of genomic DNA were

Chitin synthase gene from Tuber magnatum tested. PCR amplifications were run in a Perkin–Elmer\Cetus DNA thermal cycler using the following parameters : 95 mC 10 min (1 cycle) ; 94m 1 min, 42m 1 min, 72m 1 min (35 cycles) ; 72m 5 min (1 cycle). Genomic clone isolation, cloning and sequence analysis A T. magnatum genomic library was constructed in the EMBL4\EcoRI lambda replacement vector. For screening, plaques were blotted on Hybond-Nj membrane. The hybridisations were performed with the amplified fragment (chs4 clone) as a probe labelled with the chemiluminescent detection system (ECL Direct DNA Labelling and Detection System, Amersham) according to the manufacturer’s recommendations. Single-plaque phage DNA was purified with a Lambda Mini Kit (Qiagen) and digested with EcoRI. The fragments were inserted in the EcoRI-digested and dephosphorylated pBluescript SK II vector. Fragments of interest of the PCR reactions were purified on agarose gel with the QIAEX II Gel Extraction Kit (Qiagen). The P3–P4 amplified fragment was treated with T4 DNA polymerase and T4 polynucleotide kinase (Promega), and ligated into SmaIdigested and dephosphorylated pBluescript SK II. XL2Blue ultracompetent cells (Stratagene) were transformed and plated on selective medium following the manufacturer’s instructions. Recombinant plasmid DNAs from amplified fragments or from genomic subcloning were prepared with the Plasmid Mini Kit (Qiagen) and the sequences were conducted by the Service de Se! quencage, Universite! Laval, Que! bec, Canada. DNA and protein sequence analyses were performed with the PC\gene software (IntelliGenetics Inc., Mountain View, CA, USA), the BLAST software available through the National Center for Biotechnology Information (NCBI) and the ExPASy Molecular Biology Server (SIB). RESULTS AND DISCUSSION Identification of the chs4 clone Amplification of the genomic DNA from T. magnatum with the primers P3–P4 gave a fragment of about 900 bp. After cloning the PCR product and sequencing several recombinant plasmids, a chs homolog was identified and named chs4. The clone was 800 bp long, excluding the primer sequences. The nucleotide sequence shows 85 % of homology to the corresponding fragment from chs4 of Neurospora crassa (Din et al. 1996). In agreement with the sequence and the restriction map of the fragment, the Southern experiment on genomic DNA with the chs4 probe revealed a single hybridising band in BamHI cut and two hybridising bands in both the EcoRI and the SalI digests (Fig. 1). Isolation of the complete gene sequence for a class IV chitin synthase Screening of about 120 000 plaques of the T. magnatum library with the chs4 fragment identified two putative positive clones. These were isolated as a single plaque and phage DNAs were analysed with the restriction enzyme EcoRI. Both clones

704 displayed an identical pattern with seven bands : the two vector arms and five fragments of 6800, 3800, 2000, 1200 and 500 bp derived from the insert. A Southern blot using the chs4 fragment as probe revealed two hybridisation bands corresponding to the 1200 and 500 bp fragments (data not shown). The five fragments were cloned into the pBluescript vector and sequenced with T3 and T7 primers. New sets of internal primers were then designed and used to obtain the complete clone sequences. The chs was confined within three fragments in order 3800, 500 and 1200 bp (Fig. 2). Restriction map analysis shows colinearity with the chs4 fragment and corroborates the hybridising pattern obtained in the Southern experiment. The continuous coding sequence of 3690 nucleotides (Accession no. AJ131706) compared to other published sequences shows the highest homology with the CACHS3 and chs-4 of Candida albicans and N. crassa (Sudoh et al. 1993, Din et al. 1996) respectively. These are both class IV chs genes. By contrast with chs-4, but similarly to CACHS3, this gene does not show any intron sequence (Sudoh et al. 1993, Din et al. 1996). One hundred and sixty two nucleotides downstream from the stop codon (TAG), a putative polyadenylation signal can be recognised in the motif ATTA ; whereas, in the upstream region none of the canonical CAAT or TATA motifs can be found at the conventional distances from the ATG start codon. The putative ORF, which encodes a predicted protein of 1230 amino acids, shows a low bias for codon usage, as all the 61 possible codons are used. The closest homology at the amino acid level was found with the class IV chs-4 polypeptide of N. crassa (62 %), plus a close homology with the Chs3 of S. cerevisiae and CAChs3 of C. albicans both belonging to the class IV type chitin synthase. A multiple alignment with several chitin synthase sequences led to a dendrogram where T. magnatum chs gene was placed in class IV Chs (Fig. 5). The gene was, therefore, named TMchs4. The protein sequence shows the following typical Chs motifs (Mort-Bontemps, Gay & Fe' vre 1997) : FEYXXSXXXXK (amino acids 910–921) and SWG (1142–1144). The putative catalytic amino acids indicated by Saxena, Lin & Brown 1990) as responsible for processivity in glycosyltransferases were also identified : D (984) and QXRRW (1022–1026). These motifs correspond to those supposed to act as catalytic residues in S. cerevisiae Chs2 (Nagahashi et al. 1995). The same residues have been found in NodC proteins, hyaluronan synthase, cellulose synthase and DG42 : all of them catalyse the synthesis of oligosaccharides with β-1,4 linkages using UDP-sugar as substrate (Nagahashi et al. 1995). It has been suggested that the residues identified in the potential catalytic sites of Chs2 of S. cerevisiae are conserved in glycosyltransferases that catalyse the synthesis of β-1,4 oligosaccharides. The hydropathic plot indicated the presence of four main regions (Fig. 3) : a hydrophilic domain in the N-terminal part (aa 1–200) followed by a highly hydrophobic region in which two primary transmembrane domains are revealed. The central neutral region contains, apart from the SWG, the typical enzymatic motifs of the Chs mentioned above. Lastly, within the hydrophobic C-terminal region three primary transmembrane motifs (aminoacids 1042–1133) precede the

Lilia Garnero and others

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Figs 1–4. Fig. 1. Southern blot of T. magnatum ascomata genomic DNA digested with restriction enzymes as reported at the top. The chs4 fragment was used as probe. Size markers are indicated on the left. Fig. 2. Restriction maps of the chs4 fragment (open line, 800 bp) and of the genomic fragments of the lambda clone containing the TMchs4 gene. Dotted and dashed lines represent the sequenced regions. Restriction sites as : B, BamHI ; E, EcoRI ; H, HindIII ; K, KpnI ; P, PstI ; S, SalI ; X, XhoI. Fig. 3. Transmembrane helix plot of the predicted amino acid sequence from TMchs4. Fig. 4. Schematic presentation and relative positions within the TMChs4 of the five transmembrane regions (vertical open bars) and the four major canonical amino acid regions characteristic of Chs4 activity (F–K, Q–W, SWG represented by vertical closed bars and the D amino acid arrow), for details see text.

SWG motifs (Fig. 4). This overall structure is common to the class IV Chs polypeptides from N. crassa, S. cerevisiae and C. albicans, and in agreement with reminiscence of their membrane association derived from specific endoproteolitic cleavage (Cabib et al. 1996). Preliminary data obtained with specific amino-terminal and carbossy-terminal antibodies to TMchs4 indicating the presence of TMChs4 peptide fragments (D. Mainieri & A. Viotti, unpublished) as for the Chs3 of S. cerevisiae (Santos & Snyder 1997), strongly suggest specific functions for the various regions of the TMChs4 polypeptide. Results of Northern analysis performed with a 5h portion of the full gene TMchs4 (Figs 6–7) indicate the occurrence of two main transcripts of about 5000 and 4000 nucleotides in ascocarps showing a different maturation degree (5–50 %

of mature spores). Even if a quantitative analysis was not performed, the hybridisation signals decrease in ascocarps with a higher proportion of mature spores. By contrast, Northern analysis of total RNA from T. borchii, a related white truffle species, indicates the occurrence of a single transcript of about 4000 nucleotides (data not shown). The hybridisation results suggest that in T. magnatum more than one transcription start and termination point are simultaneously used to specify TMchs4 expression. Mycorrhizal fungi and chitin synthases Different chs genes and activities have been found in a wide range of fungal species. In S. cerevisiae, there are three genes

Chitin synthase gene from Tuber magnatum

706 NCCHS1 ELMCHS1 GVCHS1 GVCHS2 LALCHS2 COOCHS2 TMCHS2 TBCHS2 TFCHS2 TUCHS2 LALCHS1 UMCHS1 COOCHS1 ANCHSB CHS3 NCCHS4 ANCHSE TMCHS4 ANCHSD

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Fig. 5. Dendrogram showing grouping of selected chitin synthases and their class designation. NC l Neurospora crassa ; ELM l Elaphomyces muricatus ; GV l Glomus versiforme ; LAL l Laccaria laccata ; COO l Cortinarius odorifer ; TM l Tuber magnatum ; TB l T. borchii ; TF l T. ferrugineum ; TU l T. uncinatum ; UM l Ustilago maydis ; AN l Aspergillus nidulans ; CHS3 l Saccharomyces cerevisiae Chs3. Classes are indicated on the right. 5%

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Figs 6–7. Northern blot analysis of total RNA from T. magnatum ascomata. Twenty-five µg of total RNA from ascomata at different spore-maturation stages, as reported at the top, were fractionated on a formamide, formaldehyde agarose gel. Fig. 6. The filter with the blotted RNA was hybridised with the 5h portion (980 BO XhoI–EcoRI fragment, see Fig. 2) of the TMchs4 gene. Fig. 7. The ethidium bromide staining of total RNA is reported as loading control. On the right the migration of the two TMchs4 transcripts is reported. Arrow heads indicate the migration of the two larger cytoplasmic ribosomal RNAs.

that are differently activated and play specific roles during the cell cycle (Cabib et al. 1996). Less is known about these specific functions in the filamentous fungi, though a similar situation can be envisaged, since four genes have been found in N. crassa (Bowen et al. 1992, Din et al. 1996), five in Aspergillus nidulans (Specht et al. 1996) and seven in A. fumigatus (Mellado et al. 1995), and chs redundancy probably reflects the complexity of their development. A set of chs genes are also present in Glomales, endomycorrhizal fungi that form symbioses with most land plants. PCR with degenerate

primers has revealed gene portions belonging to different classes in Glomus versiforme (Lanfranco, Garnero & Bonfante 1999) and Gigaspora margarita (Lanfranco, Vallino & Bonfante 1999). Identification of TMchs4, together with the cloning of different fragments of class II genes from four Tuber species (Mehmann et al. 1994, Lanfranco et al. 1995) and the recent recovery of a class III gene detected during the screening of a cDNA and genomic library of Tuber borchii (L. Garnero, unpublished), point to an analogous redundancy in truffles. Taken as a whole, these results demonstrate that Tuber species, like other mycorrhizal fungi, possess different chs genes. This raises many questions on the role played by their products, i.e. whether specific genes are activated during mycelial growth, ascomata production and interaction with the host plant. CONCLUSIONS TMchs4 is the first gene with a known function which has so far been characterised in T. magnatum. Both its deduced amino acid sequence and its hydrophobicity pattern show that TMchs4 is closest to the class IV. Class IV chs has been suggested to play different roles : in N. crassa, it is thought to serve as an auxiliary enzyme when additional chitin is necessary, and to be related to specific growth conditions determined by environmental changes (Din et al. 1996). Alternatively, chs4 has been suggested to be responsible for the synthesis of the major fraction of cell wall chitin (Xoconostle-Ca' zares et al. 1997). Two class IV chs of an arbuscular mycorrhizal fungus Gigaspora margarita have been found to be expressed during their interactions with the host plants and not during spore germination (Lanfranco et al. 1999). The authors speculated about the existence in the host plant of a control mechanisms for chitin synthesis. T. magnatum cannot be grown in pure culture, and its mycorrhizas are found to a very limited extent (Mello et al. 1999). TMchs4 expression can, therefore, be analysed exclusively in ascomata. The positive results from the Northern analysis performed on ascomata with a different maturation degree strongly support the hypothesis that growth of T. magnatum ascomata requires new chitin deposition, which is based on chs gene activation and not only on an enzymatic activity. This is not obvious, since it has been demonstrated that chs genes are expressed at a very low level (Gooday & Scofield 1995) and a lack of correlation between chs messages and Chs activity has been often reported in Saccharomyces (Cabib et al. 1996). This has been explained by the zymogenic nature of the enzyme, which requires post-translational modifications for its activation (Gooday & Scofield 1995, Cabib et al. 1996). By contrast, molecular data on chs expression during the fruitbody development of larger fungi are not available, even if the helical arrangement of chitin microfibrils has been considered a remarkable feature of Coprinus cinereus during stipe extension (quoted in Moore 1995). Tissue expansion plays, in fact, a central role in the development of primordia of some basidiomycetes into mature fruitbodies (Moore 1998). Some observations suggest that truffles develop from minute ascomata (F. Meotto, pers. comm.), which show a shape similar to their final shape, where expansion would be

Lilia Garnero and others a major growth mechanism. We might speculate that TMchs4 is one of the growth molecular markers, which are involved in such an increase of ascomata size.

A C K N O W L E D G E M E N TS The research was funded by the Italian National Council for Research, Special project Tuber : Biotecnologia della Micorrizazione.

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