703 Lilia GARNERO 1 , Barbara LAZZARI 2 , Davide MAINIERI 2 , Angelo VIOTTI 2 and Paola BONFANTE 1  Dipartimento di Biologia Vegetale – Universita  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 photo- synthate (Smith & Read 1997). Plant carbohydrates are required for the growth of the fungus during all the steps of its life cycle : extraradical mycelium, sporophores, ecto- mycorrhizas. The molecular mechanisms controlling mor- phogenesis 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-acetyl- glucosamine into chitin chains (Cabib et al. 1996). To identify chitin synthase genes (chs) in the ecto- mycorrhizal 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 reddish- brown, 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 10). 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 Hybond- N 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 83, 50 m KCl, 15m MgCl2, 02m dNTPs, 100 pmol of each primer and 25 U of AmpliTaqGOLD DNA polymerase (Perkin–Elmer). Two concentrations (undiluted and 110) of genomic DNA were 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