Chitin synthase genes in the arbuscular mycorrhizal fungusGlomus versiforme : full sequence of a gene encoding

a class IV chitin synthase

Luisa Lanfranco a, Lilia Garnero b, Paola Bonfante a;b;*a Centro di Studio sulla Micologia del Terreno, CNR, Universitaé di Torino, Viale P.A. Mattioli 25, 10125 Torino, Italy

b Dipartimento di Biologia Vegetale, Universitaé di Torino, Viale P.A. Mattioli 25, 10125 Torino, Italy

Received 14 September 1998; received in revised form 30 October 1998; accepted 30 October 1998

Abstract

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of themolecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regionsof distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets ofdegenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase.A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to theclass IV chitin synthase from Neurospora crassa. z 1999 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Cell wall ; Morphogenesis; Chitin synthase; PCR ampli¢cation; Genomic library; Glomus versiforme

1. Introduction

Arbuscular mycorrhizal (AM) fungi are soil micro-organisms which establish a mutualistic symbiosiswith the roots of about 80% of land plants in di¡er-ent ecosystems. They undergo a complex morpho-genesis outside and inside the host root by develop-ing a number of structures (spores, germinatinghyphae, appressoria, inter- and intracellular hyphae,arbuscules and vesicles) whose morphology has beenextensively described [1]. During the development ofthese specialized structures, deep changes have been

observed in the structure and composition of theAM cell wall. Even so, chitin, the polymer of L-1,4linked N-acetylglucosamine (GlcNAc), is a constantcomponent of AM fungi, being present as highlyarched ¢brils which produce a helicoid architecturein the spores, in parallel ¢brils in hyphae and inshorter chains in the thin arbuscular branches [2].Since the biochemical pathways and molecular mech-anisms controlling the morphogenesis of AM fungiand their interactions with host plants are mostlyunknown [1^3], we speculated that understandingthe basis of chitin synthesis in these endomycorrhizalfungi might o¡er insights into their complex devel-opmental events.

The aim of the present study was to investigate the

0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 2 5 - 4

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* Corresponding author. Tel. : +39 (11) 650-2927;Fax: +39 (11) 650-7459; E-mail: bonfante@csmt.to.cnr.it

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presence of chitin synthase (chs) genes in the arbus-cular mycorrhizal fungus Glomus versiforme and tocompare them with corresponding genes from other¢lamentous fungi. Chitin synthase (Chs) enzymes aremembrane proteins which catalyze the transfer of N-acetylglucosamine from UDP-N-acetylglucosamineinto a chitin chain. A molecular approach, basedon PCR experiments with primers designed on highlyconserved Chs domains, has already successfullyidenti¢ed chs gene portions from several ¢lamentousfungi [4,5].

2. Materials and methods

2.1. Isolation of genomic DNA and PCR reactions

The spores of Glomus versiforme Karsten (Berch)(BEG 47) were surface sterilized in 4% cloramine Tand 0.004% streptomycin for 20 min. DNA fromabout 50 spores of G. versiforme was extractedwith the CTAB method [6]. The DNA was resus-pended in 30 Wl of TE bu¡er (10 mM Tris-HCl pH8.0, 1 mM EDTA).

Two set of degenerate primers were used: P3/P4reported by Specht et al. [5] and HA1 5P-CT-GAAGCTTGTTCT(CG)AC(AC)ATGTA(TC)AA-(TC)G-3P/XB1 5P-GTTCTCGAG(CT)TT(AG)TA(T-C)TCAAA(AG)TT(TC)TG-3P slightly modi¢ed fromthose described by Bowen et al. [4]. PCR reactionswere carried out in a ¢nal volume of 50 Wl containing10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mMMgCl2, 0.2 mM dNTPs, 100 pmol of each primerand 2.5 U of AmpliTaq GOLD DNA polymerase(Perkin Elmer). Several amounts of genomic DNA(undiluted and 1/10 diluted) were tested for goodampli¢cation results. PCR ampli¢cations were runin a Perkin Elmer/Cetus DNA thermal cycler as de-scribed by Bowen et al. [4]. Speci¢c oligonucleotideswere designed for each chs clone: Gvchs1 (ampli¢edproduct of 296 bp), CHS1 (5P-ATGAGTAAGA-CAGGGAAAGTTACG-3P) and CHS2 (5P-GTT-GAGCAGTCCACCACCCTCGC-3P) ; Gvchs2 (am-pli¢ed product of 153 bp) CHS3 (5P-ACAGTAAGA-CATGGGGTAAA-3P) and CHS4 (5P-TCCTT-TCCATTGACCTGCTGC-3P) ; and Gvchs3 (ampli-¢ed product of 762 bp) CHS5 (5P-CTATGGTT-CACGATCCCGAG-3P) and CHS6 (5P-TTATCA-

TCCACATCACGTAGG-3P). PCR reactions withspeci¢c primers (50 pmol of each) were carried outin the same conditions with the exception of a 50³Cannealing temperature.

2.2. Cloning, screening of the genomic library andsequence analysis

The PCR products were puri¢ed on agarose gelusing a QIAEX II Gel Extraction Kit (Qiagen,Promega) and cloned into pBluescript SK II follow-ing the standard procedures [7]. Forty thousand pla-ques of the genomic library [8] were blotted on aHybond-N� membrane (Amersham). The hybridiza-tions were performed with a chemiluminescent detec-tion system (ECL Direct DNA labeling and Detec-tion System, Amersham) according to themanufacturer's recommendations. Plasmid andphage DNAs were sequenced by the Service deSeèquenc°age, Universiteè Laval, Queèbec (Canada). Se-quences analyses were performed with the PC/genesoftware (IntelliGenetics, Mountain View, CA,USA), and the BLAST software available throughthe National Center for Biotechnology Information(NCBI).

3. Results and discussion

3.1. Isolation of three PCR clones encodingchitin synthases

PCR ampli¢cation with HA1-XB1 primers led tothe identi¢cation of two chs homologs, Gvchs1 andGvchs2. The Gvchs1 clone was 638 bp long (withoutdegenerate primers) and presented a putative intronof 59 bp between position 250 and 308 (Fig. 1). TheGvchs2 sequence had a single ORF (open readingframe) of 481 bp (Fig. 1). The alignment of the ami-no acid sequences deduced from the Gvchs1 andGvchs2 nucleotide sequences showed 63.7% of ho-mology with 102 identical residues.

The dendrogram, based on the alignment with thesame Chs region from other fungi [4^9], groupedGvChs1 and GvChs2 as class I Chs (Fig. 2). Thesedata were also supported by a databank search,showing very high homology with class I Chs, inparticular with Phaecoccomyces exophialae and Neu-

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Fig. 1. Nucleotide and deduced amino acid sequences from G. versiforme PCR clones chs1 and chs2. The putative intron in Gvchs1 is indi-cated by lower-case letters. Hyphens indicate gaps introduced in Gvchs2 to permit the alignment with Gvchs1. EMBL accession numbers:Gvchs1, AJ009628; Gvchs2, AJ009629. � indicates identical amino acids; W indicates well conserved amino acids.

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rospora crassa sequences [4] for GvChs1 and GvChs2,respectively. Other published results indicate that Zy-gomycetes are characterized by more than one chsgene belonging to the same class: Rhizopus oligospo-rus and Entomophaga auliceae possess two class IIchs genes [10,11] while multiple forms of class I, IIand IV were described in Phycomyces blakesleeanus[12].

Using the degenerate primers P3 and P4 [5], a newchs gene portion was identi¢ed, clone Gvchs3 (Fig.3). The nucleotide sequence (851 bp) presented ahypothetical intron of 73 bp (Fig. 3). A BLASTsearch showed that the closest homology of Gvchs3was with a portion of class IV Chs of N. crassa [13].

The Gvchs1 and Gvchs3 nucleotide sequences pre-sented an intron of molecular weight similar to thosefound in ¢lamentous fungi. Analysis of chs DNAfragments from several group of fungi, showed thatthe intron positions do not correlate with the classes[9]. Indeed, the Gvchs1 clone has an intron at the

same position as a class II chs from R. oligosporus[10], suggesting that these genes might be derivedfrom an ancestral gene containing the intron andcommon to the Zygomycetes. Examination from Zy-gomycetes and di¡erent sequences of more classesmay show how both the genes and their classesevolved.

The character of AM fungi as obligate biotrophslimits the amount of biological material available forSouthern blot analysis. Thus, to con¢rm the originof the three sequences, a pair of speci¢c primers wasdesigned for each clone and tested on genomic DNAfrom G. versiforme spores. An ampli¢ed product ofthe expected size was obtained for each primer pair(Fig. 4a^c).

Our knowledge on chs genes in mycorrhizal fungiis still limited. During an investigation of the molec-ular mechanisms which control the development ofsymbiotic fungi, we found that multiple chs genes arepresent in tru¥es, a group of ascomycetous ectomy-

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Fig. 2. Dendrogram showing grouping of selected chitin synthases and their class designation. EaChs1/2, Entomophaga aulicae ; RoChs1/2,Rhizopus oligosporus ; PbChs1, Phycomyces blakesleeanus ; LalChs1/2, Laccaria laccata ; CooChs1/2, Cortinarius odorifer ; RuaChs1, Russulaadulterina ; GvChs1/2, Glomus versiforme ; UmChs1/2, Ustilago maydis ; CacChs1, Cantharellus cibarius ; XbChs1, Xylohypha bantiana ; andElmChs1, Elaphomyces muricatus ; NcChs1, Neurospora crassa. Classes are indicated on the right.

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Fig. 3.

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Fig. 3 (continued).

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corrhizal fungi, in ericoid fungi as well as in anotherAM species, Gigaspora margarita (L. Garnero andcolleagues, in preparation; L. Lanfranco and col-leagues, unpublished results). Taken as a whole, theresults demonstrate that AM fungi and other mycor-rhizal fungi possess multiple chs genes. This raisesmany questions on the role played by their products,i.e. whether chs genes are di¡erentially activated dur-ing fungal morphogenesis and interaction with thehost plant.

3.2. Isolation of complete gene sequence for a classIV chitin synthase

A screening of about 40 000 plaques of the G.versiforme genomic library with the Gvchs3 fragmentled to the identi¢cation of 5 positive areas. One wasfurther puri¢ed to single plaque and phage DNAwas sequenced with CHS5 and CHS6 speci¢c prim-ers. New sets of primers were then designed andused to obtain the complete gene sequence of 4269nucleotides (Fig. 3). This appears to be the ¢rst re-port of a full genomic sequence in an AM fungus

encoding a functional gene. The open reading frameis 1194 amino acids in length, even if another twopossible ATG start codons were present at positions61 and 235. The molecular weight is similar to thatof other chitin synthases. Preliminary studies onChs2 of Saccharomyces cerevisiae suggested thatonly a small part of the protein (the C-terminal) isrequired for the catalytic activity, while the N-termi-nal may be important for other functions, i.e. mem-brane localization, binding to chitin and export ofchitin ¢bers [14].

A single putative intron of 73 nucleotides wasidenti¢ed at amino acid 1084. The ORF shows alow bias for codon usage, as all the 61 possible co-dons are used. The presence of long stretches of GC-rich sequences at the 5P-end hampered the sequenc-ing reactions so that only a 220 nucleotide sequencehas been identi¢ed. GC-rich sequences are often lo-cated in the promoter of several eukaryote geneswhere they are supposed to play regulatory func-tions. A TATA-box-like sequence was found at the369 position, whereas a CAAT-box motif occurredat 3180. The sequence determined at the 3P-end

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Fig. 3. Nucleotide and deduced amino acid sequences of Gvchs3 complete gene. The gene portion identi¢ed by PCR is underlined (from2634 to 3485) nucleotide). TATA-box and CAAT-box are boxed. The putative intron is indicated by lower-case letters. EMBL accessionnumber: AJ009630.

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(about 400 bp) presented several stop codons in allthe frames, but no canonical polyadenylation signal(AATAAA) was identi¢ed.

The hydrophobicity pro¢le of the full-length pro-tein sequence GvChs3 pointed to a structure charac-teristic of chitin synthases: a hydrophilic N-terminal,a central neutral region and a hydrophobic C-termi-nal. Three transmembrane segments were found inthe C-terminal portion (unpublished results).

The protein sequence presented the amino acidEDR (position 992^994) and QRRRW (position1031^1035) which have been indicated as putativecatalytic residues of the enzymatic activity [15]. Thesame residues have been found in NodC proteins,hyaluronan synthase, cellulose synthase and DG42:all of them catalyze the synthesis of oligosaccharideswith L-1,4 linkages using UDP-sugar as substrate[15]. It has been suggested that the residues identi¢edin the potential catalytic sites of Chs2 are conservedin glycosyltransferases that catalyze the synthesis ofL-1,4 oligosaccharides.

A databank search showed that the closest homol-ogy was with the class IV Chs from Neurospora cras-sa, since 59.2% of similarity was found when the full-length amino acid sequences were compared. Theregion of greatest similarity (79.5%) was at the C-terminal after about the amino acid 650. To de¢nethe class, the C-terminal portion (from amino acid

693 of GvChs3) was aligned with the same regionfrom other Chs: the Chs from Aspergillus nidulansbelonging to ¢ve di¡erent classes, as described inSpecht et al. [5], the class IV Chs from N. crassa[13] and the amino acid sequence corresponding tothe Chs3 of S. cerevisiae. The dendrogram derivedfrom the alignment placed the GvChs3 in class IVChs (data not shown). Furthermore the motifSWG, considered speci¢c for classes III, IV and V[5^16], was located at amino acid 1144. Since S. cere-visiae Chs3 (class IV) synthesized 90% of cell wallchitin, this class is thought to be responsible forthe synthesis of the major fraction of cell wall chitin[13]. On the other hand, gene inactivation analysisshowed that N. crassa chs4 gene product seems to bean auxiliary enzyme that is active under particulargrowth conditions [13]. These authors suggest thatother class IV Chs may compensate for the lack ofChs4 activity. However, sequence comparison withother Chs does not de¢ne the role of Gvchs3 in G.versiforme growth and development. Mutagenesisstudies are an excellent way of investigating the ac-tivity of speci¢c gene products in vivo. Notwith-standing preliminary results obtained in Gigasporarosea [17], a reliable transformation technique forAM fungi is still lacking.

In conclusion, the full gene sequence found in G.versiforme can now join recombinant protein expres-

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Fig. 4. PCR products obtained from spore DNA (lanes 1 and 2) with primers speci¢c for Gvchs1 (a), Gvchs2 (b) and Gvchs3 (c). LaneM: pUC18 digested with HaeIII. Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme : full sequence of a geneencoding a class IV chitin synthase.

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sion and antibody production to localize the proteinas a further means of investigating the growth mech-anisms of AM fungi.

Acknowledgments

The research was funded by the Italian NationalCouncil for Research.

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