Role of chitin synthase genes in Fusarium oxysporum

Magdalena Martín-Udíroz{dagger}, Marta P. Madrid and M. Isabel G. Roncero

Departamento de Genética, Campus Rabanales C5, Universidad de Córdoba, E-14071 Córdoba, Spain

Correspondence
M. Isabel G. Roncero
ge1gorom{at}uco.es


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Three structural chitin synthase genes, chs1, chs2 and chs3, were identified in the genome of Fusarium oxysporum f. sp. lycopersici, a soilborne pathogen causing vascular wilt disease in tomato plants. Based on amino acid identities with related fungal species, chs1, chs2 and chs3 encode structural chitin synthases (CSs) of class I, class II and class III, respectively. A gene (chs7) encoding a chaperone-like protein was identified by comparison of the deduced protein with Chs7p from Saccharomyces cerevisiae, an endoplasmic reticulum (ER) protein required for the export of ScChs3p (class IV) from the ER. So far no CS gene belonging to class IV has been isolated from F. oxysporum, although it probably contains more than one gene of this class, based on the genome data of the closely related species Fusarium graminearum. F. oxysporum chs1-, chs2- and chs7-deficient mutants were constructed through targeted gene disruption by homologous recombination. No compensatory mechanism seems to exist between the CS genes studied, since chitin content determination and expression analysis of the chs genes showed no differences between the disruption mutants and the wild-type strain. By fluorescence microscopy using Calcofluor white and DAPI staining, the wild-type strain and {Delta}chs2 and {Delta}chs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus, whereas the {Delta}chs1 mutant showed compartments containing up to four nuclei. Pathogenicity assays on tomato plants indicated reduced virulence of {Delta}chs2 and {Delta}chs7 null mutants. Stress conditions affected normal development in {Delta}chs2 but not in {Delta}chs1 or {Delta}chs7 disruptants, and the three chs-deficient mutants showed increased hyphal hydrophobicity compared to the wild-type strain when grown in sorbitol-containing medium. The chitin synthase mutants will be useful for elucidating cell wall biogenesis in F. oxysporum and the relationship between fungal cell wall integrity and pathogenicity.


Abbreviations: AUDPC, area under the disease progress curve; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CFW, Calcofluor white; CS, chitin synthase; DAPI, 4',6-diamidino-2-phenylindole; ER, endoplasmic reticulum; HygR, resistance to hygromycin; Pt, probability of the statistic absolute t

The GenBank/EMBL/DDBJ accession numbers for the chs1, chs2, chs3 and chs7 gene sequences reported in this paper are AY572421, AY572422, AY572423 and AY572424, respectively.

{dagger}Present address: Australian Centre for Necrotrophic Fungal Pathogens, Murdoch University, Perth, WA 6150, Australia.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chitin, an important structural cell wall component in many species of yeast and filamentous fungi but absent from plants and vertebrates, is a {beta}(1,4)-linked polymer of N-acetylglucosamine which forms a fibrous polysaccharide. This taxonomic difference provides the rationale for considering chitin as a safe and largely selective target for developing antifungal control agents (Cohen, 1990). Chitin synthases (CSs) catalyse the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine (UDPGlcNAc) to a growing chain of {beta}(1,4)-linked N-acetylglucosamine residues (chitin) (Ruiz-Herrera et al., 1992). The specific mechanism by which this polymer is synthesized in vivo by the different species appears to have selective characteristics. Fungal CSs are integral membrane-bound proteins that participate in the biosynthesis of the cell wall and are important for hyphal growth and differentiation (reviewed by Cabib et al., 1996; Roncero, 2002). Comparative analysis of the amino acid sequences deduced from fungal chs genes reveals the existence of a hydrophobic domain located towards the C-terminus, in agreement with the membrane location of these enzymes. Several authors have provided evidence for the existence of a type of specialized vesicles in the cytosol, named chitosomes, where most CS is accumulated (Bartnicki-García et al., 1984). They synthesize chitin microfibrils through an asymmetric mechanism, accepting GlcNAc residues at the cytosolic face, and delivering chitin molecules at the inner face (Ruiz-Herrera & Martínez-Espinoza, 1999). Thus, chitosomes have been regarded as the vesicles responsible for the transport of CS from the endoplasmic reticulum (ER) to the cell surface. At present, CSs from filamentous fungi are classified into six categories, class I to class VI, based on conserved regions and the presence of specific myosin motor-like domains (Bowen et al., 1992; Specht et al., 1996; Ruiz-Herrera et al., 2002; Roncero, 2002). The presence of multiple CS genes is common in a single fungal species (Munro & Gow, 2001; Roncero, 2002). In the budding yeast Saccharomyces cerevisiae, seven chs genes responsible for only three CS activities (CSI, CSII and CSIII) have been well characterized and the functions of the proteins encoded by the S. cerevisiae genes are the best understood among fungal species (Bulawa, 1993; Roncero, 2002). Structural and functional analyses of chs genes and their products have been reported for representative filamentous fungal species, as implicated in the diverse morphologies of filamentous fungi. Nevertheless, the specific roles of these enzymes have been elucidated in only a few cases (Fujiwara et al., 2000; Lee et al., 2004; Yarden & Yanofsky, 1991). In the human-pathogenic fungus Aspergillus fumigatus, the chs gene family includes at least seven different genes, members of all six CS classes (AfchsA, AfchsB, AfchsC, AfchsD, AfchsF and AfchsG) (Mellado et al., 2003). Inactivation of AfchsA (class I), AfchsB (class II), AfchsC (class III) and AfchsD (class IV) does not lead to any obvious phenotypic defect (Mellado et al., 1996a, b), whereas disruption of AfchsE (class V) and AfchsG (class III) gives rise to altered phenotypes, suggesting that class III CS functions at the apical tips of the hyphae (Mellado et al., 1995) and class V CS is responsible for cell wall structural integrity (Aufauvre-Brown et al., 1997). In Aspergillus nidulans, five CS genes [chsA, chsB, chsC, chsE (identical to chsD) and csmA] have been reported so far. Based on phenotypes present in single or double disruption mutants, the function of each chs gene has been summarized as follows. chsB is a class III CS, required for normal hyphal growth and organization (Borgia et al., 1996), and csmA (class V) seems critical for the maintenance of hyphal wall integrity and the polarized synthesis of the cell wall (Horiuchi et al., 1999). The genes chsA, chsC and chsE appear to serve redundant functions during asexual morphogenesis such as conidia formation and coniodophore development (Motoyama et al., 1996; Fujiwara et al., 2000).

Fusarium oxysporum, a vascular wilt pathogen with more than 100 specialized forms distributed worldwide, causes disease among a variety of important crop plants (Beckman, 1987). F. oxysporum has also been reported as an emerging opportunistic human pathogen in immunocompromised patients (Vartivarian et al., 1993). Between eight and twelve CS-encoding genes representative of all six categories described in filamentous fungi are present in the Fusarium graminearum genome, recently identified by in silico search of the database at Sequencing Project, Center for Genome Research (http://www.broad.mit.edu). Most of these genes have counterparts in the genomes of Neurospora crassa, A. nidulans and Magnaporthe grisea. Chitin can account for up to 10 % of the cell wall of F. oxysporum (Schoffelmeer et al., 1999) and 20 % in Aspergillus spp. (Bull, 1970), compared to only 1–2 % in the yeast S. cerevisiae (Bulawa, 1993). This difference may explain the high number of structural chs genes identified in filamentous fungal species such as A. fumigatus, A. nidulans and N. crassa, in contrast to only three genes encoding the catalytic subunits reported in S. cerevisiae (Roncero, 2002) and four genes in Candida albicans (Munro & Gow, 2001). In F. oxysporum f. sp. lycopersici a gene encoding a class V CS was isolated by random insertional mutagenesis and screening for pathogenicity mutants. The ChsV deduced protein carries a myosin domain in the N-terminal region and is required during host infection and for maintenance of cell wall integrity (Madrid et al., 2003). In this study, we report the isolation of four chs genes, chs1, chs2, chs3 and chs7 from F. oxysporum, and the construction of three targeted disruption mutants. We have characterized the deficient mutants for hyphal morphogenesis, nuclear distribution, and physiological and pathotypic behaviour.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and culture conditions.
F. oxysporum f. sp. lycopersici wild-type strain 4287 (race 2) was obtained from J. Tello, Universidad de Almería, Spain. The mutant strain deficient in the class V CS gene chsV has been described elsewhere (Madrid et al., 2003). Microconidial suspensions were stored with glycerol at –80 °C. The pathotype of the isolates was periodically confirmed by plant infection assays. For extraction of genomic DNA, mycelium was obtained from cultures grown in potato dextrose [glucose] broth (PDB, Difco) on a rotary shaker at 170 r.p.m. and 28 °C as described previously (Di Pietro & Roncero, 1998). For phenotypic analysis of colony growth inhibition or hydrophobic characteristics, microconidia were collected from PDB, washed in sterile water, counted and transferred to synthetic medium (SM) plates (Di Pietro & Roncero, 1998) supplemented or not with the following metabolites at the concentrations indicated: sorbitol, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), caffeine, {alpha}-tomatine, Congo red and hydrogen peroxide (all from Sigma). SDS sensitivity was analysed on SM plates containing 0·025 % (w/v) SDS. SM or PDA media were supplemented with hygromycin, for selection and/or maintenance of transformant phenotypes when required, at the appropriate concentrations.

Construction of gene disruption vectors.
The gene replacement vectors pDChs1 : : hyg, pDChs2 : : hyg, pDChs3 : : hyg and pDChs7 : : hyg were constructed by following the general strategy of inserting the hygromycin-resistance (HygR) cassette from plasmid pH1B (Turgeon et al., 1987), interrupting the ORF of the corresponding chs gene. In the case of the chs1 gene an internal BamHI fragment was replaced by the HygR cassette. In the case of chs2, chs3 and chs7, the HygR cassette was inserted into a BamHI internal sequence newly created by site-directed mutagenesis with the use of two divergent and complementary specific primers containing this restriction site into the ORFs (Horton et al., 1989). Linear DNA fragments containing the different interrupted chs alleles were generated by amplification of the entire constructs using primer pairs flanking both ends of the disrupted genes as indicated in Fig. 3(a).



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Fig. 3. Targeted replacement of F. oxysporum CS genes. (a) Physical maps of the genomic regions and strategy for construction of the different disruption vectors. The chs1, chs2, chs3 and chs7 coding regions are shown as black arrows with the orientation of the ORFs and the introns. Small arrows indicate the primers used for amplification of the gene replacement vectors designated pDChs1, pDChs2, pDChs3 and pDChs7. (b) Analysis of transformants {Delta}chs1, {Delta}chs2.1, {Delta}chs2.6 and {Delta}chs7 (lanes 2, 4, 5 and 7, respectively) and wild-type strain 4287 (lanes 1, 3, 6) by Southern blotting. Genomic DNAs were digested with BamHI in lanes 1 and 2, with HindIII in lanes 3, 4 and 5, with EcoRI in lanes 6 and 7, then separated in a 0·7 % agarose gel, blotted onto a nylon membrane and hybridized. The corresponding gene probes are indicated as clear horizontal bars in (a). Size markers are shown on the left.

 
Transformation-mediated gene replacement.
The final amplified constructs containing the F. oxysporum genomic DNAs with the chs coding regions interrupted with the HygR cassette were used for transformation of F. oxysporum 4287 protoplasts to hygromycin resistance according to a protocol described previously (Di Pietro & Roncero, 1998). Briefly, microconidia were germinated for 14 h in SM before being submitted to protoplasting (García-Maceira et al., 2000). Transformants were selected on hygromycin-containing plates, then purified by monoconidial isolation by two consecutive rounds of single spore isolation before being stored as microconidial suspensions at –80 °C.

Primers, PCR amplification and cloning of PCR products.
The primer pair initially used for amplification of CS domains was CHSI-1 5'-CTGAAGCTTACNATGTAYAAYGARGAY-3' and CHSI-2 5'-GTTCTCGAGYTTRTAYTCRAARTTYTG-3', designed based on highly conserved regions from different family I chs genes (Vidal-Cros & Boccara, 1998). For amplification of the F. oxysporum chs7 gene the degenerate primers CHS7-1 5'-ATHAAYGGNTTYGTNGGNTTYCAR-3' and CHS7-2 5'-TCCCARAAYTTRTANACCATCATNAC-3' were designed by comparison analysis between chs7 from S. cerevisiae (Trilla et al., 1999) and the corresponding orthologue gene identified at the A. fumigatus genome database (http://www.tigr.org/tdb/e2k1/afu1/release.shtml). F. oxysporum genomic DNA was PCR-amplified with the following conditions: first cycle of 5 min at 94 °C, then 30 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, followed by one cycle of 10 min at 72 °C. The PCR-amplified fragments obtained were analysed by gel electrophoresis and purified by using the Geneclean Turbo (Q-BIO gene). The eluted fragments were cloned into pGEM-T vector (Promega) and their identities were verified by DNA sequencing before using them as probes for screening of F. oxysporum libraries.

Nucleic acid manipulations and cloning of chs genes.
The chs1, chs2, chs3 and chs7 genes were isolated from a {lambda}-EMBL3 genomic library of F. oxysporum f. sp. lycopersici strain 4287, probed with fragments obtained by PCR amplification of F. oxysporum DNA using primers from conserved regions of different CSs as described above. A cDNA clone from chs7 was isolated from screening of a {lambda}-ZAP cDNA library (Roldán-Arjona et al., 1999). Screening of libraries, subcloning and other routine procedures were performed as described in standard protocols (Sambrook et al., 1989). Sequencing of both DNA strands was performed at the Servicio de Secuenciación de la Universidad de Córdoba, using the Dyedeoxy Terminator cycle sequencing kit (PE Biosystems) on an ABI Prism 377 genetic analyser. Analyses of sequencing data were carried out using the Lasergene programs (DNAStar). DNA and protein sequence databases were searched using the BLAST algorithm (Altschul et al., 1990) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

Total RNA and genomic DNA were extracted from F. oxysporum mycelium as described previously (Chomzczynski & Sacchi, 1987; Aljanabi & Martínez, 1997). Southern and Northern hybridization analysis and probe labelling were performed as described in standard protocols using the non-isotopic digoxigenin labelling kit (Roche Diagnostics) according to the instructions of the manufacturer. RT-PCR amplification was carried out as described previously (García-Maceira et al., 2000). First-strand cDNAs were generated from total RNA isolated from mycelia grown on SM or SM containing 1·2 M sorbitol as indicated in Fig. 4. PCR was performed using gene-specific primer pairs at the indicated amino acid positions and always flanking one intron. For amplification of chs1 transcripts, CHSI-8 5'-TCGACTTCCTCCGATCTGAT-3' (sense, from 561 to 566) and CHSI-22 5'-CCTTGTTCTTGAAGATGTCTG-3' (antisense, from 690 to 696); for amplification of chs2, CHSII-12 5'-CCTCCTCCTTCAGATGGCAT-3' (sense, from 154 to 160) and CHSII-23 5'-GTGGAGACTCGGGAACTT-3' (antisense, from 923 to 928); for amplification of chs3, CHSIII-12 5'-GTGTCATGGGGAACAAAGGG-3' (sense, from 829 to 835) and CHSIII-18 5'-CCTGTAACCCCAAAAGTATGT-3' (antisense, from nucleotides 63 to 43 after the stop codon); for amplification of chs7, CHS7-9 5'-GCTGGGCGTTATGATGGT-3' (sense, from 264 to 269) and CHS7-20 5'-GCGAGTAAGGCAGATCATAG-3' (antisense, from 319 to 325) For each independent experiment the controls included both amplifications, the actin gene for normalization of the PCR conditions, and the F. oxysporum genomic DNA for comparison with the intron-containing amplified bands. The cDNA used as template was the same for each experiment, being the amount standardized and spectrophotometrically quantified for normalization of the amplification conditions.



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Fig. 4. Detection of chs gene transcripts. RT-PCR products were obtained from wild-type and {Delta}chs1, {Delta}chs2, {Delta}chsV and {Delta}chs7 mutant strains, grown in SM or SM with 1·2 M sorbitol. PCR amplification using F. oxysporum genomic DNA as template was used as control. The DNA ladder is indicated.

 
Determination of chitin content.
Chitin content was measured by the method described by Din et al. (1996) with minor modifications (glusulase was used instead of {beta}-glucuronidase). Mycelium was collected from germlings grown for 20 h minimal medium, washed twice with deionized water and lyophilized. Ten to twenty milligrams dry weight was suspended in 1 ml 6 % (w/v) KOH and incubated for 90 min at 80 °C. Glacial acetic acid (0·1 ml) was added to each sample and the insoluble material was collected by centrifugation at 13 000 g for 15 min, washed twice with water and suspended in 0·5 ml sodium phosphate buffer (pH 6·3). A 100 µl aliquot of a 5 mg ml–1 chitinase suspension (Sigma) was added, and the samples were incubated at 37 °C for 20 h. Following centrifugation at 13 000 g for 15 min, 450 µl of the supernatant was treated with 25 µl of a 10 000 units ml–1 solution of glusulase (New England Nuclear), for 2 h at 37 °C. Portions of 0·1 ml from each sample were removed and assayed for GlcNAc content (Reissig et al., 1955).

Determination of conidiation.
Conidial suspensions from wild-type and {Delta}chs mutant strains were inoculated on PDB medium at a concentration of 5x105 spores ml–1, and incubated on a rotary shaker at 28 °C. Samples were collected at regular intervals from 0 to 70 h, and the number of microconidia present in the cultures was counted in a haematocytometer under the microscope (Olympus, BH-2).

Morphological analyses by fluorescence microscopy.
For microscopic analysis of the wild-type strain and {Delta}chs1, {Delta}chs2, {Delta}chsV, {Delta}chs7 mutants, samples from shaken cultures were diluted with 3·7 % (v/v) formaldehyde, 50 mM phosphate buffer (pH 7), 0·2 % (v/v) Triton X-100, for fixation (Harris et al., 1994), and stained with Calcofluor white (CFW) 10 µg ml–1 for 5 min, and/or 4',6-diamidino-2-phenylindole (DAPI) 0·8 µg ml–1 (all from Sigma), and observed using a fluorescence microscope (Leica, DMR) with a 20x or 40x objective and a total magnification of 200 or 400, respectively, on screen.

Pathogenicity assays on tomato plants.
Infection of tomato plants was performed as reported previously (Di Pietro & Roncero, 1998). Briefly, tomato seedlings of cv. ‘Vemar’ were inoculated with F. oxysporum f. sp. lycopersici strains by dipping the roots in a microconidial suspension, planting the seedlings in minipots with vermiculite and maintaining them in a growth chamber at 25 °C with 14 h light and 10 h dark. Plants immersed in sterile water were used as controls. For statistical analyses, the severity of disease symptoms was recorded from 1 week after the inoculation every 2 days until day 24 post-infection according to a scale ranging from 1 (healthy plant) to 5 (dead plant) (Di Pietro & Roncero, 1998). Fifteen plants were used for each treatment. The area under the disease progress curve (AUDPC) was calculated for each plant. The AUDPC means of the mutants were compared to that of the wild-type by Student's t-test. All pathogenicity assays were performed at least twice with similar results. Plant seeds were kindly provided by Syngenta Seeds (El Ejido, Almería, Spain).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of F. oxysporum genes chs1, chs2, chs3 and chs7
PCR was used to amplify genomic DNA from the wild-type strain 4287 of F. oxysporum f. sp. lycopersici, with the degenerate primers CHSI-1 (a consensus between CHSI-1/CHSI-2) and CHSI-3 (Vidal-Cros & Boccara, 1998). The products were cloned, and the resulting recombinant plasmids initially characterized by restriction mapping revealed different restriction patterns, indicating the presence of diverse PCR products. DNA sequence analysis of several plasmid inserts allowed the identification of three different CS sequences. The translation products of these F. oxysporum DNA inserts, designated fchs1, fchs2 and fchs3, displayed highest homologies to the deduced polypeptides of the genes chsI from Gibberella zeae (GenBank accession number AJ312243), chs2 from N. crassa (X77782) (Din & Yarden, 1994) and chsB from Glomerella graminicola (AY052546), respectively. Isolation of the orthologue of S. cerevisiae CHS7 was accomplished by PCR amplification of F. oxysporum genomic DNA with a pair of degenerate primers deduced from the putative A. fumigatus orthologue gene by in silico identification in the available genome database (http://www.tigr.org/tdb/e2k1/afu1/release.shtml). The product was cloned and identified by sequencing and this F. oxysporum DNA insert was designated fchs7.

The F. oxysporum {lambda}-EMBL3 genomic library available in our group was probed with the four PCR fragments (fchs1, fchs2, fchs3 and fchs7). The positive clones were subjected to PCR amplification using two {lambda} primers, {lambda}-int 5'-CGCAACTCGTGAAAGGTA-3' and {lambda}-git 5'-AAGTCCAACCCAGATAACGAT-3'. The F. oxysporum inserts present in the recombinant clones were amplified by PCR and sequenced using the strategy of DNA-walking with specific synthetic oligonucleotides (CHSI-4 to CHSI-19 for chs1, CHSII-4 to CHSII-21 for chs2, CHSIII-4 to CHSIII-19 for chs3, CHS7-3 to CHS7-15 for chs7). DNA sequences were determined and analysed by using the Lasergene Navigator and the BLAST algorithm (Altschul et al., 1990) at the NCBI. The gene sequences have been submitted to the DDBJ/EMBL/GenBank databases under the accession numbers AY572421, AY572422, AY572423 and AY572424.

Nucleotide sequences and predicted polypeptides of the chs1, chs2, chs3 and chs7 genes
The chs1 gene encodes a predicted polypeptide of 901 amino acids with a calculated mass of 102·3 kDa and a pI of 7·0, chs2 encodes a predicted polypeptide of 1041 amino acids with a calculated mass of 117·5 kDa and a pI of 7·5, and chs3 encodes a potential ORF of 978 amino acids with a calculated mass of 110·4 kDa and a pI of 7·0. The degree of identity at the amino acid level shared by the three polypeptides was 41 % between FoChs1 and FoChs2, and 29·6 % between FoChs2 and FoChs3. The N-terminal sequences of the three polypeptides diverge considerably, whereas the C-terminal sequences are more conserved as expected for the catalytic domains, with the motif QRRRW, essential for the catalytic activity (Cos et al., 1998), present in all three polypeptides. Comparison of the three predicted proteins with other fungal CSs gave the highest overall similarity with the following identified genes: FoChs1 with G. zeae Chs1 (86·8 %), FoChs2 with N. crassa Chs2 (67·9 %), and FoChs3 with G. graminicola ChsB (67·5 %) (Din & Yarden, 1994; Yarden & Yanofsky, 1991). Based on sequence similarities at the amino acid level and conservation of catalytic motifs the F. oxysporum genes chs1, chs2 and chs3 presumably belong to class I, class II and class III CSs, respectively. Fig. 1 shows the phylogenetic tree of relatedness between the complete deduced amino acid sequences of F. oxysporum chs1, chs2 and chs3, with selected members of fungal family I CS-encoding genes, obtained by CLUSTALW analysis (PAM 250). According to the dendrogram, class I and class II fungal CSs share higher degrees of identity and thus have diverged before those of class III.



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Fig. 1. Phylogenetic tree of relatedness between the predicted products of the Fochs1, Fochs2 and Fochs3 genes with fungal CSs belonging to class I, class II and class III. The deduced polypeptide sequences were aligned and subjected to CLUSTALW analysis. Species, genes and accession numbers are as follows: F. oxysporum f. sp. lycopersici, chs1 (AY572421), chs2 (AY572422) and chs3 (AY572423); F. graminearum, Fg10327, Fg12352, Fg12378 and Fg10116, http://www.broad.mit.edu; G. zeae chs1 (AJ312243); G. graminicola chsA (AY052545) and chsB (AY052546); N. crassa chs1 (M73437), chs2 (X77782) and chs3 (AF127086); Emericella nidulans, chsA (D21268), chsB (D21269) and chsC (AB023911); A. fumigatus, chsA (AAB33397), chsB (AAB33398), chsC (X94245) and chsG (X94244); Phaeosphaeria nodorum chs2 (AJ133695). The scale bar indicates relative divergence between sequences.

 
F. oxysporum chs7 encodes a predicted polypeptide of 334 amino acids with a calculated mass of 36·8 kDa and a pI of 4·9. FoChs7 has about 38·9 % identity with Chs7p from S. cerevisiae (SWISSPROT/AAB68984), responsible for a chaperone involved specifically in ScChs3p export from the ER (Trilla et al., 1999), and 73·1 % identity with a hypothetical protein deduced from the N. crassa genome database ‘assembly version 3’ (Galagan et al., 2003) (Fig. 2).



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Fig. 2. Multiple alignment of the deduced amino acid sequences of F. oxysporum chs7 (AY572424), S. cerevisiae chs7 (SWISSPROT/AAB68984) and N. crassa NCU05720 (chs7-like hypothetical protein, from http://www.broad.mit.edu/annotation/fungi/neurospora). Location of primers used for RT-PCR are shown by arrows; the discontinuous part in CHS7-20 corresponds to the amino acids absent in the Fochs7 gene product. The position of the intron is shown by an open triangle.

 
The position of introns present in the genes was located initially by comparison with other related CS polypeptides; when feasible this was confirmed by sequencing the corresponding cDNA isolated from a F. oxysporum cDNA library, as well as the transcriptional 5' terminus and the poly(A) signal site. The promoter regions of the chs genes were analysed at the nucleotide level. The AbaA Response Element (ARE) binding sequence -CATTCY- (Andrianopoulus & Timberlake, 1994) was found in the promoter of the chs2 gene at positions –807, –467 and –462 relative to the ATG codon, and in gene chs7 at position –283. The sequence -CCAAT- to which the HAP complex binds (Litzka et al., 1998) was present at position –632 in the chs1 promoter, at positions –996 and –921 in the chs2 promoter, at positions –588 in the chs3 promoter, and at position –690 in the chs7 promoter. The stress-response element binding site (STRE) -CCCCT- (Estruch, 2000) was present at positions –363, –325, –308 in gene chs1, at positions –221, –186 in gene chs2, at positions –419 and –375 in gene chs3, and at positions –752, –377, –314, –263 and –175 in gene chs7.

Targeted disruption of the chs1, chs2, chs3 and chs7 genes and molecular characterization of defective mutants
Disruption vectors for chs genes were constructed by insertion of the hygromycin-resistance cassette gene into the coding region of the corresponding genes (Fig. 3a). In the case of chs1 an internal BamHI fragment was replaced by the HygR cassette (Fusarium-disruption plasmid pDChs1). In the other three genes the HygR cassette was inserted into a BamHI internal sequence created by site-directed mutagenesis with the use of two divergent complementary specific primers containing the BamHI site (Horton et al., 1989). The resulting constructs were designated Fusarium-disruption plasmids pDChs2, pDChs3 and pDChs7. Hygromycin resistance selection allowed the isolation of transformants harbouring the disrupted version of genes chs1, chs2 and chs7. A number of these hygromycin-resistant transformants were analysed by restriction enzyme digestion of genomic DNA and Southern hybridization. Transformants originated by gene replacement were obtained for chs1, chs2 and chs7 as shown by a shift of the hybridizing DNA fragments in wild-type strain 4287, from 1 kb to 3 kb, and the absence of some hybridizing bands (in transformant {Delta}chs1), from 6·5 kb to 8·5 kb (in transformants {Delta}chs2.1 and {Delta}chs2.6), from 10 kb to 8·5 kb (in transformant {Delta}chs7) (Fig. 3b). All 45 transformants obtained with the disruption vector pDChs3 showed ectopic integration of the transforming DNA, suggesting a non-viable phenotype for deletion of this CS class III gene (data not shown).

Attempts were made to determine the transcription levels of the different chs genes in wild-type and {Delta}chs mutant strains by Northern analyses using total RNA obtained from mycelia grown on PDB or SM media with or without one of the following compounds: 1·2 M sorbitol, {alpha}-tomatine, Congo red, hydrogen peroxide or caffeine. No detectable hybridization signal was obtained with any of the probes used. Therefore RT-PCR was used to determine the expression of these genes during hyphal growth, of {Delta}chs1, {Delta}chs2 and {Delta}chs7 mutants, and the wild-type strain, in liquid media with or without osmotic stabilizer (Fig. 4). For PCR amplification of each gene transcript a pair of specific primers flanking an intron was used (as described in Methods). As shown in Fig. 4, no differences in the transcription levels of the chs genes between the different disruption mutants and the wild-type strain were observed with or without osmotic stabilizer in the growth medium.

Conidiation, septum and nuclei distribution, physiological behaviour and colony hydrophobicity of {Delta}chs mutants
Conidiation in the three {Delta}chs disruptants was examined microscopically and found to be indistinguishable from that in the wild-type strain (not shown). Initial inocula containing 1·4x105 micronidia ml–1 were germinated on PDB medium at 28 °C and 170 r.p.m. Conidiation was determined by counting spores under the microscope at different time intervals. These {Delta}chs mutants showed no significant difference in the number and the morphology of conidia produced in submerged cultures in comparison to the wild-type strain.

The positions of septa and the distribution of nuclei were examined in the wild-type and the chs-deficient mutants (Fig. 5) by fluorescence microscopy using CFW and DAPI staining. The wild-type strain, {Delta}chs2 and {Delta}chs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus (Fig. 5a, d, e), whereas in the {Delta}chs1 mutant some compartments containing up to four nuclei could be seen (Fig. 5c, c'). The class V deficient mutant ({Delta}chsV) was also included in this analysis due to its abnormal morphology showing swollen, balloon-like structures along the hyphae, previously described (Madrid et al., 2003). These structures frequently contained up to eight nuclei (Fig. 5b, b'). These results indicate that nuclear sorting/distribution seems to be affected in {Delta}chs mutants but not septum formation.



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Fig. 5. Fluorescence microscopy observation of germlings, grown for 14 h in PDB, from F. oxysporum wild-type (a) and different CS mutant strains, {Delta}chsV (b and b'), {Delta}chs1 (c and c'), {Delta}chs2 (d), {Delta}chs7 (e). DAPI and CWF staining of nuclei, hyphae and septal walls (x200 magnification; x400 in b' and c').

 
The growth of the {Delta}chs1, {Delta}chs2 and {Delta}chs7 strains was determined in the presence of the detergent SDS, which affects membrane integrity. The {Delta}chs2 mutant was at least 100 times more sensitive to SDS compared to the wild-type strain, whereas the {Delta}chs1 and {Delta}chs7 mutants were slightly more sensitive (Fig. 6a). No evidence for hyphal lysis was observed using the vital stain BCIP, as detected by the lack of a light blue zone surrounding the colonies of these three {Delta}chs mutants (data not shown). No differences in colony growth rates of mutants were observed in the presence of other compounds assayed, including the chitin-binding dye CFW, plant defence compounds ({alpha}-tomatine, caffeine or hydrogen peroxide) or Congo red, which interferes with cell wall assembly.



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Fig. 6. (a) Colony growth of F. oxysporum wild-type 4287 and mutant strains {Delta}chs1, {Delta}chs2 and {Delta}chs7, after 3 days at 28 °C on SM plates containing 0·025 % SDS. Tenfold serial dilutions starting with 105 spores (columns 1 and 2) were spotted in drops onto the SM plates. First column, control plates without SDS. (b) Hydrophobicity of colony surfaces of the wild-type strain and different CS-deficient mutants grown on synthetic medium (1 % glucose) with or without 1·2 M sorbitol.

 
To investigate whether inactivation of CS genes alters the hydrophobicity of the colony surface, drops of water were placed on the colony centre of the mutant strains {Delta}chs1, {Delta}chs2, {Delta}chs7 and the wild-type, grown in synthetic medium with or without 1·2 M sorbitol, and observed after 48 h. All the chs-deficient mutants showed greater hyphal hydrophobicity than the wild-type strain when grown in sorbitol-containing medium (Fig. 6b). In this analysis the class V deficient mutant ({Delta}chsV) was also included because of its abnormal colony morphology (Madrid et al., 2003). This deficient mutant {Delta}chsV showed the same hydrophobicity phenotype as the wild-type strain (not shown) in both media.

Chitin content of {Delta}chs1, {Delta}chs2 and {Delta}chs7 mutant strains
The total mycelial chitin content of the {Delta}chs1, {Delta}chs2 and {Delta}chs7 mutants was measured by determining the amount of GlcNAc after digestion of the cell wall with chitinase and glusulase. The chitin content of deficient mutants was found to be reduced only in {Delta}chs1 and {Delta}chs2, with a 10 % reduction in comparison to wild-type strain 4287 (Table 1). It has been previously described in A. fumigatus that only mutants defective in class III CS or homologues of this protein have a significant reduction in chitin content (Din et al., 1996), while mutants defective in the zymogen type of enzyme typically have a normal or a small reduction (10 %) in chitin content and CS activity (Mellado et al., 1996a).


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Table 1. Summary of {Delta}chs phenotypes

Disease index refers to symptoms observed on tomato plants 21 days post-inoculation (dpi) with spore suspensions from the different strains. WT, wild-type; ND, not determined. Conidiation of all the mutants was indistinguishable from that of the WT.

 
Pathotypic behaviour of {Delta}chs1, {Delta}chs2 and {Delta}chs7 mutant strains
To determine the effect of chs mutations on virulence of F. oxysporum, root infection assays with tomato plants were performed. Two-week-old plants were inoculated by immersing their roots in a microconidial suspension of the wild-type strain, or the disruptants {Delta}chs1, {Delta}chs2 and {Delta}chs7. Plants were scored for vascular wilt symptoms at different time intervals (Di Pietro & Roncero, 1998). The development of the disease is shown in Fig. 7. Plants inoculated with the wild-type strain produced characteristic wilt symptoms starting 7 days after inoculation. Disease severity increased steadily throughout the experiment, and most of the plants were dead 20 days after inoculation, except for those inoculated with {Delta}chs2 or {Delta}chs7 mutants, which were delayed in the progression of disease. Sixteen days after inoculation most of these plants showed disease symptoms with a degree of 3 in a scale of 5 compared with those inoculated with the wild-type strain (Di Pietro & Roncero, 1998). After the lag phase, the severity of wilting increased progressively, and most of the plants inoculated with wild-type and the rest of the {Delta}chs mutants were dead after 24 days except for those infected with {Delta}chs2 or {Delta}chs7 mutants. The mean response of the mutant {Delta}chs1 (AUDPC=54·27) was not significantly (Pt=0·3743) different from that of the wild-type (AUDPC=56·40). The mutants {Delta}chs2 and {Delta}chs7 significantly (Pt=0·0012, Pt=0·0350, respectively) differed in virulence towards tomato plants (AUDPC=37·10, AUDPC=45·20, respectively) compared with the wild-type.



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Fig. 7. Virulence of F. oxysporum f. sp. lycopersici chs-deficient mutants on tomato plants (cv. ‘Vemar’). Severity of disease symptoms was recorded at different times after inoculation, using an index ranging from 1 (healthy plant) to 5 (dead plant). Symbols refer to plants inoculated with the wild-type strain 4287 ({circ}), {Delta}chs1 ({blacktriangleup}), {Delta}chs2 ({triangleup}), {Delta}chs7 ({blacksquare}) and the non-inoculated control ({bullet}). Error bars indicate the standard error from 15 plants for each treatment.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Previously a class V CS-encoding gene (chsV) had been isolated and shown to be required for pathogenicity during host infection by F. oxysporum f. sp. lycopersici (Madrid et al., 2003). The results presented here report the identification of three genes, chs1, chs2 and chs3, encoding structural CSs. The high degree of conservation in CS catalytic domains allowed the design of a unique pair of degenerate primers for PCR amplification of genomic DNA. Based on deduced amino acid sequences, these newly isolated F. oxysporum chs genes presumably encode class I, class II and class III enzymes, all belonging to family I (Bowen et al., 1992). The deduced amino acid sequences of the F. oxysporum chs genes share a considerable degree of identity, ranging from 41 % to 30 % and extending mainly between amino acids positions 190 to 600. Another gene, chs7, orthologous to the S. cerevisiae CHS7 which is required for functional ScChs3p activity (class IV) and responsible for its export from the ER (Roncero, 2002), has been isolated. Thus, the complexity of chitin synthesis in this pathogenic filamentous fungus, at the sequence and protein levels, appears to be similar to that in related filamentous fungi species. Interestingly, the most perturbed mould phenotype is seen when CSs of the mould-specific class III and V are disrupted, whereas minor or no phenotypes are seen when members of class I, II and IV are inactivated (Mellado et al., 2003). In accordance with this, targeted inactivation of genes chs1 and chs2 in F. oxysporum f. sp. lycopersici, from class I and class II respectively, caused no major effects in sporulation rates, chitin content, morphology or hyphal growth, while the {Delta}chsV mutant (class V) displays morphological abnormalities and cell lysis, and is non-pathogenic and hypersensitive to plant antimicrobial defence compounds (Madrid et al., 2003); and all the attempts to disrupt the chs3 gene (class III) in F. oxysporum by gene-replacement-mediated transformation were unsuccessful, possibly due to a lethal phenotype of the null mutants. Nevertheless, in the human pathogen A. fumigatus two class III genes, chsC and chsG, have been characterized and replacement mutants independently targeted as well as double disruptants have been obtained, demonstrating the dispensable functions of the encoded proteins (Mellado et al., 1996a). In A. nidulans, disruptants in a class III gene (chsB), growing as minute colonies without conidia and producing hyphae with high degree of branching, have been isolated (Borgia et al., 1996). In spite of the numerous reports devoted to the molecular and cellular biology of chs genes and defective mutants derived therefrom, the localization and specific functions of the different CSs have not been elucidated completely in filamentous fungi. In A. nidulans the construction of single and double gene replacement mutants in CS genes, together with the use of vital reporter systems, such as {beta}-galactosidase or green fluorescent protein, enabled the demonstration that chsA (class II) is expressed specifically during asexual differentiation, whereas chsB (class III) is ubiquitous throughout the fungal body and independent of the developmental status. chsC (class I) expression is temporally and spatially regulated, being moderate during sexual development and in the early phase of vegetative growth (Specht et al., 1996; Fujiwara et al., 2000; Lee et al., 2004).

No {Delta}chs7 mutants have been described to date in filamentous fungi. S. cerevisiae {Delta}chs7 mutants have reduced levels of CSIII activity (class IV) and chitin in their cell walls, defects comparable to those observed in the Sc{Delta}chs3 mutants (Trilla et al., 1999), and stronger than those detected in Sc{Delta}chs4 (Trilla et al., 1997), Sc{Delta}chs5 (Santos et al., 1997) or Sc{Delta}chs6 mutants (Bulawa, 1993), underscoring the relevance of this gene in the control of CSIII activity. The attempts to isolate a chs gene belonging to class IV in F. oxysporum failed; however, according to an in silico search in the F. graminearum database, the presence of one or more class IV chs genes can be expected in F. oxysporum. Nevertheless, the close relationship previously reported between Scchs7 and regulation of class IV CSs (Roncero, 2002) make it possible to refer to the {Delta}chs7 F. oxysporum mutant as being defective in class IV activity.

Promoter analyses of the F. oxysporum chs DNA sequences studied identified potential recognition site motifs for different transcription activators such as AbaA, one of the key regulatory transcription factors involved in asexual development in Aspergillus spp. Several putative ARE elements are found in chs2 and chs7 genes, suggesting a developmentally regulated mechanism similar to that described for the A. nidulans chsC (Park et al., 2003). The stress response sequence STRE was present at different positions in the promoters of the four genes analysed. The chs genes might have a special role in cell proliferation and/or in maintaining the structural integrity of the cell wall and may therefore be activated in response to stress signals (Wang et al., 2002), explaining the higher sensitivity of the {Delta}chs1, {Delta}chs2 and {Delta}chs7 mutants to alterations of the membrane caused by detergents, as well as their stronger hyphal hydrophobicity when grown on medium of high osmotic pressure. These different hydrophobic properties shown by colonies of {Delta}chs1, {Delta}chs2 and {Delta}chs7 mutants on sorbitol plates, in comparison to wild-type and {Delta}chsV mutant, are reminiscent of phenotypes of mutants affected in hydrophobin assembly or production. These small secreted proteins are fundamental to the development of fungi; to date, more than 20 hydrophobin-encoding genes have been identified and proven to be ubiquitous in filamentous fungi (Wessels, 1997). The existence of hydrophobic proteins in the outer layer of conidial walls and their involvement in the construction of the conidial outer wall has been demonstrated in the human pathogen A. fumigatus, as have their interactions with the cell wall components altering the surface properties of the fungus (Paris et al., 2003).

The mutants {Delta}chs2 (class II) and {Delta}chs7 (related to class IV) show a significant reduction of virulence, and {Delta}chsV (class V) has been previously reported as non-pathogenic (Madrid et al., 2003). These results indicate the critical importance of the cell wall in the pathogenicity of F. oxysporum. The reduction in virulence of these chs mutants might be indicative of higher sensitivity to plant defence compounds caused by permeability differences in the cell wall. Accordingly, colonial growth of these mutants was more sensitive to SDS than that of the wild-type, suggesting an altered composition and structure of the cell wall that may be affecting hyphal permeability. Nevertheless, comparable levels of resistance to plant defence compounds, like {alpha}-tomatine, caffeine and hydrogen peroxide, as well as to compounds interfering with cell wall assembly, such as CFW or Congo red, were observed in {Delta}chs2 and {Delta}chs7 in comparison with wild-type (data not shown), while {Delta}chsV showed a higher sensitivity (Madrid et al., 2003). The association between the cell wall and pathogenicity has been widely reported, and also specifically with CSs. For instance, the reduction of virulence in Ustilago maydis by disruption of the genes Umchs6 (class V) and Umchs5 (class IV), and reduced virulence in a Botrytis cinerea {Delta}chs1 mutant (class I) have been reported (Garcerá-Teruel et al., 2004; Xoconostle-Cázares et al., 1997; Soulie et al., 2003).

The cell wall defects of the {Delta}chsV and {Delta}chs1 mutant strains could lead to cell cycle alterations that produce the occasional multinuclear phenotype. In A. nidulans it has been demonstrated that the NUDC protein, involved in nuclear migration, has an important role in cell wall biogenesis. Defective nudC mutants show aberrant wall deposition such as overproduction of both chitin and glucan, giving grossly abnormal cell walls uniformly distributed over the cell membrane and the formation of spherical rather than polar cells, suggesting a possible relationship between fungal cell wall biosynthesis and nuclear migration (Chiu et al., 1997). Deletion of the class V CS gene in F. oxysporum causes cell swelling and lysis (Madrid et al., 2003), producing aberrant spherical cell structures with up to eight nuclei and suggesting that altered nuclear distribution through mycelium is a consequence of this cell wall deficiency. The requirement of cell wall integrity for dynein anchoring and correct nuclear positioning has been indicated previously in related fungal systems (Chiu et al., 1997). The presence of a myosin motor-like domain in A. nidulans CsmA and F. oxysporum ChsV proteins suggests that the localization of chitin synthesis may be guided by association with cytoskeletal structures (Fujiwara et al., 1997; Madrid et al., 2003). Whereas in general the morphological phenotype of the F. oxysporum CS 1 null mutants ({Delta}chs1) was indistinguishable from wild-type, some abnormal cells with more than one nucleus were observed, supporting the occurrence of similar pleiotropic effects between cell wall structure and nuclear partitioning.

The F. graminearum genome (Fusarium graminearum Sequencing Project, Center for Genome Research, http://www.broad.mit.edu), has five representatives of the three mould-specific CS classes (class III, class V and class VI). The identification and characterization of the other components that participate in Fusarium cell wall biogenesis will be crucial for the understanding of the process at the molecular level, as well as for the elucidation of the functional relationships with pathogenicity. The results presented here together with other previously reported (Madrid et al., 2003) support the view that CSs may play a role in fungal pathogenesis, and therefore represent potential targets for antifungal intervention (Odds et al., 2003).


   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge Antonio Di Pietro (UCO) and César Roncero (USAL) for helpful discussions, Judith Lichtenzveig (ACNFP) for the statistical analyses, and Isabel Caballero (UCO) for technical assistance. This research was supported by the Ministerio de Ciencia y Tecnología (BIO2001-2601) of Spain and Junta de Andalucía (PAI-CVI-138). M. M. U. was supported by PhD fellowships from MCYT-BIO2001-2601 and PAI-Junta de Andalucía.


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Received 14 April 2004; revised 12 July 2004; accepted 19 July 2004.



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