Departamento de Genética, Campus Rabanales C5, Universidad de Córdoba, E-14071 Córdoba, Spain
Correspondence
M. Isabel G. Roncero
ge1gorom{at}uco.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
Present address: Australian Centre for Necrotrophic Fungal Pathogens, Murdoch University, Perth, WA 6150, Australia.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 12 % 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)
.
|
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 -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
-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.
|
Determination of conidiation.
Conidial suspensions from wild-type and chs mutant strains were inoculated on PDB medium at a concentration of 5x105 spores ml1, 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 chs1,
chs2,
chsV,
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 ml1 for 5 min, and/or 4',6-diamidino-2-phenylindole (DAPI) 0·8 µg ml1 (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The F. oxysporum -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
primers,
-int 5'-CGCAACTCGTGAAAGGTA-3' and
-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.
|
|
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
chs1), from 6·5 kb to 8·5 kb (in transformants
chs2.1 and
chs2.6), from 10 kb to 8·5 kb (in transformant
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 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,
-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
chs1,
chs2 and
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 chs mutants
Conidiation in the three 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 ml1 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
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,
chs2 and
chs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus (Fig. 5a, d, e
), whereas in the
chs1 mutant some compartments containing up to four nuclei could be seen (Fig. 5c, c
'). The class V deficient mutant (
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
chs mutants but not septum formation.
|
|
Chitin content of chs1,
chs2 and
chs7 mutant strains
The total mycelial chitin content of the chs1,
chs2 and
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
chs1 and
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
).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
No chs7 mutants have been described to date in filamentous fungi. S. cerevisiae
chs7 mutants have reduced levels of CSIII activity (class IV) and chitin in their cell walls, defects comparable to those observed in the Sc
chs3 mutants (Trilla et al., 1999
), and stronger than those detected in Sc
chs4 (Trilla et al., 1997
), Sc
chs5 (Santos et al., 1997
) or Sc
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
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
chs1,
chs2 and
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
chs1,
chs2 and
chs7 mutants on sorbitol plates, in comparison to wild-type and
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 chs2 (class II) and
chs7 (related to class IV) show a significant reduction of virulence, and
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
-tomatine, caffeine and hydrogen peroxide, as well as to compounds interfering with cell wall assembly, such as CFW or Congo red, were observed in
chs2 and
chs7 in comparison with wild-type (data not shown), while
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
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 chsV and
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 (
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410.[CrossRef][Medline]
Andrianopoulus, A. & Timberlake, W. E. (1994). The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol Cell Biol 14, 25032515.[Abstract]
Aufauvre-Brown, A., Mellado, E., Gow, N. A. & Holden, D. W. (1997). Aspergillus fumigatus chsE: a gene related to CHS3 of Saccharomyces cerevisiae and important for hyphal growth and conidiophore development but not pathogenicity. Fungal Genet Biol 21, 141152.[CrossRef][Medline]
Bartnicki-García, S., Ruiz-Herrera, J. & Bracker, J. (1984). Chitosomes and chitin synthesis. In Fungal Walls and Hyphal Growth, pp. 149168. Edited by J. H. Burnett & A. P. J. Trinci. Cambridge, UK: Cambridge University Press.
Beckman, C. H. (1987). The Nature of Wilt Diseases of Plants. St Paul, MN: American Phytopathological Society.
Borgia, P. T., Iartchouk, N., Riggle, P. J., Winter, K. R., Koltin, Y. & Bulawa, C. E. (1996). The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal Genet Biol 20, 193203 (erratum in Fungal Genet Biol 20, 314).[CrossRef]
Bowen, A. R., Chen-Wu, J. L., Momany, M., Young, R., Szaniszlo, P. J. & Robbins, P. W. (1992). Classification of fungal chitin synthases. Proc Natl Acad Sci U S A 89, 519523.[Abstract]
Bulawa, C. E. (1993). Genetics and molecular biology of chitin synthesis in fungi. Annu Rev Microbiol 47, 505534.[CrossRef][Medline]
Bull, A. T. (1970). Chemical composition of wild-type and mutant Aspergillus nidulans cell walls. The nature of polysaccharide and melanin constituents. J Gen Microbiol 63, 7594.[Medline]
Cabib, E., Shaw, J. A., Mol, P. C., Bowers, B. & Choi, W. J. (1996). Chitin biosynthesis and morphogenetic processes. In The Mycota. Biochemistry and Molecular Biology, vol. III, pp. 243267. Edited by R. Brambl & G. A. Marzluf. Berlin, Germany: Springer.
Chiu, Y. H., Xiang, X., Dawe, A. L. & Morris, N. R. (1997). Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal. Mol Biol Cell 8, 17351749.[Abstract]
Chomzczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156159.[CrossRef][Medline]
Cohen, E. (1990). Extracellular biopolymers as targets for pest control. In Pesticides and Alternatives. Innovative Chemical and Biological Approaches to Pest Control, pp. 2332. Edited by J. E. Casida. Amsterdam: Elsevier.
Cos, T., Ford, R. A., Trilla, J. A., Durán, A., Cabib, E. & Roncero, C. (1998). Molecular analysis of Chs3p, participation in chitin synthase III activity. Eur J Biochem 256, 419426.[Abstract]
Din, A. B. & Yarden, O. (1994). The Neurospora crassa chs-2 gene encodes a nonessential chitin synthase. Microbiology 140, 21892197.[Medline]
Din, A. B., Specht, C. A., Robbins, P. W. & Yarden, O. (1996). chs-4, a class IV chitin synthase gene from Neurospora crassa. Mol Gen Genet 250, 214222.[CrossRef][Medline]
Di Pietro, A. & Roncero, M. I. G. (1998). Cloning, expression and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum. Mol PlantMicrobe Interact 11, 9198.[Medline]
Estruch, F. (2000). Stress-controlled transcription factors, stress-induced genes and stress-tolerance in budding yeast. FEMS Microbiol Rev 24, 469486.[CrossRef][Medline]
Fujiwara, M., Horiuchi, H., Ohta, A. & Takagi, M. (1997). A novel fungal gene encoding chitin synthase with a myosin motor-like domain. Biochem Biophys Res Commun 236, 7578.[CrossRef][Medline]
Fujiwara, M., Ichinomiya, M., Motoyama, T., Horiuchi, H., Ohta, A. & Takagi, M. (2000). Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development. J Biochem 127, 359366.[Abstract]
Galagan, J. E., Calvo, S. E., Borkovich, K. A. & 74 other authors (2003). The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859868.[CrossRef][Medline]
Garcerá-Teruel, A., Xoconostle-Cázares, B., Rosas-Qui jano, R., Ortiz, L., León-Ramírez, C., Specht, C. A., Sentandreu, R. & Ruiz-Herrera, J. (2004). Loss of virulence in Ustilago maydis by Umchs6 gene disruption. Res Microbiol 155, 8797.[CrossRef][Medline]
García-Maceira, F. I., Di Pietro, A. & Roncero, M. I. G. (2000). Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum. Mol PlantMicrobe Interact 13, 359365.[Medline]
Harris, S. D., Morrell, J. L. & Hamer, J. E. (1994). Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. Genetics 136, 517532.
Horiuchi, H., Fujiwara, M., Yamashita, S., Ohta, A. & Takagi, M. (1999). Proliferation of intrahyphal hyphae caused by disruption of csmA, which encodes a class V chitin synthase with a myosin motor-like domain in Aspergillus nidulans. J Bacteriol 181, 37213729.
Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 6168.[CrossRef][Medline]
Lee, J. I., Choi, J. H., Park, B. C., Park, Y. H., Lee, M. Y., Park, H.-M. & Maeng, P. J. (2004). Differential expression of the chitin synthase genes of Aspergillus nidulans, chsA, chsB and chsC, in response to developmental status and environmental factors. Fungal Genet Biol 41, 635646.[CrossRef][Medline]
Litzka, O., Papagiannopolous, P., Davis, M. A., Hynes, M. J. & Brakhage, A. A. (1998). The penicillin regulator PENR1 of Aspergillus nidulans is a HAP-like transcriptional complex. Eur J Biochem 251, 758767.[Abstract]
Madrid, M. P., Di Pietro, A. & Roncero, M. I. G. (2003). Class V chitin synthase determines pathogenicity in the vascular wilt fungus Fusarium oxysporum f.sp. lycopersici and mediates resistance to plant defense compounds. Mol Microbiol 47, 256266.
Mellado, E., Aufauvre-Brown, A., Specht, C. A., Robbins, P. W. & Holden, D. W. (1995). A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus. Mol Gen Genet 246, 353359.[Medline]
Mellado, E., Aufauvre-Brown, A., Gow, N. A. & Holden, D. W. (1996a). The Aspergillus fumigatus chsC and chsG encode class III chitin synthases with different functions. Mol Microbiol 20, 667679.[Medline]
Mellado, E., Specht, C. A., Robbins, P. W. & Holden, D. W. (1996b). Cloning and characterisation of chsD, a chitin synthase-like gene of Aspergillus fumigatus. FEMS Microbiol Lett 143, 6976.[CrossRef][Medline]
Mellado, E., Dubreucq, G., Mol, P., Sarfati, J., Paris, S., Diaquin, M., Holden, D. W., Rodríguez-Tudela, J. L. & Latgé, J. P. (2003). Cell wall biogenesis in a double chitin synthase mutant (chsG/chsE) of Aspergillus fumigatus. Fungal Genet Biol 38, 98109.[CrossRef][Medline]
Motoyama, T., Fujiwara, M., Kojima, N., Horiuchi, H., Ohta, A. & Takagi, M. (1996). The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation. Mol Gen Genet 253, 520528.
Munro, C. A. & Gow, N. A. R. (2001). Chitin synthesis in human pathogenic fungi. Med Mycol 39, 4143.[Medline]
Odds, F. C., Brown, A. J. P. & Gow, N. A. R. (2003). Antifungal agents: mechanisms of action. Trends Microbiol 11, 272279.[CrossRef][Medline]
Paris, S., Debeaupuis, J. P., Crameri, R., Carey, M., Charles, F., Prevost, M. C., Schmitt, C., Philippe, B. & Latgé, J. P. (2003). Conidial hydrophobins of Aspergillus fumigatus. Appl Environ Microbiol 69, 15811588.
Park, B.-C., Park, Y.-H. & Park, H.-M. (2003). Activation of chsC transcription by AbaA during asexual development of Aspergillus nidulans. FEMS Microbiol 220, 241246.[CrossRef]
Reissig, J. L., Stringer, J. L. & Leloir, L. F. (1955). A modified colorimetric method for the estimation of N-acetylaminosugars. J Biol Chem 217, 959966.
Roldán-Arjona, T., Pérez-Espinosa, A. & Ruiz-Rubio, M. (1999). Tomatinase from Fusarium oxysporum f.sp. lycopersici defines a new class of saponinases. Mol PlantMicrobe Interact 12, 852861.[Medline]
Roncero, C. (2002). The genetic complexity of chitin synthesis in fungi. Curr Genet 41, 367378.[CrossRef][Medline]
Ruiz-Herrera, J. & Martínez-Espinoza, A. D. (1999). Chitin biosynthesis and structural organization in vivo. In Chitin and Chitinases, pp. 3953. Edited by P. Jolles & R. A. A. Muzzarelli. Basel, Switzerland: Birkhäuser Verlag.
Ruiz-Herrera, J., Sentandreu, R. & Martínez, J. P. (1992). Chitin biosynthesis in fungi. In Handbook of Applied Mycology, vol. 4, Fungal Biotechnology, pp. 281312. Edited by D. K. Arora, R. P. Elander & K. G. Mukerji. New York: Marcel Dekker.
Ruiz-Herrera, J., González-Prieto, J. M. & Ruiz-Medrano, R. (2002). Evolution and phylogenetic relationships of chitin synthases from yeasts and fungi. FEMS Yeast Res 4, 247256.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Santos, B., Durán, A. & Valdivieso, M. H. (1997). CHS5 a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae. Mol Cell Biol 17, 24852496.[Abstract]
Schoffelmeer, E. A. M., Klis, F. M., Sietsma, J. H. & Cornelissen, B. J. C. (1999). The cell wall of Fusarium oxysporum. Fungal Genet Biol 27, 275282.[CrossRef][Medline]
Soulie, M. C., Piffeteau, A., Choquer, M., Boccara, M. & Vidal-Cros, A. (2003). Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence. Fungal Genet Biol 40, 3846.[CrossRef][Medline]
Specht, C. A., Liu, Y., Robbins, P. W. & 8 other authors (1996). The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis. Fungal Genet Biol 20, 153167.[CrossRef][Medline]
Trilla, J. A., Cos, T., Duran, A. & Roncero, C. (1997). Characterization of CHS4 (CAL2), a gene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4. Yeast 9, 795807.[CrossRef]
Trilla, J. A., Durán, A. & Roncero, C. (1999). Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae. J Cell Biol 145, 11531163. (erratum in J Cell Biol 1999, 146, following 264).
Turgeon, B. G., Garber, R. C. & Yoder, O. C. (1987). Developmemt of a fungal transformation system based on selection of sequences with promoter activity. Mol Cell Biol 7, 32973305.[Medline]
Vartivarian, S. E., Anaissie, E. J. & Bodey, G. P. (1993). Emerging fungal pathogens in immunocompromised patients: classification, diagnosis, and management. Clin Infect Dis 17, 487491.
Vidal-Cros, A. & Boccara, M. (1998). Identification of four chitin synthase genes in the rice blast disease agent Magnaporthe grisea. FEMS Microbiol Lett 165, 103109.[CrossRef][Medline]
Wang, Q., Liu, H. & Szaniszlo, P. J. (2002). Compensatory expression of five chitin synthase genes, a response to stress stimuli, in Wangiella (Exophiala) dermatitidis, a melanized fungal pathogen of humans. Microbiology 148, 28112817.[Medline]
Wessels, J. G. (1997). Hydrophobins: proteins that change the nature of the fungal surface. Adv Microb Physiol 38, 145.[Medline]
Xoconostle-Cazares, B., Specht, C. A., Robbins, P. W., Liu, Y., León, C. & Ruiz-Herrera, J. (1997). Umchs5, a gene coding for a class IV chitin synthase in Ustilago maydis. Fungal Genet Biol 22, 199208.[CrossRef][Medline]
Yarden, O. & Yanofsky, C. (1991). Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa. Genes Dev 5, 24202430.[Abstract]
Received 14 April 2004;
revised 12 July 2004;
accepted 19 July 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |