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Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumi-dori, Sendai 981-8555,1 New Industry Creation Hatchery Center, ABE Project, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan2
Received 3 September 2006/ Accepted 11 June 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and mpk1 mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4 and swi6 mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmA, Answi4 Answi6, and mpkA strains) and analyzed mpkA and CWG transcripts after treatment with a ß-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The ß-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkA strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except 
-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell wall biogenesis and its regulation by cell signaling in the yeast S. cerevisiae have been well studied (37, 55). Fungal cells, including those of yeasts, constantly remodel their rigid structure during growth and development and under environmental stress (55). Maintaining a proper cell wall architecture in S. cerevisiae requires cell wall components such as ß-1,3-glucan, ß-1,6-glucan, chitin, and mannoproteins (28, 31). A signal transduction process in S. cerevisiae that monitors and promotes the remodeling of the cell wall has been described in detail previously (19). The sensing of cell wall perturbations requires surface sensors. Findings from genetic studies place one group of cell wall integrity and stress response genes (WSC1 to WSC4) upstream of the intracellular signal transduction pathway responsible for maintaining cell wall integrity in this yeast (10, 65). Additional cell wall stress sensors include the partially redundant Mid2p and Mlt1p cell surface proteins (48). These proteins act as mechanosensors of cell wall stress during growth or pheromone-induced morphogenesis, exposure to high temperatures, or other cell wall perturbations and transmit signals to the downstream signaling pathway (18, 30, 49, 61). The activation of the cell wall integrity signaling (CWIS) pathway proceeds through the small G protein Rho1p via Pkc1p (38) and a downstream mitogen-activated protein kinase (MAPK) cascade. Rho1p is a small GTPase upregulated by the GDP/GTP exchange factors Rom1p and Rom2p (45, 47) and downregulated by the GTPase-activating proteins Sac7p and Bem2p (46, 53). In addition to its other functions, Rho1p binds with and activates Pkc1p (27, 44), which in turn activates the MAPK cascade. The MAPK cascade is a linear pathway that is composed of a MAPK kinase kinase (encoded by BCK1) (6, 36), a pair of redundant MAPK kinases (encoded by MKK1 and MKK2) (21), and a MAPK (encoded by MPK1/SLT2) (35). CWIS is induced in response to several environmental stimuli, resulting in increased levels of expression of numerous genes, many of which encode integral cell wall proteins (glycosylphosphatidylinositol proteins, Pir [protein with internal repeats] family proteins, and others) or enzymes involved in cell wall biogenesis, including ß-1,3-glucan synthases (Fks1p and Fks2p) and chitin synthase (Chs3p) (25). Mpk1p, which is activated by means of CWIS, phosphorylates and activates the transcription factor Rlm1p, which regulates the transcription of at least 25 genes involved in cell wall biogenesis in S. cerevisiae (25). These 25 genes include MPK1, which means that the transcription of MPK1 is autoregulated in an Mpk1p-Rlm1p-dependent manner. Mpk1p has another target: the Swi4p-Swi6p complex (SBF) transcription factor. SBF also controls the transcription of cell wall-related genes in addition to that of cell cycle-related genes in the G1/S phase (23).
In filamentous fungi, including Aspergillus nidulans, A. oryzae, and Magnaporthe grisea, several genes that encode MAPKs of CWIS have been isolated (5, 41, 64). In A. nidulans, mpkA, which is a counterpart of the yeast MPK1 (SLT2), has been cloned and characterized previously (5). An A. nidulans mpkA deletion mutant (mpkA) has been constructed, and its morphological defects suggested that the kinase encoded by mpkA is involved in the germination of conidial spores and in polarized growth (5). Mizutani et al. (41) reported that the disruption of kexB, which encodes a subtilisin-like processing enzyme in A. oryzae, results in an increased level of expression of several cell wall-related genes and of an mpkA gene concomitant with high levels of phosphorylated MpkA. They further suggested the autoregulation of mpkA expression through CWIS via MpkA. During the course of the present study, Damveld et al. (8) found an A. niger rlmA gene homologous to S. cerevisiae RLM1 and reported that rlmA is involved in responses to cell wall stress. Recently, the genome databases for three Aspergillus species (A. nidulans [14], A. oryzae [39], and A. fumigatus [43]) have become available, and we searched these databases for gene homologs involved in cell wall biogenesis. In the in silico reconstruction depicted in Fig. 1, most homologs involved in yeast cell wall biogenesis in A. nidulans and the other two Aspergillus species appear to be well conserved (see Table S1 in the supplemental material). However, the in silico assignment of transcription factors is generally difficult, because conserved regions in transcription factors are often restricted to DNA-binding domains such as a zinc finger domain and a leucine zipper motif or other limited motifs. Thus, the functionality of transcription factors must be examined in vivo. Although most genes orthologous to the yeast CWIS genes are conserved in the genomes of the three Aspergillus species (14, 39, 43) and the signaling pathway seems to maintain cell wall biogenesis in these fungi as described above (5, 41), the target genes for transcription factors downstream of the CWIS via MpkA remain unclear except for those identified in a few studies, such as a study of the constitutive autoregulation of mpkA expression in an A. oryzae kexB disruptant (41) and a study of the RlmA-dependent expression of agsA, which encodes -1,3-glucan synthase, in A. niger (8).
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-1,3-glucan synthase genes is independent of RlmA or AnSwi4-AnSwi6 and rather seems to depend on non-MpkA signaling. The transcriptional regulation of mpkA and of cell wall-related genes in A. nidulans thus differs significantly from that in S. cerevisiae. |
MATERIALS AND METHODS |
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Genomic DNA isolation. Genomic DNA was extracted from mycelia frozen in liquid nitrogen and ground into a fine powder with a mortar and pestle. The powder was then resuspended in a lysing buffer (2% sodium dodecyl sulfate [SDS], 10 mM Tris·HCl [pH 7.0], 1 mM EDTA) and incubated for 2 h at 60°C. Next, an equal volume of buffer-saturated phenol was added and the mixture was centrifuged at 3,000 x g for 10 min. The supernatant was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and then with chloroform-isoamyl alcohol (24:1), followed by ethanol precipitation.
Molecular cloning and sequencing of the rlmA gene.
For subcloning, we used Escherichia coli XL1-Blue (hsdR17 supE44 recA1 endA1 gryA46 thi relA1 lac [F' proAB+ lacIqZM15::Tn10 Tetr]) cells and the pBluescript II KS+ plasmid (Toyobo Inc., Tokyo, Japan) as the host and vector, respectively, for DNA manipulation. We used the vector pGEM-T Easy (Promega Co., Tokyo, Japan) for TA cloning of PCR products. All basic molecular biology procedures were carried out as described by Sambrook and Russell (52). To clone the A. nidulans rlmA gene, we searched the A. nidulans genome database (http://www.broad.mit.edu/annotation/genome/aspergillus_nidulans/BLAST.html) for the S. cerevisiae RLM1 gene and found a sequence containing Rlm1p-like signatures such as a MADS box motif (56), a MAPK docking site (26), and an acidic amino acid region (62). DNA fragments containing the open reading frame (ORF) homologous to the yeast RLM1 gene were amplified by PCR using A. nidulans genomic DNA and the primers rlmA-1-F and rlmA-1-R. (All primers described in this paper are shown in Table 1.) The amplified fragments were subcloned into pGEM-T Easy, and DNA sequences of the cloned fragments were determined using an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit, version 3.0 (Applied Biosystems Japan Ltd., Tokyo, Japan), and an ABI PRISM 377 sequencer (Applied Biosystems Japan). Then we amplified a cDNA fragment from an A. nidulans cDNA library (13) by using PCR primers (rlmA-2-F and rlmA-2-R) designed from the genomic DNA. Each primer was designed to introduce either a HindIII site or an XhoI site. The amplified cDNA fragment was subcloned into pGEM-T Easy, and the DNA sequences of the cloned fragments were determined as described above.
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::LEU2 ura3 leu2 trp1 his4 can1) that exhibits the caffeine-sensitive phenotype for complementation analysis. Expression plasmids used in this experiment were constructed with the expression vector pYES2 (Invitrogen Co., Tokyo, Japan), in which expression is under the control of the galactose-inducible GAL1 promoter (24). A fragment containing the complete ORF of the rlmA cDNA was digested with HindIII and XhoI and ligated into the corresponding sites in pYES2, resulting in the rlmA expression vector pYESrlmA. To construct the plasmids pYESrlmA-M1 and pYESrlmA-M2, which produced mutant RlmA proteins in the MAPK docking site, we generated nucleotide substitutions in pYESrlmA by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the primers rlmA-3-F and rlmA-3-R for M1 (Leu [CTG] and Val [GTC] were each replaced by Ala [GCT]) and the primers rlmA-4-F and rlmA-4-R for M2 (Ile [ATA] and Pro [CCG] were each replaced by Ala [GCT]). Each mutation was confirmed by DNA sequencing. YEp195-RLM1 was used for the constitutive expression of yeast RLM1 as the positive control (62). We amplified an A. nidulans AN8676.3 cDNA fragment from an A. nidulans cDNA library by using PCR primers AN8676.3-F and AN8676.3-R. The amplified cDNA fragment was then subcloned into the pGEM-T Easy vector, resulting in pGEM-AN8676.3, and DNA sequences of the cloned fragments were determined as described above. We constructed plasmid pYES-AN8676.3 by the insertion of a NotI fragment containing the complete ORF from pGEM-AN8676.3 into the NotI site of pYES2. Plasmids containing rlmA cDNA, its derivatives, yeast RLM1, and AN8676.3 cDNA were used to transform S. cerevisiae GMY63-5D by the lithium acetate method (22), and we performed complementation analyses with the transformants and the wild type (GMY63-5B; MATa ura3 leu2 trp1 his4 can1) maintained on YPD medium (1% yeast extract, 2% polypepton, 2% glucose) or YPG medium (in which the glucose in YPD medium was replaced with 2% galactose) containing 7 mM caffeine at 30°C for 3 or 6 days.
Complementation analysis of mpkA in an mpk1 deletion mutant. We amplified an A. nidulans mpkA cDNA fragment from an A. nidulans cDNA library by using PCR primers mpkA-1-F and mpkA-1-R. The amplified cDNA fragment was then subcloned into the pGEM-T Easy vector, resulting in pGEMmpkA, and DNA sequences of the cloned fragments were determined as described above.
An S. cerevisiae strain (TNP46; MATa mpk1::HIS3 ura3 leu2 trp1 his3 ade2 can1) that displays an autolytic lethal phenotype at 37°C was used for the complementation analysis. The expression plasmids used in this experiment were constructed with the multicopy expression vector YEpGAP, in which expression is under the control of the constitutive GAPDH promoter. We constructed plasmid YEpGAPmpkA by inserting a NotI fragment containing the complete ORF from pGEMmpkA into the NotI site of YEpGAP. We used YCplac33-MPK1 for the constitutive expression of yeast MPK1 as the positive control (62).
Plasmids containing mpkA cDNA and yeast MPK1 were used to transform S. cerevisiae TNP46 by the lithium acetate method (22), and a complementation analysis with the transformants maintained on YPD medium at 23 and 37°C for 3 days was performed.
Construction of an A. nidulans rlmA-argB gene disruptant. A fragment containing the full length of rlmA was amplified by PCR using genomic DNA of A. nidulans FGSC A89 and the primers rlmA-5-F and rlmA-5-R and was subcloned into pGEM-T Easy, resulting in pGEMrlmA. Next, MfeI and KpnI sites were introduced into pGEMrlmA by using a QuikChange site-directed mutagenesis kit with the primers rlmA-6-F and rlmA-6-R for MfeI and rlmA-7-F and rlmA-7-R for KpnI, resulting in pTMKrlmA. A fragment of A. oryzae argB, which complements the argB2 mutant of A. nidulans, was obtained from pAORB (13) by digestion with EcoRI and KpnI. The AoargB fragment was ligated into the EcoRI and KpnI sites of pTMKrlmA, resulting in pGEMrlmA::argB. An SphI-PstI fragment bearing rlmA::argB was ligated into the SphI and PstI sites of pSOF31, containing the A. nidulans oliC31 gene, resulting in pSOFrlmA::argB. We transformed A. nidulans FGSC A89 by the protoplast method (17) with the linear form of pSOFrlmA::argB by SphI digestion. We used A. nidulans oliC31 as a selection marker for homologous recombinants, which allowed the elimination of ectopic integration of the targeting vector into chromosomes (58). A. nidulans oliC31 encodes a mutant subunit 9 of F1Fo-ATPase, and the expression of the oliC31 gene in A. nidulans indicates phenotypes that show resistance to oligomycin and hypersensitivity to triethyltin. When the targeting vector is integrated into the target site by means of homologous recombination, the fragment of oliC31 is excised and, thus, the homologous recombinants exhibit triethyltin resistance. On the other hand, when the targeting vector is integrated into an ectopic site by means of nonhomologous recombination, the fragment of oliC31 remains on the chromosome and the remaining fragment causes hypersensitivity to triethyltin. We used the following medium for positive selection: CD medium supplemented with a mixture of 0.02 µg of biotin/ml, 1% succinic acid, and 0.000025% triethyltin (Strem Chemicals, Inc., Newburyport, MA) and in which the glucose was replaced with 0.3% sucrose (58). We confirmed rlmA disruption and the integration of a single copy of rlmA::argB at the rlmA locus by Southern blot analysis. The construction of the disruptant is summarized in Fig. S1 in the supplemental material, where confirmation of the success of this disruption is also presented.
Construction of an A. nidulans mpkA-argB gene disruptant. A fragment containing the full length of mpkA was amplified by PCR using the primers mpkA-2-F and mpkA-2-R and was subcloned into pGEM-T Easy, resulting in pGEMmpkA. Next, MfeI and KpnI sites were introduced into pGEMmpkA by using a QuikChange site-directed mutagenesis kit with the primers mpkA-3-F and mpkA-3-R for MfeI and mpkA-4-F and mpkA-4-R for KpnI, resulting in pTMKmpkA. The AoargB fragment was ligated into the EcoRI and KpnI sites of pTMKmpkA, resulting in pGEMrlmA::argB. We transformed A. nidulans FGSC A89 by the protoplast method (17) with the linear form of pGEMmpkA::argB by ApaI digestion. We confirmed mpkA disruption and the integration of a single copy of mpkA::argB at the mpkA locus by Southern blot analysis. The construction of the disruptant is summarized in Fig. S1 in the supplemental material, where confirmation of the success of this disruption is also presented.
Construction of an A. nidulans Answi4::argB gene disruptant. 5'- and 3'-flanking region fragments of Answi4 were amplified by PCR using the primers Answi4-1-F (BglII), Answi4-1-R (KpnI), Answi4-2-F (MunI), and Answi4-2-R (XbaI) and digested by the indicated restriction enzymes. The two fragments and the AoargB fragment were simultaneously ligated into the BglII and XbaI sites of pSOF31, resulting in pSOFswi4::argB. We transformed A. nidulans FGSC A89 by the protoplast method (17) with the linear form of pSOFswi4::argB by ApaI digestion. We confirmed Answi4 disruption and the integration of a single copy of Answi4::argB at the Answi4 locus by Southern blot analysis. The construction of the disruptant is summarized in Fig. S1 in the supplemental material, where confirmation of the success of this disruption is also presented.
Construction of an A. nidulans Answi6::argB gene disruptant. A fragment containing the full length of Answi6 was amplified by PCR using the primers Answi6-1-F and Answi6-1-R and was subcloned into pGEM-T Easy, resulting in pGEMswi6. Next, a KpnI site was introduced into pGEMswi6 by using a QuikChange site-directed mutagenesis kit with the primers Answi6-2-F and Answi6-2-R, resulting in pTKswi6. The AoargB fragment (digested by KpnI-XbaI) was ligated into the KpnI and NheI sites of pTKmpkA, resulting in pGEMswi6::argB. An ApaI-SpeI fragment bearing Answi6::argB was ligated into the ApaI and BlnI sites of pSOF31, resulting in pSOFswi6::argB. We transformed A. nidulans FGSC A89 by the protoplast method (17) with the linear form of pSOFswi6::argB by ApaI digestion. We confirmed Answi6 disruption and the integration of a single copy of Answi6::argB at the Answi6 locus by Southern blot analysis. The construction of the disruptant is summarized in Fig. S1 in the supplemental material, where confirmation of the success of this disruption is also presented.
Fluorescence microscopy.
We inoculated conidiospores (ca. 4 x 108) of the wild-type and rlmA strains of A. nidulans into 200 ml of CD liquid medium and cultured the cells for 24 h at 30°C with shaking at 160 rpm. The cells from each culture were dried onto the glass slides for fixation. The glass slides were transferred into 3.5% formaldehyde containing a mixture of 50 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], 5 mM MgSO4, and 25 mM EGTA (PEM buffer; pH 7.0) for fixation and were incubated at room temperature for 45 min. The glass slides were washed three successive times in PEM buffer for 10 min each and further incubated in PEM buffer containing 10 µg of calcofluor white (CFW)/ml for 10 min at 25°C. After being stained with CFW, the glass slides were washed three successive times in PEM buffer for 5 min each time. Cells were observed using an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
Preparation of cell extracts from A. nidulans and immunoblot analysis.
Conidiospores (ca. 4 x 108) of the wild-type and mpkA strains of A. nidulans were inoculated into 200 ml of CD liquid medium, and the cells were cultured at 30°C with shaking at 180 rpm. After 24 h, we added micafungin (Fujisawa Pharmaceutical, Osaka, Japan), which is a ß-1,3-glucan synthase inhibitor, at a concentration of 0.01 µg/ml. We carried out the isolation of total protein samples and immunoblot analysis as described previously (14). Each sample (40 µg of protein) was subjected to SDS-polyacrylamide gel electrophoresis. The dual phosphorylation of MpkA was detected using an anti-phospho-p44/42 MAPK (Cell Signaling Technology, Inc., Beverly, MA). To detect MpkA, we used the anti-extracellular signal-regulated kinase 2 antibody (Sigma, St. Louis, MO).
Preparation of cell extracts from S. cerevisiae and immunoblot analysis. Strain W303-1A (MATa ura3 leu2 trp1 his3 ade2 can1) was grown to an A600 of 0.7 in YPD medium at 30°C, and then micafungin was added to the medium to a final concentration of 1 µg/ml. Cells were suspended in protein extraction buffer (120 mM Tris·HCl [pH 8.8], 5% SDS, 5% mercaptoethanol, 10% glycerol, and 1 mM sodium vanadate). The suspension was immediately boiled for 10 min, and then cell debris was removed by centrifugation for 10 min at 15,000 x g. Each sample (50 µg of protein) was subjected to SDS-polyacrylamide gel electrophoresis. The dual phosphorylation of Mpk1p was examined by immunoblot analysis using an anti-phospho-p44/42 MAPK (Cell Signaling Technology, Inc., Beverly, MA). To detect Mpk1p, we used the anti-Mpk1p antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was visualized using an ECL kit (Amersham) after the binding of a horseradish peroxidase-conjugated second antibody.
Preparation of total RNA from S. cerevisiae.
Wild-type (GMY63-5B) and rlm1 (GMY63-5D) strains were grown to an A600 of 0.7 in YPD medium at 30°C, and then micafungin was added to the medium to a final concentration of 1 µg/ml. We prepared total RNA from the collected cells by using Sepasol-RNA I Super according to the instructions of the manufacturer (Nacalai Tesque, Kyoto, Japan).
Northern hybridization.
We inoculated conidiospores (ca. 4 x 108) of the wild-type, Answi4, and Answi6 strains of A. nidulans into 200 ml of CD liquid medium and cultured the cells at 30°C with shaking at 180 rpm. After 24 h, we added 0.01 µg of micafungin/ml. We collected the mycelia and quickly froze them in liquid nitrogen. The frozen mycelia were ground into a fine powder with a mortar and pestle chilled with liquid nitrogen. We prepared total RNA from the powdered cells by using Sepasol-RNA I Super according to the instructions of the manufacturer (Nacalai Tesque). We isolated mRNA from the total RNA by using an Oligotex-dT30 (Super) mRNA purification kit (TaKaRa Bio Inc., Tokyo, Japan) according to the manufacturer's instructions.
We electrophoresed mRNAs (500 ng each) through an agarose-formaldehyde gel and transferred them onto Hybond-N+ nylon membranes (Amersham Biosciences Inc., Tokyo, Japan) by using 7.5 mM NaOH. Blotted membranes were hybridized with the probes for mpkA of A. nidulans. The probe for the histone H2B gene was used as a quantitative control.
We prepared the mpkA probe by PCR amplification with two primers (mpkA-1-F and mpkA-1-R) by using the A. nidulans cDNA library as the template. A probe for the histone H2B gene was prepared by PCR using the primers Histone-P-F and Histone-P-R against A. nidulans genomic DNA. These probes were labeled with [-32P]dCTP by using a Rediprime II kit (Amersham Biosciences). We then hybridized each blot at 60°C with either of the [-32P]dCTP-labeled probes, as described above. We washed the blots in a mixture of 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS at room temperature for 20 min and then twice in 1x SSC-0.1% SDS at 65°C for 15 min each time and detected the results by means of autoradiography.
Quantitative reverse transcription-PCR (RT-PCR). We reverse transcribed 250 ng of mRNA (A. nidulans) or 5 µg of total RNA (S. cerevisiae) and amplified cDNA samples by PCR. The histone H2B (A. nidulans) or RPL28 (S. cerevisiae) gene was used to standardize the mRNA levels of the target genes. Quantitative PCR analysis was performed using the DNA Engine Opticon 2 system (Bio-Rad) and the DyNAmo SYBR green quantitative PCR kit (Finnzymes, Espoo, Finland). We used the following primers: mpkA-RT-F and mpkA-RT-R for mpkA, agsA-RT-F and agsA-RT-R for agsA, agsB-RT-F and agsB-RT-R for agsB, fksA-RT-F and fksA-RT-R for fksA, gelA-RT-F and gelA-RT-R for gelA, gelB-RT-F and gelB-RT-R for gelB, chsA-RT-F and chsA-RT-R for chsA, chsB-RT-F and chsB-RT-R for chsB, chsC-RT-F and chsC-RT-R for chsC, chsD-RT-F and chsD-RT-R for chsD, csmA-RT-F and csmA-RT-R for csmA, csmB-RT-F and csmB-RT-R for csmB, gfaA-RT-F and gfaA-RT-R for gfaA, Histone-RT-F and Histone-RT-R for the histone H2B gene, MLP1-RT-F and MLP1-RT-R for MLP1, and RPL28-RT-F and RPL28-RT-R for the RPL28 gene.
Construction of a plasmid containing an mpkA-ß-glucuronidase (GUS) gene fusion. A fragment containing E. coli uidA (from pNGAG1; kindly provided by K. Gomi of Tohoku University), which has an NotI site in the 5' upstream region, was ligated into the PstI and XbaI sites of pUC142 (12), resulting in pUC(N)GUS. We constructed pAURGUS, which contains the E. coli uidA gene and the A. oryzae agdA terminator and is an autonomously replicating plasmid, as follows. The fragment containing uidA and the agdA terminator was obtained from pUC(N)GUS by digestion with BamHI and was ligated into the BamHI site of pAUR316 (TaKaRa), which has the AMA1 replication origin (1, 15) and the aureobasidin A resistance gene (33), resulting in pAURGUS. We amplified the mpkA promoter fragment from A. nidulans genomic DNA using two PCR primers (P-mpkA-F and P-mpkA-R). We subcloned the amplified promoter fragment into the pGEM-T Easy vector, resulting in pGEMmpkA(P). We then obtained the fragment containing the mpkA promoter from pGEMmpkA(P) by digestion with NotI and ligated the fragment into the NotI site of pAURGUS, resulting in pAURGUSmpkA.
GUS assay.
We transformed the A. nidulans wild-type and mpkA strains with pAURGUS or pAURGUSmpkA by the protoplast method (17). We selected the transformants on CD medium supplemented with 0.02 µg of biotin/ml and 2 µg of aureobasidin A/ml. We inoculated conidiospores (ca. 4 x 108) of the transformants into 200 ml of CD liquid medium and cultured the cells at 30°C with shaking at 180 rpm. After 48 h, we added 0.01 µg of micafungin/ml. We collected the mycelia and quickly froze them in liquid nitrogen. We ground the frozen mycelia into a fine powder with a mortar and pestle chilled with liquid nitrogen and immediately resuspended the mycelia in a protein extraction buffer (50 mM sodium phosphate [pH 7.0], 10 mM EDTA, 0.1% [wt/vol] Triton X-100, and 10 mM 2-mercaptoethanol). We placed the suspension on ice for 30 min and then removed cell debris by centrifugation for 10 min at 15,000 x g. We assayed the GUS activity of the cell extract in a buffer (50 mM sodium phosphate [pH 7.0], 0.1% Triton X-100, and 1 mM p-nitrophenyl-ß-D-glucuronide). Reactions occurred in 200-µl volumes at 37°C and were terminated by the addition of 80 µl of 1 N NaOH. We measured p-nitrophenol absorbance at 415 nm and defined 1 U as the amount of enzyme that produced 1 nmol of p-nitrophenol/min at 37°C. We determined the protein concentration of the supernatant by using a bicinchoninic acid protein assay reagent kit (Pierce).
Nucleotide sequence accession number. We have submitted the sequence data for the rlmA gene of A. nidulans to the DDBJ and the EMBL and GenBank databases under accession number AB110208.
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RESULTS |
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mutant. Additionally, we carried out complementation analysis of AN8676.3 in an rlm1 strain, but AN8676.3 failed to complement the rlm1 mutant (Fig. 2C). A. nidulans mpkA complements an S. cerevisiae mpk1 mutant. In S. cerevisiae, Rlm1p is phosphorylated and consequently activated by the MAPK Mpk1p, which belongs to the cell wall integrity pathway, resulting in the transcriptional regulation of genes coding for cell wall biogenesis by the phosphorylated Rlm1p. Bussink and Osmani (5) isolated a putative mpkA gene homologous to the yeast MPK1 gene based on the homology of the predicted amino acid sequences and constructed an mpkA gene disruptant. However, it has not yet been demonstrated whether the putative mpkA possesses the same function as MPK1. We reported previously that the A. oryzae mpkA gene, homologous to MPK1, complements the yeast mpk1 mutant (41). We examined whether the mpkA homolog would complement an S. cerevisiae mpk1 mutant that is thermosensitive at 37°C. The S. cerevisiae mpk1 mutant was transformed with the mpkA cDNA, and the mpkA gene was expressed under the control of the yeast GAPDH promoter. The mpkA gene complemented the yeast mpk1 mutant (Fig. 3). This result suggests that A. nidulans mpkA has in vivo functionality similar to that of yeast MPK1.
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) and its phenotype.
To investigate the in vivo function of rlmA, we constructed an A. nidulans rlmA strain in which part of the native rlmA gene was replaced by the A. oryzae argB selectable marker, which complements the argB2 mutation in A. nidulans. The A. nidulans rlmA strain produced conidiophores in CD liquid medium (in a 24-h culture at 30°C), but the wild type did not (see Fig. S2 in the supplemental material). The rlmA mutant grew normally on CD agar plates, as did the wild-type strain, and showed no major differences in morphological phenotypes on the plates. The rlmA strain was sensitive to a potent inhibitor of chitin synthesis (CFW) at 30 µg/ml on CD agar plates (Fig. 4), but the rlmA and wild-type strains exhibited almost the same level of sensitivity to the ß-1,3-glucan synthase inhibitor micafungin (data not shown).
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and wild-type strains. In S. cerevisiae and Cryptococcus neoformans, caspofungin, a typical ß-1,3-glucan synthase inhibitor belonging to the echinocandin group, perturbs cell wall biosynthesis and activates CWIS, resulting in increases in the phosphorylation levels of Mpk1p in S. cerevisiae or its ortholog in C. neoformans (32, 51). Micafungin is the latest echinocandin compound and exhibits a strong inhibitory effect on fungal ß-1,3-glucan synthases, including those of aspergilli. The phosphorylation of S. cerevisiae Mpk1p was induced by micafungin (Fig. 5A). Caspofungin treatment is known to upregulate the level of transcription of MLP1, which encodes a paralog of Mpk1p, in the S. cerevisiae wild-type strain but not in the rlm1 strain (25, 51). Micafungin treatment also upregulated the level of MLP1 transcription in the wild type but not in the rlm1 strain (Fig. 5B). Therefore, micafungin treatment activated CWIS in S. cerevisiae via Rlm1p as well as caspofungin treatment. When cells of the A. nidulans wild-type strain were treated with micafungin, the phosphorylation of MpkA was detected using the anti-phospho-p44/42 MAPK antibody. Levels of phosphorylation of MpkA in the wild-type strain increased within 10 min after the addition of micafungin (Fig. 5C), suggesting that micafungin treatment can activate CWIS in A. nidulans and S. cerevisiae. We analyzed the time course of levels of mpkA transcription when MpkA was phosphorylated by CWIS triggered by the micafungin treatment. Transcription levels of mpkA reached a plateau by 30 min after micafungin treatment, then decreased (Fig. 6A).
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strain.
After cells of the wild-type and rlmA strains were treated with micafungin for 0, 30, 60, or 120 min, we analyzed the levels of transcription of mpkA and of the cell wall-related genes agsA and agsB (two -1,3-glucan synthase genes); fksA (a ß-1,3-glucan synthase gene); gelA and gelB (two ß-1,3-glucanosyl transferase genes orthologous to A. fumigatus GEL1 and GEL2); chsA, chsB, chsC, chsD, csmA, and csmB (chitin synthase genes); and gfaA (the glutamine-fructose-6-phosphate amidotransferase gene) by quantitative RT-PCR. Levels of transcription of mpkA, fksA, gelA, gelB, gfaA, and all the chitin synthase genes in the two strains were upregulated within 30 min by treatment with micafungin (Fig. 6A and D to M). Levels of gfaA transcription in the A. nidulans rlmA strain after micafungin treatment were lower than those in the wild-type strain (P < 0.009) (Fig. 6M). Levels of agsA transcripts were significantly lower in the wild-type strain with or without micafungin treatment. However, levels of agsA transcription in the rlmA strain increased in a time-dependent manner after micafungin treatment (Fig. 6B). On the other hand, the transcription of agsB in the wild type but not in the rlmA strain was markedly enhanced after micafungin treatment, suggesting that the transcription of agsB depends on RlmA (Fig. 6C).
The rlmA and wild-type strains had similar levels of transcripts of mpkA, gelA, gelB, and all chitin synthase genes before and after micafungin treatment (Fig. 6A, E, and F). The two strains exhibited similar changes in levels of fksA transcripts after micafungin treatment (i.e., an initial increase, followed by a decrease), but the levels in the rlmA strain were lower than those in the wild type during the time course analysis (Fig. 6D). These observations differ significantly from those in a previous report that the S. cerevisiae rlm1 strain lost transcriptional regulation of at least 25 cell wall-related genes, including MPK1 (25). Hence, these results suggest that RlmA is not a major transcription factor regulated by the CWIS pathway in A. nidulans.
Transcriptional analysis of mpkA and cell wall-related genes in the mpkA strain.
Since the rlmA mutation did not alter the levels of transcription of mpkA or of the cell wall-related genes that we examined (except gfaA, agsA, and agsB), other, unknown transcription factors may be involved in the transcriptional regulation of mpkA and the cell wall-related genes. We propose the following two possibilities. First, MpkA that is activated by CWIS may phosphorylate unidentified transcription factors that would then regulate the transcription of mpkA and other genes involved in cell wall biogenesis. Second, transcription factors that are not under the control of MpkA CWIS may regulate the levels of transcription of mpkA and some of the other cell wall-related genes. To examine these possibilities, we constructed an mpkA mutant and analyzed the levels of transcription of other cell wall-related genes. Bussink and Osmani (5) reported that the A. nidulans mpkA mutant shows significant defects in the germination of conidiospores and in the polarized growth of mycelia, and we observed similar results with our A. nidulans mpkA strain. Our mpkA strain exhibited sensitivity to micafungin (10 ng/ml) and CFW (10 µg/ml) (Fig. 7). After 0, 30, 60, and 120 min of micafungin treatment, we compared the levels of transcription of the mpkA, agsA, agsB, fksA, gelA, gelB, chsA, chsB, chsC, chsD, csmA, csmB, and gfaA genes in the wild-type and mpkA strains (Fig. 6).
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strains (Fig. 6D to F and M). However, levels of gfaA transcription in the mpkA strain after micafungin treatment were apparently lower than those in the wild-type strain (P < 0.003) (Fig. 6M). Levels of the ags transcripts differed most obviously between the mpkA and wild-type strains. As described above, agsA transcripts in the wild type remained at low levels regardless of micafungin treatment but the transcription of agsB in the wild type was apparently upregulated after micafungin treatment. In contrast, the transcription of agsA in the mpkA strain was upregulated regardless of micafungin treatment and the transcription of agsB was maintained at low levels with or without micafungin treatment (Fig. 6B and C). Changes in the levels of transcription of the other cell wall-related genes in the mpkA and wild-type strains after micafungin treatment were similar.
Transcription of mpkA is autoregulated by MpkA.
To examine whether the mpkA promoter is autoregulated by CWIS via MpkA, we constructed an mpkA promoter fused with the E. coli uidA gene, which encodes GUS, as a reporter gene and introduced the reporter into the wild-type and mpkA strains. We then analyzed the expression of GUS in the presence or absence of micafungin. As shown in Fig. 8, high basal levels of GUS activity in the wild-type strain were found and micafungin treatment increased GUS activity. However, the mpkA strain showed little GUS activity, with or without micafungin treatment. These results indicate that the transcription of mpkA is autoregulated by MpkA that has been activated by CWIS.
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and Answi6 strains.
In S. cerevisiae, Mpk1p is known to activate another transcription regulatory complex (SBF, composed of Swi4p and Swi6p) in a manner that depends on the stage of the cell cycle. Because RlmA was not a major transcription factor for A. nidulans mpkA, there are two other possibilities for a putative transcription factor that would control the transcription of mpkA through CWIS: (i) orthologs (AnSwi4 and AnSwi6) of S. cerevisiae SBF (Swi4p-Swi6p) or (ii) other, unknown transcription factors. S. cerevisiae Swi4p possesses an APSES (Asm-1, Phd1, StuA, Efg1, and Sok2) DNA-binding domain (2, 48) and ankyrin (ANK) repeats that are thought to be involved in protein-protein interactions (54), whereas Swi6p has only ANK repeats (63). We searched the A. nidulans genome database for genes encoding proteins homologous to S. cerevisiae Swi4p and Swi6p and found two genes (AN3154.3 and AN6715.3) that encode proteins with similarity scores (E [expect] values) of 10–20 for Swi4p and 10–36 for Swi6p. The two putative proteins contained both APSES domains and ANK repeats. We also searched the S. cerevisiae genome database for genes encoding proteins homologous to the two putative A. nidulans proteins, and these two putative proteins had the highest E values for Swi4p and Swi6p. Therefore, we designated AN3154.3 Answi4 and AN6715.3 Answi6. Since we could isolate cDNA of Answi4 and Answi6 from A. nidulans cDNA libraries, the two genes were expressed in A. nidulans (data not shown). To examine whether AnSwi4 and AnSwi6 regulate the transcription of mpkA, we constructed Answi4 and Answi6 disruptants (Answi4 and Answi6 strains). We deleted most of the ORFs, including those corresponding to the N termini, DNA-binding APSES domains, and ANK repeats, by replacing them with the A. oryzae argB gene. Both the Answi4 and Answi6 strains formed conidiospores that were lighter in color than those of the wild type but exhibited no other obvious phenotypic changes. When we treated the two disruptants with micafungin to activate CWIS, levels of mpkA transcription in the two mutants and in the wild type were similarly upregulated (Fig. 9). The levels of mpkA transcription in Answi4 and Answi6 strains appeared to be higher than those in the wild type, especially at 120 min, but the reason was not clear. Double mutants in which both Answi4 and Answi6 were disrupted also grew normally and did not show any significant phenotypic changes except the previously mentioned color of the conidiospores (data not shown). These results suggest that the transcription of mpkA is not regulated by Answi4 or Answi6 through CWIS and depends on other, unknown transcription factors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-1,3-glucan, ß-1,3-glucan, and chitin in the filamentous fungi differs from that in S. cerevisiae. Moreover, the organization of upstream sensing and signaling machineries (G protein-coupled receptors, G protein subunits, and two-component signaling proteins) in the aspergilli appears to be more complex than that found in S. cerevisiae (39). Although the filamentous fungi are morphologically and developmentally distinctive from S. cerevisiae, the comparative in silico reconstruction of the system of genes for CWIS revealed that most homologs of the genes coding for the proteins organizing the Mpk1p-dependent CWIS in S. cerevisiae are well conserved in the three Aspergillus species examined, including A. nidulans (Fig. 1). The transcription factor Rlm1p activated by the MAPK Mpk1p via CWIS plays a central role in the maintenance of cell wall integrity in S. cerevisiae. Taken together, the conservation of the genes coding for CWIS components and the diversity of cell wall-related genes in the aspergilli raise the following questions: which cell wall-related genes are under the control of the MpkA MAPK cascade and whether (and/or how) the conserved RlmA and MpkA proteins are physiologically crucial for cell wall integrity in the aspergilli. In the present study, we considered the potentially critical role of CWIS in A. nidulans from the viewpoints of comparative transcriptional analyses of cell wall-related genes in the wild-type, rlmA, and mpkA strains. A. nidulans rlmA and mpkA are functional orthologs of S. cerevisiae RLM1 and MPK1. Although the A. niger rlmA (8) and A. nidulans mpkA (5) genes were isolated previously, it has not been shown whether the two genes are indeed functional orthologs of S. cerevisiae RLM1 and MPK1, respectively. In the present study, we first carried out complementation experiments with rlm1 and mpk1 null mutants and A. nidulans rlmA and mpkA cDNA. The A. nidulans rlmA gene suppressed the caffeine sensitivity of the yeast rlm1 mutant, but the rlmA mutated genes encoding RlmA derivatives in which amino acid residues inside the MpkA docking site were replaced with alanine did not (Fig. 2B), suggesting the in vivo functionality of A. nidulans RlmA in S. cerevisiae. Although the amino acid sequence of RlmA shows low similarity to that of Rlm1p (only 13%), except in the MADS domain, the small docking site in RlmA is required and is sufficient for CWIS in vivo. We also cloned the A. nidulans mpkA gene (cDNA) and confirmed that its expression suppressed the temperature sensitivity of the S. cerevisiae mpk1 mutant (Fig. 3), suggesting the in vivo functionality of A. nidulans MpkA in S. cerevisiae. The complementation analyses clearly demonstrate that A. nidulans rlmA and mpkA are functional orthologs of RLM1 and MPK1, respectively.
In vivo functionalities of rlmA and mpkA in A. nidulans markedly differ from those of their orthologs in S. cerevisiae.
In order to investigate the functionalities of rlmA and mpkA in A. nidulans, we further analyzed the transcriptional responses of mpkA and other cell wall-related genes to treatment with micafungin in the wild-type, rlmA, and mpkA strains (Fig. 6 and 8). These transcriptional analyses suggested the following four major principles of the transcriptional regulation of mpkA and other cell wall-related genes (Fig. 10): (i) mpkA is MpkA dependent but not RlmA dependent; (ii) agsA and agsB are MpkA dependent and partly dependent on RlmA; (iii) gfaA is partly dependent on both MpkA-RlmA and unidentified non-MpkA systems; and (iv) fksA, gelA, gelB, chsA, chsB, chsC, chsD, csmA, and csmB are independent of the MpkA system. In S. cerevisiae, the transcription of 25 cell wall-related genes (including MPK1, CHS3, and FKS1) is known to depend on Rlm1p activated by Mpk1p (25), suggesting that Rlm1p is the major transcription factor under the control of CWIS via Mpk1p. Since the prominent role of Rlm1p in the transcriptional regulation of cell wall-related genes is critical in the model of CWIS and cell wall biogenesis in S. cerevisiae, it should be considered whether RlmA and MpkA occupy positions comparable to those of the yeast orthologs in respect to CWIS and cell wall biogenesis in A. nidulans. The in vivo functionalities of rlmA and mpkA in A. nidulans with respect to transcriptional analyses of cell wall-related genes are further discussed in detail below.
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-1,3-Glucan synthase genes.
-1,3-Glucan is one of the major cell wall polysaccharides in A. fumigatus (3, 40) and probably in other aspergilli, including A. niger (9). A. niger possesses five -1,3-glucan synthase genes (agsA to agsE) (9), and A. fumigatus is reported to have three -1,3-glucan synthase genes (AGS1, AGS2, and AGS3) (3, 40) corresponding to A. niger agsE, agsD, and agsA, respectively. According to the A. nidulans genome database, A. nidulans has only two putative -1,3-glucan synthase genes (agsA and agsB), which are the counterparts of A. niger agsD and agsE, respectively (9). A. nidulans does not possess the ortholog of A. niger agsA, the transcription of which depends partly on the transcription factor RlmA (8). A. niger agsA disruptants (agsA) and A. fumigatus disruptants mutated in AGS3 (the ortholog of A. niger agsA) were generated, but the disruptants did not show obviously different phenotypes under normal growth conditions, and the -1,3-glucan contents were the same as those of the wild-type strains (3, 9). Since an A. fumigatus AGS1 disruptant forms hyperbranched hyphal tips and has a lower -1,3-glucan content than the wild type (3), the AfAGS1 class of genes seems to be important for the biosynthesis of -1,3-glucan. Levels of transcripts of A. nidulans agsB were increased by micafungin treatment (Fig. 6C), just as levels of transcripts of A. niger agsE were increased by CFW treatment (9). Although the levels of transcription of agsB were upregulated after micafungin treatment, those in the rlmA strain were significantly decreased before and after micafungin treatment, and the transcription of agsB was more severely diminished in the mpkA strain (Fig. 6C). These results suggest that the transcription of A. nidulans agsB depends mainly on MpkA-RlmA signaling. A. nidulans agsA is an ortholog of A. niger agsD and A. fumigatus AGS2. Because the disruption of A. fumigatus AGS2 causes hyperbranched hyphal tips (as does the disruption of AfAGS1), this class of ags genes also seems to be important for morphogenesis in Aspergillus species (3). agsA transcripts were maintained at low levels in the wild type even after micafungin treatment. However, the levels of transcripts of A. nidulans agsA in the rlmA strain increased in a time-dependent manner after micafungin treatment, and the levels of agsA transcripts in the mpkA strain were upregulated regardless of micafungin treatment (Fig. 6B). The transcriptional analyses of agsA and agsB among the wild-type, rlmA, and mpkA strains suggest that agsB is involved in the response to cell wall stress through MpkA-RlmA signaling whereas agsA functions in cells in which MpkA-dependent CWIS is suppressed. CWIS seems to be important for the biosynthesis of -1,3-glucan through the transcriptional regulation of agsA and agsB in a reciprocal fashion. However, it remains unclear why the transcription of agsA was not enhanced even when MpkA was activated through CWIS by micafungin treatment, and thus, it remains unclear whether RlmA is directly or indirectly involved in the transcriptional regulation of agsA.
gfaA gene.
In S. cerevisiae, levels of transcription of GFA1, which encodes a probable rate-limiting enzyme of chitin biosynthesis, are regulated by Rlm1p (34, 59). When the mycelia of A. niger are treated with caspofungin, CFW, or SDS, the transcription of gfaA (an ortholog of S. cerevisiae GFA1) is upregulated, and consequently, chitin biosynthesis is stimulated (50). Since the levels of transcripts of gfaA in the mpkA strain were significantly lower than those in the wild type after micafungin treatment and those in the rlmA strain were moderately lower than those in the wild type (P < 0.015) (Fig. 6M), we hypothesize that the transcription of gfaA depends on MpkA-RlmA as well as on other, unknown transcription factors.
The 5' untranslated regions (ca. 1,000 bp) of A. niger gfaA and agsA contain consensus sequences [TA(A/T)4TAG] that are similar to S. cerevisiae Rlm1p-binding sequences, and the consensus sequences are thought to be involved in the transcriptional regulation of the two genes of A. niger through RlmA (8). However, we could not identify such Rlm1p-binding consensus sequences in the 5' untranslated regions of A. nidulans gfaA, agsA, or agsB that seem to be regulated by CWIS through RlmA. RlmA-binding consensus sequences in A. nidulans may thus differ from those in S. cerevisiae or A. niger. Another possibility is that RlmA is indirectly involved in the transcriptional regulation of the three genes.
Chitin synthase genes.
The levels of transcription of all six chitin synthase genes (chsA, chsB, chsC, chsD, csmA, and csmB) in the wild-type cells increased after micafungin treatment (Fig. 6G to L). The basal levels of transcripts of all chitin synthase genes and changes in their levels after micafungin treatment were similar in the wild-type, rlmA, and mpkA strains. These results suggest that the basal levels of transcription of these genes and the increases in the transcription of the genes after micafungin treatment are independent of CWIS via MpkA and RlmA.
ß-1,3-Glucan-related genes.
The fksA, gelA, and gelB genes are involved in ß-1,3-glucan biogenesis in Aspergillus species (11, 42). The transcription of S. cerevisiae FKS1 and FKS2 genes, which encode subunits of ß-1,3-glucan synthase, is partly regulated by Rlm1p activated via CWIS (25). In A. nidulans, fksA is the only gene that encodes ß-1,3-glucan synthase (29). Levels of fksA, gelA, and gelB transcription changed in the rlmA and mpkA strains after micafungin treatment, and the changes were similar to those observed in the wild type (Fig. 6D to F). These results suggest that the regulation of the transcription of fksA, gelA, and gelB is independent of CWIS via MpkA and RlmA. Overall, the transcriptional regulation of most genes involved in the biosynthesis of ß-1,3-glucan and chitin (except gfaA, the transcriptional levels of which were decreased in the mpkA strain) seems to be regulated by an unknown signaling mechanism activated by micafungin treatment rather than by CWIS via MpkA.
Autoregulation of mpkA transcription.
The finding that the levels of transcripts of mpkA were almost the same in the wild-type and rlmA strains (Fig. 6A) suggests that the transcriptional upregulation of the gene is independent of RlmA, even under conditions in which MpkA is phosphorylated via CWIS as a result of micafungin treatment (Fig. 5C). In contrast to the production of GUS under the control of the mpkA promoter in the wild type, GUS activity in the mpkA strain was detected at very low levels and did not increase, even after micafungin treatment (Fig. 8); thus, the expression of mpkA seems to be autoregulated by CWIS via MpkA. We hypothesize that transcription factors other than RlmA are required as the targets of MpkA for the transcription of mpkA.
In S. cerevisiae, SBF (Swi4p-Swi6p), which is activated by Mpk1p, is a G1/S-specific transcription complex for cell wall biogenesis, but the details of SBF activation by the cell cycle remain unclear (20). Answi4 and Answi6 disruption mutants both grew as well as the wild type and did not exhibit significantly different phenotypes, with the exception that the mutants formed lighter-colored conidiospores. When the two mutants and the wild type were treated with micafungin to activate CWIS, changes in mpkA transcription levels were similar among the three strains (Fig. 9). The results of single and double mutations of Answi4 and Answi6 suggest that unknown transcription factors other than RlmA, AnSwi4, and AnSwi6 regulate the transcription of mpkA in A. nidulans.
As shown in Fig. 1, the Crz1p protein of S. cerevisiae is another transcription factor controlling the ß-1,3-glucan synthase gene FKS2 and other cell wall-related genes through Ca2+ signaling in response to several environmental stimuli (7, 57). We previously isolated an A. nidulans crzA gene that
