Repression of chsB expression reveals the functional importance of class IV chitin synthase gene chsD in hyphal growth and conidiation of Aspergillus nidulans

Masayuki Ichinomiyaa,1, Takayuki Motoyamaa,b,1, Makoto Fujiwarab,1, Masamichi Takagi1, Hiroyuki Horiuchi1 and Akinori Ohta1

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan1

Author for correspondence: Hiroyuki Horiuchi. Tel: +81 3 5841 5170. Fax: +81 3 5841 8015. e-mail: ahhoriu{at}mail.ecc.u-tokyo.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The functions of two previously identified chitin synthase genes in Aspergillus nidulans, chsB and chsD, were analysed. First, a conditional chsB mutant was constructed in which the expression of chsB is under the control of a repressible promoter, the alcA promoter, of A. nidulans. Under repressing conditions, the mutant grew slowly and produced highly branched hyphae, supporting the idea that chsB is involved in normal hyphal growth. The involvement of chsB in conidiation was also demonstrated. Next, double mutants of chsB and chsD were constructed, in which chsB was placed under the control of the alcA promoter and chsD was replaced with the argB gene of A. nidulans. These double mutants grew more slowly than the chsB single mutant under high-osmolarity conditions. The hyphae of the double mutant appeared to be more disorganized than those of the chsB single mutant. It was also found that ChsD was functionally implicated in conidiation when the expression of chsB was limited. These results indicate the importance of the ChsD function in the absence of chsB expression. The roles of ChsB and ChsD in hyphal growth and in conidiation were supported by the analysis of the spatial expression patterns of chsB and chsD, using lacZ of Escherichia coli as a reporter gene.

Keywords: cell wall, conidiation, filamentous fungi

Abbreviations: AP, alkaline phosphatase; RACE, rapid amplification of cDNA ends

The GenBank accession number for the the new version of the chsD nucleotide sequence is D83246.

a These authors contributed equally to this work.

b Present address: The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chitin is a major component of the cell walls of most filamentous fungi and a minor component of the cell walls of many yeasts. Chitin synthase genes have been isolated from many filamentous fungi and yeasts, and have been classified into at least five classes, I–V, according to sequence similarity (Bowen et al., 1992 ; Beth Din et al., 1996 ; Specht et al., 1996 ; Motoyama et al., 1996 ; Aufauvre-Brown et al., 1997 ; Xoconostle-Cázares et al., 1997 ). In Aspergillus nidulans, four chitin synthase genes, chsC, chsA, chsB and chsD, which belong to class I, class II, class III and class IV, respectively, and a gene, csmA, which encodes chitin synthase with a myosin motor-like domain, have been cloned in our laboratory (Yanai et al., 1994 ; Motoyama et al., 1994a , 1996 ; Fujiwara et al., 1997 ) Our chsD is also referred to as ‘chsE’ by Specht et al. (1996) . The chitin synthase domain of CsmA, which belongs to class V, is similar to the product of ‘chsD’ as reported by Specht et al. (1996) .

The functions of chitin synthases in filamentous fungi have been analysed by gene disruptions or gene deletions. The respective null mutants of class I (Motoyama et al., 1994a ), class II (Beth Din & Yarden, 1994 ; Yanai et al., 1994 ) and class IV (Beth Din et al., 1996 ; Motoyama et al., 1996 ; Specht et al., 1996 ) chitin synthase genes of filamentous fungi exhibited no obvious phenotypic defect under standard conditions with the exception of that studied by Beth Din & Yarden (2000) . Recently, it was reported that a class II chitin synthase gene (CHS1) of the dimorphic yeast Candida albicans was essential even for the growth of the yeast form (Sudoh et al., 2000 ; Munro et al., 2001 ). Disruptions or deletions of class III (Yarden & Yanofsky, 1991 ; Yanai et al., 1994 ; Borgia et al., 1996 ; Mellado et al., 1996 ) or class V (Specht et al., 1996 ; Aufauvre-Brown et al., 1997 ; Horiuchi et al., 1999 ) chitin synthase genes, with the exception of two class III genes (Gold & Kronstad, 1994 ; Mellado et al., 1996 ), caused some phenotypic changes. These observations indicate that the respective chitin synthase genes (not all genes) alone do not bear essential functions for hyphal growth and conidiation, and that the class of chitin synthase is not necessarily related to the function and importance of these genes among different fungal species.

To reveal the function of individual chitin synthase genes, double and triple disruption mutants were constructed and analysed. We previously found that the class II and class IV chitin synthases of A. nidulans have some overlapping function(s) in conidiation (Motoyama et al., 1996 ), and that the class I and class II chitin synthases of A. nidulans share important roles in hyphal growth and conidiation (Fujiwara et al., 2000 ). A partial redundancy of function of class II and class IV chitin synthases was also reported by another group (Culp et al., 2000 ). From these results, we speculated that chitin synthases in class I, class II and class IV may play important roles in growth or differentiation, and that these synthases cooperate with each other. In the yeast Saccharomyces cerevisiae, the class IV chitin synthase is involved in most of the cellular chitin synthesis and serves redundant functions with class II chitin synthase in growth (Valdivieso et al., 1991 ; Shaw et al., 1991 ). These results suggest that fungal chitin is synthesized by the overlapping and cooperative function of multiple chitin synthase genes.

In our previous study, we showed that A. nidulans chsD, which encodes a class IV chitin synthase, is expressed in the growing hyphae (Motoyama et al., 1996 ), and chsB, which encodes a class III chitin synthase, plays essential roles in normal hyphal growth. These results suggest that ChsD and ChsB have some related functions in chitin synthesis and other activities of growing hyphae. In this study, we focused on the functional relationship between ChsB and ChsD in hyphal growth and in conidiation. The construction of double deletion mutants of chsB and chsD seemed to be difficult because null mutants of chsB are heavily damaged in hyphal growth (Yanai et al., 1994 ; Borgia et al., 1996 ). Therefore, we constructed conditional double mutants in which chsD was replaced with the argB gene of A. nidulans, and chsB was placed under the control of a repressible promoter (the alcA promoter). We then analysed the functional relationship between the two chitin synthases.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, media and transformation.
The A. nidulans strains used are shown in Table 1. Complete medium (CM) and minimal medium (MM) for A. nidulans described by Rowlands & Turner (1973) were used. YGU medium [0·5% yeast extract (Difco), 1% glucose, 0·1% trace elements (Rowlands & Turner 1973 ), 10 mM uridine] was also used. When the expression of the alcA promoter was induced, we used 100 mM threonine and 0·1% fructose as carbon sources instead of glucose (MMFT). Transformation was done as described by May (1992) . When the expression of the alcA promoter was induced in the transformation experiments, we used 1·2 M sorbitol as an osmotic stabilizer instead of sucrose. Transformants were grown in MM with appropriate supplements.


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Table 1. A. nidulans strains used in this study

 
Plasmids were amplified in Escherichia coli MV1190 [{Delta}(lac–proAB) thi supE {Delta}(srl–recA)306::Tn10 (Tetr) (F' traD36 proAB lacIq Z{Delta}M15)]. E. coli was grown in LB and transformation was done by standard methods (Sambrook et al., 1989 ).

Total DNA isolation and Southern analysis.
Total DNA of filamentous fungi was extracted as described by Oakley et al. (1987) . Southern analysis was done with ECL direct nucleic acid labelling and detection systems (Amersham Pharmacia Biotech). DNA was separated by electrophoresis on agarose gels and further manipulation was done as described in the manufacturer’s instructions.

Total RNA isolation and Northern analysis.
Total RNA was prepared from mycelia by using the RNeasy Mini Kit (QIAGEN). Five micrograms of total RNA were separated by electrophoresis on a formaldehyde-agarose gel, and blotted onto Hybond-XL membrane (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Blots were hybridized to 32P-labelled probes prepared by using Random Primer DNA Labelling Kit Version 2 (TaKaRa). As a probe for chsB expression, the 1·6 kb NcoI fragment from pchsB was used.

PCR-amplification of DNA fragments.
PCR-amplification was done by using a thermal cycler (model 480, Perkin-Elmer/Cetus) and Taq DNA polymerase (Boehringer Mannheim) or the Expand High Fidelity PCR system (Boehringer Mannheim).

RT-PCR amplification.
Twenty micrograms of total RNA were treated with deoxyribonuclease (RT Grade) (NIPPON GENE), then reverse transcriptions were performed by using 3 µg DNA-free RNA and ReverTra Ace reverse transcriptase (TOYOBO) according to the manufacturer’s instructions. The primer used for reverse transcriptions was chsB.3072as (5'-GGCATCCAGTCTAGGTTGC-3'). For PCR amplifications, 5 µl samples from 20 µl of reverse transcription reactions were used. The primers used were chsB.2055s (5'-CATCATGGATCTGGTAGG-3') and chsB.3075as (5'-ATACATACAATTGCAAGGC-3'). Since we designed the primer set to amplify the region that contains an intron, the products of RT-PCR were expected to be 57 bp smaller than the PCR products amplified from genomic DNA by using the same primer set. PCR products and 100 bp DNA ladder (New England Biolabs) were electrophoresed on a 1·0% agarose gel.

Plasmid constructions.
Plasmids used in this paper are shown in Table 2. To make the strain in which the expression of chsB is under the control of the alcA promoter, the plasmid p5BP-ALCB was constructed as follows. The alcA promoter was amplified from the genomic DNA of strain AU1 by the PCR method using the two primers, M16-1 (5'-AAAATCGATGGCGGGGCGGAAATTGACA-3') and M16-2 (5'-AAAAAGCTTTGAGGCGAGGTGATAGGAT-3'), according to the sequence reported by Kulmburg et al. (1992) . Using these two primers, 35 cycles were run consisting of a 94 °C 1 min melting step, a 56 °C 2 min annealing step, and a 72 °C 3 min extension step. The 5·5 kb HindIII fragment containing the chsB gene was prepared from the DNA purified from the phage clone that contains the chsB gene (Yanai et al., 1994 ) and ligated with HindIII-digested and bacterial alkaline phosphatase (AP)-treated pUC118 (Takara Shuzo) to yield pchsB and pchsBR. The direction of chsB transcription was from the HindIII site to the EcoRI site on the multiple cloning site of pchsB, and the direction of the chsB insert of pchsBR was reverse compared to pchsB.


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Table 2. Plasmids used in this study

 
The HindIII site (indicated by underlining, see below) was introduced on the plasmid pchsBR just in front of the initiation codon (indicated by boldface, see below) by a site-directed mutagenesis kit (Bio-Rad) using the primer KY-X (5'-GCCATGGTTAAGCTTGTATGTGC-3') to yield pchsB5.5H. The ClaI- and EcoRI-digested and AP-treated pDJB1 (Ballance & Turner, 1985 ), the ClaI- and HindIII-digested 0·5 kb PCR fragment which contains the alcA promoter, and the 1·7 kb HindIII–EcoRI fragment which contains a region of chsB, from its initiation codon to about half of ORF, derived from pchsB5.5H, were ligated to yield pDJB-ALC-B{Delta}. The blunted 0·9 kb HindIII–EcoRI fragment, which contains the upstream region of the chsB promoter, derived from pchsB5.5H, was ligated with XbaI-digested, blunted, and AP-treated pP1, a plasmid bearing the Neurospora crassa pyr4 gene (Motoyama et al., 1994a ), to yield p5BP. The direction of chsB transcription was from the EcoRI site to the HindIII site on the multiple cloning site. The blunted 1·7 kb ClaI fragment, which contains the alcA promoter and part of the chsB ORF, derived from pDJB-ALC-B{Delta}, was ligated with Sse8387I-digested, blunted, and AP-treated p5BP to yield p5BP-ALCB.

The plasmid pSS-chsB, for chsB expression, was constructed by ligating the 5·5 kb HindIII fragment, which contains the chsB gene derived from pchsB, and BamHI-digested, blunted, and AP-treated pSS1 (Motoyama et al., 1994a ).

Plasmids for the analysis of promoter activities in A. nidulans with lacZ of E. coli as a reporter gene were constructed as follows. The 3·0 kb BamHI fragment which contains lacZ of E. coli derived from pMC1871 (Casadaban et al., 1983 ) was ligated with BamHI-digested and AP-treated pSS1 to yield pSS-LZ. The blunted 0·9 kb SphI fragment, which is expected to contain the terminator region of chsB, derived from pchsB5.5H, was ligated with XbaI-digested, blunted, and AP-treated pSS-LZ to yield pSS-TB-LZ. The 1·8 kb HindIII–NaeI fragment, which contains the chsB promoter (1·3 kb) and a part of the chsB ORF (0·5 kb), derived from pchsB, was blunted and ligated with SmaI-digested and AP-treated pSS-TB-LZ to yield pB-LAC. The 1·8 kb NspV fragment, which contains the chsD promoter (1·4 kb) and a part of the chsD ORF (0·4 kb), derived from pchsD, was blunted and ligated with SmaI-digested and AP-treated pSS-TB-LZ to yield pD-LAC. Linkage points were confirmed by sequencing with a primer, prm-LZ2 (5'-TTCTGGTGCCGGAAACCA-3'). We found some sequencing errors in the chsD and the new version of the chsD nucleotide sequence is registered with accession no. D83246. Consequently, total amino acid residues of ChsD were changed from 1086 to 1184 due to the extension of 96 aa residues in its N-terminus. From the results of 5'-RACE experiments, the transcriptional start point(s) were estimated approximately 530 bp upstream of the revised ATG initiation codon and an intron of 151 bp was identified from -6 to -157 when the first A of the revised initiation codon was designated +1.

Construction of A. nidulans strains by transformation.
All the strains originated from AU1 or ABPU1. The argB gene of A. nidulans and the pyr4 gene of N. crassa were used as selectable markers to complement arginine- and uridine-auxotrophy, respectively.

Conditional mutants of chsB were made as follows. The promoter of chsB in the genome of A. nidulans strain ABPU1 was interrupted with the alcA promoter by transformation with the 3·9 kb DraI–ClaI fragment from p5BP-ALCB. By Southern analysis of HindIII-digested or PstI-digested total DNA of ABPU1 transformants probed with the 1·3 kb HindIII fragment (Fig. 1a, probe) from pchsB5.5H, homologous recombination at the chsB locus was confirmed in strains named BM-3, BM-4 and BM-12 by a shift of the signal size from 5·5 kb to 1·9 kb and from 1·6 kb to 3·8 kb, respectively (Fig. 1b, lanes 1–8). By the same strategy, the promoter of chsB in the genome of the chsD null mutant D3-2 (Table 1, Motoyama et al., 1996 ) was exchanged with the alcA promoter to yield strains DB-13 and DB-19. The exchange of these promoters was confirmed by Southern analysis (data not shown). Strain BB-3 was created by transformation of strain BM-3 with pSS-chsB. The strains ABPU/A and BM-3/A1 were constructed by transformation of strains ABPU1 and BM-3, respectively, with pSS1. chsD of strain BM-3 was replaced with the argB gene by using pD{Delta}A7 as described previously (Motoyama et al., 1996 ), generating a strain designated as BD-2.



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Fig. 1. Constructions of conditional chsB mutant strains. (a) Construction of strains BM-3, BM-4 and BM-12 by inserting the 3·9 kb DraI–ClaI fragment from p5BP-ALCB into the promoter region of chsB. Abbreviations: H, HindIII; P, PstI. (b) Southern analysis of transformants. HindIII-digested total DNA of strain BM-3 (lane 1), strain BM-4 (lane 2), strain BM-12 (lane 3) and strain ABPU1 (lane 4) was hybridized with the 1·3 kb HindIII fragment (Fig. 1a, probe) from pchsB5.5H. PstI-digested total DNA of strain BM-3 (lane 5), strain BM-4 (lane 6), strain BM-12 (lane 7) and strain ABPU1 (lane 8) was hybridized with the same 1·3 kb HindIII fragment.

 
The strain to analyse the promoter activity of chsB was constructed as follows. AU1 was transformed with BglII-digested pB-LAC. By Southern analysis of XbaI-digested total DNA of a transformant, BL-1, probed with the 1·1 kb HindIII–StuI fragment (Fig. 2a, probe) from pSS1, homologous recombination at the argB locus was confirmed by a shift of the signal size from 3·5 kb to 14 kb (Fig. 2b, lane 2). Only one signal was found by Southern analysis of total DNA of this transformant digested with three different restriction enzymes and probed with the 3·0 kb BamHI fragment which contains lacZ from pMC1871, indicating that one copy of the plasmid was integrated into this transformant (Fig. 2b, lanes 3–5). The strain to analyse the promoter activity of chsD was constructed as follows. ABPU1 was transformed with NcoI-digested pD-LAC. By Southern analysis of XbaI-digested total DNA of a transformant, DL-4, probed with the 1·1 kb HindIII–StuI fragment (Fig. 2c, probe) from pSS1, homologous recombination at the argB locus was confirmed by a shift of the signal size from 3·5 kb to 6·6 kb and 7·2 kb (Fig. 2d, lane 2). Only one signal was found by Southern analysis of total DNA of this transformant digested with three different restriction enzymes and probed with the 3·0 kb BamHI fragment which contains lacZ from pSS-TB-LZ, indicating that one copy of the plasmid was integrated into this transformant (Fig. 2d, lanes 3–5).



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Fig. 2. Construction of strains to analyse promoter activities of chsB and chsD. (a) Construction of strain BL-1, which produces ß-galactosidase under the control of the chsB promoter. Strain AU1 was transformed with BglII-digested pB-LAC. Abbreviations: B, BglII; X, XbaI. (b) Southern blot analysis of XbaI-digested (lanes 1–3), StuI-digested (lane 4) and HindIII-digested (lane 5) total DNAs from strain AU1 (lane 1) and from strain BL-1 (lanes 2–5) probed with the 1·1 kb StuI–HindIII fragment from pSS1 (lanes 1 and 2) or probed with the 3·0 kb BamHI fragment of lacZ from pMC1871 (lanes 3–5). (c) Construction of strain DL-4, which produces ß-galactosidase under the control of the chsD promoter. Strain ABPU1 was transformed with NcoI-digested pD-LAC. Abbreviations: N, Nco I; X, XbaI. (d) Lanes 1 and 2, Southern blot analysis of XbaI-digested total DNA from strains ABPU1 (lane 1) and DL-4 (lane 2) probed with the 1·1 kb StuI–HindIII fragment from pSS1. Lanes 3–5, Southern blot analysis of BamHI- (lane 3), HindIII- (lane 4) and XbaI- (lane 5) digested total DNA of strain DL-4 probed with the 3·0 kb fragment from pSS-TB-LZ.

 
Calcofluor white staining.
Conidia were spread on a YGU plate, and incubated for 24 h at 37 °C. Hyphae on a piece of agar (approx. 15 mmx15 mm) were cut off, stained with 0·01% calcofluor white (fluorescent brightener 28, Sigma), and observed under a fluorescent microscope.

Carbon source exchange experiment.
Conidial suspensions of A. nidulans were inoculated into liquid MMFT (200 ml, fructose and threonine as carbon sources) in a 500 ml Erlenmeyer flask to a final concentration of 2x106 conidia ml-1. After agitating the flasks on a rotary shaker for 17 h at 37 °C, mycelia were collected by filtration through G3 glass filter, washed by liquid MM (glucose as a carbon source), and spread on MM plates (glucose as a carbon source). After 25 h incubation at 37 °C, conidia were collected from the plates and the numbers were counted using a haemocytometer. All the MMFT and MM used were supplemented with 0·20 mg arginine ml-1, 0·02 µg biotin ml-1, 0·50 µg pyridoxine ml-1 and 10 mM uridine. To examine the abundance of the chsB transcript, total RNA was prepared from BM-3/A1 (chsB mutant) mycelia taken at 0, 5, 15 and 25 h after transfer onto a MM plate.

Chitin content determination.
Chitin content was determined as described previously (Fujiwara et al., 2000 ), except that strains were grown in 100 ml YGU medium for 18 h at 37 °C, and that the cell-wall-containing pellets were sonicated in distilled water to a fine suspension before enzymic treatment. The protein levels, by which we normalized the amount of N-acetylglucosamine, were not very different between the cell extracts from different strains.

In situ staining of the ß-galactosidase activity.
ß-Galactosidase activity was stained in situ by a modification of a method reported previously (Adams & Timberlake, 1990 ; Aguirre et al., 1990 ). In brief, 20 µl of a conidial suspension (108 conidia ml-1) was spread on a MM plate (supplemented with biotin, pyridoxine and uridine). After incubating for 13 or 36 h at 37 °C, hyphae on a piece of agar (approx. 15 mmx15 mm) were cut off, and treated with chloroform vapour for 20 min at room temperature. The piece was transferred into a staining solution (0·05 M sodium phosphate, pH 7·5, 0·02% X-Gal), incubated for 2 h at 37 °C, and cells were observed under a microscope.

Microscopy.
Samples were observed and photographed by using a light microscope (model BHS-RFK, Olympus) equipped with an automatic camera (model PM-10ADS, Olympus) attachment.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of conditional mutants of chsB
We constructed chsB-conditional mutants in which chsB was placed under an inducible promoter, namely the alcohol dehydrogenase gene (alcA) promoter of A. nidulans (Kulmburg et al., 1992 ), and we analysed the functions of ChsB using these mutants. In these mutants, the expression of chsB is expected to be induced by ethanol or threonine and repressed by glucose. By introducing the 3·9 kb DraI–ClaI fragment from p5BP-ALCB into strain ABPU1, we obtained three transformants in which the promoter of chsB in the genome was interrupted by the alcA promoter. We designated these three transformants BM-3, BM-4 and BM-12 (Fig. 1, Table 1). We used strain BM-3 for further experiments.

Effect of chsB repression on growth and differentiation
We analysed the effect of the repression of chsB expression on the growth of A. nidulans by comparing the radial growth rate of the chsB-repressed strain with that of the wild-type strain. Since auxotrophic markers could affect the growth rate, BM-3/A1 was constructed by the transformation of BM-3 with pSS1, a plasmid containing the argB gene, and the growth rate of BM-3/A1 was compared with that of the wild-type strain ABPU/AU. Conidia of each strain were point-inoculated on solid medium and incubated for 72 h at 37 °C. The colony diameter was then determined (Table 3). On MM plates [alcA(p)-repressing condition], the colony diameter of BM-3/A1 (15·5±4·3 mm) was much smaller than that of ABPU/AU (34·5±2·9 mm). Under this condition, dark brown pigmentation of hyphae in the agar was observed in BM-3/A1. Borgia et al. (1996) reported that the chsB disruptant colonies became brown. Significant reduction in the growth rate of BM-3/A1 was also observed on YGU plates [alcA(p)-repressing condition]. Since the growth defects of some cell-wall-deficient mutants can occasionally be remedied by the addition of osmotic stabilizers into the media, we examined the effects of osmotic stabilizers. The growth defect of strain BM-3/A1 on YGU plates was not suppressed by the addition of 0·6 M KCl. On MMFT plates [alcA(p)-inducing condition], the colony diameter of BM-3/A1 (31·8±0·8 mm) was slightly smaller than that of ABPU/AU (36·7±0·9 mm). Taken together, these results suggest that the growth defect of BM-3/A1 on MM and YGU plate was caused by the repression of chsB expression.


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Table 3. Comparison of growth rate of the wild-type strains and mutants

 
We next analysed the morphology of BM-3/A1 under the chsB-repressing condition. BM-3/A1, ABPU/AU and BB-3, an alcA(p)::chsB strain bearing an extra copy of the intact chsB gene, were incubated for 24 h on YGU plates. Hyphal growth and conidiophore development were then observed (Fig. 3). BM-3/A1 displayed defects in hyphal growth (Fig. 3d, e); specifically, its hyphae branched more frequently than those of ABPU/AU, and the positions of branch formation were aberrantly close to the tips of the hyphae or to each other. The lateral walls lacked smoothness. Hyphal tips, branching points close to tips, and septa, which appeared to be formed normally, were intensely stained with calcofluor white, which is known to bind to chitin (Fig. 3e). Conidiophores developed rather normally on BM-3/A1 colonies, although fewer of them and of the conidia produced on each of them were produced than in ABPU/AU (Fig. 3f, and data not shown). The morphology of BB-3 was almost the same as that of ABPU/AU (Fig. 3i, j, k). These results suggest that the defects of BM-3/A1 were caused by the repression of the chsB expression.



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Fig. 3. Morphology of the chsB single mutant and the chsB chsD double mutant. The wild-type strain (ABPU/AU) (a–c), the chsB mutant (BM-3/A1) (d–f), the chsB chsD double mutant (DB-13) (g, h) and the chsB mutant transformed with the chsB gene (BB-3) (i–k) were grown for 24 h on YGU plates. Hyphae were stained with calcofluor white to visualize chitin distribution (b, e, h and j). Bar, 30 µm.

 
Involvement of chsB in conidiation
In the previous section, we showed that BM-3/A1 has some defects in asexual development. It is possible that ChsB is directly involved in conidiophore development and conidia formation, or that ChsB is involved only in hyphal growth. In the latter case, the defect in conidiophores and conidia formation could be caused by the partial inability to produce conidiation-competent hyphae (Miller et al., 1991 ). To clarify which possibility is the case, we performed a carbon source exchange experiment (see Methods). Conidiation-competent hyphae of ABPU/AU, BM-3/A1 and BB-3, cultured in alcA(p)-inducing liquid MMFT, were transferred onto alcA(p)-repressing MM plates on which conidiation was induced. The numbers of conidia, which were expected to be produced without additional chsB expression, were then counted (Table 4). BM-3/A1 could form only 45% of the number of conidia produced by ABPU/AU. The efficiency of the conidiation of BB-3 was recovered up to 75% of that of ABPU/AU, suggesting that a part of the reduction in the conidiation efficiency of BM-3/A1 could be due to the absence of chsB expression during developmental induction. The reason why the conidiation efficiency of BB-3 could not reach the same level as that of the wild-type strain is not clear. It is possible that the expression of the extra copy of chsB is not sufficient due to the effect of the position where chsB was integrated.


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Table 4. Conidia formation efficiency of the wild-type strain and mutants

 
In comparison with the previous report by Borgia et al. (1996) , in which they showed that the chsB disruptants did not conidiate, the reduction in conidiation efficiency of BM-3/A1 was rather moderate. It is possible that the repression of chsB transcription is incomplete on MM plates, and the incompleteness could relieve the conidiation defect to some extent. We examined the abundance of the chsB transcripts after the shift to MM plate by Northern analysis (Fig. 4a). chsB mRNA was easily detectable in the sample prepared from the mycelium cultured for 17 h in liquid MMFT (Fig. 4a, 0 h). After shift to a MM plate, chsB mRNA decreased drastically by 5 h after shift, and was almost undetectable at 15 h after shift (Fig. 4a). At 25 h after shift, a low level of chsB expression was again detected (Fig. 4a). This resumption of chsB expression may be due to the decrease of glucose level and the concomitant derepression of the alcA promoter. By performing RT-PCR, we obtained a clearer result that shows the presence of the chsB transcripts at 5 h and 25 h after shift (Fig. 4b). These results imply that the conidiation defect of BM-3/A1 was underestimated in the carbon source exchange experiment because of the stability of chsB mRNA and the leaky expression of chsB.



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Fig. 4. Expression of chsB after the shift from inducing conditions to repressing conditions. (a) Northern blot analysis of chsB. Total RNA was prepared from the mycelia of the chsB mutant (BM-3/A1) cultured for 0, 5, 15 and 25 h after the shift onto MM plates. Equal loading of total RNA was evaluated by ethidium bromide staining (rRNA). (b) RT-PCR amplification of chsB. The RNA samples used for Northern analysis were treated with DNase, and subjected to RT-PCR. To assure the amplification from cDNA, PCR was also performed by using total DNA from strain ABPU1 (wild-type) as a template (lane D). The DNA standard (100 bp ladder) is in lane M.

 
Functional importance of ChsD in the chsB-conditional mutant
To examine the functional importance of ChsD under the ChsB-repressing condition, we generated double mutants of chsB and chsD. We transformed the chsD null mutant, D3-2 (Motoyama et al., 1996 ), by using the same strategy as the BM-3 construction, generating strains DB-13 and DB-19 (see Methods). Since DB-19 showed almost the same phenotype as DB-13 (data not shown), data on DB-13 are given for the following experiments. We also generated a double mutant by deleting the chsD gene of BM-3. The resulting strain, BD-2, exhibited almost the same phenotype as DB-13 under the conditions tested (data not shown). We first examined the growth rate of the double mutant (Table 3). On MMFT plates, the colony diameter of DB-13 was almost identical to that of the wild-type strain ABPU/AU and was larger than that of BM-3/A1. The colony diameter of the double mutant was about half that of BM-3/A1 on MM plates, whereas it was almost the same as that of BM-3/A1 on plates of YG, another alcA(p)-repressing medium. We found the osmolarity of YGU medium to be lower than that of minimal medium (data not shown). Supplementing YGU plates with 0·6 M KCl caused the colony diameter of DB-13 to decrease to half of that of BM-3/A1. The colony diameters of the chsD null mutant (D3-2) were slightly larger than those of the relevant wild-type strain (ABPU/A) under all conditions tested. These results indicate that the importance of the ChsD function in hyphal growth increased under high osmolarity conditions when the expression of chsB was repressed.

When the hyphal morphology of DB-13 on YGU plate was observed microscopically, the hyphae were seen to be even more disorganized than those of BM-3/A1 (Fig. 3g, h). The number of conidiophores was much smaller than that of BM-3/A1 (data not shown). The number was not greatly increased by 24 h of additional incubation (data not shown).

In the carbon source exchange experiment, it was shown that DB-13 generated approximately 60% of the number of conidia generated by BM-3/A1 (Table 4). Taking into account that the chsD single mutants show no significant reduction in conidiation efficiency (Motoyama et al., 1996 ; Culp et al., 2000 , in which chsD is referred to as chsE), it is suggested that the importance of the ChsD function in conidiation also increased under the chsB-repressing condition.

We measured the chitin content of BM-3/A1 and DB-13 (Table 5). The chitin contents of BM-3/A1 in YGU and in YGU containing 0·6 M KCl were both approximately 70% of those of the wild-type strain (ABPU/AU), whereas those of BB-3 were almost the same as those of the wild-type strain. The chitin contents of DB-13 were approximately half those of BM-3/A1. Since the chitin contents of D3-2 under these conditions were not very different from those of the relevant wild-type strain (ABPU/A), the effect of the chsD deletion on chitin content was considered to be larger in the chsB-conditional mutant than in the wild-type strain. Taking these results together, we concluded that the chsB repression increased the importance of the ChsD functions in hyphal growth, conidiation and chitin content maintenance.


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Table 5. Chitin contents of the wild-type strains and mutants

 
Expression of chsB and chsD
Transcriptional regulation of chitin synthase genes has been shown in other fungi (Chen-Wu et al., 1992 ; Pammer et al., 1992 ; Sudoh et al., 1993 ; Choi et al., 1994 ; Motoyama et al., 1994b ; Xoconostle-Cázares et al., 1996 , 1997 ; Munro et al., 1998 ; Wang & Szaniszlo, 2000 ), and seems to be one of the regulatory mechanisms for chitin synthesis. To investigate the involvement of ChsB and ChsD in hyphal growth and conidiation in another way, we analysed the expression pattern of chsB and chsD using lacZ of E. coli as a reporter gene. We constructed strains BL-1 and DL-4 using plasmids pB-LAC and pD-LAC, respectively (Fig. 2, see Methods). In strain BL-1, it was predicted that the ChsB–LacZ fusion protein, which contains a 10 aa linker between the N-terminal 132 aa of ChsB and LacZ, would be expressed under the control of the chsB promoter. In strain DL-4, it was predicted that the ChsD–LacZ fusion protein, which contains a 10 aa linker between the N-terminal 126 aa of ChsD and LacZ, would be expressed under the control of the chsD promoter (see Methods). To avoid a position effect on expression, both pB-LAC and pD-LAC were integrated into the same argB locus. The growth rates of strains BL-1 and DL-4 were almost the same as those of the relevant wild-type strains AU/A and ABPU/A, respectively, indicating that expression of the fusion proteins is not toxic (data not shown). We analysed the spatial expression pattern of these genes by staining ß-galactosidase activity using X-Gal as a substrate (Fig. 5). In strain BL-1, blue staining was observed in the hyphae (Fig. 5a), as well as in the conidiophore, especially in the metulae, phialides and conidia (Fig. 5b). A similar staining pattern was observed in strain DL-4 (Fig. 5c, d), while no staining was detected in the wild-type strains, AU/A and ABPU/A (data not shown). These expression data further support the proposition that ChsB and ChsD function not only in hyphal growth, but also in conidiation.



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Fig. 5. Spatial expression patterns of chsB and chsD. Conidia from strains BL-1 (a, b) and DL-4 (c, d) were incubated for 13 h (a, c) or for 36 h (b, d) on MM plates, and mycelia were stained with X-Gal and observed under a microscope. Bar, 25 µm.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we found by using a conditional mutant of chsB that ChsB functions not only in hyphal growth but also in conidiation. By using double mutants of chsB and chsD, we found evidence that the functional importance of ChsD increases in hyphal growth and in conidiation when the expression of chsB is repressed. The involvement of ChsB and ChsD in both hyphal growth and conidiation was supported by the expression analysis using lacZ of E. coli as a reporter gene.

Our laboratory reported previously that a chsB disruptant could grow only as a heterokaryon containing both wild-type and disrupted chsB alleles (Yanai et al., 1994 ). The hyphae of a haploid chsB disruptant stopped growing immediately after the germination of conidia, with their hyphal tips swelling. Borgia et al. (1996) obtained viable homokaryons carrying the disrupted chsB allele. Their mutants grew very slowly with a high degree of branching, and contained normal septa. The colonies did not form conidiophores and conidia, and became brown after about 4 days incubation. These phenotypes of the Borgia group’s chsB disruptants are very similar to those of the repressed chsB conditional mutant generated in this study (Fig. 3, and our unpublished result). These results strongly suggest that chsB is necessary not only for hyphal growth but also for conidiation. The lethality of the chsB disruptant reported by Yanai et al. (1994) might be caused not only by the disruption of chsB, but also by the production of the C-terminally truncated ChsB, which could be expressed from the disrupted chsB locus. As shown in Fig. 4 and will also be mentioned below, the repression of alcA(p)::chsB is imperfect in nature, and the viability of the conditional chsB mutant BM-3/A1 may depend on this imperfection. While Borgia et al. (1996) reported that the chitin content of their chsB disruptant was almost the same as that of the parental strain, the chitin contents of the mutant BM-3/A1 in YGU and in YGU containing 0·6 M KCl were both approximately 70% of those of the wild-type strain (Table 5). It is possible that these differences were simply derived from the differences in the genetic backgrounds of the strains employed.

The results in Table 4 clearly show the defect of chsB mutants in conidiation, though the reduction in conidiation efficiency seems rather moderate. When we use repressible promoters for the analysis of the functions of gene products, we should pay attention to two points. First, when the repression of the transcription of the gene is leaky, effects milder than gene disruptions or deletions would be observed under the repressing condition. It has been reported that the expression from the alcA promoter is not completely repressed in minimal medium (Som & Kolaparthi, 1994 ; McGoldrick et al., 1995 ). The results in Fig. 4 show that the chsB transcripts are actually present even at 25 h after the shift from MMFT to MM plate. Second, when transcripts and/or products of the gene are stable after the shift to the repressive condition, the effects of transcriptional repression would be observed only after prolonged cultivation. As shown in Fig. 4 (5 h lane), this was also the case. Taking these precautions into account, it is possible that the contribution of ChsB to conidiation was underestimated in the carbon source exchange experiment (Table 4).

The defects in the hyphal growth of BM-3/A1 after the shift to a chsB-repressing MM plate might have resulted in a reduction of the amount of conidiation-competent hyphae. However, in combination with the chsB expression in conidiophores and conidia (Fig. 5), we take our result to suggest that the function of ChsB is not limited to the generation of conidiation-competent hyphae but is directly involved in conidiation.

We noticed that the colony diameter of BM-3/A1 on MMFT plates is smaller than that of the wild-type strain. This finding might be caused by the dominant negative effect of the chsB overexpression. However, the chsB chsD double mutant DB-13 showed almost the same colony diameter as the wild-type strain. Therefore, it is likely that the chsB overexpression per se does not cause growth inhibition, but the simultaneous expression of chsD disturbs the fully wild-type growth.

A reduction in the growth rate of DB-13 compared to that of BM-3/A1 was observed on MM plates and YGU plates containing 0·6 M KCl, but occurred only slightly on YGU plates (Table 3). This result indicates that, when chsB expression is repressed, the importance of the ChsD function in hyphal growth increases under conditions of higher osmolarity. ChsD seems to be dispensable for hyphal growth under conditions of low osmolarity in the absence of chsB expression. It is possible that ChsD either does not function or its loss can be compensated for by other chitin synthases when the osmolarity of the culture media is low. However, the hyphal morphology of DB-13 was more disorganized than that of BM-3/A1 on YGU plates (Fig. 3g, h), suggesting that ChsD functions and contributes to hyphal morphology even under low-osmolarity conditions. To measure the growth rates on solid media, we inoculated many conidia at one point on a plate. It is possible that, as the colony diameter and hyphal density increase and glucose is consumed, leaky expression of chsB occurs around the centre of a colony. Thus, it is likely that we have underestimated the effect of the chsD deletion on the growth rate on YGU. Supplementation of 0·6 M KCl did not greatly affect the ratio of the chitin content between DB-13 and BM-3/A1 (Table 5), suggesting that chitin content does not necessarily correlate with the degree of physiological disorder.

The conidiation efficiency of DB-13 was approximately 60% that of BM-3/A1 in the carbon source exchange experiment (Table 4). In this experiment, we used as media MMFT and MM, which have higher osmolarity than YGU. Since the deletion of chsD affected hyphal growth under these conditions (Table 3), the defect in conidiation may have been derived from some defects in hyphal growth. Furthermore, since we have obtained evidence that ChsD is present in conidiophore structures of the wild-type strain (Fig. 5), we propose that ChsD may also be directly involved in conidiation.

Our analyses of DB-13 revealed the importance of the chsD function under chsB-repressing conditions. Beth Din et al. (1996) suggested that Chs4, the class IV chitin synthase of N. crassa, may serve as an auxiliary enzyme that supplements chitin synthesis when additional chitin is necessary. ChsD, also a class IV chitin synthase, may contribute to chitin synthesis in response to various conditions, including the chsB-repressing condition. Since the chsD deletion in the chsA mutants reduced the conidiation efficiency of the mutants (Motoyama et al., 1996 ; Culp et al., 2000 ), it is possible that the chsA deletion may also be the condition that increases ChsD activity. Alternatively, ChsD may have some redundant functions with ChsA and ChsB.

The phenotypes of the conditional chsB mutants have some similarity to the phenotypes of the null mutants of the class III chitin synthase genes of other filamentous fungi, namely chs-1 of N. crassa and chsG of A. fumigatus. Further analysis of the chitin synthase functions using the conditional mutation in combination with mutations of other related genes will help to elucidate their coordination for fungal morphogenesis and differentiation, and to develop anti-fungal reagents effective against filamentous fungi.


   ACKNOWLEDGEMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. M.I. was financially supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists. This work was performed using the facilities of the Biotechnology Research Center, The University of Tokyo.


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Received 8 October 2001; revised 7 January 2002; accepted 10 January 2002.



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