disordered cell integrity signaling caused by disruption ...molecular cloning and sequencing of...

EUKARYOTIC CELL, Aug. 2004, p. 1036–1048 Vol. 3, No. 41535-9778/04/$08.00�0 DOI: 10.1128/EC.3.4.1036–1048.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Disordered Cell Integrity Signaling Caused by Disruption of thekexB Gene in Aspergillus oryzae†

Osamu Mizutani, Akira Nojima, Morimasa Yamamoto, Kentaro Furukawa, Tomonori Fujioka,Youhei Yamagata, Keietsu Abe,* and Tasuku Nakajima

Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School ofAgricultural Science, Tohoku University, Sendai 981-8555, Japan

Received 8 January 2004/Accepted 12 April 2004

We isolated the kexB gene, which encodes a subtilisin-like processing enzyme, from a filamentous fungus,Aspergillus oryzae. To examine the physiological role of kexB in A. oryzae, we constructed a kexB disruptant(�kexB), which formed shrunken colonies with poor generation of conidia on Czapek-Dox (CD) agar plates andhyperbranched mycelia in CD liquid medium. The phenotypes of the �kexB strain were restored under highosmolarity in both solid and liquid culture conditions. We found that transcription of the mpkA gene, whichencodes a putative mitogen-activated protein kinase involved in cell integrity signaling, was significantly higherin �kexB cells than in wild-type cells. The �kexB cells also contained higher levels of transcripts for cell wall-related genes encoding �-1,3-glucanosyltransferase and chitin synthases, which is presumably attributable tocell integrity signaling through the increased gene expression of mpkA. As expected, constitutively increased lev-els of phosphorylated MpkA were observed in �kexB cells on the CD plate culture. High osmotic stress greatlydownregulated the increased levels of both transcripts of mpkA and the phosphorylated form of MpkA in �kexBcells, concomitantly suppressing the morphological defects. These results suggest that the upregulation oftranscription levels of mpkA and cell wall biogenesis genes in the �kexB strain is autoregulated by phosphory-lated MpkA as the active form through cell integrity signaling. We think that KexB is required for preciseproteolytic processing of sensor proteins in the cell integrity pathway or of cell wall-related enzymes under tran-scriptional control by the pathway and that the KexB defect thus induces disordered cell integrity signaling.

Some secretory proteins of eukaryotic cells are convertedfrom the precursor proteins to the mature proteins after mod-ification through processes such as various glycosylation reac-tions and limited proteolysis within the Golgi apparatus. Themodification process is well conserved from yeast to mammals,and the target proteins of the process are expanded to peptidehormones, neuropeptides, serum proteins, cell growth factors,and cell growth factor receptors. Therefore, elucidation of theprotein modification process serves a clue to understand thephysiological meaning of the posttranslational process. Kexinis a Ca2�-dependent transmembrane serine protease thatcleaves the secretory proproteins on the carboxyl side of Lys-Arg and Arg-Arg in a late Golgi compartment (15, 46). Kexin-like enzymes have been found from yeast to mammals (16, 19,54, 65). Because filamentous fungi, including Aspergillus spe-cies, secrete large amounts of proteins that are predicted to bemodified through proteolysis in a Golgi compartment, kexinsalso are thought to be key enzymes of the proteolytic processin the Golgi compartment of the fungi. Aspergillus kexins arefound in Aspergillus nidulans (39) and A. niger (29).

Disruption of the A. niger kexB gene causes various morpho-logical alterations such as shorter and multibranched hyphae(29, 60). Recent studies have shown that Kex2p in the dimor-

phic yeast Candida glabrata is required for cell surface integrity(3). A bioinformatics approach, using the C. albicans genomedatabase, assigned 147 open reading frames (ORFs) as encod-ing potential substrates for Kex2p in C. albicans (56). Amongthese ORF products, some predicted Kex2p targets were cellwall-related proteins and sensor proteins including GAS1 andWSC2 homologs. In light of the prediction from the bioinfor-matics approach using C. albicans and the morphological de-fects of the A. niger kexB disruptant, kexin activity in Aspergillusspecies also seems to be concerned primarily with formation ofthe cell wall and morphogenesis. Therefore, maintenance ofmorphologenesis in fungi is likely to be one of the biologicallyimportant functions in which kexin plays some roles. However,the mechanism underlying the involvement of kexB in morpho-genesis in fungi is still unclear.

Although the Saccharomyces cerevisiae kex2-null mutant(kex2�) does not exhibit morphological deffects compared withthe phenotypes of other filamentous fungi, additional geneticdefects in MPK1 (SLT2), together with the kex2� mutation,cause lethality (62). Since the yeast MPK1 gene encodes amitogen-activated protein (MAP) kinase in the cell integritypathway that plays an essential role in maintaining the biogen-esis and integrity of the cell wall (41), kexin seems to beinvolved in cell wall integrity in S. cerevisiae. The cell integritypathway is induced in response to several environmental stim-uli, resulting in the increased expression of numerous genes,many of which encode integral cell wall proteins (glycosylphos-phatidylinositol proteins, PIR [proteins with internal repeats]family proteins, and others) or enzymes, including �-1,3-glucansynthases (Fks1p and Fks2p) and chitin synthases, required for

* Corresponding author. Mailing address: Laboratory of Enzymol-ogy, Department of Molecular and Cell Biology, Graduate School ofAgricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumi-dori,Aobaku, Sendai 981-8555, Japan. Phone: 81-22-717-8776. Fax: 81-22-717-8778. E-mail: [email protected].

† Supplemental material for this article may be found at http://ec.asm.org.

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cell wall biogenesis in S. cerevisiae (13, 31, 67, 68). The A.nidulans mpkA gene that is a counterpart of the yeast MPK1(SLT2) was cloned and characterized (7). An mpkA deletionmutant (�mpkA) was constructed, and its morphological de-fects suggested that the kinase is involved in germination ofconidial spores and polarized growth. As described above, theapparent morphologic changes observed in the A. niger kexBdisruptant suggest the possibility that involvement of kexin incell integrity is more significant in Aspergillus fungi than S.cerevisiae. Consequently, our studies focus on gaining an in-sight into the roles of kexin in morphogenesis and cell wallintegrity in Aspergillus fungi.

A. oryzae, which is an economically important filamentousfungus, as well as A. niger, is used in the manufacture of fer-mented foods and in the production of enzymes for medicaland food-grade use (10, 27, 71). Because its industrial impor-tance has fortunately accelerated the establishment of A. ory-zae genomics, including an expressed sequence tag (EST) da-tabase and genome sequence information, A. oryzae genomicsis now one of the most advanced platforms of fungal genomicsand thus is also valuable for cell biology research on filamen-tous fungi (43; http://www.aist.go.jp/RIODB/ffdb/index.html).As a part of the A. oryzae genome projects, we manufacturedA. oryzae cDNA microarrays consisting of approximately 2,000cDNA clones by using information in the A. oryzae EST data-base (44). In the present study, we isolated an A. oryzae kexBknockout (�kexB) strain that had remarkable morphologicaldefects, and the mutant phenotypes, surprisingly, were sup-pressed under highly osmotic conditions. We investigated thefunction of kexB in morphogenesis by using the kexB knockoutstrain and our cDNA microarrays. Here we report (i) cloningof the A. oryzae kexin gene (kexB) and enzymatic characteriza-tion of KexB in the membrane fraction isolated from a KexB-overexpressing A. oryzae strain, (ii) construction of the �kexBstrain and observation of its phenotype, (iii) comparison ofgene expression profiles between the �kexB strain and the wildtype under solid-culture conditions by using our cDNA mi-croarrays and Northern blot analysis, and (iv) demonstrationof constitutive upregulation of both transcription levels of thempkA gene and phosphorylation levels of MpkA in the A.oryzae �kexB strain on the solid culture. Our results suggestthat disruption of kexB in A. oryzae leads to morphologicalchanges attributable to disordered cell integrity signaling. Wediscuss the contribution of A. oryzae kexB to the cell integritypathway and morphogenesis.

MATERIALS AND METHODS

Strains, media, and growth conditions. We used A. oryzae RIB40 (ATCC42149) as the wild type and for constructing the kexB knockout mutant. Thisstrain was also used for the A. oryzae EST and genome sequencing projects (43).A. oryzae niaD300 (niaD), a niaD mutant derived from RIB40, was used as arecipient strain for transformation and protein expression. These strains weregrown in YPD complete medium (1% yeast extract, 2% polypeptone, 2% dex-trin) or Czapek-Dox minimal medium (CD) (52). Instead of glucose, maltose wasadded to CD medium as an inducer for overexpression by recombinant A. oryzae.CD medium supplemented with 0.1 �g of pyrithiamine (TaKaRa Bio Inc., To-kyo, Japan) per ml was used as the selection medium for the kexB knockoutderivatives of A. oryzae. To isolate niaD mutants from the kexB knockout strainof A. oryzae, 500 mM chlorate and either nitrite (NO2), hypoxanthine, glutamate,or ammonium chloride as a nitrogen source were added to CD medium.

Molecular cloning and sequencing of kexB. For subcloning, Escherichia coliXL1-blue (hsdR17 supE44 recA1 endA1 gryA6 thi relA1 lac [F� proAB� lacIq

lacZ�M15::Tn10, Terr]) cells and pBluescript II SK(�) plasmid (TOYOBO Inc.,Tokyo, Japan) were used as host and vector, respectively, for DNA manipulation.The vector pGEM-T Easy (Promega Co., Tokyo, Japan) was used for TA cloningof PCR products. All basic molecular biology procedures were carried out asdescribed by Sambrook et al. (63). To clone the kexin gene of A. oryzae, wesearched the A. nidulans EST database of the University of Oklahoma (http://www.genome.ou.edu/fungal.html) with the yeast (S. cerevisiae) KEX2 gene andfound an approximately 600-bp homologous sequence. The nucleotide identitybetween the yeast KEX2 gene and the obtained A. nidulans DNA fragment wasabout 40%. PCR primers (5�-CGCTTCTGGGAAGATCTACAAATC-3� and5�-GGCCCGAACTTCTCCCGCGCATC-3�) were designed on the basis of thesequence of the fragment, and PCR was performed using genomic DNA fromA. nidulans as the template. We obtained a 581-bp fragment, which we used asa probe for screening an A. nidulans cDNA library. Three positive clones wereobtained from 2,000 plaques, and the positive clone with the longest insert wassequenced. The insert contains a 2,460-bp ORF encoding a single polypeptidecomprising 819 amino acid residues. A FASTA search against the A. oryzae ESTdatabase (http://www.aist.go.jp/RIODB/ffdb/index.html) was performed with thefull-length sequence of the A. nidulans kexB gene (DDBJ/EMBL/GenBank ac-cession no. AB056726). Clone 6-58 in the A. oryzae EST database was homolo-gous, and the DNA sequencing of this clone was completed using the ABIPRISM BigDye Terminator cycle-sequencing ready reaction kit version 2.0 (Ap-plied Biosystems Japan Ltd., Tokyo, Japan) and an ABI PRISM 377 sequencer(Applied Biosystems Japan). The initiation codon of the A. oryzae kexB gene waspredicted by comparison with that of the A. niger kexB gene (29) and on the basisof the discovery of a stop codon 33 bp upstream of the initiation codon in theA. oryzae kexB cDNA.

Creation of the kexB-overexpressing strain. The overexpression plasmidpNAKX1 was constructed as follows. The kexB gene was PCR amplified usingZ-Taq DNA polymerase (TaKaRa) and a pair of primers, 5�-AAGCTTATAATGCGGCTTTCCGAAAG-3� and 5�-AAGCTTGTAGAAGCAAATGCAAAGCC-3�. Each primer was designed to introduce a HindIII site (underlined). ESTclone 6-58 of A. oryzae was used as the template. The amplified fragment wasinserted into the pGEM-T Easy vector (Promega) and sequenced. The plasmidwas digested with HindIII, and the digested fragment was ligated into theHindIII sites of the pNGA142 vector that has the glaA142 promoter (24). Theconstructed expression plasmid was named pNAKX1. Transformation of A. ory-zae niaD300 (niaD) was performed using the modified protoplast-polyethyleneglycol method (20) and pNAKX1 digested with BamHI. The BamHI-digestedpNGA142 also was introduced into the niaD300 strain as a control (pNGAstrain). For protoplast formation, 5 mg of lysing enzyme (Sigma Chemical Co.,St. Louis, Mo.) per ml, 10 mg of cellulase Onozuka R-10 (Yakult Co., Tokyo,Japan) per ml, and 10 mg of Yatalase (TaKaRa) per ml were used. Transfor-mants were subcultured at least three times on CD agar plates to obtain homo-karyotic strains.

Enzyme assay. The cells were grown in 50 ml of CD liquid medium withshaking for 2 days at 30°C. They were then transferred to CD liquid mediumcontaining 2% maltose and cultured for 17 h at 30°C. They were collected usingglass filters (Asahi Techno Glass Co., Funabasi, Japan) and ground in a mortaron ice. The ground cells were resuspended in 0.2 M HEPES (pH 7.6) containing1 mM EDTA and centrifuged at 10,000 � g for 10 min at 4°C. The supernatantwas further centrifuged at 100,000 � g for 90 min at 4°C. The precipitate wasresuspended in 50 mM HEPES (pH 7.6) containing 1 mM EDTA, 50 mM NaCl,2% sodium deoxycholate, and 20% glycerol, and the mixture was centrifuged for90 min at 4°C and 100,000 � g. The supernatants were pooled as the membraneprotein fraction. Enzyme assays were performed as described previously (45), but20 mM Tris-HCl (pH 7.0) was used as the assay buffer.

Creation of the kexB disruption mutant. The plasmid for kexB gene disruption(pP�kexB-stop) was created as follows. The kexB fragment was obtained by PCRwith primers 1 (5�-TAATGCGGCTTTCCGAAAcTGCAgCGGTAG-3�) and 2(5�-TAGGGTAGAAGCAAATGCAAAGCtTAGTCC-3�) by using EST clone6-58 of A. oryzae as a template. These primers were designed to introduce PstIand HindIII sites (underlined; nucleotides that were changed to create therestriction sites are lowercase), respectively. The fragment was digested with PstIand HindIII and ligated into the PstI-HindIII fragment of the pPTRI vector(TaKaRa), which contains the pyrithiamine resistance gene (ptrA). The con-structed plasmid was named pP�kexB. In addition, a stop codon linker (5�-CTAGTGCTGTGATCACTAGGATCCTTACA-3�) was inserted into the NheIsite of pP�kexB. The linker had terminator codons in every frame, and theresulting plasmid was named pP�kexB-stop (Fig. 1A). Transformation of A.oryzae RIB40 (wild type) was performed as described above by using pP�kexB-stop digested with XhoI (Fig. 1B). A. oryzae transformants were screened for

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resistance to pyrithiamine (0.1 �g/ml) and were subcultured at least three timeson CD agar plates containing pyrithiamine.

Candidates for the knockout strain were selected by colony PCR by usingprimer sets 1 (�-sense 1 [5�-GTTGGGTAACGCCAGGGTTTTCCC-3�] and�-antisense 1 [5�-GACTGACAACGAAAACTAAATTACGGGAGC-3�]) and2 (�-sense 2 [5�-CCCATAATGCGGCTTTCCGAAAGTGC-3�] and �-anti-sense 1) and A. oryzae genomic DNA as a template. A. oryzae genomic DNA wasisolated from mycelia grown in CD medium for 40 h at 30°C as describedpreviously (66). If the XhoI-digested pP�kexB-stop fragment was inserted intothe kexB coding region, a 1,861-bp fragment was amplified by using primer set 1,and if the fragment was not inserted into the kexB coding region, a 1,783-bpproduct was amplified by using primer set 2. Recombinants were confirmed bySouthern analysis. Two probes were prepared. One was the 1.0-kb EcoRI frag-ment of A. oryzae EST clone 6-58 (kexB probe), and the other was a full-lengthptrA fragment PCR amplified using primers 5�-GATTTCGGTCTATTGGCTAGCAAATGAGCT-3� and 5�-CACCAGCGTTTCTGGGCTAGCAAAAACAGG-3� and pPTRI as a template (ptrA probe).

Electron microscopy. Samples were prepared for electron microscopy by amodification of the method of Nakamura et al. (53). �kexB and wild-type colo-nies grown on CD plates for 4 days at 30°C were fixed with 2.5% (vol/vol)glutaraldehyde, washed in 0.1 M sodium phosphate buffer (pH 7.0), and dehy-drated in 50 to 100% (vol/vol) ethanol and then in 100% isoamyl alcohol.Residual isoamyl alcohol in the dehydrated colonies was removed by the critical-point drying method. Dried colonies were coated with platinum-vanadium andobserved under an S-700 scanning electron microscope (Hitachi Ltd., Tokyo,Japan) at an accelerating voltage of 15.0 kV.

mRNA isolation from mycelia in solid and liquid culture. Spores (104) wereinoculated on a sterile nylon mesh filter (Nippon Rikagaku Kikai Co. Ltd.,Tokyo, Japan) on a CD plate and grown at 30°C for 4 days. The nylon mesh filterwith mycelia of A. oryzae was then removed from the plate and frozen in liquidN2. The mycelia were collected with a spatula and ground to a fine powder in amortar. Total RNA was prepared from powdered cells by using Isogen (Nippon-gene Co. Ltd., Tokyo, Japan) as specified by the manufacturer. mRNA wasisolated from the total RNA by using Message Maker (Invitrogen Co., Tokyo,Japan) as specified by the manufacturer. mRNA also was prepared from liquidcultures. Conidia (5 � 106) were inoculated into 100 ml of CD medium andcultured at 30°C for 22 h, and mRNA was isolated as described above.

Microarray analysis. Details of A. orzyae cDNA microarrays were described byMaeda et al. (44), and 2,000 cDNA clones on the cDNA microarray were chosenas highly expressed genes from approximately 5,000 nonredundant EST clones ofthe A. oryzae EST libraries (http://www.aist.go.jp/RIODB/ffdb/index.html) (43).Comparative microarray analyses between the �kexB and wild-type strains cul-tured on CD agar plates and on CD agar plates containing 0.8 M NaCl wereperformed. First, we examined the transcription levels of the histone H2B andH4 genes by using Northern blot analysis. Under both conditions, the transcrip-tion levels of the histone genes were the same in both the �kexB and wild-typestrains (data not shown), suggesting that these genes are appropriate controls formicroarray analyses. Fluorescently labeled cDNA was prepared from eachmRNA by using Cy3-dUTP or Cy5-dUTP (Amersham Biosciences Inc., Tokyo,Japan) and the CyScribe first-strand cDNA labeling kit (Amersham Biosciences)as specified by the manufacturer. Cy3- and Cy5-labeled cDNAs (20 �l each) weremixed and purified using the Microcon-30 sample reservoir (Millipore Co.,Tokyo, Japan). Human Cot-1 DNA (60 �g; Invitrogen) was added to the cDNA,which was then concentrated to 28 �l. Blocking reagents, including 4 �l of yeasttRNA (10 �g/�l; Invitrogen), 16 �l of poly(dA) (1 �g/�l; Invitrogen), 10.2 �l of20� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and 1.8 �l of10% sodium dodecyl sulfate (SDS), were added to the mixture and boiled for1 min. The mixture was cooled to room temperature. The labeled DNA wasapplied to a microarray slide (Asahi Techno Glass Co.) in a cassette chamber,and the chamber was incubated at 55°C for 9 h. The slide was washed sequen-tially with 2� SSC to 0.1% SDS at room temperature, 0.2� SSC–0.1% SDS at40°C, and 0.2� SSC at room temperature for 5 min per wash. The slide was driedby centrifugation for 1 min at 300 � g.

Fluorescent DNA bound to the microarray was detected and quantified witha GenePix 4000B microarray scanner (Axon Instruments, Foster City, Calif.),using the GenePix Pro 3.0 software package to locate spots in the microarray.Data are means of triplicate determinations from three different experiments.Spots were quantified as the median of all pixel values in the spot region. Thebackground was quantified for each spot separately as the median of all pixelvalues in the background region. Background values were subtracted from therespective spot values to obtain net signal values. Net signal values of 1 and belowwere set to 1. Probes with net signal values below 1.000 for both channels wereeliminated from further calculation. Spots that showed net signal values less than

5% of that of histone H2 were also omitted. The merged signal values were usedfor subsequent comparisons and assessed with the Genomic Profiler softwarepackage (Mitsui Knowledge Industry, Tokyo, Japan) and Student’s t test by themethod of Arfin et al. (2). We performed both global normalization and internalnormalization with histones H2B and H4 as the internal standards and detectedonly a small difference between the results from the two normalization methods(44).

Northern blot hybridization. mRNAs were prepared as described above fromthe kexB disruptant and wild-type strains cultured on CD agar plates at 30°C for50, 70, and 105 h or a CD agar plate plus 0.8 M NaCl at 30°C for 105 h. mRNAs(500 ng each) were electrophoresed through an agarose-formaldehyde gel (64)and transferred to Hybond-N� nylon membranes (Amersham Biosciences) byusing 7.5 mM NaOH. Blotted membranes were hybridized with the probes forchsC, chsB, gelA, or gelB of A. oryzae. The probe for the histone H2B gene wasused as a quantitative control. Probes for chsC, chsB, gelA, and gelB wereprepared by PCR using the primers 5�-GAGTCTTATGGGGAGGACGACCATAGTC-3� (forward) and 5�-CTTGGTATTCATCGCTGTAGGGATCGGG-3�(reverse), 5�-ATGGCCTACCAACCGCCTGGTAAAG-3� (forward) and 5�-ATGTTTCACTGCGAGCGTAGGGAGAAGC-3� (reverse), 5�-ATATTAAATATACTTCAGACGGATCTCGCC-3� (forward) and 5�-TCGAAGAAATTCATGAAACAACAATGACCC-3� (reverse), and 5�-CGATATAGATCCACAGATTACCCGAAAAGG-3� (forward) and 5�-GAAAAGCAATACATGAGGTTAATTCAACGC-3� (reverse), respectively, against A. oryzae genomic DNA. ThempkA probe was prepared from the cDNA library (17) of A. nidulans. The A.oryzae probe for the histone H2B gene was prepared by PCR using the M13primer set (TaKaRa) against A. oryzae EST clone JZ5168 (http://www.aist.go.jp/RIODB/ffdb/). All probes were labeled with [�-32P]dCTP by using the Re-diprime II kit (Amersham Biosciences). Each blot was hybridized at 60°C (55°Cfor the mpkA probe) with [�-32P]dCTP-labeled probe. The blots were washed in2� SSC–0.1% SDS at room temperature for 20 min and then twice in 1�SSC–0.1% SDS for 15 min at 60°C (55°C for the mpkA probe) and detected byautoradiography.

Molecular cloning and sequencing of mpkA and plasmid construction. AFASTA search against the A. oryzae EST database (http://www.nrib.go.jp/ken/EST/db/index.html) was performed with the full-length sequence of the A. ni-dulans mpkA gene (7) (DDBJ/EMBL/GenBank accession no. AAD24428).AoEST02842 in the A. oryzae EST database was found to be homologous to theA. nidulans mpkA gene. We predicted the initiation codon of A. oryzae mpkA incomparison with that of the A. nidulans mpkA gene (7). The mpkA gene wasamplified by the PCR method using Z-Taq DNA polymerase (TaKaRa) and apair of primers, 5�-AAGCTTATGGCTGATCTACGTAAGATC-3� and 5�-TCTAGATTATTGTACGTCCATTCCACGC-3�. Each primer was designed tohave a HindIII or XbaI site at the 5� end (underlined). The A. oryzae cDNAlibrary, which was kindly provided by Katsuya Gomi, was used as the template.After TA cloning of the PCR products, the DNA sequencing of this clone wascompleted using the ABI PRISM BigDye Terminator cycle-sequencing readyreaction kit version 2.0 and an ABI PRISM 377 sequencer. To examine the invivo functionality of A. oryzae MpkA, S. cerevisae strain TNP46 (MATa mpk1D::HIS3 ura3 leu2 trp1 his3 ade2 can1) with a temperature-sensitive MPK1 allele(41) was used for complementation analysis. Expression plasmids used in thisexperiment were constructed with the expression vector pYES2 (Invitrogen), inwhich expression is under the control of the galactose-inducible GAL1 promoter(30). A fragment containing the complete ORF of the A. oryzae mpkA cDNA wasdigested with HindIII and XbaI and ligated into the HindIII-XbaI fragment ofpYES2, resulting in the mpkA expression vector pYmpkA.

Creation of the mychis-tagged MpkA-expressing strain. The plasmids(pNmpkAmh and pYmpkAmh) for expression of MpkA possessing a c-myc epi-tope and a polyhistidine tag (mychis-tagged) were created as follows. To con-struct the mychis-tagged vectors, a mychis fragment was digested with XhoI andAgeI from pPITZ�C (Invitrogen) and ligated to the fragment of pSL1180 (Am-ersham Biosciences) digested with XhoI and AgeI, resulting in pSLmychis. Themychis fragment derived from pSLmychis by SphI-SpeI double digestion wasligated to the fragment from pNGA142 digested with SphI and SpeI to generatepNGmychis. The mpkA fragment was obtained by PCR with primers 5�-AAGCTTATGGCTGATCTACGTAAGATC-3� and 5�-CGGCCCTCTACTagtTTGTACGTCCATTCC-3� (stop codon [TAA] replaced by the codon for Thr [ACT];nucleotides that were changed to remove a nonsense codon are lowercase).These primers were designed to introduce HindIII and SpeI sites (underlined),respectively. Plasmid pYmpkA was used as the template. The amplified fragmentwas inserted into the pGEM-T Easy vector and sequenced. The resulting plasmidwas digested with HindIII and SpeI, and the digested fragment was ligated intothe HindIII and SpeI sites of the pNGmychis vector, resulting in the vectorpNmpkAmh expressing mychis-tagged MpkA. The fragment containing the

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ORF for mychis-tagged MpkA was digested with HindIII and NheI from thepNmpkAmh and ligated into the HindIII and XbaI sites of pYES2, resulting inthe vector pYmpkAmh expressing mychis-tagged MpkA.

To add a niaD mutation to the A. oryzae �kexB strain, the nitrite auxotrophsderived from the �kexB strain were screened for resistance to chlorate by usinga CD agar plate containing nitrite (NO2) instead of nitrate (NO3) as a nitrogensource in the presence of 500 mM chlorate. We further screened the niaD-mutating �kexB strains that were unable to utilize nitrate but could use nitrite,hypoxanthine, glutamate, or ammonium chloride as nitrogen sources. Strains(�kexB niaD) obtained from the selection were subcultured at least three timeson the above-mentioned selection agar plates. Transformations of A. oryzae�kexB niaD and niaD300 (niaD) strains were performed as described aboveby using pNmpkAmh digested with BamHI. A. orzyae transformants (�kexB-mpkAmh and wt-mpkAmh strains) were screened as prototrophs.

Transformants, �kexB-mpkAmh and wt-mpkAmh strains of A. oryzae, wereobtained by colony PCR by using primers 5�-GCAAACGAAGTCGAAGCAGTCG-3� and 5�-GTAGAATCACGAATGAGACCTTTGACGACC-3� to con-firm that the BamHI-digested pNmpkAmh fragment was inserted into the niaDlocus of the genome. Genomic DNAs isolated from the transformants as de-scribed above were used as templates for colony PCR. When the BamHI-digested pNmpkAmh fragment was inserted into the niaD locus, a 2,218-bpfragment was amplified by these primers.

Preparation of cell extracts and immunoblot analysis. Cell extracts wereprepared by the same method as described for the mRNA isolation from solidcultures of the �kexB, �kexB-mpkAmh, wild-type, and wt-mpkAmh strains grownon CD agar plates at 30°C for 50, 70, and 105 h or a CD agar plate plus 0.8 MNaCl at 30°C for 105 h. The mycelia were ground to a fine powder in a mortarand immediately suspended in prewarmed SDS sample buffer (120 mM Tris HCl[pH 8.8], 5% SDS, 5% mercaptoethanol, 10% glycerol, 1 mM sodium vanadate)without dye. The samples were vortexed at 10 s quickly and boiled at 100°C for10 min, and the cell debris was removed by centrifugation for 10 min at 15,000� g. Each sample (50 �g of protein) was subject to SDS-polyacrylamide gelelectrophoresis analysis. Protein concentrations were determined by the methodof Schaffner and Weissmann (64) with bovine serum albumin as a standard. Afterthe transfer of proteins to a Pall Fluoro Trans W membrane (NIPPON GeneticsCo. Ltd., Tokyo, Japan) using a semidry blotting apparatus (Bio-Rad), themembrane was used for immunoblotting with anti-phospho-p44/42 MAP kinase(Cell Signalling Technology, Inc., Beverly, Calif.) antibodies followed by immu-noconjugation with the alkaline phosphatase-conjugated goat anti-rabbit immu-noglobulin G antibody (NACALAI TESQUE, Inc., Kyoto, Japan) and visual-ization of the immune complexes with the chromogenic alkaline phosphatasesubstrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. To detectthe MpkA MAP kinase containing the mychis tag, immunoblots were probedwith the anti-myc monoclonal antibody or anti-His monoclonal antibody (Co-vance Laboratories Inc., Vienna, Va.) and then an alkaline phosphatase-conju-gated horse anti-mouse IgG antibody (Vector Laboratories Inc., Burlingame,Calif.). Immune complexes were detected as described above.

Nucleotide sequence accession number. The sequence data of the kexB genesof A. oryzae and A. nidulans have been submitted to the DDBJ/EMBL/GenBankdatabase under accession no. AB056727 and AB056726, respectively. The se-quence data of the mpkA gene of A. orzyae have been submitted to the DDBJ/EMBL/GenBank database under accession no. AB167718.

RESULTS

Cloning and characterization of the kexB gene from A. ory-zae. To clone the kexin gene of A. oryzae, we searched for A.oryzae EST sequences homologous to the A. nidulans kexin-like gene by using the A. oryzae EST database (http://www.aist.go.jp/RIODB/ffdb/) and identified clone 6-58. We determinedthe complete nucleotide sequence of this EST clone, whichcontained a 2,511-bp ORF encoding a protein of 836 aminoacid residues. The putative amino acid sequence showed about70% identity to those of the putative kexin identified in A.nidulans (AB056726) and A. niger KexB (29), and thus theA. oryzae and A. nidulans genes both were designated kexB.The catalytic triad, Ser-Asp-His, and several amino acids up-stream and downstream from the catalytic domain were wellconserved. The putative A. oryzae KexB also has a P-domain,

which is necessary for enzymatic activities in other proteases(42, 55). Hydropathy analysis suggested the presence of twohighly hydrophobic regions that are a signal peptide and trans-membrane domain required for anchoring to Golgi mem-branes, similar to those in the amino acid sequence of yeastkexin. A propeptide with a Lys-Arg dibasic autocleavage sitefollows the signal peptide. In the putative cytoplasmic tail, 15amino acids downstream from the transmembrane domain, wefound the conserved peptide sequence YDFEMI, similar tothat found in KexB of A. niger (29). The underlined amino acidresidues are identical to the late-Golgi retention signal (con-sensus, YXFXXI) in the cytoplasmic tail of the S. cerevisiaeKex2p (69).

To confirm whether the A. oryzae kexB gene product hasprotein-processing activities like those of known kexins, kexBwas integrated at the niaD locus in the A. oryzae niaD300 strainand expressed using the A. oryzae glaA142 promoter. Southernblot analysis confirmed, in addition to the authentic kexB locus,the integration of an extra copy of kexB at the niaD locus in thetransformant (data not shown). The transformed strain inwhich kexB was overexpressed did not show a detectable ornoteworthy phenotype for hyphal development or morphologyunder induced conditions in either liquid or agar plate culture.We fractionated a cell lysate of the kexB-overexpressing strainand found kexin-like enzymatic activities in the membranefraction. The solubilized membrane fraction from the kexB-overexpressing strain showed higher hydrolysis of fluorogenicpeptides than did that from the control strain harboring theexpression vector lacking the kexB insert (Table 1). In partic-ular, A. oryzae KexB has a preference for peptides containingdibasic residues, as follows: Boc-Gln-Arg-Arg-MCA (4-meth-ylcoumaryl-7-amide), Boc-Leu-Lys-Arg-MCA, Boc-Leu-Arg-Arg-MCA, and Boc-Arg-Val-Arg-Arg-MCA. The sub-strate specificity of A. oryzae KexB is similar to that of yeastKex2p (5). KexB of A. oryzae also recognizes Arg-Arg andLys-Arg at the P1 and P2 positions but hardly ever recognizesLys-Lys. According to the substrate specificity, we concludedthat the cloned kexB gene encodes kexin of A. oryzae.

Construction and characterization of the kexB disruptant.To further study the in vivo functionality of A. oryzae kexB, weconstructed the kexB disruptant (�kexB) strain by homologousrecombination with pP�kexB-stop (Fig. 1A), which containedptrA and a truncated kexB fragment carrying a stop codon

TABLE 1. Enzymatic activities for peptidyl-MCA substrates

Peptide substratea Cleavage activity (nkat/ml)

P4 P3 P2 P12 P1� pNGA142 pNAKX1

Boc-Gln-Arg-Arg-MCA 0.07 3.47Boc-Leu-Lys-Arg-MCA 0.06 3.15Boc-Leu-Arg-Arg-MCA 0.07 2.91

Boc-Arg-Val-Arg-Arg-MCA 0.05 2.39Boc-Val-Pro-Arg-MCA 0.04 1.74Boc-Gly-Lys-Arg-MCA 0.03 1.30Boc-Gly-Arg-Arg-MCA 0.01 0.56

Boc-Leu-Ser-Thr-Arg-MCA 0.02 0.55Boc-Leu-Gly-Arg-MCA 0.00 0.11Boc-Gln-Gly-Arg-MCA 0.01 0.20Boc-Glu-Lys-Lys-MCA 0.00 0.01

a The arrow indicates the cleavage site by A. oryzae KexB. Boc, t-butyloxycar-bonyl-; MCA, 4-methylcoumaryl-7-amide. Basic amino acids are in bold.



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linker downstream of the XhoI site (Fig. 1B). The stop codonlinker had multiple stop codons to cover all reading frames.The �kexB strain was isolated from about 400 colonies of thetransformants by the colony PCR method as described by vanZeijl et al. (66) with primers 1 and 2, as described in Materialsand Methods. The �kexB candidate was further confirmed byPCR and Southern analysis (Fig. 1C). The probes for bothkexB and ptrA indicated the expected two hybridization signalsto digested genomic DNA isolated from the �kexB candidateand the single hybridization signal to that from the wild-typestrain, suggesting that the homologous recombination success-fully took place at the authentic kexB locus. Reverse transcrip-tion-PCR failed to reveal transcripts derived from kexB in the�kexB strain (data not shown).

The �kexB strain formed shrunken colonies and scarcelydifferentiated conidia on CD agar plates (Fig. 2A). The de-tailed morphology of the �kexB strain was further comparedwith that of the wild type by using scanning electron micros-copy (Fig. 2C to G). �kexB cells grown for 4 days on CD agarplates formed neither conidiophores nor conidia. The hyphaeof the �kexB strain became finer, denser and hyperbranched incomparison with those of the wild type. Hyphal tips of the�kexB strain were thicker and multibranched (Fig. 2F). Thedisruptant grown in CD liquid culture medium also showedhighly branched mycelia (data not shown). We carried out arescue experiment in which the mutant phenotypes of the A.oryzae �kexB (�kexB niaD) strain were almost fully restored bytransformation with the pNAKX1 plasmid containing the wild-type kexB gene (see Fig. S1 in the supplemental material).However, the A. oryzae �kexB (�kexB niaD) strain transformedwith the pNGA142 vector without the kexB insert maintainedthe mutant phenotypes (see Fig. S1 in the supplemental ma-terial). These results suggested that the phenotypes of the�kexB strain are attributable to the defect of the kexB gene.Surprisingly, although the �kexB strain exhibited the variousdescribed morphological defects on CD agar plates, the defectswere suppressed on high-osmolarity CD agar plates containing0.8 M sodium chloride (Fig. 3), 0.8 M potassium chloride, or1.2 M sorbitol (data not shown). The morphological defects ofthe �kexB strain also were restored in a high-osmolarity CDliquid culture medium (data not shown).

cDNA microarray analysis of the �kexB disruptant. Becausethe �kexB strain showed the various described phenotypes, wewondered how transcription profiles reflected the phenotypesand therefore compared the gene expression profiles of the�kexB and wild-type strains by using A. oryzae cDNA microar-rays. We analyzed transcripts from the two strains cultured onCD agar plates for 105 h at 30°C as well as on CD agar platescontaining 0.8 M NaCl (Table 2). On CD agar plates, a largenumber of genes were more upregulated in the �kexB cellsthan in wild-type cells whereas gene expression levels of the�kexB strain were similar to those of the wild type in thepresence of high osmotic pressure (�0.8 M NaCl). The �kexB

FIG. 1. Generation of the kexB gene disruptant. (A) Constructionof the plasmid for kexB gene disruption. The plasmid contains a trun-cated kexB gene and ptrA, which encodes a thiazole biosynthetic en-zyme (37). The open box indicates a stop codon linker inserted in thekexB ORF. (B) Strategy for homologous recombination of A. oryzae forkexB gene disruption. The solid box indicates the kexB ORF with thestop codon linker. The gray bars indicate the hybridization positions ofthe probes to confirm gene disruption by Southern blot analysis. Theupper bars indicate the kexB probe. The lower bar indicates the ptrAprobe. (C) Southern blot analyses of the genomic DNA from trans-formant. Each lane contained 20 �g of restriction enzyme-digestedgenomic DNA of the �kexB disruptant (lanes 1, 2, 5, and 6) and wild

type (lanes 3, 4, 7, and 8). The enzymes used were PstI and SphI (lanes1 and 3), ApaLI and NspV (lanes 2 and 4), SpeI (lanes 5 and 7), andPstI (lanes 6 and 8). Hybridization was performed with the kexB probe(see Materials and Methods) (left) and the ptrA probe (right).



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strain exhibited 4.9- and 55-fold-lower levels of brlA (70) androdA (57) transcripts, respectively, than did the wild type. Be-cause brlA encodes a transactivator to promote the formationof conidia and rodA encodes a hydrophobic protein necessaryto form conidiophores, the downregulation of brlA expressionin the �kexB strain might be one of the reasons for its poorgeneration of conidia. We were unable to assign identities toother genes that showed markedly reduced transcript levels in

the �kexB strain, because the annotations of some genes in thecDNA microarray remain unknown.

Because of the morphological defects of the �kexB strain, wepaid further attention to the following eight genes which areinvolved in cell wall biogenesis: chsC (50), chsA (unpublisheddata; DDBJ/ENBL/GenBank accession no. BAB85683), chsB(51), chsY, chsZ (9), gelA, gelB (48), and fksA (34) (Fig. 4).chsC, chsA and -B, chsY, and chsZ encode type I, III, V, and VIchitin synthases, respectively (9). According to tBlastx analysis,gelA and gelB are predicted to be the A. oryzae counterparts ofthe A. fumigatus gel1 and gel2 genes, which encode putativeglucanosyltransferases that are thought to be involved in cellwall biosynthesis (49). fksA is the putative A. oryzae �-1,3-glucan synthase gene (34). On CD agar plates, transcription ofchsC, chsB, and gelB of the �kexB strain was markedly upregu-lated and their transcription levels were 3.2, 4.6, and 4.3 timeshigher, respectively, than those of the wild type. In contrast,high osmotic pressure (0.8 M NaCl) in the culture plate sup-pressed the upregulation of these three genes in the �kexBstrain.

Northern blot analysis of the �kexB strain. Cell wall bio-genesis in S. cerevisiae is thought to be under the control of

FIG. 2. Morphological phenotypes of the �kexB strain on CD agarplates. �kexB (A, D, and F) and wild-type (B, C, E, and G) cells werecultivated on CD agar plates at 30°C for 4 days. (A and B) Colonygrowth of the �kexB (A) and wild-type (B) strains. (C through G)Scanning electron micrographs. Conidiophores and conidia of the wildtype are shown (C and E, respectively); the �kexB strain did not formconidiophores or conidia. Hyphae of the �kexB and wild-type strainsare visible (D and G, respectively); hyphal tips of the �kexB strain canalso be seen (F).

FIG. 3. Restoration of the �kexB phenotypes on CD agar plateswith high osmotic pressure. �kexB (A) and wild-type (B) strains werecultivated on CD agar plus 0.8 M NaCl at 30°C for 4 days. Conidio-phores of �kexB and wild-type strains (C and E, respectively) andconidia of �kexB and wild-type strains (D and F, respectively) are alsovisible.

TABLE 2. Number of genes whose expression levels were alteredby deletion of kexB in A. oryzae

Relative expression ratios(�kexB/wild type) (fold)

No. of genes in cells grown ona:

CD CD � 0.8 M NaCl

4.0 68 02.0–4.0 291 250.5–2.0 1,087 1,736

0.25–0.5 48 10.25 27 0

a After three independent analyses, 1,521 spots on the cDNA microarrays forCD plates and 1,762 spots on the cDNA microarrays for CD plates with 0.8 MNaCl were statistically verified by using the Genomic profiler program.



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signal transduction pathways including the cell integrity path-way. The S. cerevisiae MPK1 (SLT2) gene encodes a MAPkinase in the cell integrity pathway (26). In S. cerevisiae, whencell wall biosynthesis is inhibited or the cell wall is damaged,transcription levels of MPK1 and genes required for cell wallbiogenesis are simultaneously upregulated by the transcriptionfactor Rlm1p, which is a phosphorylation target of Mpk1p (13,31, 67, 68). Bussink et al. (7) isolated the A. nidulans mpkAgene which is the counterpart of yeast MPK1. Recently weconfirmed that expression of A. nidulans mpkA cDNA in atemperature-sensitive S. cerevisiae mpk1 disruptant suppressedthe mpk1 disruption mutation, suggesting that A. nidulans

mpkA functionally complements yeast MPK1 (T. Fujioka, K.Furukawa, O. Mizutani, K. Abe, and T. Nakajima, unpublisheddata). We also found an A. oryzae mpkA gene homolog in theA. oryzae EST database and A. oryzae genome informationthrough homology searches with the nucleotide sequence ofA. nidulans mpkA. Because our cDNA microarray analysesshowed upregulation of the transcription levels of genes in-volved in cell wall biogenesis in A. oryzae �kexB, we furtherexamined whether transcription of mpkA is also upregulated inthis strain. We performed transcriptional analyses of mpkAand cell wall-related genes such as chsC, chsB, gelA, and gelB byNorthern blotting at various times after inoculation (Fig. 5Aand B). The wild-type strain began to form conidiophores andconidia 70 h after inoculation and had formed fully matureconidiophores with plenty of conidia at 105 h on CD plates.Transcription of the histone H2B gene seemed to be constitu-tive and was used as a control at all time points. Transcriptionlevels of chsC, chsB, and gelB were higher in the �kexB strainthan in the wild type at the three time points assayed. Asexpected, the transcription of mpkA in the �kexB strain alsowas increased at all time points. However, the transcriptionlevels of gelA were almost same in both strains at all timepoints. Because cell integrity signaling is inactivated by highosmotic stress in S. cerevisiae (11, 25, 26), we examined whetherhigh osmotic stress downregulates the high level of transcrip-tion of mpkA in the �kexB strain to the transcription level ofmpkA in the wild-type strain. We performed transcriptionalanalyses of mpkA by Northern blotting under conditions ofhigh osmosis (Fig. 5C). As expected, high osmotic stress ap-parently downregulated the transcription level of mpkA in the�kexB strain to a level similar to that in the wild-type strain.Therefore, the morphological defects concomitant with themarked upregulation of mpkA and the osmoresponsive sup-pression of the morphological defects with the simultaneousdownregulation of transcription of mpkA and genes for cellwall biogenesis may indicate that kexB is involved in a signaltransduction pathway, particularly cell integrity signaling.

Cloning of the mpkA gene from A. oryzae and time course ofMpkA phosphorylation in the �kexB strain. Since transcrip-tion levels of the mpkA gene in the �kexB strain are consti-tutively upregulated under normal culture conditions anddownregulated in the presence of osmotic stress, we furtherexamined phosphorylation levels of MpkA protein in the�kexB strain under normal and high-osmosis conditions. Fromthe A. oryzae cDNA library, we isolated a positive clone byusing PCR. We determined the complete nucleotide sequenceof this clone, which contained a 1,272-bp ORF encoding anMpkA protein of 423 amino acid residues. The catalytic do-main of MpkA possesses all the subdomains found in proteinkinases (23). In addition, MpkA has a TEY tripeptide dualphosphorylation motif characteristic of MAP kinases, which isknown to be required for activation of MAP kinases. Theputative amino acid sequence showed about 90% identity tothat of the putative MpkA identified in A. nidulans (7), andthus the A. oryzae gene was designated mpkA.

Because MpkA of A. oryzae is also related to the yeastMpk1p, we investigated the in vivo functionality of MpkA andits derivative mychis-tagged MpkA by using an S. cerevisiaempk1 mutant. We confirmed that expression of both A. oryzaempkA and mpkAmh genes in the S. cerevisae mpk1 disruptant

FIG. 4. Expression levels of genes involved in cell wall biogenesis.The bar graphs indicate the expression levels of the genes encoding cellwall synthesis-related proteins from wild-type and �kexB strains grownon CD agar plates (A) and CD agar plates containing 0.8 M NaCl (B).The gray and white bars indicate the relative intensities of transcrip-tion of the genes in the wild-type and �kexB strains, respectively, onthe basis of the cDNA microarray analyses. The relative intensities ofthe examined genes were calculated using the intensity of histone H2Bas an internal standard (1.0). The genes were as follows: 1, chsC (chitinsynthase C) (50); 2, chsA (chitin synthase A) (our unpublished data;DDBJ/ENBL/GenBank accession no. BAB85683); 3, chsB (chitin syn-thase B) (51); 4, chsY (chitin synthase Y) (9); 5, chsZ (chitin synthaseZ) (9); 6, gelA (glycosylphosphatidylinositol-anchored glucanosyltrans-ferase) (48); 7, gelB (glycosylphosphatidylinositol-anchored glycanosyl-transferases) (48); and 8, fksA [�(1-3) glucan synthesis] (34).



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suppressed the temperature sensitivity attributed to the mpk1mutation, suggesting that both mpkA and mpkAmh function-ally complement the yeast MPK1 (data not shown). To dem-onstrate whether MpkA is phosphorylated in the �kexBstrain, we examined the phosphorylation levels of MpkA andMpkAmh expressed in �kexB cells grown on the CD agarplates. We detected a significant increase in the phosphoryla-tion levels of both MpkA and MpkAmh in the A. oryzae �kexBcells grown for 105 h on the CD agar plates by using anti-phospho-p44/42 MAP kinase antibodies (Fig. 6A, lanes 1 and2), although MpkA in the wild-type strain was scarcely phos-phorylated, regardless of osmotic stress (lanes 3, 4, 7, and 8).The phosphorylation levels of MpkA and MpkAmh in the

�kexB strain were apparently downregulated by high osmoticstress (lanes 5 and 6), with concomitant downregulation of thetranscription levels of mpkA (Fig. 5C). Phosphorylation ofMpkA was observed when the wild-type cells were subjectedto hypotonic stress (Fig. 6A, lanes 9 and 10), indicating thatMpkA is capable of transducing the same types of stresssignals through the putative cell integrity pathway as well asthat of S. cerevisiae. The �kexB-mpkAhm and wt-mpkAhmstrains used as controls of expression quantity of MpkAshowed the same phenotypes as the �kexB and the wild-typestrains on the CD agar plate culture, respectively. Further-more, the phosphorylation levels of MpkAmh and the authen-tic MpkA in the �kexB-mpkAmh cells significantly increased atthe three culture time points examined whereas MpkA and itsderivative in the wt-mpkAmh strain were poorly phosphory-lated (Fig. 6B). The phosphorylation levels of MpkAmh andthe authentic MpkA in the �kexB-mpkAmh strains at 70 and105 h of cultivation were higher than those at 50 h. Althoughthe phosphorylation levels of MpkAs at each growth stage

FIG. 5. Gene expression analysis of chsC, chsB, gelA, gelB, andmpkA by Northern blotting. (A) Time course of the phenotypic changeof the �kexB and wild-type strains on CD agar plates. The left, middle,and right panels show colonies cultivated for 50, 70, and 105 h, respec-tively. (B) Gene expression analysis of chsC, chsB, gelA, gelB, andmpkA over time (50, 70, and 105 h) by Northern blotting. A histonegene was used as a control. wt, wild type. (C) Gene expression analysisof mpkA on CD agar plates plus 0.8 M NaCl for 105 h by Northernblotting. A histone gene was used as a control.



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were different in the �kexB-mpkAmh cells, the MpkAs in the�kexB-mpkAmh were remarkably phosphorylated at all timepoints after inoculation, suggesting that the KexB defect inA. oryzae causes constitutive activation of the cell integritypathway.

DISCUSSION

In the present study, we cloned the A. oryzae kexB gene,which encodes 836 amino acid residues and confirmed that theencoded protein has processing activity (Table 1). To studykexB function, we constructed a kexB gene disruptant strain(�kexB; Fig. 1). The �kexB strain showed shrunken colonieswith poor generation of conidia on CD agar plates (Fig. 2), andhyperbranched mycelia occurred in liquid culture. Interest-ingly, the morphological defects derived from the �kexB geno-

type were restored under conditions of high osmosis (Fig. 3).In A. oryzae �kexB, the transcription levels of genes for cellwall biogenesis and an mpkA homolog that presumably en-codes a MAP kinase involved in cell integrity signaling werehigher than those in the wild type (Table 2; Fig. 4 and 5), andhigh osmotic pressure downregulated the transcription levelsof A. oryzae mpkA and the cell wall-related genes in the �kexBstrain to levels similar to those in the wild type (Fig. 4 and 5).Then we cloned the A. oryzae mpkA gene and confirmed thatits expression suppressed the temperature sensitivity of the S.cerevisiae mpk1 disruptant (data not shown), suggesting the invivo functionality of A. oryzae MpkA. As expected, constitu-tively elevated phosphorylation levels of MpkA in �kexB cellson the CD agar plate culture were demonstrated by usinganti-phospho-p44/42 MAP kinase antibodies, and high osmoticstress downregulated the increased phosphorylation levels ofMpkA in the �kexB strain to the same as those observed in thewild type (Fig. 6). These results suggest that the upregulationof transcription levels of mpkA and cell wall-related genes inthe �kexB strain is mediated by phosphorylated MpkA as anactive form through cell integrity signaling.

The phenotypes of A. oryzae �kexB are different from thoseof kexB disruptants of A. niger (29, 60) and A. nidulans (K.Furukawa, O. Mizutani, T. Fujioka, Y. Yamagata, K. Abe, K.Gomi, and T. Nakajima, unpublished data). The A. niger kexBdisruptant formed conidiophores and conidia on agar plates(29). The A. nidulans �kexB strain showed shrunken colonieson agar plates but had differentiated conidiophores andconidia. According to the phenotypes of the �kexB strains ofthese three Aspergillus species, the function of the kexB gene inA. oryzae is probably more essential for cell growth and espe-cially for cell wall biogenesis. The differences of the �kexBphenotypes among these three Aspergillus species may be at-tributable to the processing targets of KexB in each species butnot to the substrate specificity of each KexB protein, becausethe substrate specificity of the A. oryzae product was similar tothose of A. niger (29) and A. nidulans (39). Moreover, it isnoteworthy that the various morphological phenotypes derivedfrom the �kexB genotype in A. oryzae were restored under highosmotic stress in both solid and liquid culture. However, thenumber of conidia in the �kexB strain was 70% of that in thewild type even under high osmotic pressure, suggesting thatosmotic suppression of �kexB phenotypes is incomplete. Al-though high osmotic pressure did not suppress the phenotypesof kexin gene disruptants of S. cerevisiae or C. albicans (36, 56),the osmotic restoration of �kexB phenotypes occurs in A. ory-zae and A. nidulans (Furukawa et al., unpublished). It remainsunknown whether the �kexB phenotypes of A. niger are sup-pressed under high osmotic conditions.

In our comprehensive and comparative analysis of gene ex-pression between the A. oryzae �kexB and wild-type strains byusing cDNA microarrays, the transcription levels of a largenumber of genes were higher in the �kexB strain than in thewild type on CD agar plates (Table 2). These results imply thatdisruption of the kexB gene affects the transcription levels of abroad range of genes and that KexB of A. oryzae probablyprocesses key proteins required for maintenance of normalmorphogenesis and cell growth. In addition, we found that theexpression levels of chsC, chsB, and gelB were markedly higherin the A. oryzae �kexB strain than in the wild type (Fig. 4).

FIG. 6. The cell integrity pathway MAP kinase MpkA is constitu-tively phosphorylated in the A. oryzae �kexB strain on the CD plate.Upper panels show immunoblotting with anti-phospho-p44/42 MAPkinase antibodies (Anti-p44/42). Lower panels show immunoblottingwith anti-Myc antibodies (Anti-myc). (A) �kexB (lanes 1 and 5), �kexBwith mpkAmh (�kexB-mpkAmh; lanes 2 and 6), wild type (wt; lanes 3and 7) and wild type with mpkAmh (wt-mpkAmh; lanes 4 and 8) weregrown for 105 h at 30°C on the CD plate (CD) and CD plate with anosmolyte (�NaCl) before preparation of cell extracts (see Materialsand Methods). As controls, wild-type (lane 9) and wt-mpkAmh (lane10) cells were exposed for 1 h to a hypotonic condition (Hypo) byaddition of water after cultivation for 50 h on the CD plate. (B) The�kexB-mpkAmh (lanes 1 to 3) and wt-mpkAmh (lanes 4 to 6) strainswere grown at 30°C on the CD plate for 50, 70, and 105 h, respectively,before preparation of cell extracts.



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Cell wall biogenesis in S. cerevisiae is thought to be under thecontrol of various signal transduction pathways including thecell integrity pathway (26). Cell integrity signaling is activated,with different timing and kinetics, by hypoosmotic shock (11,32), by heat shock (32), during bud emergence (72), on expo-sure to mating pheromone (6, 72), and on various treatmentsleading to perturbation of the cell wall (4, 35, 67). The pathwayorganizes changes in cellular morphology by controlling theexpression of genes encoding enzymes involved in cell wallmetabolism. The central pathway concerned with cell integrityis the Mpk1p MAP kinase cascade, and Mpk1p phosphorylatesand consequently activates the transcription factor Rlm1p,whose transcriptional targets are genes encoding cell wall bio-genesis proteins, cell wall proteins, and Mpk1p itself (13, 31,68). Northern blot analyses of cell wall-related genes andmpkA revealed that transcription levels of the examined geneswere simultaneously upregulated in A. oryzae �kexB. Westernblot (immunoblot) analysis by using anti-phospho-p44/42 MAPkinase antibodies also revealed the constitutive phosphoryla-tion of MpkA in A. oryzae �kexB. If cell integrity signalingsimilar to that of S. cerevisiae is functional in Aspergillus fungi,including A. oryzae, the results shown in Fig. 4 to 6 suggest that

cell integrity signal transduction is constitutively activated bydisruption of the kexB gene in A. oryzae.

We propose the following two scenarios to explain why andhow cell integrity signaling is activated in the A. oryzae �kexBstrain. In the first scenario, if KexB processes enzymes re-quired for cell wall biogenesis, then the �kexB strain fails toprocess these enzymes, resulting in perturbation of cell wallassembly. Various sensor proteins might detect perturbation ofthe cell wall as a stressor in A. oryzae �kexB and consequentlyactivate the cell integrity pathway that upregulates the tran-scription of some cell wall-related genes required for restora-tion of the damaged cell wall. Sensor proteins such as Mid2pand Wsc1p in S. cerevisiae are thought to directly sense alter-ations of certain cell wall properties to mediate the activationof the cell integrity pathway via G-protein reactions (26). Pu-tative ORFs homologous to the yeast sensors occur among A.oryzae genome sequences (data not shown). In the secondscenario, if KexB processes putative cell surface sensors of thecell integrity pathway, incorrect processing of the sensor pro-teins by the �kexB mutation might be responsible for the con-stitutive activation of cell integrity signaling and for the con-sequent upregulation of the transcription levels of cell wall-

FIG. 7. Schematic model for implication of the cell integrity pathway in the �kexB (A) and wild-type (B) strains. The cell integrity MAP kinasepathway is shown by the dotted box. pkcA (47), mpkA (7), and rlmA (Fujioka et al., unpublished; accession no. BAD01583) were isolated fromAspergillus fungi. The yeast BCK1 and MKK1 orthologs have not yet been isolated from Aspergillus species; however, putative ORFs (bckA, mkkA)homologous to the two genes are found in A. oryzae genome sequences (O. Mizutani, K. Abe, and T. Nakajima, unpublished data). The cellintegrity pathway in the �kexB strain is constitutively activated (A), although the pathway is not activated (resting) unless the wild-type strain sensessome stress such as hypoosmolarity (B). A. oryzae KexB is predicted to be required for precise proteolytic processing of sensor proteins in the cellintegrity pathway and/or of cell wall-related enzymes whose genes are under transcriptional control by the pathway.



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related genes. The morphological defects of the �kexB strainmight be caused by upregulation of cell wall-related genesthrough activation of cell integrity signaling. A bioinformaticsapproach using the C. albicans genome database assigned 147ORFs as putative Kex2p substrates, and these ORFs includeda gene encoding a Wsc2p homologue that seems to be a sensorprotein in the cell integrity pathway (56). Applying this analogyto A. oryzae, the sensor/signal-like proteins of the pathway in A.oryzae might be processed by KexB.

Although KexB may process cell wall-related enzymesand/or cell surface sensors (Fig. 7), we currently prefer the firstscenario in light of the following predictions. Although theyeast KEX2 disruptant (kex2�) has no noteworthy morpholog-ical phenotype, the Pkc1-Mpk1 pathway is known to be acti-vated in the kex2� strain, resulting in perturbation of cell wallstructure (62). Consequently, additional defects of MPK1, to-gether with the kex2� mutation, cause cell death (62). Thissynthetic lethality suggests that the perturbation of cell wallassembly in the kex2� strain is probably suppressed by activa-tion of cell integrity signaling, which consequently upregulatesthe transcription of cell wall-related genes required for resto-ration and maintenance of the putative damaged cell wall (12,31, 61). Furthermore, disruption of genes encoding various cellwall-related enzymes causes activation of the pathway in S.cerevisiae (8, 40), whereas, to our knowledge, evidence foractivation of the cell integrity pathway by impaired sensors isunavailable even for S. cerevisiae (21, 28, 35, 67). However, wecannot exclude the possibility that the transcription level ofmpkA might be upregulated by the influence of another signaltransduction pathway.

The kex2� mpk1� phenotype of S. cerevisiae is rescued bygrowth on high-osmolality medium (62). The phenotype of theA. oryzae �kexB strain also was restored under high osmoticpressure (Fig. 3). We propose the following two explanationsfor why the phenotypes of the �kexB strain were suppressedunder high osmotic pressure. First, instead of KexB, perhapsother processing proteases inducible under high osmotic pres-sure suppress the �kexB mutation. YPS1 and YPS2, whichencode aspartyl proteases, are multicopy suppressors of thekex2� mutation in S. cerevisiae (14, 36). The expression levelof YPS1 in S. cerevisiae cells treated for 10 min with 0.4 MNaCl is 15.7 times higher than that in cells without the NaCltreatment (58). We found two genes homologous to YPS1and YPS2 in A. oryzae (38), and NaCl may induce thesehomologs and suppress the �kexB mutation. Second, therestoration of the �kexB phenotypes might depend on mech-anisms mediated by osmotic stress, such as a cascade similarto the yeast Hog1 MAP kinase pathway (22, 26, 33, 59). Thecell integrity pathway in S. cerevisiae is thought to be nega-tively regulated by osmotic pressure (11, 25, 26), and acti-vation of the Hog1p pathway mediates adaptive rearrange-ments in cell wall composition and architecture in S.cerevisiae and C. albicans (1, 18). A similar scenario mightexplain the osmotic restoration of the �kexB phenotypes inA. oryzae. To verify restoration of the altered phenotypes byincreased osmotic pressure in �kexB, in vivo functional stud-ies, such as exploration of suppressors of the �kexB muta-tion or construction of a mutant in which the HOG pathwayis constitutively activated in the �kexB genetic background,are necessary and in progress.

In conclusion, we predict that A. oryzae KexB is required forprecise proteolytic processing of sensor proteins in the cellintegrity pathway or of cell wall-related enzymes whose genesare under transcriptional control by the pathway. Thus, theKexB defect leads to disordered cell integrity signaling.

ACKNOWLEDGMENTS

We thank Katsuya Gomi and Hiroyuki Horiuchi for helpful sugges-tions. We also thank Osamu Hatamoto, Hiroshi Maeda, Junichi Ma-ruyama, Turuji Satou, Takamitsu Maruyama, Yoshihiko Matsuda,Kanako Suzuki, Motoaki Sano, and Masayuki Machida for helpfuldiscussions and/or technical assistance.

This work was supported in part by a Grant-in-Aid (Bio DesignProgram) from the Ministry of Agriculture, Forestry and Fisheries ofJapan (BDP-03-VI-1-7).

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