Presence and Functionality of Mating Type Genes in the SupposedlyAsexual Filamentous Fungus Aspergillus oryzae

Ryuta Wada,a Jun-ichi Maruyama,a Haruka Yamaguchi,a Nanase Yamamoto,a Yutaka Wagu,b Mathieu Paoletti,c David B. Archer,c

Paul S. Dyer,c and Katsuhiko Kitamotoa

Department of Biotechnology, The University of Tokyo, Tokyo, Japana; Bio’c Co., Ltd., Toyohashi, Japanb; and School of Biology, University Park, University of Nottingham,Nottingham, United Kingdomc

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MATgenes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of acomplementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the devel-opment of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed,including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 andMAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type.MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expressionwas compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MATgenes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or theMAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulategene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1expression specifically upregulated an �-pheromone precursor gene, but the functions of most of the genes affected were un-known. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexualcycle are discussed.

The filamentous fungus Aspergillus oryzae is known as a kojimold and plays an important role in the Japanese food indus-

try, where it has long been used for traditional fermentative foodproduction, such as the manufacture of sake, soy sauce, and miso(33). More recently, the ability of the species to secrete largeamounts of proteins has extended its industrial use to includeheterologous protein production (28, 46, 63, 64). A. oryzae has noknown sexual cycle and has traditionally been classified within theFungi Imperfecti (the Deuteromycota), or “mitosporic” fungi(35). Therefore, it has not been possible to utilize the sexual cyclefor strain improvement purposes, preventing, for example, theoutcrossing of independent strains with industrially useful char-acteristics. However, sequencing of the A. oryzae genome has re-vealed the presence of a series of genes implicated in sexual repro-duction, suggesting the possibility of cryptic sexuality in thissupposedly asexual fungus (19, 39). Phylogenetic analysis alsoclearly demonstrates that A. oryzae, along with other asexualAspergillus species, is a member of the fungal subphylum Pezizo-mycotina, which is defined by sexual reproduction involving theproduction of sexual ascospores within asci (15, 52).

Sexual reproduction in pezizomycete fungi typically occurs intwo distinct manners. Homothallic fungi are self-fertile and cancomplete the sexual cycle without the need for a partner, whereasheterothallic fungi require a partner of a complementary matingtype. Research over the past 20 years has revealed that the differentforms of sexual breeding systems are determined principally bythe presence within the genome of key “mating type” (MAT)genes (11). Heterothallic species contain a single MAT locus, andcomplementary mating types have different genes encoding mem-bers of the high-mobility-group (HMG) superfamily of proteins(41). MAT1-1 isolates characteristically contain a MAT1-1 geneencoding a protein with a MAT�_HMG domain, whereas

MAT1-2 isolates characteristically contain a MAT1-2 gene encod-ing a protein with a MATA_HMG domain (11, 41). In contrast,homothallic species typically contain MAT genes encoding bothMAT�_HMG and MATA_HMG domain proteins within thesame genome (11, 50). These mating type proteins are thought toact as master regulatory transcription factors, controlling cellidentity, for example, by regulating the expression of pheromoneprecursor and receptor genes (3, 4, 9, 11, 61). Mating type genesare also thought to regulate a wider series of genes together withlater stages of sexual development, but only a limited number ofinvestigations into such roles have been made for the Pezizomy-cotina, as reported by Pöggeler et al. (56), Klix et al. (34), andBidard et al. (3).

With respect to the aspergilli, mating type genes have beenidentified from a series of both known sexual and asexual speciesof Aspergillus (19, 49, 50, 51, 58, 59). Within the aspergilli, MATgenes were first shown to be functional in the homothallic speciesAspergillus nidulans, in which deletion of the resident MAT1 andMAT2 genes led to loss of sexual reproduction, while their over-expression suppressed vegetative growth and stimulated sexualdifferentiation under conditions normally unfavorable for sex(50). Subsequently, the MAT1-1 and MAT1-2 products of A. fu-migatus were also shown to be functional by their ability, through

Received 27 September 2011 Accepted 24 January 2012

Published ahead of print 10 February 2012

Address correspondence to Katsuhiko Kitamoto, akitamo@mail.ecc.u-tokyo.ac.jp.

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

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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heterologous complementation, to restore fertility to MAT delet-ant strains of A. nidulans, at a time when A. fumigatus was consid-ered asexual (22, 57). Following the discovery of a sexual cycle inA. fumigatus (47), the MAT1-1 and MAT1-2 genes were shown tobe required for sexual development in A. fumigatus, as evidencedby the sterility of MAT deletant strains (62). However, the geneticmechanisms by which mating type genes regulate sexual repro-duction in the aspergilli still remain largely unknown, although arecent study has provided insight into the regulatory mechanismscontrolling the expression of A. nidulans MAT2 (a synonym ofmatA) during the sexual cycle (10). In the aspergilli as a whole,more than 80 genes, including the MAT genes, have been shown tobe involved in the regulation of sexual reproduction (12, 14). Sig-nificantly, the recovery of isolates of complementary MAT1-1 andMAT1-2 identities (containing MAT1-1 and MAT1-2 genes, re-spectively, in a heterothallic arrangement) for the species A. fu-migatus, A. flavus, A. parasiticus, and A. nomius, previously con-sidered “asexual,” has led to the discovery of functional sexualcycles in all of these species as a result of the setup of laboratorycrosses between isolates of compatible mating types under appro-priate conditions (15, 25, 26, 27, 47).

Regarding A. oryzae, the reference strain RIB40, used for ge-nome sequencing, was found to contain a MAT1-1 �_HMG do-main gene, i.e., to be a MAT1-1 strain (19). It was not determined,however, whether complementary MAT1-2 strains required forheterothallic mating might exist in nature. In this study, we firstidentified a MAT1-2 strain of A. oryzae containing the comple-mentary MAT1-2 HMG gene at the MAT locus. Using a PCRdiagnostic assay, we then demonstrated the existence of bothMAT1-1 and MAT1-2 genotypes among a variety of reference andindustrial strains used in the manufacture of sake, soy sauce, andmiso. Finally, to explore the possible functionality of these matingtype genes, we constructed a MAT replacement strain in which theoriginal MAT1-1 gene was replaced by the MAT1-2 gene. Thisenabled us to use DNA microarray methodology to analyze pos-sible differential expression by genes of opposite mating typeswithin the same genetic background—the first such analysis ofMAT gene expression in a supposedly asexual filamentous asco-mycete fungus.

MATERIALS AND METHODSStrains and media. A. oryzae strains RIB40 (39) and AO6 (CBS108.24)(43) were used as DNA donors. Escherichia coli DH5� was used as the hostfor DNA manipulations. A. oryzae strain NSPlD1 (MAT1-1 niaD� sC�

adeA� �argB::adeA� �ligD::argB �pyrG::adeA) (42) was used as the hoststrain for replacement and disruption of MAT genes. For the initial mat-ing type analysis of A. oryzae, the industrial strains AO1 (RIB128),RIB177, RIB430, RIB609, and RIB647 from the NRIB (National ResearchInstitute of Brewing, Hiroshima, Japan) culture collection and strainsIAM2609, IAM2735, IAM2959, and IAM2960 from the IAM culture col-lection (Japan Collection of Microorganisms, RIKEN BioResource Cen-ter, Wako, Japan) were used. In addition, previously described A. oryzaereference strains AO1 (ATCC 22788), AO2 (IAM 13883), AO3 (CBS466.91), AO4 (ATCC 10196, ATCC 13791), AO5 (IFO 4278) (43), andAO8 from the University of Nottingham culture collection were used ininitial MAT screening. In subsequent experiments, 164 further strains oftane-koji (koji mold starters; isolate codes starting with KBN) used in themanufacture of sake, soy sauce, and miso (Bio’c Co., Ltd., Toyohashi,Japan) were also analyzed. The strains studied are summarized in TablesS1 and S2 in the supplemental material. DPY medium (2% dextrin, 1%polypeptone, 0.5% yeast extract, 0.5% KH2PO4, and 0.05% MgSO4 ·7H2O), potato dextrose (PD) medium supplemented with 0.5% uridine,

0.2% uracil, and 1.5% agar, and Aspergillus complete medium (ACM)(49) were used to grow the A. oryzae strains. M medium [0.2% NH4Cl,0.1% (NH4)2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05%MgSO4 · 7H2O, 0.002% FeSO4 · 7H2O, and 2% glucose (pH 5.5)] supple-mented with 0.5% uridine and 0.2% uracil was used as a selective mediumin A. oryzae transformation (all percentages are weight per volume). DNAextraction from A. oryzae isolates and transformation of A. oryzae wereachieved according to previously reported methods (33). The pyrGmarker gene, used in the replacement and disruption of MAT genes, wasexcised using PD medium supplemented with 1 mg/ml 5-fluorooroticacid (5-FOA), 0.5% uridine, 0.2% uracil, and 1.5% agar according to amethod described previously (42).

Identification of the MAT1-2 locus. A PCR fragment was amplifiedfrom genomic DNA of A. oryzae strain AO6 using the degenerate primerpair MAT5-4 and MAT3-2 according to the method of Seymour et al.(60). These primers were designed to amplify conserved HMG domain-encoding sequences within pezizomycete MAT1-2 genes (60). A gene walktoward both ends of the idiomorph was then initiated by using the GeneWalker kit from Stratagene (La Jolla, CA) according to the manufacturer’sinstructions. Gene-specific primer sequences are provided in Table S3 inthe supplemental material. The gene walk reached the end of the id-iomorph upstream of the MAT1-2 gene but not downstream. A fragmentcontaining the MAT1-2 gene and its flanking sequences was then ampli-fied from strain AO6 genomic DNA by PCR using Expand Long TemplateTaq DNA polymerase (Roche, Rotkreuz, Switzerland) and primersAOmat-2R and AOmat-2F, based on the conserved region sequencesof DNA lyase (gene identification [ID] AO080527000086) and Aoend4(gene ID AO080527000085) (http://nribf21.nrib.go.jp/CFGD/search.cgi?dspQO�1&prj�02201), respectively. The amplified fragmentwas sequenced to confirm its identity.

Southern blot analysis. After electrophoresis, the digested genomicDNAs were transferred to Hybond N� membranes (GE Healthcare, Pis-cataway, NJ). ECL (enhanced chemiluminescence) direct nucleic acid la-beling and detection (GE Healthcare) and a LAS-4000miniEPUV lumi-nescent image analyzer (Fuji Photo Film, Tokyo, Japan) were used fordetection. Probes were generated using the A. oryzae strain RIB40 genomeas a template, and the primers are listed in Table S3 in the supplementalmaterial.

PCR analysis of mating type genes. The mating type of isolates wasdetermined by a diagnostic PCR using primers MAT1-F and MAT1-R forthe MAT1-1 gene and primers MAT2-F and MAT2-R for the MAT1-2gene (see Table S3 in the supplemental material). PCR conditions in-volved the use of 20 ng genomic DNA in a 20-�l reaction volume with 5.0U Ex Taq polymerase (TaKaRa, Otsu, Japan), 0.5 �M primers, 0.2 mMdeoxynucleoside triphosphate (dNTP) mixture, and cycle parameters ofinitial denaturation at 94°C for 5 min, followed by 25 cycles of 94°C for 30s, 53°C for 30 s, and 72°C for 1 min before storage at 4°C (all at a ramp rateof 1.0 kb/min). MAT diagnostic PCR was performed using genomic DNAfrom 164 tane-koji strains as templates (see Table S2 in the supplementalmaterial). These strains were distinguished by several morphologicalcharacteristics, such as levels of conidiation, pigmentation and color, andenzyme productivity (data not shown). Also, the length of aerial hyphaewas assessed, since this can be an important consideration in choosingtane-koji strains for particular commercial processes. Given the numberof strains involved, a 5-point scale based on visual observation was used. Ascore of 1 indicated an aerial hypha length of �0.5 mm; 2, 0.5 mm to 1.5mm; 3, 1.5 mm to 2.5 mm; 4, 2.5 mm to 3.5 mm; 5, �3.5 mm (see TableS2). The two-tailed Mann-Whitney U test, available from the VassarStatswebsite (http://faculty.vassar.edu/lowry/VassarStats.html), was used tocompare possible differences in the length of aerial hyphae between testgroups.

Construction of plasmids for MAT gene replacement and disrup-tion. All plasmids used for A. oryzae transformation in this study wereconstructed by the MultiSite Gateway cloning system (Invitrogen, Carls-bad, CA). All primers used for constructing the plasmids and PCR prod-

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ucts described below are listed in Table S3 in the supplemental material.The 1.5-kb 3= end of the DNA lyase gene in RIB40 was amplified by PCRusing primers aB4�lyase-2.5kF and MAT2�lyase-R, while the MAT1-2locus in AO6 was amplified using primers lyase�MAT2dF andaB1�MAT2uR. Those two DNA fragments were united by fusion PCRaccording to previously described methods (29, 65) using primersaB4�lyase-2.5kF and aB1�MAT2uR. The 0.3-kb 3= end of the MAT1-2locus in AO6 was amplified by PCR using primers aB2�MAT2uF andfla�MAT2uR, while a 0.2-kb downstream region flanking the Aoend4(SLA2) gene in RIB40 was amplified by PCR using primers MAT2�fla-Fand aB3�SLA-0.1kR. Those two DNA fragments were united by fusionPCR using primers aB2�MAT2uF and aB3�SLA-0.1kR. The former andlatter fusion PCR products were cloned by a BP Clonase reaction into thepDONR P4-P1R and pDONR P2R-P3 entry vectors, respectively. Theresultant 5= and 3= entry clones and the center entry clone, pgEpG (con-taining the pyrG marker gene) (42), were subjected to an LR Clonasereaction in the presence of pDEST R4-R3 (the destination vector) to pro-duce plasmid pgpM2. The replacement fragment for the MAT1-1 genewas amplified by PCR using plasmid pgpM2 as a template and primersaB4�lyase-2.5kF and aB3�SLA-0.1kR. The 1.5-kb 5= region upstream ofthe MAT1-1 gene was amplified by PCR using primers aB4-5MAT1-1_Fand aB1r-5MAT1-1_R. The 0.3-kb 5= upstream region of the MAT1-1gene was amplified by PCR using primers aB2r-MAT1-1_F and f-MAT1-1�p_R, while the 1.5-kb 3= downstream region of the MAT1-1 gene wasamplified by PCR using primers f-MAT1-1�p_F and aB3-MAT1-1_R.Those two DNA fragments were united by fusion PCR using primersaB2r-MAT1-1_F and aB3-MAT1-1_R. The 1.5-kb 5= upstream region ofthe MAT1-1 gene and the fusion PCR product were cloned by a BP Clo-nase reaction into the pDONR P4-P1R and pDONR P2R-P3 entry vec-tors, respectively. The resultant 5= and 3= entry clones and the center entryclone pgEpG were subjected to the LR Clonase reaction in the presence ofpDEST R4-R3 to produce plasmid pgdM1pG. The disruption fragmentfor the MAT1-1 gene was amplified by PCR using plasmid pgdM1pG as atemplate and primers aB4-5MAT1-1_F and aB3-MAT1-1_R.

RNA isolation and qRT-PCR analysis. Approximately 2 � 106

conidia were inoculated into 100 ml DPY liquid medium supplementedwith 0.5% uridine and 0.2% uracil. After 24 h of growth, 200 mg of my-celia was shifted onto DPY agar medium covered with a cellulose acetatemembrane (Membrane Filter; Advantec Toyo, Tokyo, Japan) and wasfurther cultivated for 48 h. Total RNA was extracted as described previ-ously (32), followed by treatment with DNase I (TaKaRa, Otsu, Japan).First-strand cDNAs were synthesized with ReverTra Ace reverse trans-criptase (Toyobo, Osaka, Japan) using oligo(dT)12–18 (Invitrogen) as theprimer. Quantitative real-time PCRs (qRT-PCRs) were performed using aLightCycler FastStart DNA Master SYBR green I system (Roche Diagnos-tics, Indianapolis, IN) as instructed by the manufacturer. The primersused in this study are listed in Table S3 in the supplemental material.Transcript levels were quantified by qRT-PCR, and the means from threeindependent experiments were taken. Transcript levels were normalizedagainst actin as the internal standard.

DNA microarray analysis. Total RNA was extracted from MAT1-1(NSPlD1) and MAT1-2 (NSPlD-M2) strains as described above using theRNeasy Plant minikit (Qiagen Sciences, Germantown, MD) and was dis-solved in RNase-free water. DNA microarray experiments were conductedaccording to the Affymetrix GeneChip manual with an A. oryzae DNAchip(Affymetrix, Madison, WI) containing probe sets for 13,857 genes and 1,704expressed sequence tags (ESTs) from A. oryzae strain RIB40 (48). The geneIDs used were those registered in the Comparative Fungal Genome Database(http://nribf21.nrib.go.jp/CFGD/gnm.cgi?prj�02201&gnm�aor01).The ESTs selected for the DNA microarray do not overlap with the genesregistered in the genome database (Aspergillus oryzae EST DataBase [http://nribf21.nrib.go.jp/EST2/index.html]). Briefly, one cycle of cDNA syn-thesis was performed, and then biotin-labeled cRNA was amplified, fol-lowed by fragmentation. The fragmented cRNA was hybridized with the25-mer oligonucleotide probe sets, each containing 16 probe pairs con-

sisting of a perfect-match probe (PM) and a mismatch probe (MM). Afterwashing and staining, the probes were scanned with a GeneChip Scanner3000 (Affymetrix). Microarray data were normalized using intensity anddetection P values. The trimmed mean signal of the array was scaled to thetarget signal of 500 with the “all probe sets” scaling option. Statisticalanalysis for signal intensities and “detection P values” was performed withthe algorithm of GeneChip Operating Software (GCOS), version 1.4 (Af-fymetrix). Detection of the transcripts was assessed by the “detection call”(P, present; A, absent; M, marginal) on the basis of detection P values. Thearray data of the MAT1-1 and MAT1-2 strains were compared with eachother. The comparative analysis was performed for each strain by GCOS,which calculates the “signal log ratio” and “change P values” to determinethe “change call” (I, increasing; MI, marginally increasing; NC, no change;MD, marginally decreasing; D, decreasing). For each strain, genes with adetection call of “P” (expression detected) and a change call of “I” wereselected as upregulated genes. A detection call P value of �0.001111 wasregarded as indicating an increase (I); a P value of �0.001111 and�0.001333 or of �0.998667 and �0.998889 indicated marginal (M) re-sults; a P value of �0.001333 and �0.998667 indicated no change (NC);and a P value of �0.998889 indicated a decrease (D). Fold changes wereconverted from the signal log ratio for each strain by using MicrosoftExcel, and for the purposes of the present study, we investigated further allgenes showing at least a 10-fold difference in expression between MAT1-1and MAT1-2 strains as those subject to possible MAT regulation (seeTables 2 and 3). A detection P value of �0.04 was regarded as indicatingthe presence (P) of a gene; a P value of �0.04 and �0.06 was considered toindicate a marginal (M) result; and a P value of �0.06 was considered toindicate the absence (A) of a gene. Upregulated genes with detection callsof “A” in both strains were omitted.

Microarray data accession number. Our DNA microarray data weresubmitted to the Gene Expression Omnibus public database (http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE33542.

RESULTSIdentification of a MAT1-2 gene in A. oryzae. PCR using degen-erate primers MAT5-4 and MAT3-2, designed to anneal to a se-quence in the conserved HMG box of fungal MAT1-2 genes (60),led to the amplification of an approximately 300 bp fragment fromA. oryzae strain AO6 genomic DNA but not from strain RIB40genomic DNA harboring the MAT1-1 idiomorph. Sequencing ofthis 300-bp fragment confirmed homology to known MAT1-2HMG genes (results not shown). A gene walk that reached the endof the idiomorph upstream of the MAT1-2 gene was thereforeinitiated, but the kit failed to yield any products downstream ofthe MAT1-2 gene. Given that gene synteny is fairly well conservedaround the MAT locus in the aspergilli and many pezizomycetefungi (11, 12), primers AOmat-2R and AOmat-2F, located in theflanking DNA lyase region, predicted to be present downstream ofthe MAT1-2 gene (12), and in the upstream Aoend4 (SLA2) re-gion, respectively, were designed. This primer pair successfullyamplified the whole MAT1-2 idiomorph along with some con-served flanking sequences. A schematic diagram of the MAT lociof strains RIB40 and AO6 is shown in Fig. 1A. The locations of thetwo mating type genes at the MAT locus in A. oryzae are similar tothose in other heterothallic aspergilli (12, 19, 49, 58). The pre-dicted MAT1-2 gene sequence (1,069 bp, including two introns)was contained within a 3,276-bp MAT1-2 idiomorph, comparedto a 3,130-bp MAT1-1 idiomorph (including the 1,168-bpMAT1-1 gene) located between the DNA lyase and Aoend4 (SLA2)genes (DDBJ accession number AB617942). An amino acid se-quence alignment of MAT1-2 proteins from representative asper-gilli is shown in Fig. 1B. The predicted A. oryzae MAT1-2 proteinconsists of 321 amino acids and contains a MATA_HMG domain,

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as conserved in other fungal MAT1-2 proteins. The amino acidsequence shows 100%, 63.0%, and 51.3% identity with theMAT1-2 mating type proteins of A. flavus, A. fumigatus, and A.nidulans, respectively. Sequencing of a limited number of 8 iso-lates, 4 from each mating type, suggested the presence of somefixed polymorphic differences within the flanking sequences (upto 1 kb away) between MAT1-1 and MAT1-2 isolates, which couldindicate either a lack of sexual reproduction or the suppression ofrecombination at/around the MAT locus. However, sequencing ofa higher number of isolates is required to confirm this observa-tion.

Distribution of mating types in industrial A. oryzae strains.Using the DNA fragments of MAT1-1 and MAT1-2 genes from A.oryzae strains RIB40 and AO6, respectively, as probes, Southernblot analysis was carried out to identify the mating types of 11industrial A. oryzae strains from the RIB and IAM culture collec-tions (see Table S1 in the supplemental material). As shown in Fig.2, a single MAT locus was detected, and four of five strains used forsake production (AO1 [RIB128], RIB177, RIB430, RIB609, andRIB647) were identified as MAT1-1 strains, while IAM2959, usedfor soy sauce production, was identified as a MAT1-2 strain. Thenucleotide sequences of the MAT1-1 and MAT1-2 open readingframes (ORFs) from these strains were 100% identical to those ofstrains RIB40 and AO6, respectively (data not shown). Slight dif-

ferences in the sizes of bands were observed (Fig. 2), presumablydue to sequence divergence in the MAT locus region outside theMAT ORFs themselves. In parallel, initial screenings using themating type diagnostic PCR (employing primers designed to am-plify sequences from either MAT gene) determined that three ofthe reference strains belonged to the MAT1-1 genotype (AO1,AO2, and AO5) and three belonged to the MAT1-2 genotype(AO3, AO4, and AO8).

In order to examine whether the mating type differs accordingto the use of the source strains for the production of sake, soysauce, or miso, we further analyzed the distribution of matingtypes in 164 strains from tane-koji (koji mold starter) strains uti-lizing the MAT diagnostic PCR test. Isolates of the MAT1-1 andMAT1-2 genotypes were found to be present among strains usedfor the production of sake, soy sauce, and miso (Table 1). Strainsused for sake and miso production showed a near-1:1 ratio ofmating types, with no statistically significant bias (P, 0.20 and 0.43,respectively) from a balanced ratio. In contrast, tane-koji strainsused for soy sauce showed a statistically significant dominance ofthe MAT1-2 mating type (P, 0.0019). Although the overall distri-bution of the two mating types in the tane-koji strains was close toa 1:1 ratio, there was a slight bias toward the MAT1-2 mating type(Table 1). When the height of aerial hyphae was scored on a5-point scale (from a score of 1 for the shortest to 5 for the longest

FIG 1 Mating type genes of Aspergillus oryzae. (A) Schematic arrangements of the MAT loci in A. oryzae strains RIB40 and AO6. Arrows represent putative ORFsin regions adjoining the MAT loci. (B) Amino acid sequence alignment of MAT1-2 proteins from A. oryzae, A. flavus, A. fumigatus, and A. nidulans. The conservedHMG domain is boxed. Arrowheads indicate the locations of conserved introns.

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[see Materials and Methods]), the aerial hyphae of MAT1-2 mat-ing type strains were significantly (P � 0.005) shorter thanthose of MAT1-1 mating type strains on average (2.38 0.11 and2.96 0.13, respectively; see Table S2 in the supplemental mate-rial). These data may be correlated with the fact that strains withshorter aerial hyphae had been selected for manufacturing soysauce.

Generation of MAT1-2 and �MAT strains. In order to exam-ine the function of mating type genes, and especially for the detec-tion of MAT-specific gene expression by use of DNA microarrayand qRT-PCR analyses, it is very important to generate strains ofopposite mating types with the same genetic background that dif-fer only in their MAT loci. Hence, we generated a mating typereplacement strain in which the original MAT1-1 gene was re-placed with a MAT1-2 gene. The mating type genes were replacedby marker recycling techniques (42). A DNA fragment containingthe MAT1-2 gene from A. oryzae strain AO6 and a pyrG markergene was introduced into A. oryzae strain NSPlD1 (referred tobelow as the MAT1-1 strain), derived from strain RIB40 by ho-mologous recombination (Fig. 3A). The pyrG marker gene wasjoined to direct repeats of a 0.3-kb region from strain AO6, whichwas located at the end of the MAT1-2-specific region. Integrationof the MAT1-2 gene with the pyrG marker gene into the MATlocus of the resultant strain NSlD-pM2 was verified by Southernblot analysis (Fig. 3B). Subsequently, conidia of the NSlD-pM2strain were spread onto PD agar medium containing 5-FOA, uri-dine, and uracil for positive selection of strains in which the pyrG

FIG 2 Existence of both the MAT1-1 and MAT1-2 mating types in industrial A. oryzae strains from the RIB and IAM culture collections. (A) Construct forSouthern blot analysis. (B) Mating type identification by Southern blot analysis. DNA fragments from the MAT1-1 and MAT1-2 genes were used as probes.MAT1-1 strains were RIB40, AO1 (RIB128), RIB177, RIB430, RIB609, and IAM2609. MAT1-2 strains were AO6, IAM2735, IAM2959, IAM2960, and RIB647.

TABLE 1 Distribution of mating types in A. oryzae tane-koji strains asdetermined by mating type diagnostic PCR

Strain group

% (no.) of strains ofmating type:

�2 (df)Contingency�2 (df)MAT1-1 MAT1-2

All tane-koji strains 40.6 (67) 59.4 (97) 5.49a (1)Sake 43.2 (38) 56.8 (50) 1.64 (1)Soy sauce 27.5 (14) 72.5 (36) 9.68b (1)Miso 57.7 (15) 42.3 (11) 0.62 (1) 7.05a (2)a Significant (P � 0.05).b Significant (P � 0.01).

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marker gene was excised by homologous recombination with thedirect repeats (Fig. 3A). Excision of the pyrG marker gene in the mat-ing type replacement strain NSPlD-M2 (referred to below as theMAT1-2 strain) was verified by Southern blot analysis (Fig. 3C).

In order to clearly define mating type-specific gene expression,we also generated a control strain in which the MAT1-1 gene hadbeen disrupted (referred to below as the �MAT strain). A disrup-tion fragment containing the 1.5-kb upstream and downstreamregions of the MAT1-1 ORF and the pyrG marker gene was intro-

duced into strain NSPlD1 (see Fig. S1A in the supplemental ma-terial), and its presence was verified in the �MAT1-1::pyrG strain(NSlD-pdM1) by Southern blot analysis (see Fig. S1B). By positiveselection using a medium containing 5-FOA, the �MAT strain(NSPlD-dM1) was obtained, and excision of the pyrG markergene was verified by Southern blot analysis (Fig. S1C).

No apparent phenotypic differences in growth and conidiation(on DPY, PD, and M agar media, each supplemented with 0.5%uridine and 0.2% uracil, at 30°C for 5 days) were observed be-

FIG 3 Replacement of the MAT1-1 gene. (A) Strategy for replacement of the MAT1-1 gene with MAT1-2 by pyrG marker recycling. pyrG was excised byhomologous recombination with direct repeats of the 0.3-kb upstream region of the MAT1-2 gene. (B) Verification of MAT gene replacement by Southern blotanalysis. DNA fragments from MAT1-1 and MAT1-2 were used as probes. (C) Southern blot analysis of the strain from which pyrG had been excised. A DNAfragment from the MAT1-2 gene was used as a probe.

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tween the MAT1-1 (NSPlD1), MAT1-2 (NSPlD-M2), and �MATstrains (data not shown). Measurement of the height of aerialhyphae showed no significant difference, suggesting that this phe-notype was not linked to MAT1-2 expression (data not shown).

Analysis of the MAT1-1 and MAT1-2 strains for differentialgene expression. To analyze possible differential gene expressionthat is dependent on the mating type in A. oryzae, DNA microar-ray analysis was performed using the MAT1-1 (NSPlD1) andMAT1-2 (NSPlD-M2) strains. First, we determined the cultureconditions under which the A. oryzae strains expressed matingtype genes. When analyzed by semiquantitative RT-PCR, expres-sion of the MAT1-1 and MAT1-2 genes could not be observedwhen strains were grown for 24 h or 48 h in DPY liquid medium(data not shown), but the “culture shift” method, in which themycelia were transferred to an agar medium, allowed us to detectMAT gene expression by both semiquantitative RT-PCR andqRT-PCR (data not shown). DNA microarray analyses weretherefore carried out using RNA extracted from cultures subjectedto the same conditions.

The resulting microarray analysis revealed a series of 1,155 genesthat were upregulated �2-fold relative to each other in either theMAT1-1 (NSPlD1) or the MAT1-2 (NSPlD-M2) strain (596 geneswere upregulated for MAT1-1 and 559 genes for MAT1-2). Fullresults are available from the Gene Expression Omnibus publicdatabase (http://www.ncbi.nlm.nih.gov/geo/), accession numberGSE33542. For the purposes of this study, particular attention wasgiven to those genes showing �10-fold upregulation, to reduce therisks of indirect changes in expression. Using these criteria, a total of

33 genes (listed in Tables 2 and 3) were identified. This marked dif-ference in expression revealed that both the MAT1-1 and MAT1-2genes are functional in regulating gene expression in A. oryzae. A.oryzae has genes encoding an �-pheromone precursor (AoppgA[AO080503000008]) and putative �- and a-pheromone receptors(AogprA [AO080550000138] and AogprB [AO080551000023], re-spectively). Among 24 genes upregulated in the MAT1-1 strain (Ta-ble 2), the mating type-specific gene AoppgA showed remarkable up-regulation (39.4-fold) compared to the MAT1-2 strain, but mostgenes encoded predicted proteins of unknown function according tothe annotation. The results from the microarray analysis of AoppgAexpression were verified by qRT-PCR experiments. Transcripts of theputative �-pheromone precursor gene AoppgA were indeed moreabundantly detected in the MAT1-1 strain than in the MAT1-2 and�MAT (NSPlD-dM1) strains (Fig. 4), although the differences inexpression were not as marked as those detected by microarray anal-ysis (by qRT-PCR analysis, AoppgA transcript levels were about 5times greater in the MAT1-1 strain than in the MAT1-2 strain), pos-sibly due to overlap in the probes used, resulting in higher microarraysignals. In contrast, no significant differences in the transcript levels ofputative pheromone receptor genes were observed between theMAT1-1 and MAT1-2 strains by DNA microarray analysis (data notshown). This is consistent with previous reports of analyses of pher-omone receptor gene expression, which did not show differential ex-pression in isolates of other heterothallic Pezizomycotina with differ-ent mating types (34, 49, 62).

Meanwhile, 9 genes were markedly (�10-fold) upregulated inthe MAT1-2 (NSPlD-M2) strain (Table 3). These included twogenes (AO080505000070 and AoEST5207) showing very highrises in expression (85- and 64-fold, respectively). However, thesetwo genes were predicted to encode proteins of unknown func-tion, as were all but one of the other genes (Table 3). It should benoted that no a-pheromone precursor gene has been identified yetin the aspergilli, most likely due to sequence divergence, becausethe a-factor pheromone sequence is poorly conserved in the Pe-zizomycotina (12, 14, 54). The results of microarray expressionexperiments were verified by qRT-PCR analysis for theAO080505000070 and AoEST5207 genes, which confirmed signif-icant upregulation of these genes in the MAT1-2 strain comparedto the MAT1-1 and �MAT (NSPlD-dM1) strains (Fig. 4).

DISCUSSION

Mating type (MAT) genes have been shown to be key regulators ofsexual identity throughout the fungal kingdom and appear to be a

TABLE 2 Genes upregulated in the MAT1-1 mating type strain basedon DNA microarray analysis

Gene IDFold change(MAT1-1/MAT1-2) Proteina

AO080503000328 55.7 Acyl-CoA dehydrogenasesAO080508000390 55.7 Predicted proteinAO080503000008 39.4 AoPpgA, putative �-pheromone

precursorAO080508000389 27.9 Predicted proteinAO080541000465 26.0 Predicted proteinAO080523000564 24.3 Predicted proteinAO080511000446 21.1 Probable taurine catabolism

dioxygenaseAO080521000309 19.7 Predicted proteinAO080502000077 18.4 Predicted proteinAO080521000174 18.4 NptB, neutral protease NPIIAO080564000028 18.4 NADP FAD-dependent

oxidoreductaseAO080523000782 16.0 Predicted proteinAoEST3991 16.0 Predicted proteinAO080501000145 14.9 Predicted proteinAO080513000200 14.9 Predicted proteinAO080541000466 14.9 Predicted proteinAO080531000014 13.9 Cytochrome P450AoEST3853 13.9 Predicted proteinAO080508000337 13.0 Predicted proteinAO080523000711 13.0 Cytochrome P450AO080541000488 13.0 Predicted proteinAO080521000051 12.1 Predicted proteinAO080523000026 11.3 ChitosanaseAO070333000246 10.6 Predicted proteina Acyl-CoA, acyl coenzyme A; FAD, flavin adenine dinucleotide.

TABLE 3 Genes upregulated in the MAT1-2 mating type strain basedon DNA microarray analysis

Gene IDFold change(MAT1-2/MAT1-1) Protein

AO080505000070 84.4 Predicted proteinAoEST5207 64.0 Predicted proteinAO080515000084 18.4 Predicted proteinAO080532000139 18.4 Vesicle coat complex COPII,

subunit Sec31AoEST6582 17.1 Predicted proteinAO080523000595 14.9 Predicted proteinAO080508000372 14.9 Predicted proteinAO080511000543 11.3 Predicted proteinAO080553000049 11.3 Predicted protein

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prerequisite for sexual reproduction (36). Therefore, many stud-ies assessing the asexual nature of particular fungal species havefirst investigated the presence of MAT genes as a possible indica-tion of cryptic sexuality, and MAT loci have been detected in aseries of supposedly asexual fungi (35). It should be noted, none-theless, that the presence of mating type genes alone does notnecessarily confer the ability to undergo sexual reproduction;many other genes are also required for mating, fruiting body de-velopment, and meiosis. Indeed, at least 75 genes have been shownto be involved in sexual reproduction in pezizomycete fungi suchas the aspergilli (14).

In the present study, we therefore investigated the possiblepresence and functionality of MAT genes in A. oryzae as part oflong-term studies aiming to induce sexuality in this supposedlyasexual species. The results were 3-fold. First, we were able toidentify an isolate, AO6, containing a MAT1-2 gene encoding aMATA_HMG domain protein that was present at the same id-iomorphic MAT locus as the MAT1-1 gene, previously identifiedby genome sequencing of A. oryzae strain RIB40, which encodes aMAT�_HMG domain protein (19, 39, 41). Both the MAT1-1 andMAT1-2 genes appeared to encode putative functional proteinswith no frameshift or deletion mutations, in contrast to, for ex-ample, the asexual species Candida parapsilosis, in which a defec-tive MAT allele is present (38). The MAT genes had 100% se-quence identity to those of the known sexual species Aspergillus

(Petromyces) flavus (25), which is very closely related to A. oryzaeaccording to phylogenetic analysis (43, 52). This first result is sig-nificant because it demonstrates that A. oryzae has the character-istic MAT locus organization of known sexual heterothallic pe-zizomycete species, an important indication of potential for sexualreproduction (15, 16).

Second, we developed a MAT diagnostic PCR assay and used itto determine the mating types of �180 reference and industrialstrains of A. oryzae, including tane-koji strains used in the manu-facture of sake, soy sauce, and miso. The overall results showed thepresence of the MAT1-1 and MAT1-2 genotypes in a near-1:1distribution, again consistent with either current potential for sex-ual reproduction or evidence of historic sexuality, or both (15, 16),although a slight bias toward the MAT1-2 genotype was observedin strains used for soy sauce production. MAT1-2 strains overallexhibited shorter aerial hyphae, although this is likely to be anartifact of the preponderance of the MAT1-2 mating type amongsoy sauce production strains, in which shorter aerial hyphae arefavored. This morphological feature avoids the formation ofhighly entangled masses of hyphae, which can be inconvenient,since they may lower enzyme activity due to increases in temper-ature and moisture levels and may pose an increased risk of con-tamination by other microorganism, such as Bacillus species. Wecaution that some of the A. oryzae isolates might have been clonal;therefore, the total number of independent isolates assayed may in

FIG 4 Analysis of gene expression in the MAT1-1, MAT1-2, and �MAT strains by qRT-PCR. qRT-PCR was performed for the AoppgA gene, putatively involvedin the mating process, and for genes upregulated in the MAT1-2 strain (AO080505000070 and AoEST5207) to confirm the changes in gene expression detectedby DNA microarray experiments. The actin transcript levels were used as internal standards, and the transcript levels for MAT1-1 were set at 100%. The datashown are means standard errors from three independent experiments.

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reality have been smaller. However, almost all isolates were ob-tained from different sources and exhibited morphological varia-tion, and in addition, some were proven to be genetically distinctby amplified fragment length polymorphism (AFLP) fingerprint-ing (43). Thus, it is considered likely that the majority of isolateswere nonclonal. It is noteworthy that the tane-koji starter culturessold to brewing companies usually consist of strains that aremixed beforehand, and it is therefore possible that strains of op-posite mating types might meet under industrial growth condi-tions. There have been similar reports of the presence of bothMAT1-1 and MAT1-2 isolates in populations of other “asexual”pezizomycete fungi, including Alternaria species (2), Aspergillusspecies (49, 58), Rhynchosporium species (37, 66), Cercospora spe-cies (21), Coccidioides species (18, 40), and Penicillium chrysoge-num (23, 24). The only exception has been the asexual speciesAcremonium chrysogenum, used to produce cephalosporin, forwhich only the MAT1-1 genotype could be found in a preliminarysurvey (55).

Third, and finally, we investigated whether the MAT genesidentified in A. oryzae might prove to be functionally active inregulating gene expression. Using a MAT1-1 strain as the host, anisogenic strain was created in which the MAT1-1 idiomorph wasreplaced with the newly identified MAT1-2 idiomorph, includingthe MAT1-2 gene encoding a MATA_HMG domain protein.Comparisons in gene expression were then made between thesetwo strains by use of DNA microarray and qRT-PCR methodolo-gies under growth conditions under which the MAT genes wereknown to be expressed. It was found by microarray analysis that1,155 genes were upregulated �2-fold in either the MAT1-1 hoststrain or the MAT1-2 gene replacement strain relative to eachother. Of these, 33 genes were found to be upregulated more than10-fold in either the MAT1-1 host strain or the MAT1-2 genereplacement strain, and upregulation was confirmed for certaingenes by qRT-PCR. This showed that both the MAT1-1 andMAT1-2 genes are functional in regulating gene expression in A.oryzae, although our experimental design did not distinguish be-tween direct and indirect transcriptional activation of these genes.Furthermore, the MAT genes acted in a mating type-dependentmanner, suggesting that A. oryzae MAT genes potentially act assexual determinants. Consistent with this hypothesis, MAT1-1specifically upregulated the putative �-pheromone precursorgene AoppgA, in agreement with reports of MAT genes differen-tially regulating pheromone production in other heterothallic pe-zizomycete fungi (9, 11). However, most of the other genes up-regulated in either the MAT1-1 or the MAT1-2 strain were ofunknown function, so their role(s) in determining sexual identityand regulating sexual development remains to be investigated.Those showing major upregulation according to the microarrayanalysis (e.g., the 4 genes with �50-fold differences in expression[Tables 2 and 3]) are particularly interesting subjects for futurestudy. Despite the anticipation that an a-pheromone precursorgene would be upregulated in the MAT1-2 strain, we failed to findany candidate genes encoding a C-terminal CAAX motif thatmight be part of an a-pheromone precursor (6, 7, 9). Thus, thesearch for the elusive putative a-pheromone precursor gene in theaspergilli continues (14).

Parallel studies using microarrays and RT-PCR to determinegenes regulated by MAT expression have been reported from pe-zizomycete fungi with known sexual cycles. Pöggeler et al. (56)found that more than 100 genes were differentially regulated in a

�Smta-1 mating type mutant strain of the homothallic speciesSordaria macrospora. These included a pheromone precursor geneand elements of signal transduction pathways, with a cutoff valueof �2-fold selected for significance. In subsequent experimentalwork by Klix et al. (34), additional �SmtA-1 and �SmtA-2 matingtype mutants of S. macrospora were created, and microarray anal-yses showed altered expression of �900 genes, including phero-mone precursor genes, with a �2-fold level again chosen for sig-nificance. Meanwhile, Bidard et al. (3) recently reported agenomewide analysis of the effects of MAT gene expression in theheterothallic species Podospora anserina. They found that 157genes were differentially transcribed in isolates of different matingtypes (�2-fold), including known sex-related genes for produc-tion of pheromone precursors and receptors, and for enzymaticprocessing of propheromones. However, to our knowledge, thepresent study is the first to use DNA microarray analysis to exam-ine differential gene expression that is dependent on mating typegenes in a supposedly asexual filamentous ascomycete.

Our data, together with the discovery of a complement of genesthat are likely to be required for sexual reproduction in the ge-nome of A. oryzae (19), are good indicators of the potential forsexual reproduction in A. oryzae. We have also observed the ex-pression of pheromone precursor and receptor genes, and ele-ments of a pheromone response mitogen-activated protein(MAP) kinase signal transduction pathway, in two MAT1-1(AO46 and AO10) and two MAT1-2 (AO6 and AO8) A. oryzaeisolates of complementary mating types (M. Paoletti, E. U.Schwier, and P. S. Dyer, unpublished data). In addition, there isevidence of previous sexual reproduction by A. oryzae based onanalysis of repeat-induced point mutations (RIP) in the genome(44), as also observed for the asexual species Aspergillus niger andPenicillium chrysogenum (5). Furthermore, sexual cycles have re-cently been detected in both A. flavus and A. parasiticus (25, 27),which are phylogenetically very closely related to A. oryzae (43,52), and it has been suggested that A. flavus is an ancestor of A.oryzae (20). As a result, mating experiments are now being startedusing the congenic MAT1-1 and MAT1-2 strains constructed inthe present study.

However, a major obstacle remains to be overcome before asexual cycle might be induced in A. oryzae. Sexual reproduction inAspergillus section Flavi (teleomorph genus, Petromyces), whichincludes A. oryzae, A. flavus, and A. parasiticus, requires the for-mation of sclerotia, spherical resistance structures formed for sur-vival under adverse conditions (14). One of the key differencesbetween A. flavus and A. oryzae is that most strains of A. oryzaehave lost the ability to form sclerotia or have a much lower abilitythan A. flavus (45), perhaps as a result of domestication. There-fore, it will be necessary to reliably induce the formation of scle-rotia in A. oryzae before sexuality may be realized. This mightinvolve the manipulation of culture conditions, given that sclero-tial formation is dependent on environmental conditions (14) andgiven the exacting demands of some aspergilli for sexual repro-duction (14, 47), and/or the use of genetic manipulation tech-niques to induce sclerotial formation. At present, only one A.oryzae gene, encoding the bHLH transcription factor SclR (forsclerotium regulator), involved in the production of sclerotia hasbeen identified (14, 30, 31). Deletion of sclR in certain strainsobserved to form sclerotia was found to result in sparse sclerotialproduction, while overexpression of sclR led to �5-fold produc-tion of sclerotia with increased formation of branched aerial hy-

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phae (31). In related work, deletion of a zinc finger calcineurinresponse gene, crzA, was found to result in the production ofmainly immature sclerotia in A. parasiticus (8), so a homologmight also be required for the maturation of sclerotia in A. oryzae.Genes linked to sclerotial formation have also been identified inthe pezizomycete fungi Botrytis cinerea and Sclerotinia sclerotio-rum; these might provide insight for studies with the aspergilli (1).

Within an industrial setting, sexual reproduction has beenused to breed strains with desirable characteristics, to generatenovel variation allowing selection for significantly increased me-tabolite production, to restore the vigor of impaired strains, and toinvestigate the genetic basis of traits of interest. Specific examplesinclude selection for flocculation in Saccharomyces cerevisiae, in-creased alkaloid production in Claviceps purpurea, improved ther-motolerance in Pleurotus species, and investigation of resistance tofungicides in Tapesia yallundae (13, 17, 53). It is hoped that futureresearch with A. oryzae might lead to a “sexual revolution” in thisspecies, as seen for other aspergilli previously assumed to be asex-ual (15, 47), thus providing an important new method for strainimprovement in this widely utilized industrial fungus.

ACKNOWLEDGMENTS

M.P., D.B.A., and P.S.D. thank the Biotechnology and Biological SciencesResearch Council (United Kingdom) for providing funding to supportthis research. J.M. thanks the Japan Society for the Promotion of Sciencefor providing funding for Grant-in-Aid Challenging Exploratory Re-search.

M.P., D.B.A., and P.S.D. thank Elke Schwier for experimental assis-tance with preliminary screening work.

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