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Fungal Genetics and Biology 45 (2008) 878889 www.elsevier.com/locate/yfgbi

A defect of LigD (human Lig4 homolog) for nonhomologous end joining signicantly improves eciency of gene-targeting in Aspergillus oryzae
Osamu Mizutani a,1,2, Youhei Kudo a,1, Akemi Saito a, Tomomi Matsuura a, Hirokazu Inoue b, Keietsu Abe c, Katsuya Gomi a,*
Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan b Laboratory of Genetics, Department of Regulation Biology, Faculty of Science, Saitama University, Shimo-okubo 255, Sakura-ku, Saitama 338-8570, Japan c Laboratory of Enzymology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan Received 10 October 2007; accepted 27 December 2007 Available online 11 January 2008
a

Abstract Gene-targeting by homologous recombination occurs rarely during transformation since nonhomologous recombination is predominant in Aspergillus oryzae. To develop a highly ecient gene-targeting system for A. oryzae, we constructed disrupted strains harboring a gene (ligD) encoding human DNA ligase IV homolog that is involved in the nal step of DNA nonhomologous end joining. The A. oryzae ligD disruptants showed no apparent defect in vegetative growth and/or conidiation, and exhibited increased sensitivity to high concentration of methyl methansulfonate causing double-stranded DNA breaks compared with that of wild-type strain, but not to ethyl methanesulfonate and phleomycin. Gene replacement of the prtR, a gene encoding a transcription factor which regulates extracellular proteolytic genes, using the Aspergillus nidulans sC gene as the selectable marker resulted in 100% of gene-targeting eciency in the ligD disruptant, compared to less than 30% for a wild-type, when the length of the homologous anking sequences used was longer than 0.5 kb. Similarly, gene-targeting eciency was as high as 100% for aspartic protease-encoding gene (pepA). Furthermore, using this ligD disruptant system of A. oryzae, we readily succeeded in disrupting ve mitogen-activated protein kinase (MAPK) genes, namely mpkA, mpkB, hogA, mpkC and A. oryzae unique MAPK (mpkD). Such results show that the ligD disruptant system is an extremely convenient genetic background for gene-targeting in A. oryzae. 2008 Elsevier Inc. All rights reserved.
Keywords: Aspergillus oryzae; DNA ligase IV (ligD); Gene-targeting; Homologous recombination; Mitogen-activated protein kinase (MAPK)

1. Introduction In all living organisms from archaebacteria to plants and mammals, two major recombination pathways have been identied for the repair of DNA Double-Strand
Corresponding author. Fax: +81 22 717 8902. E-mail address: gomi@biochem.tohoku.ac.jp (K. Gomi). 1 These authors contributed equally to this work. 2 Present address: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan. 1087-1845/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2007.12.010
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Breaks (DSBs) which are induced by endogenous and exogenous causes and they are the most detrimental DNA lesions (Haber, 2000). These pathways dier as to whether they require DNA sequence homology and distinct sets of protein factors (Wang et al., 2003). Homologous recombination (HR) utilizes intact genetic information from an undamaged homologous region as a template for repair of DSBs, whereas nonhomologous end-joining (NHEJ) rejoins DSBs by direct ligation of the strand ends without any requirement for sequence homology. These repair mechanisms have been conserved throughout evolution

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and operate in a wide range of organisms. Also in fungi, HR and NHEJ pathways require various proteins, for example, Rad51 epistasis group in the HR and Ku protein and DNA ligase IV in the NHEJ (see reviews Daley et al., 2005; Krappmann, 2007). Saccharomyces cerevisiae utilizes primarily the HR pathway in DSB repair and exogenous DNA fragments are mainly integrated at homologous sites in the genome, while most lamentous fungi preferentially repair DSBs by NHEJ and exogenous DNA can be integrated at ectopic sites in the genome. Therefore, gene-targeting-mediated through HR can be readily accomplished in S. cerevisiae (Schiestl et al., 1994), but the eciency of gene-targeting tends to be limiting in most lamentous fungi. However, Ninomiya et al (2004) have recently demonstrated that deletion of mus-51 and/or mus-52 (homologs of S. cerevisiae YKU70 and YKU80, and human KU70 and KU80, respectively) in a lamentous fungus Neurospora crassa greatly increases the eciency of gene-targeting as much as 100%, compared with 20% in wild-type. Thus mus51- and/or mus-52-decient strains are quite suitable as recipients for transformation to disrupt gene(s) of interest, and consequently have been used in the development of a high-throughput gene knockout application (Colot et al., 2006). Based on this outstanding observation, ecient gene-targeting has also been reported in KU-decient mutants of Aspergillus fumigatus (da Silva Ferreira et al., 2006; Krappmann et al., 2006), Aspergillus nidulans (Nayak et al., 2006), Aspergillus sojae, Aspergillus oryzae (Takahashi et al., 2006a; Takahashi et al., 2006b), Aspergillus niger (Meyer et al., 2007), Sordaria macrospora (Po ggeler and Ku ck, 2006) and Cryptococcus neoformans (Goins et al., 2006), indicating that KU-decient strains are ideal recipients for gene-targeting in ascomycetous and basidiomycetous fungi. Disappointingly, gene-targeting frequencies in A. sojae and A. oryzae ku disruptants were found to be lower [up to 70% (Takahashi et al., 2006b)] than A. fumigatus, A. nidulans, and A. niger ku mutants with routine frequencies of 90% (da Silva Ferreira et al., 2006; Krappmann et al., 2006; Nayak et al., 2006; Meyer et al., 2007). The complete genome sequence of A. oryzae, a fungus which is used extensively in the manufacture of fermented foods and in the production of commercial enzymes for food processing (Christensen et al., 1998; Ichishima, 2000; Yaver et al., 2000), is known (Machida et al., 2005). The 38 Mb of A. oryzae genome contains 12,074 genes, and therefore contains a signicantly larger genome than A. fumigatus and A. nidulans (Galagan et al., 2005; Nierman et al., 2005). The functions of many of these extra genes are unknown or poorly uncharacterized. Therefore, a host strain available for highly ecient gene-targeting more than A. oryzae ku disruptants is mandatory for comprehensive and high-throughput functional genomics. In this study, based on the result of N. crassa for gene-targeting work (Ishibashi et al., 2006) where deletion of mus-53 [a homolog of human DNA ligase IV (LIG4)] resulted in a gene-targeting eciency of 100%, even when the DNA

sequence homologous to the targeted region is very short (100-bp), we have created an A. oryzae disruptant of a gene encoding LIG4 homolog (ligD) and subsequently demonstrated that the eciency of gene-targeting is as high as 100% also in the gene disruptant (DligD). In order to examine the usefulness of this A. oryzae DligD strain, we generated knockout mutants of all MAP kinase genes including mpkA, mpkB, hogA, mpkC and A. oryzae unique MAP kinase (mpkD). As a result, we succeeded in targeted deletions in loci encoding the ve A. oryzae MAP kinases. The DligD strain, therefore, is crucial for the functional analysis of individual gene as well as the comprehensive study of functional A. oryzae genome. 2. Materials and methods 2.1. Strains, media and molecular biological techniques Standard Escherichia coli manipulations were performed as described previously (Sambrook and Russell, 2001). E. coli strain DH5a (TaKaRa Bio Inc., Otsu, Japan) was used for plasmid propagation. Standard yeast genetic manipulations were performed as described by Adams et al. (1998). S. cerevisiae strain BY4741 (MATa his3D leu2D met15D ura3D) was used for in vivo plasmid construction (see below). A. oryzae genomic DNA was isolated as described previously (Chigira et al., 2002). A. oryzae RIB40 (National Research Institute of Brewing Stock Culture and ATCC42149) that was used for the genomesequencing project (Machida et al., 2005) was used as a donor of genomic DNA. A. oryzae NS4 (Yamada et al., 1997) carrying double selectable markers of niaD and sC, derived from RIB40, was used as a recipient strain for construction of ligD knockout mutants. These strains were grown in YPD complete medium (1% yeast extract, 2% polypeptone, 2% glucose) or CDME medium which is CzapekDox (CD) minimal medium (Nakajima et al., 2000) supplemented with 30 lg/ml methionine and 70 mM monosodium glutamate instead of sodium nitrate (NaNO3) as a sulfur and nitrogen source, respectively, for preparation of the conidial suspension. CDME medium supplemented with 0.1 lg/ml pyrithiamine (TaKaRa) or CDE medium which is CDME medium devoid of methionine was used as the selection medium for the ligD knockout derivatives of A. oryzae. 2.2. Construction of ligD disruption mutants Two plasmids, DligD::sC/pYES2 carrying the A. nidulans sC gene and DligD::ptrA/pYES2 carrying pyrithiamine resistance gene (ptrA) of A. oryzae (Kubodera et al., 2000), used for ligD gene disruptions were constructed as follows. The DligD::sC/pYES2 was generated by using the methods of Oldenburg et al. (1997) and Colot et al. (2006). All primers described in this paper are provided in Supplemental Table 1. The 50 and 30 fragments of the ligD gene were obtained by PCR with primers, designated 5ligDFw + 5lig-

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DRv and 3ligDFw + 3ligDRv (Supplemental Table 1), by using genomic DNA of A. oryzae RIB 40 as a template. The 5ligDFw primer incorporated an AatII site (underlined in Supplemental Table 1) to mutate an initiation codon of the ligD gene. The sC cassette was prepared from pUSC (Yamada et al., 1997) digested with BamHI and PstI. A yeast vector, pYES2 (Invitrogen Co., Tokyo, Japan), was digested with EcoRI and BamHI. These four DNA fragments were assembled in S. cerevisiae using the endogenous homologous recombination system, resulting in DligD::sC/pYES2. To replace the sC cassette in the DligD::sC/pYES2 with the ptrA cassette, another NdeI site was deleted from DligD::sC/pYES2 having two NdeI sites by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with the primers QCFw and QCRv (Supplemental Table 1), resulting in DligD::sC-N/ pYES2. A fragment of the pyrithiamine resistance gene (ptrA) was obtained from pPTRI (TaKaRa) by digestion with MunI and NdeI. The ptrA fragment was ligated into 7.9-kb MunI and NdeI fragment of DligD::sC-N/pYES2, resulting in DligD::ptrA/pYES2. A. oryzae NS4 was transformed with DligD::sC/pYES2 digested with BamHI and NotI as previously described (Gomi et al., 1987). A. oryzae transformants were screened for sulfate prototrophy and puried by subculturing at least three times on CDE agar plates. Knockout candidates were selected for colony-PCR using primer sets 1 (ligDP1and ligD-P2) and 2 (ligD-P1 and ligD-P3) (Supplemental Table 1) and A. oryzae genomic DNA as a template. Colony-PCR was performed according to the method as described previously by van Zeijl et al. (1997). When the DligD::sC fragment was inserted into the targeted ligD locus, a 1.4-kb fragment was amplied using primer set 1, while a 1.5-kb resulting fragment was amplied using primer set 2, indicating that the fragment was inserted into the ectopic locus or the candidate was still heterokaryotic. A single correct homologous integration resulting in the replacement of the resident ligD gene with the DligD::sC was conrmed by Southern analysis. A probe used for hybridization was the 0.8-kb MscI and NotI fragment of DligD::sC/pYES2. Similarly, the DligD::ptrA fragment from DligD::ptrA/ pYES2 after digestion with XhoI and KpnI was transformed into the A. oryzae DligD::sC strain and candidates where the selectable marker sC was replaced with ptrA were selected by colony-PCR using primer sets 3 (ligD-P4 and ligD-P5) (Supplemental Table 1) and 2, and A. oryzae genomic DNA as a template. Gene replacement with DligD::ptrA was also conrmed by Southern analysis by using the same probe as described above. 2.3. Mutagen sensitivity Sensitivity to chemical mutagen toxicity was analyzed by spot tests (Kato et al., 2004). Ethyl methanesulfonate (EMS) and phleomycin (PLM) were added to CDME agar medium at nal concentrations of 0.2% and 0.0005%,

respectively. Methyl methanesulfonate (MMS) was added to CDME agar medium at nal concentrations of 0, 0.01, 0.02, 0.025, 0.05, and 0.1%. Approximately 104 conidiospores were spotted onto these plates and grown at 30 C for 4 days. 2.4. Plasmid constructions and transformations for determination of gene-targeting eciency To determine eciencies of integration and targeting to the prtR locus encoding a transcription factor (Accession No. AB333776) in the DligD::sC strain, DligD::sC and wild-type strains were transformed with the prtR-disruption plasmid pDprtRTAX (T. Matsuura and K. Gomi, unpublished data) digested with SpeI and XhoI. pDprtRTAX carries the selectable marker, A. oryzae ptrA, that anked on each side with 1 kb of the 50 and 30 regions of the prtR gene. Similarly, DligD::ptrA and wild-type strains were transformed with the prtR-disruption plasmid DprtR::sC/pYES2, which was constructed as described below, digested with BamHI and NotI. The 1 kb fragments of 50 and 30 regions of the prtR were obtained by PCR with primers, designated 5prtRFw, 5prtRRv, 3prtRFw and 3prtRRv (Supplemental Table 1), by using genomic DNA of A. oryzae RIB40 as a template. The sC cassette was prepared from pUSC digested with BamHI and PstI. The pYES2 was digested with EcoRI and BamHI. These four DNA fragments were assembled in S. cerevisiae using the endogenous homologous recombination system, resulting in DprtR::sC/pYES2. In addition, to examine the eect of the length of homologous regions on the targeting eciency, DprtR::sC500/pTopo, DprtR::sC100/pTopo and DprtR::sC50/pTopo, which have 500 bp, 100 bp, and 50 bp of the prtR 50 and 30 regions on each side of the sC, respectively, were constructed as follows. The fragments of 500 bp, 100 bp, and 50 bp of the prtR 50 and 30 regions were PCR amplied using pairs of primers, 500Fw and 500Rv, 100Fw and 100Rv, and 50Fw and 50Rv (Supplemental Table 1), respectively. DprtR::sC/ pYES2 was used as the template. The amplied fragments were cloned using a TOPO TA cloning kit (Invitrogen) and sequenced. DligD::ptrA and wild-type strains were transformed with DprtR::sC500/pTopo, DprtR::sC100/pTopo and DprtR::sC50/pTopo digested with SpeI and NotI. Furthermore, to measure the eciency of targeting to the pepA locus, which encodes an aspartic protease (Gomi et al., 1993), in the DligD::ptrA strain, DligD::ptrA and wild-type strains were also transformed with the pepA-disruption plasmid DpepA::sC/pYES2, which was constructed as described below, digested with BamHI and NotI. The 50 and 30 fragments of the pepA gene were obtained by PCR with primers, designated 5pepAFw, 5pepARv, 3pepAFw and 3pepARv (Supplemental Table 1), by using a genomic DNA of A. oryzae RIB40 as a template. The sC cassette was prepared from pUSC digested with BamHI and PstI. The pYES2 was digested with EcoRI and BamHI. These four DNA fragments were assembled in S. cerevisiae by

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endogenous homologous recombination, resulting in DpepA::sC/pYES2. To conrm whether homologous integration event occurred in primary transformants, the colony-PCR was carried out by using primer set 4 (prtR-P1 and ligD-P4) for DprtR::ptrA, 5 (prtR-P1 and ligD-P2) for DprtR::sC, and 6 (pepA-P1 and ligD-P2) for DpepA::sC candidates (Supplemental Figure 1). PCR was also carried out by using primer set 1 for DligD::sC, 3 for DligD::ptrA, and 2 for the wild-type, as positive controls for PCR amplication. 2.5. Constructions of ve MAP kinase gene disruptants 2.5.1. DmpkA The 50 and 30 fragments of the mpkA gene were obtained by PCR with primers, designated 5mpkAFw + 5mpkARv and 3mpkAFw + 3mpkARv (Supplemental Table 1), by using of A. oryzae RIB40 genomic DNA as a template. The initiation codon of the mpkA gene was replaced by the codon for Ile [ATC (underlined in Supplemental Table 1)] with the 5mpkARV primer (Supplemental Table 1). The sC cassette was obtained from pUSC digested with BamHI and PstI. The pYES2 was digested with HindIII and XbaI. These four DNA fragments were assembled in S. cerevisiae via the endogenous homologous recombination, resulting in DmpkA::sC/pYES2. 2.5.2. DmpkB, DhogA, DmpkC and DmpkD The 50 and 30 fragments of the mpkB, hogA, mpkC, and mpkD gene were obtained by PCR with primers, designated for mpkB (5mpkBFw + 5mpkBRv and 3mpkBFw + 3mpkBRv), hogA (5hogAFw + 5hogARv and 3hogAFw + 3hogARv), mpkC (5mpkCFw + 5mpkCRv and 3mpkCFw + 3mpkCRv) and mpkD (5mpkDFw + 5mpkDRv and 3mpkDFw + 3mpkDRv) (Supplemental Table 1) with the genome of A. oryzae RIB40 as the template, respectively. The sC cassette was made from pUSC digested with BamHI and PstI. The pYES2 was digested with HindIII and XbaI. These each four DNA fragments were assembled in S. cerevisiae by means of the endogenous homologous recombination, resulting in DmpkB::sC/pYES2, DhogA::sC/pYES2, DmpkC::sC/pYES2, and DmpkD::sC/ pYES2, respectively. Transformation of A. oryzae DligD::ptrA and wild-type were performed as described above with (i) the DmpkA::sC fragment from DmpkA::sC/pYES2 digested with PstI and SpeI, (ii) DmpkB::sC fragment from DmpkB::sC/pYES2 digested with MluI and SpeI, (iii) DhogA::sC fragment from DhogA::sC/pYES2 digested with Aor51HI and MluI, (iv) DmpkC::sC fragment from DmpkC::sC/pYES2 digested with MluI and SphI, and (v) DmpkD::sC fragment from DmpkD::sC/pYES2 digested with NcoI and SpeI. Eciencies of homologous integration in primary transformants were determined by colony-PCR with corresponding primer sets. These are (i) 6 (mpkA-P1 and Sc-P2) for DmpkA, (ii) 7 (mpkB-P1 and Sc-P2) for DmpkB, (iii) 8 (hogA-P1

and Sc-P2) for DhogA, (iv) 9 (mpkC-P1 and Sc-P2) for DmpkC and (v) 10 (mpkD-P1 and Sc-P2) for DmpkD. The homokaryotic disruptants (DmpkA, DmpkB, DhogA, DmpkC and DmpkD) obtained were conrmed by PCR and Southern analysis. Each probe used for hybridizations was obtained by PCR with primer sets, which are mpkAprobeF and mpkAprobeR for DmpkA, mpkBprobeF and mpkBprobeR for DmpkB, hogAprobeF and hogAprobeR for DhogA, mpkCprobeF and mpkCprobeR for DmpkC, and mpkDprobeF and mpkDprobeR for DmpkD (Supplemental Table 1). The genomic DNA of A. oryzae RIB40 was used as the template for PCR amplication of each probe. 3. Results 3.1. Identication of the A. oryzae LIG4 homolog gene We searched the A. oryzae genome database (http:// www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao) for the ortholog of N. crassa MUS-53, which is a homolog of human LIG4. The search revealed that A. oryzae possesses a single MUS-53 homolog (AO090120000322 in the A. oryzae genome database) consisting of 1006 amino acid residues, and thus we designated this gene ligD. According to the prediction of introns in the A. oryzae database, the open reading frame (ORF) of the ligD gene consisting of a 3363-bp has six introns, which were located at 81148, 22542305, 24382494, 26282679, 27862833, and 32733337. Genes encoding putative Lig4 orthologs were also found in the A. nidulans, A. fumigatus, and A. niger genome sequences. A. oryzae LigD shares 71.8%, 71.2% and 71.9% identity with A. nidulans, A. fumigatus and A. niger Lig4 homologs, respectively (Fig. 1). 3.2. Construction and characterization of the ligD disruptants To conrm whether the LigD is involved in NHEJ pathway in A. oryzae, we initially constructed a ligD disruptant (DligD::sC) by homologous recombination with the DligD::sC fragment, which contained A. nidulans sC marker gene (Fig. 2A). Approximately 50 DligD::sC transformants were identied by colony-PCR using primer sets 1 and 2 described in the Section 2. In addition, we selected the ptrA gene, which confers resistance to pyrithiamine toxicity (Kubodera et al., 2000), as a selectable marker for deletion of the ligD gene. To obtain a DligD::ptrA mutant of A. oryzae, the sC heterologous selectable marker of the DligD::sC was replaced by the ptrA marker gene (Fig. 2B). The DligD::ptrA transformants were identied by colony-PCR using primers set 3 and 2. These DligD::sC and DligD::ptrA candidates were further conrmed by PCR and Southern blot analysis. These analyses revealed the expected hybridization signal at 2.7, 3.5 and 5.5 kb in digested genomic DNAs isolated from the DligD::sC, and the DligD::ptrA candidate transformants as well as the

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Fig. 1. Phylogenetic tree of DNA ligase IV proteins. The tree was created with a sequence analysis software DNAMAN using default settings for gap and extension penalties. Protein aligned are as follows; AfLigD (Aspergillus fumigatus LigD), EAL91408; AbLigD (Aspergillus niger LigD), CAK44569; AnLigD (Aspergillus nidulans LigD), EAA65275; AoLigD (Aspergillus oryzae LigD), BAE62914; CaLig4 (Candida albicans Lig4), EAK96312; CgLig4 (Candida glabrata Lig4), CAG58725; CnLig4 (Cryptococcus neoformans Lig4), AAW46139; NcLig4 (Neurospora crassa Lig4), EAA33632; ScLig4 (Saccharomyces cerevisiae Lig4), CAA99193; and SpLig4 (Schizosaccharomyces pombe Lig4), CAA21085.

wild-type NS4 strain, respectively (Fig. 2C). Such results showed that gene replacement had occurred successfully at the resident ligD locus. Since Lig4 is involved in nonhomologous end-joining repair of DSBs in phylogenetically diverse organisms (Hopfner et al., 2002; Lisby and Rothstein, 2004) and in telomere maintenance in some organisms (Hande, 2004), we examined DligD::sC and DligD::ptrA strains for growth characteristics and sensitivities to mutagen toxicity. Growth rates of DligD::sC and DligD::ptrA were not significantly reduced compared with those of wild-type on the plate culture and in the liquid culture (Fig. 3A control and C). The DligD strains showed normal conidiation and germination rate similar to the wild-type strain. Furthermore, no signicant dierence in protoplast recovery was observed. In N. crassa, deletion of the LIG4 homolog mus-53 and the KU70 homolog mus-51 resulted in increased sensitivity to chemical agents which cause double-stranded DNA breaks such as EMS and MMS (Ninomiya et al., 2004; Ishibashi et al., 2006). A. oryzae DligD::sC and DligD::ptrA strains did not show altered sensitivity to low concentrations of MMS (0.02%), but displayed increased sensitivity to high concentration of MMS (0.05%) (Fig. 3B). However, no growth dierence was observed in DligD::sC and DligD::ptrA to EMS or phleomycin, relative to the wild-type strain (Fig. 3A). These phenotypes were similar to those of A. fumigatus

Fig. 2. Generation of the ligD gene disruptants. (A) Strategy for homologous recombination of A. oryzae for ligD gene disruption using A. nidulans sC gene as a selectable marker. An asterisk indicates a stop codon replacing the initiation codon of the ligD ORF. The gray bar indicates the hybridization positions of the probe to conrm the gene replacement by Southern blot analysis. (B) Strategy for homologous recombination of A. oryzae DligD::sC strain for ligD gene disruption using the ptrA, which confers pyrithiamine resistance (Kubodera et al., 2000), as a selectable marker. The asterisk and the gray bar indicate as in (A). (C) Southern blot analysis of the genomic DNA from transformants. Each lane contained 20 lg of restriction enzyme-digested genomic DNA of the wild-type (lane 1), DligD::sC (lane 2) and DligD::ptrA (lane 3) cut with SalI and SpeI.

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ku mutants (da Silva Ferreira et al., 2006; Krappmann et al., 2006). 3.3. Targeting the prtR gene in the DligD::sC and DligD::ptrA strains To examine whether the gene-targeting eciency is increased in A. oryzae DligD::sC and DligD::ptrA as well as in N. crassa mus-53 (lig4 disruptant) (Ishibashi et al., 2006), the prtR gene was selected as a target for gene disruption experiments. The prtR gene is an ortholog of the A. niger prtT (Accession No. CAK44694; C. Hjort, C.A. van den Hondel, P.J. Punt, and F.H. Schuren, 9 May 2007, European Patent Oce), and encodes a transcription factor involved in gene expression of extracellular proteolytic enzymes including aspartic protease (PepA), neutral protease (NptA and NptB) and alkaline protease (AlpA) according to DNA microarray data derived from a PrtR overexpression strain (T. Matsuura and K. Gomi, unpublished data). Initially, we examined the targeting eciency of the DligD::sC strain with the A. oryzae ptrA gene as a selectable marker. The targeting eciencies were estimated by colony-PCR as described above in the Section 2 (Supplemental Figure 1). When gene replacement was performed with 1-kb of homologous anking sequences at both ends of the selectable marker gene, the targeting eciency was signicantly improved and achieved 96% in the DligD::sC, while that in the wild-type was approximately 8% (Table 1), suggesting that the ligD gene is involved in the NHEJ pathway in A. oryzae as well as in N. crassa. In contrast to N. crassa, however, gene-targeting eciency in the A. oryzae DligD::sC did not attain 100%. We presumed that homologous integration of the target fragment at the genomic ptrA (wild-type allele, thiA) locus could have occurred, and that targeted integration at the resident prtR locus failed to take place in a few transformants. Therefore, then we selected the A. oryzae DligD::ptrA strain and the A. nidulans sC gene as a heterologous selectable marker for the replacement of the prtR. As the result of the experiment where the same procedure as in DligD::sC was employed, targeting frequency in DligD::ptrA reached as high as 100% (Table 2). Furthermore, gene replacement of the pepA gene encoding an aspartic protease, Aspergillopepsin O (Gomi et al., 1993; Ichishima, 2000), again with the A. nidulans sC gene as the selectable marker,

resulted in 100% of gene-targeting eciency in the DligD::ptrA strains, while the gene-targeting eciency in the wild-type was 23% (Table 3).

"
Fig. 3. Phenotypes of the ligD disruptants. (A) Sensitivity of wild-type, DligD::sC and DligD::ptrA strains to ethyl methanesulfonate (EMS, 0.2% v/v), and phleomycin (PLM, 0.0005% w/v) toxicity. Wild-type, DligD::sC and DligD::ptrA cells (1 104) were cultivated on each plate at 30 C for 4 days. (B) Growth rates of wild-type, DligD::sC and DligD::ptrA strains on methyl methanesulfonate (MMS). Conidiospores (1 104) were grown on plates containing indicated concentrations of MMS at 30 C for 4 days. (C) Growth of wild-type and DligD::sC and DligD::ptrA strains in liquid medium. Wild-type and DligD::sC and DligD::ptrA cells (1 106) were grown in the YPD liquid medium at 30 C.

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Table 1 Frequency of homologous integration at the prtR locus in NS4 (wild-type) and DligD::sC disruptant Host strains NS4 DligD::sC Transformants tested 60 55 Homologous integrants 5 53 Gene-targeting eciency (%)a 8 96

Transformation was performed with the targeting plasmid harboring A. oryzae ptrA gene as a selectable marker. Homologous integration at the prtR locus was conrmed by colony-PCR. a The eciency of gene-targeting was estimated by the ratio of the number of homologous integrants to the number of the transformants tested.

Table 2 Frequency of homologous integration at the prtR locus in NS4 (wild-type) and DligD::ptrA disruptant with the targeting DNA fragments carrying dierent lengths of homologous anking regions Host strains Length of homology (bp) 1000 500 100 50 1000 500 100 50 Transformants tested 50 52 50 65 49 46 1b 0b Homologous integrants 14 7 0 0 49 46 0 Gene-targeting eciency (%)a 28 13 0 0 100 100 0

the prtR gene, and transformed the wild-type and DligD::ptrA strains. Whereas gene-targeting eciency decreased in the wild-type when homologous anking sequences were 500-bp long, in the DligD::ptrA targeting eciency remained around 100% (Table 2). On the other hand, when homologous anking regions were 100-bp or 50-bp long, no targeted transformant was obtained in both the wildtype and the DligD::ptrA mutant. Interestingly, the number of transformants in the wild-type did not decrease when homologous anking sequences were less than 100-bp, but few transformants appeared in DligD::ptrA even after three transformation experiments. These results indicated that the ligD disruptant is a suitable host for highly ecient gene-targeting when the length of homologous anking regions in the targeting cassettes is longer than 500-bp. Moreover, since very few transformants were obtained in the ligD disruptant when homologous anking regions were very short, integration event might occur predominantly through HR pathway in the ligD disruptant. 3.4. Generation of disruptants for MAPK genes by using the DligD::ptrA strain To validate whether the ligD gene disruptant is available more generally as a host for highly ecient gene-targeting, we undertook disruptions of mitogen-activated protein kinase (MAPK) genes by using the DligD::ptrA strain. From the A. oryzae genome database we identied ve MAPK genes, designated mpkA (Accession No. AB167718) (Mizutani et al., 2004), mpkB (N. crassa MAK-2 homolog, AO090003000402 in the A. oryzae genome database) (Pandey et al., 2004), hogA (AB185922) (A. nidulans HogA/SakA homolog) (Han and Prade, 2002; Kawasaki et al., 2002; Furukawa et al., 2005), mpkC (A. nidulans MpkC homolog, AO090020000466) (Furukawa et al., 2005), and mpkD (AO090701000642) which is unique in A. oryzae (Kobayashi et al., 2007). Cassettes for disruptions were constructed by using endogenous homologous recombination system in S. cerevisiae as described by Colot et al. (2006). These cassettes contained (in order, 50 30 ) 1-kb of 50 sequence anking the target MAPK gene ORF, the A. nidulans sC as a heterologous selectable marker, and 1-kb of 30 -downstream sequence for the target gene. The primary transformants were subjected to colony-PCR analyses to verify homologous recombination integration had occurred at the resident target MAPK gene loci. The gene-targeting eciencies of mpkA, mpkB, hogA, mpkC and mpkD in the DligD::ptrA strain were 86%, 100%, 100%, 100% and 80%, whereas those of these genes in the wild-type strain were 5%, 14%, 0%, 2% and 42%, respectively (Table 4). After representative disruptants were subcultured at least twice on CDE agar plates to obtain homokaryons, PCR and Southern blot analyses were carried out to conrm that the homologous recombination successfully took place at the target gene loci, and revealed that all transformants examined were homokaryons carrying only a disrupted single copy

NS4

DligD::ptrA

Transformation was carried out with the targeting plasmid harboring A. nidulans sC gene as a heterologous selectable marker. Homologous integration at the prtR locus was conrmed by colony-PCR. a The eciency of gene-targeting was estimated by the ratio of the number of homologous integrants to the number of the transformants tested. b Total number of transformants obtained with three transformation experiments.

Table 3 Frequency of homologous integration at the pepA locus in NS4 (wild-type) and DligD::ptrA disruptant Host strains NS4 DligD::ptrA Transformants tested 48 50 Homologous integrants 11 50 Gene-targeting eciency (%)a 23 100

Transformation was done with the targeting plasmid harboring A. nidulans sC gene as a heterologous selectable marker. Homologous integration at the pepA locus was conrmed by colony-PCR. a The eciency of gene-targeting was estimated by the ratio of the number of homologous integrants to the number of the transformants tested.

Ishibashi et al. (2006) reported that the mus-53 (lig4 mutant) in N. crassa had a gene-targeting eciency of 100% even if homologous anking sequences were very short, unlike mus-51 (ku70 mutant) and mus-52 (ku80 mutant). Hence, we constructed three targeting cassettes in which the sC marker was anked on both sides with either 500-bp, 100-bp, or 50-bp of 50 - and 30 -regions of

O. Mizutani et al. / Fungal Genetics and Biology 45 (2008) 878889 Table 4 Frequency of homologous integration at loci of the MAPK genes in NS4 (wild-type) and DligD::ptrA disruptant Host strains NS4 Targeted genes mpkA mpkB hogA mpkC mpkD mpkA mpkB hogA mpkC mpkD Transformants tested 37 74 43 57 12 22 4 15 22 10 Homologous integrants 2 10 0 1 5 19 4 15 22 8 Gene-targeting eciency (%)a 5 14 0 2 42 86 100 100 100 80

885

DligD::ptrA

Transformation was carried out with the targeting plasmid harboring A. nidulans sC gene as a heterologous selectable marker. Homologous integration at each MAPK gene locus was conrmed by colony-PCR. a The eciency of gene-targeting was estimated by the ratio of the number of homologous integrants to the number of the transformants tested.

of the targeted gene (Fig. 4). Following this, homokaryotic MAPK disruptants were cultivated on CDME agar plates (standard growth condition) and CDME agar plates containing 0.8 M NaCl (hypertonic growth condition) to examine disruptant phenotypes (Fig. 5). In this regard, DmpkA mutant strain displayed a remarkable growth defect, namely, shrunken colonies and scarcely dierentiated conidia under both standard and hypertonic growth conditions. This diered from the phenotype exhibited by the A. nidulans mpkA disruptant (Bussink and Osmani, 1999; Fujioka et al., 2007), which was found to be restored under hypertonic growth conditions. The DmpkB and DmpkC strains showed no apparent growth defect grown in standard or hypertonic culture regimes, though the DmpkB strain showed slightly reduced conidia formation when grown in standard culture conditions. In contrast, the DmpkD strain showed a reduced vegetative growth on CDME agar plates, and furthermore displayed a pronounced growth defect under hypertonic conditions. The DhogA strain showed no apparent growth defect on standard growth medium, but showed a reduced vegetative growth under hypertonic condition. Since we have readily succeeded in obtaining targeted disruptants of all ve MAPK genes as described here, the disruptant of ligD gene is indeed available as an ecient host for gene-targeting in A. oryzae. 4. Discussion To develop a highly ecient gene-targeting method for A. oryzae, we generated disruptants of a gene (ligD) encoding DNA ligase IV homolog involved in the nal step of nonhomologous end joining (NHEJ). The ligD disruptants showed no apparent growth defect and a similar sensitivity to toxicity by chemical agents which cause DNA damage,

Fig. 4. Generation of disruptants for MAPK genes. (AE) Strategies for homologous recombination of A. oryzae for MAPK genes disruptions using A. nidulans sC gene as a selectable marker. The gray bars indicate the probes used for hybridization to conrm the gene replacement by Southern blot analysis. (FJ) Southern blot analyses of the genomic DNA of the DligD::ptrA (lanes 1, 3, 7, 10 and 13), DmpkA (lane 2), DmpkB 1, 2 and 3 (lanes 4, 5 and 6), DhogA 1 and 2 (lanes 8 and 9), DmpkC 1 and 2 (lanes 11 and 12) and DmpkD 1 and 2 (lanes 14 and 15). The enzymes used are XbaI (lanes 1 and 2), NdeI (lanes 3, 4, 5 and 6), BamHI (lanes 7, 8 and 9), HindIII (lanes 10, 11 and 12), and MfeI (lanes 13, 14 and 15).

with exception for higher concentrations of MMS. Gene replacement of the prtR gene encoding a transcription factor for extracellular proteolytic genes using A. nidulans sC gene as a heterologous selectable marker resulted in 100%

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Fig. 5. Phenotypes of ve MAP kinase gene disruptants. Standard growth condition indicates CDME agar culture and hypertonic condition indicates CDME agar containing 0.8 M NaCl culture. Control strain (DligD::ptrA), DmpkA, DmpkB, DmpkC, DmpkD and DhogA cells (1 104) were cultivated on CDME solid media and CDME solid media with high osmotic pressure (0.8 M NaCl) at 30 C for 4 days.

of gene-targeting eciency in the ligD disruptant (DligD::ptrA) when the length of the homologous anking regions was at least longer than 0.5 kb. In addition, we have readily succeeded in creating disruptions of all ve MAPK genes present in the A. oryzae genome, by means of the DligD::ptrA strain. Based on these results, the ligD mutants should be potentially very useful and convenient tools for gene knockouts in this industrially important fungus. The gene-targeting eciencies in the ligD disruptant were as high as 100% for 5 of the 7 genes tested in this study, which were much higher than that (<70%) of the ku disruptant in A. oryzae (Takahashi et al., 2006b). Takahashi et al (2006b) discussed the possibility of the existence of the ku-independent pathway to explain why genetargeting frequencies in the A. oryzae ku disruptant did not achieve 100% levels. Molecular genetic analyses of N. crassa suggested that chromosomal integration of exogenous DNA is achieved via four possible pathways: two involving MEI-3-dependent and MEI-3-independent homologous integration (HI) and two involving MUS-52dependent and MUS-52-independent nonhomologous integration (NHI) (Ishibashi et al., 2006). The main route of NHI in N. crassa is the MUS-52-dependent and the minor pathway is MUS-11-dependent but MUS-52-independent NHI. In this context, the minor pathway which is involved in the Ku-independent NHI in A. oryzae may dominate in other Aspergillus species and N. crassa, resulting in the relatively low eciency of gene-targeting. MUS-53 (LIG4) is required for both major MUS-52-dependent and minor MUS-11-dependent NHI pathways. Therefore it would appear that gene-targeting eciency of ligD disruptants in A. oryzae is signicantly higher than that of the ku70 disruptant in A. oryzae (Takahashi et al., 2006b). This notion is supported by the observation that transformation eciencies decreased dramatically in ligD disruptants with very short homologous anking regions, demonstrating that the transformation/integration pathway is exclusively HI in the ligD disruptant. The eciency of gene-targeting in the A. oryzae ligD disruptant remained as high as 100% when the homologous regions that anked on either side of the heterologous selectable marker were 500-bp in length, whereas that in the ku70 mutant of A. sojae the frequency was only 14% (Takahashi et al., 2006b), suggesting that the ligD disruptant is preferable as host for gene-targeting in A. oryzae. Post-functional genomics requires improvement of genetargeting eciency, and thus disruption of the ligD ortholog is one of the best ways to make strains in many groups of microorganisms including fungi. On the other hand, in nkuA deletion mutants of A. nidulans, heterologous integration event occurred at a very low frequency (1 out of 83) (Nayak et al., 2006). In this regards, the possibility that extra heterologous integrations rarely occur could not be ruled out in the ligD disruptants. As described above, however, since the transformants are exclusively from HI in the ligD disruptants, it would be expected that heterologous

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integration event occurs, if any, much less than does in ku mutant. For post-genomic exploitation of A. oryzae, the results reported in this article are immensely important. Genetic analyses in A. oryzae lag far behind other Aspergillus species from the following diculties: (i) the low eciency of gene-targeting, (ii) small numbers of marker genes available, (iii) multiple nuclei in not only their vegetative cells but also their reproductive conidiospores (Ushijima and Nakadai, 1987), in contrast to a model fungus A. nidulans that possesses uninuclear conidiospores (Harris et al., 1994), and (iv) diculty of inter-strain crosses since there is no sexual life cycle. The high gene-targeting eciency (almost 100%) in A. oryzae ligD disruptants reported here is a signicant breakthrough in functional genomics. Its importance is exemplied by rapid disruption of all ve A. oryzae MAPK genes using the ligD disruptant. MAPK pathways are highly conserved signaling units found in eukaryotes, and play essential roles in the response to environmental signals and hormones, growth factors, and cytokines. Genome sequences of the three Aspergillus species (A. oryzae, A. nidulans and A. fumigatus) have revealed that the numbers of MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK) are three of each throughout the three Aspergilli, and the numbers of MAPK are four for A. nidulans and A. fumigatus, and ve for A. oryzae (Kobayashi et al., 2007). Thus, A. oryzae has the largest number of MAP kinase genes (designated each hogA/sakA, mpkA, mpkB, mpkC and mpkD) among the three Aspergilli. To examine the utility of the ligD disruptant for comprehensive functional genomics, we attempted rstly the deletion of these ve MAPK genes and were successful in isolation of the disruptants for each gene. In particular, although we were unable to obtain a hogA transformant with targeted integration in the wildtype, all primary transformants possessed the gene replacement in the DligD strain. In addition, mpkC showed the same pattern. In two cases, mpkA and mpkD, however, the targeting eciencies were also dramatically improved but failed to attain 100%. We suspected the relationship between gene-targeting eciencies and GC content in anking regions used, but the GC content of each anking region was almost the same. The reason is still unclear, although this might be due to the presence of a LigD-independent NHEJ pathway in A. oryzae. Notwithstanding, taking together such results indicate that the use of DligD strain and procedure is very promising for A. oryzae high-throughput gene disruption. Since A. oryzae has multinuclear conidiospores, primary transformants with the targeted integration were found to be heterokaryons. Hence, homokaryotic disruptants need to be puried and this is carried out conveniently through successive transfer on selective medium. Such putative homokaryotic strains were examined phenotypically on standard growth medium in the absence or presence of salt. Vegetative growth defect was observed in the DmpkA strain irrespective of salt, whilst DmpkD displayed signicantly

reduced growth under hypertonic growth condition in addition to relatively reduced vegetative growth in standard culture conditions. MpkD whose orthologs were not observed in A. nidulans or A. fumigatus, is a likely paralog of HogA in A. oryzae (Kobayashi et al., 2007). Interestingly, the mpkD-decient mutant displayed a signicant growth defect under hypertonic growth condition compared to the DhogA strain, suggesting that the MpkD may be predominantly involved in the osmotic response pathway in A. oryzae, although it has been reported that the hogA/sakA encodes the stress-activated kinase that responds to hypertonic stress, reactive oxygen species, and heat shock in A. nidulans, A. fumigatus, and N. crassa (Han and Prade, 2002; Kawasaki et al., 2002; Zhang et al., 2002; Xue et al., 2004; Furukawa et al., 2005; Du et al., 2006; Noguchi et al., 2007). To understand in detail the role of each MAPK played in response to environmental stimuli in A. oryzae, more rigorous analyses of these disruptants are necessary and indeed are in progress. For precise functional analyses, although the DligD mutant showed no obvious growth defect so far, a rescue of the wild-type ligD should be taken into account for the possibility of a synthetic interaction of deletion of the ligD in the gene replacement experiments prior to functional analyses. In addition to increased sensitivity to high concentrations of MMS, the ligD mutants might have defect in telomere maintenance, since Nielsen et al. (2007) have noted that an nkuA deletion mutant of A. nidulans showed shortened telomeres. In this context, it would be desirable to restore the NHEJ activity following the successful disruption of a target gene in the ligD mutant, and for this purpose a transient disruption system for NHEJ pathway developed by Nielsen et al. (2007) is especially eective in A. oryzae. In conclusion, we succeeded in signicantly improving gene-targeting eciency in A. oryzae by using the ligD disruptants, which is comparable to N. crassa mus-53 (lig4) decient strain (Ishibashi et al., 2006). In this respect, ve MAPK gene disruptants were promptly and conveniently generated via the ligD disruptant. Such results encourage us to create a comprehensive gene disruption library by using the A. oryzae ligD-decient strain and the project is now underway.

Acknowledgments We thank Takahiro Shintani and Kentaro Furukawa for helpful suggestions and Yoshimi Watanabe, Akira Yoshimi, Jun-ichiro Marui and Tomonori Fujioka for helpful discussions and/or technical assistance. We are also grateful to James R. Kinghorn for critical reading of the manuscript. This work was supported by Grant-in-Aid for Scientic Research on Priority Areas Applied Genomics (No. 17019001) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K.G. and also by Grant-in-Aid for Scientic Research (B) (No.

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