A Defect of LigD (Human Lig4 Homolog) for Nonhomologous

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A defect of LigD (human Lig4 homolog) for nonhomologousend joining significantly improves efficiency 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,*

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, Japanb 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 2007Available online 11 January 2008

Abstract

Gene-targeting by homologous recombination occurs rarely during transformation since nonhomologous recombination is predom-inant inAspergillus oryzae. To develop a highly efficient gene-targeting system forA.oryzae, we constructed disrupted strains harboring agene (ligD) encoding human DNA ligase IV homolog that is involved in the final step of DNA nonhomologous end joining. The A. ory-zae ligDdisruptants showed no apparent defect in vegetative growth and/or conidiation, and exhibited increased sensitivity to high con-centration 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 extracellularproteolytic genes, using theAspergillus nidulans sCgene as the selectable marker resulted in 100% of gene-targeting efficiency in theligDdisruptant, compared to less than 30% for a wild-type, when the length of the homologous flanking sequences used was longer than0.5 kb. Similarly, gene-targeting efficiency was as high as 100% for aspartic protease-encoding gene (pepA). Furthermore, using thisligDdisruptant system ofA.oryzae, we readily succeeded in disrupting five mitogen-activated protein kinase (MAPK) genes, namely mpkA,mpkB,hogA,mpkCand A.oryzaeunique MAPK (mpkD). Such results show that the ligDdisruptant system is an extremely convenientgenetic 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 plantsand mammals, two major recombination pathways havebeen identified for the repair of DNA Double-Strand

Breaks (DSBs) which are induced by endogenous and exog-enous causes and they are the most detrimental DNAlesions (Haber, 2000). These pathways differ as to whetherthey require DNA sequence homology and distinct sets ofprotein factors (Wang et al., 2003). Homologous recombi-nation (HR) utilizes intact genetic information from anundamaged homologous region as a template for repairof DSBs, whereas nonhomologous end-joining (NHEJ)rejoins DSBs by direct ligation of the strand ends withoutany requirement for sequence homology. These repairmechanisms have been conserved throughout evolution

1087-1845/$ - see front matter 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.fgb.2007.12.010

* Corresponding author. Fax: +81 22 717 8902.E-mail address:[email protected](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.

www.elsevier.com/locate/yfgbi

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and operate in a wide range of organisms. Also in fungi,HR and NHEJ pathways require various proteins, forexample, Rad51 epistasis group in the HR and Ku proteinand DNA ligase IV in the NHEJ (see reviews Daley et al.,2005; Krappmann, 2007).

Saccharomyces cerevisiaeutilizes primarily the HR path-

way in DSB repair and exogenous DNA fragments aremainly integrated at homologous sites in the genome, whilemost filamentous fungi preferentially repair DSBs byNHEJ and exogenous DNA can be integrated at ectopicsites in the genome. Therefore, gene-targeting-mediatedthrough HR can be readily accomplished in S. cerevisiae(Schiestl et al., 1994), but the efficiency of gene-targetingtends to be limiting in most filamentous fungi. However,Ninomiya et al (2004) have recently demonstrated thatdeletion ofmus-51and/ormus-52(homologs ofS.cerevisi-ae YKU70 and YKU80, and human KU70 and KU80,respectively) in a filamentous fungus Neurospora crassagreatly increases the efficiency of gene-targeting as much

as 100%, compared with 20% in wild-type. Thus mus-51- and/or mus-52-deficient strains are quite suitable asrecipients for transformation to disrupt gene(s) of interest,and consequently have been used in the development of ahigh-throughput gene knockout application (Colot et al.,2006). Based on this outstanding observation, efficientgene-targeting has also been reported in KU-deficientmutants of Aspergillus fumigatus (da Silva Ferreira et al.,2006; Krappmann et al., 2006),Aspergillus nidulans(Nayaket al., 2006), Aspergillus sojae, Aspergillus oryzae (Takah-ashi et al., 2006a; Takahashi et al., 2006b),Aspergillus niger(Meyer et al., 2007), Sordaria macrospora (Poggeler and

Kuck, 2006) and Cryptococcus neoformans (Goins et al.,2006), indicating that KU-deficient strains are ideal recipi-ents for gene-targeting in ascomycetous and basidiomyce-tous fungi. Disappointingly, gene-targeting frequencies inA. sojae and A. oryzae ku disruptants were found to belower [up to 70% (Takahashi et al., 2006b)] than A. fumig-atus,A.nidulans, and A.niger kumutants with routine fre-quencies of 90% (da Silva Ferreira et al., 2006; Krappmannet al., 2006; Nayak et al., 2006; Meyer et al., 2007).

The complete genome sequence of A. oryzae, a funguswhich is used extensively in the manufacture of fermentedfoods and in the production of commercial enzymes forfood 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 12,074genes, and therefore contains a significantly larger genomethan A. fumigatus and A. nidulans (Galagan et al., 2005;Nierman et al., 2005). The functions of many of these extragenes are unknown or poorly uncharacterized. Therefore, ahost strain available for highly efficient gene-targetingmore than A.oryzae kudisruptants is mandatory for com-prehensive and high-throughput functional genomics. Inthis study, based on the result ofN. crassafor gene-target-ing work (Ishibashi et al., 2006) where deletion ofmus-53[ahomolog of human DNA ligase IV (LIG4)] resulted in a

gene-targeting efficiency 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 agene encoding LIG4 homolog (ligD) and subsequentlydemonstrated that the efficiency of gene-targeting is as highas 100% also in the gene disruptant (DligD). In order toexamine the usefulness of this A. oryzae DligD strain, we

generated knockout mutants of all MAP kinase genesincludingmpkA, mpkB, hogA, mpkCand A. oryzaeuniqueMAP kinase (mpkD). As a result, we succeeded in targeteddeletions in loci encoding the five A. oryzaeMAP kinases.The DligD strain, therefore, is crucial for the functionalanalysis of individual gene as well as the comprehensivestudy of functionalA. oryzae genome.

2. Materials and methods

2.1. Strains, media and molecular biological techniques

StandardEscherichia colimanipulations were performed

as described previously (Sambrook and Russell, 2001).E. colistrain DH5a (TaKaRa Bio Inc., Otsu, Japan) wasused for plasmid propagation. Standard yeast geneticmanipulations were performed as described by Adamset al. (1998). S. cerevisiae strain BY4741 (MATa his3Dleu2D met15D ura3D) was used forin vivoplasmid construc-tion (see below). A. oryzae genomic DNA was isolated asdescribed previously (Chigira et al., 2002). A. oryzaeRIB40 (National Research Institute of Brewing Stock Cul-ture and ATCC42149) that was used for the genome-sequencing project (Machida et al., 2005) was used as adonor 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 con-struction of ligD knockout mutants. These strains weregrown in YPD complete medium (1% yeast extract, 2%polypeptone, 2% glucose) or CDME medium which is Cza-pekDox (CD) minimal medium (Nakajima et al., 2000)supplemented with 30lg/ml methionine and 70 mMmonosodium glutamate instead of sodium nitrate (NaNO3)as a sulfur and nitrogen source, respectively, for prepara-tion of the conidial suspension. CDME medium supple-mented with 0.1 lg/ml pyrithiamine (TaKaRa) or CDEmedium which is CDME medium devoid of methioninewas used as the selection medium for the ligD knockoutderivatives ofA. oryzae.

2.2. Construction of ligD disruption mutants

Two plasmids, DligD::sC/pYES2 carrying the A. nidu-lans sCgene andDligD::ptrA/pYES2 carrying pyrithiamineresistance gene (ptrA) ofA.oryzae(Kubodera et al., 2000),used forligDgene disruptions were constructed as follows.TheDligD::sC/pYES2 was generated by using the methodsofOldenburg et al. (1997) and Colot et al. (2006). All prim-ers described in this paper are provided in SupplementalTable 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), byusing genomic DNA of A. oryzae RIB 40 as a template.The 5ligDFw primer incorporated an AatII site (under-lined in Supplemental Table 1) to mutate an initiationcodon of the ligDgene. ThesCcassette was prepared frompUSC (Yamada et al., 1997) digested with BamHI and

PstI. A yeast vector, pYES2 (Invitrogen Co., Tokyo,Japan), was digested with EcoRI and BamHI. These fourDNA fragments were assembled in S. cerevisiae using theendogenous homologous recombination system, resultingin DligD::sC/pYES2. To replace the sC cassette in theDligD::sC/pYES2 with the ptrAcassette, another NdeI sitewas deleted from DligD::sC/pYES2 having two NdeI sitesby using a QuikChange site-directed mutagenesis kit (Strat-agene, La Jolla, CA, USA) with the primers QCFw andQCRv (Supplemental Table 1), resulting in DligD::sC-N/pYES2. A fragment of the pyrithiamine resistance gene(ptrA) was obtained from pPTRI (TaKaRa) by digestionwith MunI and NdeI. The ptrA fragment was ligated into

7.9-kb MunI and NdeI fragment ofDligD::sC-N/pYES2,resulting in DligD::ptrA/pYES2.

A. oryzae NS4 was transformed with DligD::sC/pYES2digested with BamHI and NotI as previously described(Gomi et al., 1987).A. oryzaetransformants were screenedfor sulfate prototrophy and purified by subculturing atleast three times on CDE agar plates. Knockout candidateswere selected for colony-PCR using primer sets 1 (ligD-P1and ligD-P2) and 2 (ligD-P1 and ligD-P3) (SupplementalTable 1) and A. oryzae genomic DNA as a template. Col-ony-PCR was performed according to the method asdescribed previously by van Zeijl et al. (1997). When the

DligD::sC fragment was inserted into the targeted ligDlocus, a 1.4-kb fragment was amplified using primer set 1,while a 1.5-kb resulting fragment was amplified using pri-mer set 2, indicating that the fragment was inserted intothe ectopic locus or the candidate was still heterokaryotic.A single correct homologous integration resulting in thereplacement of the resident ligD gene with the DligD::sCwas confirmed by Southern analysis. A probe used forhybridization was the 0.8-kb MscI and NotI fragment ofDligD::sC/pYES2.

Similarly, the DligD::ptrA fragment from DligD::ptrA/pYES2 after digestion with XhoI and KpnI was trans-formed into the A. oryzae DligD::sCstrain and candidateswhere the selectable marker sC was replaced with ptrAwere selected by colony-PCR using primer sets 3 (ligD-P4and ligD-P5) (Supplemental Table 1) and 2, and A. oryzaegenomic DNA as a template. Gene replacement withDligD::ptrA was also confirmed by Southern analysis byusing the same probe as described above.

2.3. Mutagen sensitivity

Sensitivity to chemical mutagen toxicity was analyzed byspot tests (Kato et al., 2004). Ethyl methanesulfonate(EMS) and phleomycin (PLM) were added to CDME agar

medium at final concentrations of 0.2% and 0.0005%,

respectively. Methyl methanesulfonate (MMS) was addedto CDME agar medium at final concentrations of 0, 0.01,0.02, 0.025, 0.05, and 0.1%. Approximately 104 conidiosp-ores were spotted onto these plates and grown at 30 C for4 days.

2.4. Plasmid constructions and transformations fordetermination of gene-targeting efficiency

To determine efficiencies of integration and targeting tothe prtR locus encoding a transcription factor (AccessionNo. AB333776) in the DligD::sC strain, DligD::sC andwild-type strains were transformed with the prtR-disrup-tion plasmid pDprtRTAX (T. Matsuura and K. Gomi,unpublished data) digested with SpeI and XhoI. pDprtR-TAX carries the selectable marker, A. oryzae ptrA, thatflanked on each side with 1 kb of the 50 and 30 regions ofthe prtRgene. Similarly, DligD::ptrAand wild-type strainswere transformed with the prtR-disruption plasmidDprtR::sC/pYES2, which was constructed as describedbelow, digested with BamHI and NotI. The 1 kb fragmentsof 50 and 30 regions of theprtRwere obtained by PCR withprimers, designated 5prtRFw, 5prtRRv, 3prtRFw and3prtRRv (Supplemental Table 1), by using genomic DNAofA.oryzaeRIB40 as a template. The sCcassette was pre-pared from pUSC digested with BamHI and PstI. ThepYES2 was digested with EcoRI and BamHI. These fourDNA fragments were assembled in S. cerevisiae using theendogenous homologous recombination system, resultingin DprtR::sC/pYES2. In addition, to examine the effect ofthe length of homologous regions on the targeting effi-

ciency, DprtR::sC500/pTopo, DprtR::sC100/pTopo andDprtR::sC50/pTopo, which have 500 bp, 100 bp, and50 bp of the prtR 50 and 30 regions on each side of thesC, respectively, were constructed as follows. The frag-ments of 500 bp, 100 bp, and 50 bp of the prtR 50 and 30

regions were PCR amplified using pairs of primers,500Fw and 500Rv, 100Fw and 100Rv, and 50Fw and50Rv (Supplemental Table 1), respectively. DprtR::sC/pYES2 was used as the template. The amplified fragmentswere cloned using a TOPO TA cloning kit (Invitrogen) andsequenced. DligD::ptrA and wild-type strains were trans-formed with DprtR::sC500/pTopo, DprtR::sC100/pTopoandDprtR::sC50/pTopo digested with SpeI and NotI. Fur-thermore, to measure the efficiency of targeting to the pepAlocus, which encodes an aspartic protease (Gomi et al.,1993), in the DligD::ptrAstrain, DligD::ptrAand wild-typestrains were also transformed with the pepA-disruptionplasmid DpepA::sC/pYES2, which was constructed asdescribed below, digested with BamHI and NotI. The 50

and 30 fragments of the pepA gene were obtained by PCRwith primers, designated 5pepAFw, 5pepARv, 3pepAFwand 3pepARv (Supplemental Table 1), by using a genomicDNA of A. oryzae RIB40 as a template. The sC cassettewas 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 Dpe-pA::sC/pYES2.

To confirm whether homologous integration eventoccurred in primary transformants, the colony-PCR wascarried 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 byusing primer set 1 for DligD::sC, 3 for DligD::ptrA, and 2for the wild-type, as positive controls for PCRamplification.

2.5. Constructions of five MAP kinase gene disruptants

2.5.1. DmpkA

The 50 and 30 fragments of thempkAgene were obtainedby PCR with primers, designated 5mpkAFw + 5mpkARvand 3mpkAFw + 3mpkARv (Supplemental Table 1), byusing of A. oryzae RIB40 genomic DNA as a template.

The initiation codon of the mpkA gene was replaced bythe codon for Ile [ATC (underlined inSupplemental Table1)] with the 5mpkARV primer (Supplemental Table 1). ThesCcassette was obtained from pUSC digested with BamHIand PstI. The pYES2 was digested with HindIII and XbaI.These four DNA fragments were assembled in S.cerevisiaevia the endogenous homologous recombination, resultingin DmpkA::sC/pYES2.

2.5.2. DmpkB, DhogA, DmpkC andDmpkD

The 50 and 30 fragments of the mpkB, hogA, mpkC, andmpkDgene were obtained by PCR with primers, designated

for mpkB (5mpkBFw + 5mpkBRv and 3mpkBFw +3mpkBRv), hogA (5hogAFw + 5hogARv and 3hogAF-w + 3hogARv), mpkC (5mpkCFw + 5mpkCRv and3mpkCFw + 3mpkCRv) and mpkD (5mpkDFw +5mpkDRv and 3mpkDFw + 3mpkDRv) (SupplementalTable 1) with the genome ofA. oryzae RIB40 as the template,respectively. ThesCcassette was made from pUSC digestedwith BamHIand PstI. The pYES2 was digested with HindIIIand XbaI. These each four DNA fragments were assembledin S. cerevisiae by means of the endogenous homologousrecombination, resulting in DmpkB::sC/pYES2, Dho-gA::sC/pYES2, DmpkC::sC/pYES2, and DmpkD::sC/pYES2, respectively.

Transformation ofA. oryzae DligD::ptrA and wild-typewere performed as described above with (i) the DmpkA::sCfragment from DmpkA::sC/pYES2 digested with PstI andSpeI, (ii) DmpkB::sC fragment from DmpkB::sC/pYES2digested with MluI and SpeI, (iii) DhogA::sC fragmentfrom DhogA::sC/pYES2 digested with Aor51HI and MluI,(iv)DmpkC::sCfragment fromDmpkC::sC/pYES2 digestedwith MluI and SphI, and (v) DmpkD::sC fragment fromDmpkD::sC/pYES2 digested with NcoI and SpeI. Efficien-cies of homologous integration in primary transformantswere determined by colony-PCR with corresponding pri-mer 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) forDmpkC and (v) 10 (mpkD-P1 and Sc-P2) for DmpkD.The homokaryotic disruptants (DmpkA, DmpkB, DhogA,DmpkC and DmpkD) obtained were confirmed by PCRand Southern analysis. Each probe used for hybridizationswas obtained by PCR with primer sets, which are mpkAp-

robeF and mpkAprobeR for DmpkA, mpkBprobeF andmpkBprobeR for DmpkB, hogAprobeF and hogAprobeRfor DhogA, mpkCprobeF and mpkCprobeR for DmpkC,and mpkDprobeF and mpkDprobeR for DmpkD(Supple-mental Table 1). The genomic DNA of A. oryzae RIB40was used as the template for PCR amplification of eachprobe.

3. Results

3.1. Identification 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 ofN.crassaMUS-53, which is a homologof human LIG4. The search revealed that A. oryzae pos-sesses a single MUS-53 homolog (AO090120000322 inthe A. oryzae genome database) consisting of 1006 aminoacid residues, and thus we designated this gene ligD.According to the prediction of introns in the A. oryzaedatabase, the open reading frame (ORF) of the ligD geneconsisting of a 3363-bp has six introns, which were locatedat 81148, 22542305, 24382494, 26282679, 27862833,and 32733337. Genes encoding putative Lig4 orthologswere 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. fumigatusand A. niger Lig4 homologs, respectively (Fig. 1).

3.2. Construction and characterization of the ligD

disruptants

To confirm whether the LigD is involved in NHEJ path-way inA.oryzae, we initially constructed aligDdisruptant(DligD::sC) by homologous recombination with theDligD::sCfragment, which contained A. nidulans sCmar-ker gene (Fig. 2A). Approximately 50 DligD::sCtransfor-mants were identified by colony-PCR using primer sets 1and 2 described in the Section 2. In addition, we selectedtheptrAgene, which confers resistance to pyrithiamine tox-icity (Kubodera et al., 2000), as a selectable marker fordeletion of the ligD gene. To obtain a DligD::ptrAmutantof A. oryzae, the sC heterologous selectable marker ofthe DligD::sC was replaced by the ptrA marker gene(Fig. 2B). The DligD::ptrA transformants were identifiedby colony-PCR using primers set 3 and 2. These DligD::sCandDligD::ptrAcandidates were further confirmed by PCRand Southern blot analysis. These analyses revealed theexpected hybridization signal at 2.7, 3.5 and 5.5 kb indigested genomic DNAs isolated from the DligD::sC, and

the DligD::ptrA candidate transformants as well as the

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ku mutants (da Silva Ferreira et al., 2006; Krappmannet al., 2006).

3.3. Targeting the prtR gene in the DligD::sC and

DligD::ptrA strains

To examine whether the gene-targeting efficiency isincreased in A. oryzae DligD::sC and DligD::ptrA as wellas in N. crassa mus-53 (lig4 disruptant) (Ishibashi et al.,2006), the prtR gene was selected as a target for gene dis-ruption experiments. The prtR gene is an ortholog of theA. niger prtT (Accession No. CAK44694; C. Hjort, C.A.van den Hondel, P.J. Punt, and F.H. Schuren, 9 May2007, European Patent Office), and encodes a transcrip-tion factor involved in gene expression of extracellularproteolytic enzymes including aspartic protease (PepA),neutral protease (NptA and NptB) and alkaline protease(AlpA) according to DNA microarray data derived froma PrtR overexpression strain (T. Matsuura and K. Gomi,

unpublished data). Initially, we examined the targetingefficiency of the DligD::sC strain with the A. oryzae ptrAgene as a selectable marker. The targeting efficiencieswere estimated by colony-PCR as described above inthe Section 2 (Supplemental Figure 1). When genereplacement was performed with 1-kb of homologousflanking sequences at both ends of the selectable markergene, the targeting efficiency was significantly improvedand achieved 96% in the DligD::sC, while that in thewild-type was approximately 8% (Table 1), suggestingthat the ligD gene is involved in the NHEJ pathway inA. oryzae as well as in N. crassa. In contrast to N. crassa,

however, gene-targeting efficiency in the A. oryzaeDligD::sCdid not attain 100%. We presumed that homol-ogous integration of the target fragment at the genomicptrA (wild-type allele, thiA) locus could have occurred,and that targeted integration at the resident prtR locusfailed to take place in a few transformants. Therefore,then we selected the A. oryzae DligD::ptrA strain andthe A. nidulans sCgene as a heterologous selectable mar-ker for the replacement of the prtR. As the result of theexperiment where the same procedure as in DligD::sCwasemployed, targeting frequency in DligD::ptrA reached ashigh as 100% (Table 2). Furthermore, gene replacementof the pepA gene encoding an aspartic protease, Asperg-illopepsin O (Gomi et al., 1993; Ichishima, 2000), againwith the A. nidulans sC gene as the selectable marker,

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

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

grown in the YPD liquid medium at 30C.

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Ishibashi et al. (2006) reported that the mus-53 (lig4mutant) in N. crassa had a gene-targeting efficiency of100% even if homologous flanking sequences were veryshort, unlike mus-51 (ku70 mutant) and mus-52 (ku80mutant). Hence, we constructed three targeting cassettesin which the sC marker was flanked on both sides with

either 500-bp, 100-bp, or 50-bp of 50

- and 30

-regions of

theprtRgene, and transformed the wild-type and DligD::p-trA strains. Whereas gene-targeting efficiency decreased inthe wild-type when homologous flanking sequences were500-bp long, in the DligD::ptrA targeting efficiencyremained around 100% (Table 2). On the other hand, whenhomologous flanking regions were 100-bp or 50-bp long,

no targeted transformant was obtained in both the wild-type and theDligD::ptrAmutant. Interestingly, the numberof transformants in the wild-type did not decrease whenhomologous flanking sequences were less than 100-bp,but few transformants appeared in DligD::ptrA even afterthree transformation experiments. These results indicatedthat theligDdisruptant is a suitable host for highly efficientgene-targeting when the length of homologous flankingregions in the targeting cassettes is longer than 500-bp.Moreover, since very few transformants were obtained inthe ligD disruptant when homologous flanking regionswere very short, integration event might occur predomi-nantly through HR pathway in the ligDdisruptant.

3.4. Generation of disruptants for MAPK genes by using the

DligD::ptrA strain

To validate whether theligDgene disruptant is availablemore generally as a host for highly efficient gene-targeting,we undertook disruptions of mitogen-activated proteinkinase (MAPK) genes by using the DligD::ptrA strain.From the A. oryzae genome database we identified fiveMAPK genes, designated mpkA (Accession No.AB167718) (Mizutani et al., 2004), mpkB (N. crassaMAK-2 homolog, AO090003000402 in the A. oryzae gen-

ome 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) (Furuka-wa et al., 2005), and mpkD (AO090701000642) which isunique in A. oryzae (Kobayashi et al., 2007). Cassettesfor disruptions were constructed by using endogenoushomologous recombination system in S. cerevisiae asdescribed byColot et al. (2006). These cassettes contained(in order, 5030) 1-kb of 50 sequence flanking the targetMAPK gene ORF, the A. nidulans sC as a heterologousselectable marker, and 1-kb of 30-downstream sequencefor the target gene. The primary transformants were sub-jected to colony-PCR analyses to verify homologousrecombination integration had occurred at the resident tar-get MAPK gene loci. The gene-targeting efficiencies ofmpkA, mpkB, hogA, mpkC and mpkD in the DligD::ptrAstrain were 86%, 100%, 100%, 100% and 80%, whereasthose of these genes in the wild-type strain were 5%, 14%,0%, 2% and 42%, respectively (Table 4). After representa-tive disruptants were subcultured at least twice on CDEagar plates to obtain homokaryons, PCR and Southernblot analyses were carried out to confirm that the homolo-gous recombination successfully took place at the targetgene loci, and revealed that all transformants examined

were homokaryons carrying only a disrupted single copy

Table 2Frequency of homologous integration at theprtRlocus in NS4 (wild-type)and DligD::ptrAdisruptant with the targeting DNA fragments carryingdifferent lengths of homologous flanking regions

Host strains Length ofhomology

(bp)

Transformantstested

Homologousintegrants

Gene-targetingefficiency (%)a

NS4 1000 50 14 28500 52 7 13100 50 0 0

50 65 0 0

DligD::ptrA 1000 49 49 100500 46 46 100100 1b 0 0

50 0b

Transformation was carried out with the targeting plasmid harboringA. nidulans sC gene as a heterologous selectable marker. Homologousintegration at the prtRlocus was confirmed by colony-PCR.a The efficiency of gene-targeting was estimated by the ratio of the

number of homologous integrants to the number of the transformantstested.b Total number of transformants obtained with three transformation

experiments.

Table 3Frequency of homologous integration at the pepAlocus in NS4 (wild-type)and DligD::ptrAdisruptant

Hoststrains

Transformantstested



NS4 48 11 23DligD::ptrA 50 50 100

Transformation was done with the targeting plasmid harboringA. nidulanssCgene as a heterologous selectable marker. Homologous integration atthe pepAlocus was confirmed by colony-PCR.a The efficiency of gene-targeting was estimated by the ratio of the

number of homologous integrants to the number of the transformantstested.

Table 1Frequency of homologous integration at the prtRlocus in NS4 (wild-type)and DligD::sCdisruptant

Hoststrains

Transformantstested



NS4 60 5 8

DligD::sC 55 53 96

Transformation was performed with the targeting plasmid harboring A.oryzae ptrA gene as a selectable marker. Homologous integration at the

prtRlocus was confirmed by colony-PCR.a The efficiency of gene-targeting was estimated by the ratio of the




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of the targeted gene (Fig. 4). Following this, homokaryoticMAPK disruptants were cultivated on CDME agar plates(standard growth condition) and CDME agar plates con-taining 0.8 M NaCl (hypertonic growth condition) toexamine disruptant phenotypes (Fig. 5). In this regard,DmpkA mutant strain displayed a remarkable growthdefect, namely, shrunken colonies and scarcely differenti-ated conidia under both standard and hypertonic growthconditions. This differed from the phenotype exhibited bythe A. nidulans mpkA disruptant (Bussink and Osmani,

1999; Fujioka et al., 2007), which was found to be restoredunder hypertonic growth conditions. The DmpkB andDmpkC strains showed no apparent growth defect grownin standard or hypertonic culture regimes, though theDmpkB strain showed slightly reduced conidia formationwhen grown in standard culture conditions. In contrast,the DmpkDstrain showed a reduced vegetative growth onCDME agar plates, and furthermore displayed a pro-nounced growth defect under hypertonic conditions. TheDhogA strain showed no apparent growth defect on stan-dard growth medium, but showed a reduced vegetativegrowth under hypertonic condition. Since we have readily

succeeded in obtaining targeted disruptants of all fiveMAPK genes as described here, the disruptant ofligDgeneis indeed available as an efficient host for gene-targeting inA. oryzae.

4. Discussion

To develop a highly efficient gene-targeting method forA.oryzae, we generated disruptants of a gene (ligD) encod-ing DNA ligase IV homolog involved in the final step ofnonhomologous end joining (NHEJ). The ligDdisruptantsshowed no apparent growth defect and a similar sensitivity

to toxicity by chemical agents which cause DNA damage,

with exception for higher concentrations of MMS. Genereplacement of the prtRgene encoding a transcription fac-tor for extracellular proteolytic genes using A. nidulans sC

gene as a heterologous selectable marker resulted in 100%

Table 4Frequency of homologous integration at loci of the MAPK genes in NS4(wild-type) and DligD::ptrAdisruptant

Hoststrains

Targetedgenes

Transformantstested



NS4 mpkA 37 2 5mpkB 74 10 14

hogA 43 0 0mpkC 57 1 2mpkD 12 5 42

DligD::ptrA mpkA 22 19 86

mpkB 4 4 100hogA 15 15 100mpkC 22 22 100mpkD 10 8 80

Transformation was carried out with the targeting plasmid harboring

A. nidulans sC gene as a heterologous selectable marker. Homologousintegration at each MAPK gene locus was confirmed by colony-PCR.a The efficiency of gene-targeting was estimated by the ratio of the


Fig. 4. Generation of disruptants for MAPK genes. (AE) Strategies forhomologous recombination of A. oryzae for MAPK genes disruptionsusing A. nidulans sCgene as a selectable marker. The gray bars indicatethe probes used for hybridization to confirm the gene replacement bySouthern blot analysis. (FJ) Southern blot analyses of the genomic DNAof the DligD::ptrA(lanes 1, 3, 7, 10 and 13), DmpkA(lane 2), DmpkB1, 2and 3 (lanes 4, 5 and 6), DhogA 1 and 2 (lanes 8 and 9), DmpkC1 and 2(lanes 11 and 12) and DmpkD1 and 2 (lanes 14 and 15). The enzymes usedare XbaI (lanes 1 and 2), NdeI (lanes 3, 4, 5 and 6), BamHI (lanes 7, 8 and9), HindIII (lanes 10, 11 and 12), and MfeI (lanes 13, 14 and 15).



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of gene-targeting efficiency in theligDdisruptant (DligD::p-trA) when the length of the homologous flanking regionswas at least longer than 0.5 kb. In addition, we have readilysucceeded in creating disruptions of all five MAPK genespresent in theA.oryzaegenome, by means of the DligD::p-trAstrain. Based on these results, the ligDmutants should

be potentially very useful and convenient tools for geneknockouts in this industrially important fungus.The gene-targeting efficiencies in the ligD disruptant

were as high as 100% for 5 of the 7 genes tested in thisstudy, which were much higher than that (


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integration event occurs, if any, much less than does in kumutant.

For post-genomic exploitation ofA. oryzae, the resultsreported in this article are immensely important. Geneticanalyses in A. oryzae lag far behind other Aspergillus spe-cies from the following difficulties: (i) the low efficiency of

gene-targeting, (ii) small numbers of marker genes avail-able, (iii) multiple nuclei in not only their vegetative cellsbut also their reproductive conidiospores (Ushijima andNakadai, 1987), in contrast to a model fungus A. nidulansthat possesses uninuclear conidiospores (Harris et al.,1994), and (iv) difficulty of inter-strain crosses since thereis no sexual life cycle. The high gene-targeting efficiency(almost 100%) in A. oryzae ligD disruptants reported hereis a significant breakthrough in functional genomics. Itsimportance is exemplified by rapid disruption of all fiveA. oryzae MAPK genes using the ligDdisruptant.

MAPK pathways are highly conserved signaling unitsfound in eukaryotes, and play essential roles in the

response to environmental signals and hormones, growthfactors, and cytokines. Genome sequences of the threeAspergillus species (A. oryzae, A. nidulans and A. fumiga-tus) have revealed that the numbers of MAPK kinase(MAPKK) and MAPK kinase kinase (MAPKKK) arethree of each throughout the three Aspergilli, and the num-bers of MAPK are four for A. nidulans and A. fumigatus,and five for A. oryzae (Kobayashi et al., 2007). Thus, A.oryzaehas the largest number of MAP kinase genes (desig-nated each hogA/sakA, mpkA, mpkB, mpkC and mpkD)among the three Aspergilli. To examine the utility of theligD disruptant for comprehensive functional genomics,

we attempted firstly the deletion of these five MAPK genesand were successful in isolation of the disruptants for eachgene. In particular, although we were unable to obtain ahogA transformant with targeted integration in the wild-type, all primary transformants possessed the gene replace-ment in the DligD strain. In addition, mpkC showed thesame pattern. In two cases, mpkA and mpkD, however,the targeting efficiencies were also dramatically improvedbut failed to attain 100%. We suspected the relationshipbetween gene-targeting efficiencies and GC content inflanking regions used, but the GC content of each flankingregion was almost the same. The reason is still unclear,although this might be due to the presence of a LigD-inde-pendent NHEJ pathway in A. oryzae. Notwithstanding,taking together such results indicate that the use ofDligDstrain and procedure is very promising for A. oryzaehigh-throughput gene disruption.

SinceA. oryzaehas multinuclear conidiospores, primarytransformants with the targeted integration were found tobe heterokaryons. Hence, homokaryotic disruptants needto be purified and this is carried out conveniently throughsuccessive transfer on selective medium. Such putativehomokaryotic strains were examined phenotypically onstandard growth medium in the absence or presence of salt.Vegetative growth defect was observed in the DmpkAstrain

irrespective of salt, whilst DmpkD displayed significantly

reduced growth under hypertonic growth condition inaddition to relatively reduced vegetative growth in stan-dard culture conditions. MpkD whose orthologs were notobserved in A. nidulansor A. fumigatus, is a likely paralogof HogA in A. oryzae (Kobayashi et al., 2007). Interest-ingly, the mpkD-deficient mutant displayed a significant

growth defect under hypertonic growth condition com-pared to the DhogA strain, suggesting that the MpkDmay be predominantly involved in the osmotic responsepathway in A. oryzae, although it has been reported thatthe hogA/sakA encodes the stress-activated kinase thatresponds 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 therole of each MAPK played in response to environmentalstimuli in A. oryzae, more rigorous analyses of these dis-ruptants are necessary and indeed are in progress. For pre-

cise functional analyses, although the DligD mutantshowed no obvious growth defect so far, a rescue of thewild-type ligD should be taken into account for the possi-bility of a synthetic interaction of deletion of the ligD inthe gene replacement experiments prior to functional anal-yses. In addition to increased sensitivity to high concentra-tions of MMS, the ligD mutants might have defect intelomere maintenance, since Nielsen et al. (2007) havenoted that an nkuAdeletion mutant ofA. nidulans showedshortened telomeres. In this context, it would be desirableto restore the NHEJ activity following the successful dis-ruption of a target gene in the ligD mutant, and for this

purpose a transient disruption system for NHEJ pathwaydeveloped byNielsen et al. (2007) is especially effective inA. oryzae.

In conclusion, we succeeded in significantly improvinggene-targeting efficiency in A. oryzaeby using theligDdis-ruptants, which is comparable to N. crassa mus-53 (lig4)deficient strain (Ishibashi et al., 2006). In this respect, fiveMAPK gene disruptants were promptly and convenientlygenerated via the ligD disruptant. Such results encourageus to create a comprehensive gene disruption library byusing the A. oryzae ligD-deficient strain and the project isnow underway.

Acknowledgments

We thank Takahiro Shintani and Kentaro Furukawafor helpful suggestions and Yoshimi Watanabe, AkiraYoshimi, Jun-ichiro Marui and Tomonori Fujioka forhelpful discussions and/or technical assistance. We are alsograteful to James R. Kinghorn for critical reading of themanuscript. This work was supported by Grant-in-Aidfor Scientific Research on Priority Areas Applied Genom-ics(No. 17019001) from the Ministry of Education, Cul-ture, Sports, Science and Technology of Japan to K.G.

and also by Grant-in-Aid for Scientific Research (B) (No.



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18370001) from Japan Society for the Promotion of Science(JSPS) to H.I.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.fgb.2007.12.010.

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http://dx.doi.org/10.1016/j.fgb.2007.07.003http://dx.doi.org/10.1016/j.fgb.2007.07.003

A Defect of LigD (Human Lig4 Homolog) for Nonhomologous

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