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Improved gene targeting in Magnaporthe grisea by inactivationof MgKU80 required for non-homologous end joining q

Francois Villalba a,1, Jerome Collemare b,1, Patricia Landraud b, Karine Lambou b,Viviane Brozek a, Benedicte Cirer a, Damien Morin b, Christophe Bruel b,

Roland Beffa a, Marc-Henri Lebrun b,*

a Biochemistry, Bayer CropScience, Lyon, Franceb UMR2847 Centre National de la Recherche Scientifique/Bayer CropScience, 14 Rue Pierre Baizet, 69263 Lyon Cedex 09, France

Received 7 March 2007; accepted 19 June 2007Available online 10 July 2007

Abstract

The ascomycete Magnaporthe grisea is a model species for the study of plant fungal interactions. As in many filamentous fungi, tar-geted gene replacement occurs at low frequency in M. grisea (average 7%). mus52/KU80 is a gene essential for non-homologous end join-ing (NHEJ) of DNA double-strand breaks. Its deletion increases the frequency of targeted gene replacement in fungi [Ninomiya, Y.,Suzuki, K., Ishii, C., Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for non-homologous end joining.Proc. Natl. Acad. Sci. USA 101(33), 12248–53]. M. grisea KU80 deletion mutants were constructed and displayed wild-type phenotypesregarding pathogenicity, growth, sporulation and mating. MgADE4 targeted gene replacement frequency was increased in Dku80

mutants (80% vs 5%) and high frequencies (>80%) were observed at seven other loci. However, the deletion of MgKU80 did not increasethe frequency of ACE1 replacement indicating that this locus has an intrinsic reduced ability for gene replacement. These results open theway to large-scale reverse genetics experiments in M. grisea facilitating the study of the infection process.� 2007 Elsevier Inc. All rights reserved.

Keywords: Magnaporthe grisea; KU80; Homologous recombination; Non-homologous end joining

1. Introduction

The recent development of genomics in fungi (Galaganet al., 2005; Xu et al., 2006) has highlighted a significantnumber of candidate genes potentially involved in pathoge-nicity on plants (Dean et al., 2005; Xu et al., 2006). Theirstudy is hampered by the difficulty in constructing nullmutants by targeted gene replacement, since its frequencyis low (<5%) in most filamentous fungi (Weld et al.,2006). Targeted gene replacement requires the cellular

machinery involved in the repair of DNA double-strandbreaks through two main pathways: (1) repair by homolo-gous recombination, and (2) repair of DNA double-strandbreaks independently of sequence homology using the non-homologous end joining pathway (NHEJ, Aylon andKupiec, 2004). In the yeast Saccharomyces cerevisiae,homologous recombination is the main pathway involvedin repairing DNA double-strand breaks (Takita et al.,1997), whereas in mammals NHEJ is the most active path-way (Tachibana, 2004). NHEJ involves the binding of a Kucomplex (K70–Ku80 heterodimer) to broken DNA ends. ADNA-dependent protein kinase (DNA-PKcs) is recruitedby the Ku complex and phosphorylates the DNA exonucle-ase Artemis. This complex stimulates the binding of DNAligase IV—Xrcc4 at the broken DNA ends (Hefferin andTomkinson, 2005). The Ku70 and Ku80 proteins were alsoidentified in insects, plants, and fungi (Hefferin and

1087-1845/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.fgb.2007.06.006

q Submitted for the ‘‘Technical Advances’’ section of Fungal Geneticand Biology.

* Corresponding author. Fax: +33 472 852 297.E-mail address: marc-henri.lebrun@bayercropscience.com (M.-H.

Lebrun).1 These authors contributed equally to the work.

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Fungal Genetics and Biology 45 (2008) 68–75

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Tomkinson, 2005). In the ascomycete Neurospora crassa,these proteins are encoded by mus-51 and mus-52, respec-tively. The deletion of these genes leads to a large increasein the frequency of targeted gene replacement suggestingthat the NHEJ pathway competes with homologousrecombination for the integration of exogenous DNA frag-ments (Ninomiya et al., 2004; Ishibashi et al., 2006).Increased frequency (70–100%) of targeted gene replace-ment was also observed in other fungal KU70 or KU80

deletion mutants (Aspergillus fumigatus, da Silva Ferreiraet al., 2006 and Krappmann et al., 2006; Cryptococcus neo-

formans, Goins et al., 2006; Aspergillus sojae/oryzae,Takahashi et al., 2006; Aspergillus nidulans, Nayak et al.,2006; Sordaria macrospora, Poggeler and Kuck, 2006).These Dku70 and Dku80 mutants had a normal growth,mycelium morphology, ascospores or basidiospores (C.

neoformans) and were as pathogenic on mice as corre-sponding wild-type strains (A. fumigatus and C. neofor-

mans). These mutants have stimulated large-scale geneknock-out experiments in N. crassa (Colot et al., 2006).Extending these techniques to plant pathogenic fungiwould facilitate deciphering mechanisms involved in path-ogenicity through large-scale reverse genetics experiments.

The ascomycete Magnaporthe grisea is responsible forthe rice blast disease and this fungus is a model organismfor the study of plant fungal interactions. Its genomicsequence is known (Dean et al., 2005) and extensive studieshave been performed to identify the molecular mechanismsinvolved in the infection process (Xu et al., 2007). How-ever, targeted gene replacement occurs at low frequency(average 7%) in this fungal species (Talbot and Foster,2001). In this study, we assayed the effect of the inactiva-tion of MgKU80 involved in the NHEJ pathway on the fre-quency of targeted gene replacement in M. grisea. TheMgKU80 gene orthologous to N. crassa mus-52 (Ninomiyaet al., 2004) was identified in M. grisea genome. MgKU80

was deleted in two different M. grisea isolates (P1.2,Guy11) by targeted gene replacement. This deletionincreases the frequency of targeted gene replacement ateight independent loci tested (>80%), but not at theACE1 locus.

2. Material and methods

2.1. Fungal strains and growth conditions

Wild-type M. grisea isolates (P1.2 and Guy11) patho-genic on rice and barley were obtained from the Centre deCooperation Internationale pour la Recherche Agronomi-que et le Developpement (CIRAD). For sporulation,Guy11 and P1.2 were grown on ‘‘Rice-Guy11’’ agar (20 g/L rice flour, 10 g/L glucose, 2 g/L KH2PO4, 3 g/L KNO3,agar 15 g/L, adjusted to pH 6) and ‘‘Rice-P1.2’’ agar(20 g/L rice flour, 2 g/L yeast extract, agar 15 g/L), respec-tively. Other media have the following composition. TNK-CP derived from Tanaka-B medium (Ou, 1985): 10 g/Lglucose, 2 g/L yeast extract, 2 g/L NaNO3, 2 g/L KH2PO4,

0.5 g/L MgSO4Æ7H2O, 0.1 g/L CaCl2Æ2H2O, 0.004 g/LFeSO4Æ7H2O, microelements as Tanaka-B, adjusted to pH5.5–5.8. TNK-CP-Hyg contains 0.12 g/L hygromycin(Sigma–Aldrich, Saint-Louis, USA). TNK-MM: TNK-CPwithout yeast extract supplied with thiamine 1 mg/L andbiotine 1 lg/L after sterilization. TNK-CP-Su-Hyg: sameas TNK-CP supplied with 200 g/L sucrose instead of glucoseand 0.24 g/L hygromycin. SW-Basta derived from Sweigardet al. (1997): 1.6 g/L YNB w/o amino-acid, 2 g/L aspara-gine, 1 g/L NH4NO3, 1 g/L glucose, 0.035 g/L glufosinate(Basta, Cluzeau Info Labo, Ste Foy la Grande, France)adjusted to pH 6. All solid media contained agar at 15 g/Lexcept TNK-MM supplied with 8 g/L agarose.

2.2. Construction of the gene replacement cassettes

Upstream and downstream regions of the genes of inter-est were obtained by PCR using genomic DNA (100 ng) astemplate, primers pairs KO1gene/KO2gene and KO3gene/KO4gene and 0.5 U of DNA polymerase Phusion (Finn-zymes, Espoo, Finland). The primers are listed in (Supple-mentary Table S1) and the strategy used for constructingthe gene replacement cassettes is derived from Kamper(2004). Purified amplicons were digested by SfiI whoserestriction sites are present in primers KO2gene (SfiIa) andKO3gene (SfiIb). BAR or HPH cassettes (Sweigard et al.,1997) conferring resistance to glufosinate or hygromycinwere excised from plasmid pFV9 or pFV8, respectively(pB2KS� vectors carrying BAR or HPH cassettes flankedby SfiIa and SfiIb sites). Following ligation, the genereplacement cassette was obtained by amplification usingprimers KO5gene and KO6gene. For the SER3 gene,KO2gene and KO3gene contained a BamHI and a PstIrestriction site, respectively, and the HPH cassette wasexcised from a pUC-HPH vector by digestion with thesetwo enzymes. The ACE1 deletion cassette was constructedby Bohnert et al. (2004).

2.3. M. grisea transformation

Three micrograms of transforming DNA suspended in20 ll water were mixed with 20 ll STC (200 g/L sucrose,50 mM Tris–HCl, pH 7.5, 50 mM CaCl2Æ2H2O). Frozenprotoplasts (107 in 200 ll) prepared as described (Bohnertet al., 2004) were thawed on ice and added to the bufferedDNA. After 15-min incubation at room temperature, 1 mLof 60% PEG-4000 (NBS Biologicals, Huntingdon, UK) in0.6 M sorbitol, 10 mM MOPS was slowly added to the pro-toplasts. Following gentle mixing and 20 min incubation atroom temperature, 3 mL of TB3 (200 g/L sucrose, 3 g/Lyeast extract) were added. The suspension was shaken(110 rpm) at 26 �C for 3 h (hygromycin) or overnight (Bas-ta/glufosinate). The protoplasts were concentrated to 1 mLby centrifugation at 3500 rpm, mixed to 4 ml of meltedTB3-agar containing 5 g/L bacteriological agar (Oxoid,Basingstoke, UK), overlaid onto either TNK-CP-Su-Hygor SW-Basta plates and incubated at 30 �C for 5–7 days.

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Growing colonies were transferred to new TNK-CP-Hygor SW-Basta plates for assessing resistance. Resistant colo-nies were further grown on rice agar media for 7–10 days at26 �C, purified by single-spore isolation, tested for resis-tance and stored as dried filter paper disks at �20 �C.

2.4. Molecular analysis of the transformants

Genomic DNA, prepared as described in Bohnert et al.(2004) from lyophilised mycelium, was used as template(100 ng) for PCR amplifications with the primers describedin Table S1. Geneexon5 0 and Geneexon3 0 were used to amplifythe gene of interest in wild-type and ectopic transformants.BAR (or HPH) and KO4gene pairs or the TRPC/KO1gene

pair were used to amplify DNA fragments correspondingto respectively the 5 0- and 3 0-cassette-locus junctions intransformants resulting from targeted gene replacement.For Southern hybridization, genomic DNA (5 lg) digestedwith BamHI was subjected to 1% agarose gel electrophore-sis, transferred to positive nylon membrane (Q-BIOgene,Irvine, USA) and hybridized at 65 �C in Denhardt’s bufferwith [32P]-labelled probes. The radioactive signals wererevealed using a phosphoimager (Molecular Dynamics,Sunnyvale, USA).

2.5. Phenotypic analysis

Following central inoculation of rice, TNK-CP or PDAagar media with calibrated mycelium plugs (4 mm diameter),radial growth (colony diameter) was measured daily duringincubation at 26 �C in the dark. Auxotrophy was assessed bytransferring mycelial plugs onto TNK-MM agar medium.Control plates were supplemented with 1 mM adenine, ser-ine, histidine or threonine and methionine according to theexpected auxotrophic defect. Cold sensitivity was assessedby incubating 3 days old rice plates for 7, 15, 30 and 45 daysat 4 �C followed by incubation at 26 �C and monitoringradial growth daily. Conidia were collected from 10–14 daysold mycelium cultures grown on rice media. Conidia germi-nation was assayed using 103 spores/ml suspensions depos-ited onto 3% agar plates. Spore germination wasmonitored by observing spores with germ tubes under a ste-reomicroscope after 16h of incubation at 26 �C. Seedlingsfrom susceptible (C039, Sariceltik) or Pi33-resistant (Bala,C101Lac) rice cultivars were cultivated for 20 days (70% rel-ative humidity; 25 �C day, 20 �C night). Four-leaf stageplants were fed with 15 mL ammonium sulphate (12 g/L),sprayed with a suspension (10 mL/pot) of 3 104 spores/mlcontaining 3 g/L gelatine, incubated overnight in a humidchamber (25 �C, 99% humidity, dark) and transferred to agreenhouse for 6 days (25 �C, 50% humidity, 12-h light).Lesions were scored at 7 days after inoculation.

2.6. Bioinformatics and genes studied

Blast and protein domains searches were performedat the Broad Institute (M. grisea database: http://www.

broad.mit.edu), EBI (Interproscan: http://www.ebi.ac.uk/interproscan) and NCBI (Blast—nr: http://www.ncbi.nlm.nih.gov/blast/). Alignments were performed with ClustalWand displayed using Genedoc. Phylogenetic analyses wereperformed using Mega 3.1. MgKU80 (XP_365937.1,MGG_10157.5, chromosome I) is orthologous to N. crassamus-52. MgADE4 encoding an amido-phosphoribosyl-transferase (XP_362173.1, MG_04618.5, chromosome I),MgHOM2 encoding an aspartate-semialdehyde dehydro-genase (XP_366975.1, MGG_03051.5, chromosome VII),MgSER3 encoding a 3-phosphoglycerate dehydrogenase(XP_367282, MGG_07207.5, chromosome I) and MgHIS1

encoding an ATP phosphoribosyl-transferase (XP_361911.1,MGG_04356.5, chromosome I) are required for the bio-synthesis of adenine, threonine/methionine, serine and his-tidine, respectively. MGG_02061.5 (XP_365359.1,chromosome II), MGG_ 02065.5 (XP_365363.1, chromo-some II), MGG_08113.5 (XP_362530.1, chromosome I)and MGG_13913.5 (chromosome III), encode respectivelya putative protein kinase, a putative protein with a tetrat-ricopeptide repeat domain (TRP, PF00515), a pseudo-tet-raspanin and a novel tetraspanin. The ACE1 gene(CAG28797.1, MGG_12447.5, chromosome I) encodes aPKS-NRPS. Phylogenetic analysis was conducted usingPhyML v2.4.4 (maximum-likelihood algorithm; Guindonand Gascuel, 2003). The resulting tree was drawnusing MEGA version 3.1 (Kumar et al., 2004). Numbersat each node indicates percentage of 100 bootstrapreplicates. MgKu80: M. grisea (XP_365937.1); MUS-52:N. crassa (BAD16623.1); CHGG_05790: Chaetomiumglobosum (XP_001221885.1); AcKu80: Aspergillus clavatus

(EAW11519.1); AfKu80: A. fumigatus (XP_749397.1);AN4552.2: A. nidulans (XP_662156.1); AoKu80: A. oryzae

(BAE78503.1); AsKu80: A. sojae (BAE78504.1); AtKu80:Aspergillus terreus (XP_001209605.1); FG06721.1: Fusar-

ium graminearum (XP_386897.1); CIMG_01804: Coccidioi-

des immitis (EAS36450.1); BC1G_10199: Botrytis cinerea;SS1G_07128: Sclerotinia sclerotiorum; SNOG_11900: Pha-eosphaeria nodorum (EAT80944.1); HDF2: Saccharomyces

cerevisiae (CAL35976.1).

3. Results

3.1. Identification and deletion of the M. grisea KU80 gene

The N. crassa Mus52 protein sequence (Ninomiya et al.,2004) was used to perform a BlastP search of the M. griseaprotein database at the Broad Institute web site (http://www.broad.mit.edu), highlighting MGG_10157.5 as a sin-gle hit (e-value 1e-167). MGG_10157.5 displayed 55% iden-tity and 72% similarity with N. crassa Mus52, its closestrelative, and clusters with other known fungal KU80 genes(Fig. 1). Two characteristic Ku80 functional domains, theN-terminal a/b domain (PF03731) and the central b-barreldomain (PF02735) were detected in MGG_10157.5 proteinsequence. To delete the corresponding gene designated asMgKU80, we constructed a gene replacement cassette in

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which BAR is flanked on each side by 1.5 kb of upstreamand downstream genomic sequences from the MgKU80

locus (Fig. 2a). This gene replacement cassette wasintroduced into M. grisea wild-type isolates Guy11 andP1.2 by protoplast transformation. KU80 or possiblevector junction fragments were amplified by PCR inpurified resistant transformants. 17 of 79 (21.5%) P1.2transformants and 12 of 35 Guy11 transformants (34%)displayed PCR amplification products characteristic of aDku80::bar gene replacement event. Some of these puta-tive P1.2 and Guy11 Dku80::bar mutants were analyzedby Southern hybridization and all their hybridizationprofiles were congruent with a single gene replacementevent (Fig. 2b).

3.2. Phenotypic characterisation of the M. grisea Dku80::bar

mutants

Deletion of MgKU80 in either P1.2 or Guy11 had noeffect on mycelial growth, as deduced from the monitoringof radial growth on three different agar media (rice, TNK-

CM, PDA). We did not observe significant differencesbetween parental strains and their corresponding Dku80::-

bar mutants with respect to sporulation and spore germina-tion rates (Table 1). Sexual reproduction was unaffected inthe KU80 deletion mutants, as crosses with tester strainsfrom opposite mating type led to the formation of matureperithecia and ascospores (data not shown). Sensitivity ofDku80::bar mutants to cold treatment was analyzed as pri-mary transformants are stored at 4 �C while their purifica-tion and resistance testing process is performed. Up to45 days at 4 �C, both mutant and wild-type isolates wereable to resume growth, showing that the Dku80::bar

mutants were as cold resistant as wild-type isolates.Finally, pathogenicity of Dku80::bar mutants on plantswas assayed by inoculating spore suspensions on suscepti-ble rice (CO39, Sariceltik) or barley (Plaisant) cultivars.The KU80 deletion mutants were qualitatively and quanti-

Fig. 1. Phylogenetic tree of fungal Ku80 proteins. Alignment wasperformed using ClustalW and edited in GeneDoc (SupplementaryFig. S1). Phylogenetic analysis was restricted to ascomycetes and yeastand conducted with PhyML v2.4.4 (maximum-likelihood algorithm;Guindon and Gascuel, 2003). The resulting tree was plotted using MEGAversion 3.1 (Kumar et al., 2004). Numbers at each node indicatespercentage of 100 bootstrap replicates. MgKu80: Magnaporthe grisea

(XP_365937.1); Mus52: Neurospora crassa (BAD16623.1); CHGG_05790:Chaetomium globosum (XP_001221885.1); AcKu80: Aspergillus clavatus

(EAW11519.1); AfKu80: A. fumigatus (XP_749397.1); AN4552.2: A.

nidulans (XP_662156.1); AoKu80: A. oryzae (BAE78503.1); AsKu80: A.

sojae (BAE78504.1); AtKu80: A. terreus (XP_001209605.1); FG06721.1:Fusarium graminearum (XP_386897.1); CIMG_01804: Coccidioides imm-

itis (EAS36450.1); BC1G_10199: Botrytis cinerea; SS1G_07128: Scleroti-

nia sclerotiorum; SNOG_11900: Phaeosphaeria nodorum (EAT80944.1);HDF2: Saccharomyces cerevisiae (CAL35976.1).

Fig. 2. Targeted replacement of MgKU80 in M. grisea. (a) Constructionof the MgKU80 replacement cassette. Genomic DNA sequences flankingMgKU80 were amplified by PCR, digested by SfiI and ligated to a Basta/glufosinate resistance cassette (bar). The ligation product was used as atemplate to amplify the gene replacement cassette by PCR using internalprimer (KO5, KO6). (b) Analysis of transformants by Southern hybrid-isation. Wild-type MgKU80 and expected Dku80::bar loci are shown onthe left with BamHI genomic restriction sites. DNA fragments used asprobes are indicated as thick lines. On the right, BamHI hybridisationprofiles of four Dku80::bar mutants (1–4), wild-type P1.2 strain (P) and atransformant exhibiting a single ectopic integration of the gene replace-ment cassette (5).

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tatively as pathogenic as their corresponding wild-type iso-lates, as illustrated for rice in Fig. 3. Avirulence to resistantrice cultivars carrying the Pi33 resistance gene (Bala,C101Lac) was also assayed for the Guy11 Dku80::bar

mutant, revealing an avirulent behaviour similar to paren-tal Guy11 isolate (Bohnert et al., 2004). Altogether, theseresults show that the deletion of MgKU80 does not affectmycelial growth, sporulation, mating and pathogenicity/avirulence of M. grisea.

3.3. Improved gene targeting in M. grisea Dku80::bar strains

To assay the effect of MgKU80 deletion on the fre-quency of targeted gene replacement, we first used theMgADE4 gene (MGG_04618.5) that is orthologous to S.

cerevisiae ADE4 required for adenine biosynthesis. TheMgADE4 replacement cassette was constructed with 1.5-and 0.76-kb upstream and downstream genomic sequencesflanking a HPH hygromycin resistance cassette. 223 hygro-mycin-resistant transformants were tested for their auxot-

rophy by transfer onto minimal medium (TNK-MM)supplemented or not with adenine (Fig. 4). When wild-typestrains P1.2 and Guy11 were used as receivers in the trans-formation experiment, 5–11% of the transformants wereauxotrophic for adenine (no growth on minimal medium).When the corresponding Dku80::bar mutants were used asreceivers, the percentage of adenine auxotrophs increasedto 83–97% (Table 2). These results show that the deletionof MgKU80 increased the frequency of targeted genereplacement at the MgADE4 locus in both P1.2 andGuy11 genetic backgrounds. Three other M. grisea geneswhich disruption should lead to auxotrophy were selectedfor further gene targeting experiments in the P1.2-Dku80::-

bar mutant. MgHOM2 (MGG_03051.5), MgSER3(MGG_07207.5) and MgHIS1 (MGG_04356.5) are ortho-logue genes of S. cerevisiae HOM2, SER3 and HIS1

required for the biosynthesis of threonine/methionine, ser-ine and histidine, respectively. The three correspondinggene replacement cassettes were constructed and intro-duced by transformation into the P1.2-Dku80::bar mutant.For MgHOM2, 12 of 15 hygromycin-resistant transfor-mants were auxotrophic for threonine/methionine (Table2). PCR analysis confirmed the replacement of HOM2 bythe HPH resistance cassette in the 12 auxotrophic mutantsanalyzed. Similar results were obtained for the targetedgene replacement of MgSER3 and MgHIS1, as 87% and78% of hygromycin-resistant transformants were auxo-troph for serine and histidine, respectively (Table 2).PCR analysis confirmed targeted gene replacement atMgSER3 and MgHIS loci in the 15 and 9 auxotrophictransformants tested. In addition, four genes that couldbe involved in pathogenicity according to their biochemicalfunctions (see below) were selected for assessing the fre-quency of targeted gene replacement in the P1.2-Dku80::-

Table 1Sporulation and conidia germination in Dku80::bar mutants

Strains

P1.2 P1.2-Dku80 Guy11 Guy11-Dku80

Sporulation 5.2 ± 1.7 4.6 ± 1.8 5.6 ± 0.7 7.4 ± 1· 106 conidia/mL · 105 conidia/mL

Germination (%) 100 98 98 98

Wild-type P1.2 and Guy11 isolates and their corresponding Dku80::bar

mutants were grown for 11 days on rice agar media. Spores were collectedfrom the mycelium, plated onto 3% water agar medium and incubatedovernight. Spore germination was monitored by observing spores withgerm tubes under a stereomicroscope (100 conidia per sample in triplicateexperiments).

Fig. 3. Pathogenicity of Dku80::bar mutants on rice. Spores from wild-type P1.2 and Guy11 isolates and their corresponding Dku80::bar mutantswere spray-inoculated on susceptible rice cultivar CO39 (four-leaf stage).Grey–brown foliar lesions formed upon infection were observed 7 daysafter inoculation and scored (10 different leaves for each strain).

Fig. 4. Adenine auxotrophic mutants obtained by targeted replacement ofMgADE4. MgADE4 replacement cassette was introduced by transforma-tion into the wild-type isolate Guy11 and its corresponding Dku80::bar

mutant. Hygromycin-resistant transformants growing on complete med-ium (TNK-CP) were transferred onto (left) minimal medium (TNK-MM)or minimal medium supplemented with 1 mM adenine (right). Wild-type islocated at top left corners (white square). Dade4::hph mutants are visibleas transformants that do not grow on minimal medium while growingnormally on minimal medium supplemented with adenine. Ectopictransformants carrying the gene replacement DNA cassette integrated atanother locus than the target gene behave as wild-type.

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bar mutant. These genes, MGG_02061.5, MGG_02065.5,MGG_08113.5 and MGG_07922.5, encode respectively aputative protein kinase, a protein with a TRP domain(PF00515), a pseudo-tetraspanin and a novel tetraspaninunrelated to Pls1. Nine to fifteen hygromycin-resistanttransformants were isolated for each experiment and testedfor gene replacement by PCR. As shown in Table 2, a highfrequency of gene replacement (78–100%) was observed atthese four loci. These results demonstrate that the deletionof MgKU80 dramatically increases the efficiency of tar-geted gene replacement at ADE4 locus in M. grisea andleads to a high frequency of gene replacement at 7 otherloci.

3.4. Targeted gene replacement at the ACE1 locus in the

Guy11-Dku80::bar mutant

ACE1 is a M. grisea avirulence gene required for theproduction of a signal recognized by rice cultivars carryingthe Pi33 resistance gene (Bohnert et al., 2004). Virulentace1 null mutants were obtained by targeted replacementof Guy11 ACE1 wild-type allele, although at low frequency(1.3%). We investigated the possible impact of MgKU80

deletion on ACE1 replacement frequency. A previouslyconstructed ACE1 replacement cassette conferring hygro-mycin resistance (Bohnert et al., 2004) was used to trans-form protoplasts of a Guy11 Dku80::bar mutant. TheACE1 locus of purified hygromycin-resistant transfor-mants was analysed by PCR. Surprisingly, the wild-typeACE1 allele was amplified in the 55 transformants tested.These results indicate that none of the tranformantsresulted from gene replacement, suggesting that the fre-quency of gene replacement at the ACE1 locus is as lowin the KU80 deletion mutant as in the parental Guy11 iso-late (Table 2). Gene replacement frequency was notincreased in Dku80::bar mutant at two other loci(MGG_08391.5 and MGG_08380.5) located close toACE1 (Collemare et al., 2007). These results suggest thatthe ACE1 genomic region has a reduced ability for homol-

ogous recombination that cannot be improved by the sup-pression of NHEJ mediated by the deletion of MgKU80.

3.5. Pathogenicity of deletion mutants obtained in M. grisea

Dku80

The eight mutants obtained in P1.2 Dku80 isolate werefurther characterized for their pathogenicity on barleyand rice (data not shown). Dade4 mutant displayed a verystrong reduction in pathogenicity since only small necroticspots were observed 7 days after infection. These spots didnot further enlarge to wild-type lesions size. Dhom2 andDhis1 mutants were moderately reduced in pathogenicityas they produced small lesions at 7 days that enlarged towild-type size at 11 and 14 days after rice infection, respec-tively, suggesting that these mutants are only delayed inpathogenicity on rice. However, Dhis1 is strongly reducedon barley with few lesions 7 days after infection that didnot enlarge. Dser3 mutant is as pathogenic as wild-typeon barley and rice. Similarly, the four deletion mutantsof the candidate pathogenicity genes (MGG_02061.5,MGG_02065.5, MGG_08113.5 and MGG_07922.5) wereas pathogenic as wild-type on barley. These four genesare likely not involved in the infection process or are onlyminor pathogenicity factors.

4. Discussion

Inactivation of the NHEJ DNA repair pathway throughthe deletion of KU80 or KU70 genes greatly increases thefrequency of targeted gene replacement in fungi such asN. crassa (Ninomiya et al., 2004), Aspergillus spp. (Takah-ashi et al., 2006; Nayak et al., 2006), C. neoformans (Goinset al., 2006) and S. macrospora (Poggeler and Kuck, 2006).We have developed a similar strategy for the ascomyceteM. grisea. A single MgKU80 gene orthologous to mus-52

from N. crassa was identified in M. grisea(MGG_10157.5). The protein sequence deduced fromMgKU80 has a high degree of homology to fungal Ku80

Table 2Increased frequency of targeted gene replacement using Dku80::bar mutants

Genes Frequency of targeted gene replacement

P1.2 P1.2-Dku80 Guy11 Guy11-Dku80

1. MGG_04618.5 (MgADE4) 4.9% (223) 97% (60) 11.5% (96) 83% (93)2. MGG_03051.5 (MgHOM2) 80% (15)3. MGG_07207.5 (MgSER3) 87% (15)4. MGG_04356.5 (MgHIS1) 78% (9)5. MGG_02061.5 (kinase) 80% (10)6. MGG_02065.5 (TPR protein) 78% (9)7. MGG_08113.5 (pseudo-tetraspanin) 82% (11)8. MGG_13913.5 (novel tetraspanin) 100% (15)9. MGG_12447.5 (ACE1, PKS-NRPS) 1.3% (150) 0% (55)

Gene replacement cassettes were constructed for four M. grisea genes involved in the biosynthesis of adenine or amino-acids (1–4), four candidatepathogenicity genes (5–8) and the ACE1 avirulence gene (9). Hygromycin-resistant tranformants were obtained with the wild-type P1.2 and Guy11isolates, or their corresponding Dku80::bar mutants. The targeted genomic loci were analysed by PCR as indicated in the text. For genes 1–4, replica-plating of the transformants onto minimal medium deprived of adenine or amino acid was also performed. The frequencies of gene replacement are givenin percentages. The total number of transformants analysed is shown in brackets.

F. Villalba et al. / Fungal Genetics and Biology 45 (2008) 68–75 73

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proteins defining a family of orthologous fungal genes(Fig. 1). Ku80 is involved in NHEJ DNA repair, interactsphysically with telomeric regions in human (Hsu et al.,1999), and plays a role in telomere length maintenance inyeast, protozoa, plants and mammals (Manolis et al.,2001; Janzen et al., 2004; Riha et al., 2002; Bailey et al.,1999). To ensure that the absence of this protein does notimpair M. grisea developmental processes or pathogenicity,we have performed a detailed phenotypic comparison oftwo independent M. grisea KU80 deletion mutants withtheir corresponding wild-type parental isolates. P1.2 is astrain that has been developed in our laboratory for itsenhanced fertility in crosses and sporulation rate, whileGuy11 is a reference isolate widely used for molecular stud-ies. The comparison of the two corresponding KU80 dele-tion mutants allowed us to assess whether its deletion hasstrain-dependent effect or not. Our results demonstrate thatthe two Dku80::bar mutants are similar to their parentswith regards to mycelium growth, sporulation, spore ger-mination rate and mating. Their pathogenicity was unaf-fected on susceptible and resistant rice cultivars. Theseresults are similar to those obtained in other fungi forwhich the absence of KU80 does not affect mycelial growth,sporulation or mating (Ninomiya et al., 2004; da SilvaFerreira et al., 2006; Krappmann et al., 2006; Poggelerand Kuck, 2006; Nayak et al., 2006). Deletion of KU80

in fungi pathogenic on human such as A. fumigatus andC. neoformans does not impair their pathogenicity on mice(da Silva Ferreira et al., 2006; Goins et al., 2006). Ourresults extend the use of KU80 deletion mutants describedin fungal saprobes or human pathogens to a fungal speciespathogenic on plants.

In M. grisea, the frequency of targeted gene replacementobtained using the transformation of protoplasts with linearDNA cassettes, is generally low (average 8%, Talbot andFoster, 2001), and varies according to the locus (0–40%).Deletion of MgKU80 increases targeted gene replacementfrequency from 5–10% to 80–100% at ADE4 locus. High fre-quencies of gene replacement (78–100%) were also obtainedat seven other loci in the Dku80::bar mutant background.Such high frequencies have never been observed at any lociin M. grisea as the average rate of targeted gene replacementis about 7% (Talbot and Foster, 2001). In our experiment,average frequency of targeted gene replacement at theseseven loci is over 90%. Therefore, we attributed this high fre-quency of targeted gene replacement to the deletion ofKU80. The eight loci tested are located on different chromo-somes (chromosomes I, II, III and VII), suggesting that thisproperty of KU80 deletion mutants is relevant for mostother M. grisea loci. This property has important practicalconsequences as only few transformants are now needed toobtain mutants by targeted gene replacement. This repre-sents a significant improvement and should facilitate highthroughput reverse genetics in M. grisea.

This study also identify a locus (ACE1) for which tar-geted replacement frequency is not increased in a KU80

deletion mutant. As ACE1 is not located close to the chro-

mosome I centromere or telomeres, this result suggests thatthe ACE1 locus has a reduced ability for homologousrecombination that cannot be improved by the absence ofthe NHEJ DNA repair pathway. In fungi, the frequencyof targeted gene replacement greatly varies according toloci (Hasty et al., 1994; Weld et al., 2006). These variationsmay be related to locus-specific properties that could inhi-bit or stimulate homologous recombination locally. In par-ticular, differences in chromatin structure could modify theaccessibility of nuclear proteins to DNA, locally affectingthe efficiency of DNA double-strand break repair andhomologous recombination (Moore and Krebs, 2004). Itis tempting to speculate that the low frequency of targetedgene replacement at the ACE1 locus, regardless of the pres-ence or absence of Ku80 and NHEJ, results from a partic-ular chromatin status. One additional argument supportingthis hypothesis is that ACE1 belongs to a cluster of genesinvolved in secondary metabolism (Bohnert et al., 2004;Collemare et al., 2007). Genes from such clusters are sus-pected to have a particular chromatin status when theyare not expressed (Yu and Keller, 2005; Bok et al., 2006).Although additional experiments are needed to furtherexplore the mechanisms involved in the inhibition of tar-geted gene replacement at ACE1 locus, this example sug-gests that some loci, likely few, are insensitive to thepositive effect of KU80 deletion.

In conclusion, we have improved targeted gene replace-ment in M. grisea using KU80 deletion mutants defectivefor NHEJ DNA repair. In this genetic background, theconstruction of null mutants becomes a high-throughputprocess that could be applied to a large number of genes.This strategy will be very useful for the genetic analysisof complex biological processes such as pathogenicity onplants for which a large number of candidate genes hasbeen identified through the analysis of M. grisea genomeor differential gene expression patterns during infection.

Acknowledgments

We thank D. Tharreau and J. Milazzo (BGPI, CIRAD,Montpellier, France) for wild-type M. grisea strains andmating analysis of Dku80::bar mutants. The help of G.de Gesualdis (Bayer Cropscience, Lyon, France) for ricecultivation is also greatly appreciated.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.fgb.2007.06.006.

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