Functional identification of high-affinity iron permeases from Fusarium graminearum

Fungal Genetics and Biology 43 (2006) 273–282 www.elsevier.com/locate/yfgbi

Functional identiWcation of high-aYnity iron permeases from Fusarium graminearum Yong-Sung Park a, Il-Dong Choi a, Chang-Min Kang a, Mun-Sik Ham a, Ji-Hyun Kim a, Tae-Hyoung Kim b, Sung-Hwan Yun c, Yin-Won Lee d, Hyo-Ihl Chang a, Ha-Chin Sung a, Cheol-Won Yun a,¤ b

a School of Life Sciences and Biotechnology, Korea University Anam-dong, Sungbuk-gu, Seoul, Republic of Korea Chosun University School of Medicine, Department of Biochemistry and Molecular Biology, 375 Seosuk-Dong, Dong gu, Gwangju, Republic of Korea c Division of Life Sciences, Soonchunhyang University, Asan 336-745, Republic of Korea d School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Republic of Korea

Received 14 July 2005; accepted 19 December 2005 Available online 7 February 2006

Abstract The ScFTR1 gene encodes an iron permease in Saccharomyces cerevisiae. Its homologues, FgFtr1 and FgFtr2, were identiWed from Wlamentous pathogenic plant fungus, Fusarium graminearum. Homologies between the deduced amino acid sequences of ScFtr1p and FgFtr1 and FgFtr2 were 56 and 54%, respectively, and both had REXXE sequences, which form the conserved amino acid sequence of ScFtr1p. FgFtr1 expression increased under iron depletion, and although FgFtr2 mRNA was not detected in the wild-type strain, it was detected in the fgftr1 strain in the iron-depleted condition. When the FgFtr1 and FgFtr2 were deleted, the amount of growth was found not to be diVerent from the wild-type in iron-depleted media. However, the mRNA of FgSid, a homologue of the SIDA of Aspergillus fumigatus, was dramatically increased in the fgftr1/fgftr2 strain and in an iron-depleted condition. FgFtr1 and FgFtr2 genes act as functional complements when they are introduced into the S. cerevisiae Scftr1 strain. The Scftr1 strain, which contains either the FgFtr1 or FgFtr2, grew well in iron-depleted media. Moreover, speciWc alteration of the REXXE consensus sequence of FgFtr1 and FgFtr2 did not allow for sustained growth of the Scftr1 strain on iron-depleted medium. The iron uptake activity was recovered when FgFtr1 and FgFtr2 genes were introduced into the Scftr1 strain. Though the Fet3p in S. cerevisiae was found on the intracellular vesicle in the Scftr1 strain, Fet3p was found on the plasma membrane when FgFtr1 or FgFtr2 was introduced into the ftr1 strain. An infection test was carried out with deletion strains; however, no change in the ability of these strains to cause disease was observed. These results suggest that FgFtr1 and FgFtr2 may function as iron permeases in the reductive iron uptake pathway and that they do not play major roles in the pathogenicity of F. graminearum. © 2005 Elsevier Inc. All rights reserved. Keywords: Fusarium graminearum; Saccharomyces cerevisiae; Iron; Ftr1; Fet3; Aft1

1. Introduction All living organisms require trace elements, such as metals and other factors, which are essential for their growth. Iron is one of these trace elements; because of its fast oxidation–reduction reactions, iron performs important func-

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1087-1845/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2005.12.005

tions in cellular electron transport, enzyme function, and many other physiological pathways. Though iron is one of the most abundant metals in the world, it exists in an insoluble salt form which restricts its use. On the other hand, iron can be very toxic to living organisms because, when present in excess, it induces the formation of hydroxyl radicals (Fenton et al., 1979). Therefore, living organisms have developed unique ways of regulating cellular iron concentrations, and these mechanisms are conserved in many life forms, from microorganisms to mammals.

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The mechanisms of iron metabolism have been studied using Saccharomyces cerevisiae as a model organism. There are two diVerent pathways that allow iron uptake from the environment: the reductive iron uptake pathway and the siderophore-mediated uptake pathway (Yun et al., 2001; Yun et al., 2000a,b). The reductive iron uptake pathway has been well studied and is mediated by Fet3p, a multicopper oxidase (Dancis et al., 1994) and Ftr1p, an iron permease localized on the plasma membrane (Bonaccorsi di Patti et al., 2005). For the uptake of iron from the environment to occur in the reductive iron uptake pathway, iron must be reduced from its insoluble salt form by surface reductase (Askwith et al., 1996; Lesuisse et al., 1996; Shatwell et al., 1996) localized on the plasma membrane; the iron is then taken up by the speciWc iron permease complex, Fet3/Ftr1p. Notably, gene transcripts involved in the reductive iron transport system are regulated by the Aft1p transcription factor (Yamaguchi-Iwai et al., 1995; Yamaguchi-Iwai et al., 1996). It has been reported that Aft1p regulates the expression of the genes involved in the copper transporter (Dancis, 1998; Martins et al., 1998), as well as copper carrier proteins (Chen and Kaplan, 2000). These results show the closely linked relationship between the iron and copper transport systems. Saccharomyces cerevisiae also use a siderophore-mediated mechanism for the uptake of iron from the environment (Kim et al., 2005; Moore et al., 2003; Park et al., 2005). Although S. cerevisiae cannot synthesize siderophore (Neilands, 1995), it uses siderophore synthesized by other microorganisms and takes it up in a complex of siderophore/iron. In S. cerevisiae, four siderophore transporter proteins have been identiWed; the expression of those proteins is regulated by Aft1p and the cellular iron state. The mechanism of siderophore uptake is quite complex, and siderophore-iron complex uptake occurs in two diVerent ways. One form involves use of the reductive iron uptake pathway, which depends on the Fet3p/Ftr1p complex. In brief, iron bound to siderophore is reduced by surface reductase, and released iron is taken up by the Fet3/Ftr1p permease complex. The other form of uptake is the endocytosis-dependent pathway, which is carried out by the Arn1 protein. It has recently been reported that reductive iron uptake and siderophore-mediated iron uptake are important factors in the virulence of many pathogenic fungi, but are not factors in the virulence of Ustilago maydis (Mei et al., 1993; Schrettl et al., 2004). In Wlamentous fungi, the siderophoremediated iron uptake pathway plays an important role in iron metabolism, and the gene products which are involved in iron metabolism are regulated by GATA-factor (Haas, 2003). The reductive iron assimilation pathway of Wlamentous fungi has been reported. Its impact in virulence has been studied, and in the Fusarium graminearum work, it was found to have no inXuence. Its expression also has been studied. For example, Aspergillus fumigatus is a pathogenic animal fungus, and if the gene encoding SidA, which synthesizes the intermediate of siderophore synthesis, is

deleted, the virulence is decreased. However, it was not decreased in the ftrA of the ScFTR1 homologue. In fact, it is diYcult to identify the reductive iron transport system in fungi because of the highly eYcient synthesis of siderophore (Banuett, 1995). Fusarium graminearum is a pathogenic plant fungus that causes head blights of wheat, ear rots of maize, tuber dry rot of potato, bakanae disease of rice, and pitch canker of Pinus species (Han et al., 2005; Rubini et al., 2005; Schmolke et al., 2005). The biological importance of F. graminearum has made the F. graminearum genome project possible; this genome database can now be freely accessed (http://www.broad.mit.edu/annotation/fungi/fusarium/ index.html). We recently identiWed the ScFTR1 homologous genes from F. graminearum; interestingly, two diVerent copies of these homologous genes exist. In this study, we have identiWed a function of the gene products of the ScFTR1 homologues, FgFtr1 and FgFtr2 and the results suggest that they may function as iron transporter in F. graminearum. 2. Materials and methods 2.1. Strains and culture conditions The S. cerevisiae strains used in this study were BY4741 (MATa his31 leu20 met150 ura30) and its derivatives. S. cerevisiae strains were grown in 1% yeast extract, 2% peptone, 2% glucose (YPD), or synthetic deWned (SD) medium (6.7 g/L yeast nitrogen base, Q-BIO gene, USA) supplemented with auxotrophic requirements. Media with deWned iron concentrations were prepared using a modiWed SD minimal medium containing 0.67% yeast nitrogen base without iron and copper, 2% dextrose, and 25 mM MES buVer (pH 6.1), and amino acids with auxotrophic requirements. The iron-limited condition was made by the addition of 100 M bathophenanthroline disulfonate (BPS) and ferrous ammonium sulfate (FAS) at various concentrations. Recombinant Escherichia coli strains were grown on Luria–Bertani agar or liquid medium supplemented with 100 g/ml ampicillin. F. graminearum (lineage 7) wild-type strain Z03643 and all mutant strains generated in this study were cultured and maintained at 25 °C. Fungal strains were maintained and cultured in potato dextrose medium. For fungal transformation, the fungi were grown in CMC liquid medium (15 g carboxylmethyl cellulose, 1 g yeast extract, 0.5 g MgSO4, 1 g NH4NO3, and 1 g KH2PO4 per liter) and YPG liquid medium (3 g yeast extract, 10 g peptone, and 20 g glucose per liter). Plasmid constructions were carried out using E. coli strain XL1-Blue (Stratagene, La Jolla, CA). 2.2. Plasmid construction Total RNA was extracted using the TRIZOL (Invitrogen, USA) method from F. graminearum. Cloning of cDNA fragments and other methods were performed according to

Y.-S. Park et al. / Fungal Genetics and Biology 43 (2006) 273–282

standard protocols. cDNA clones of FgFtrs were obtained by RT-PCR reaction using the following primers: for FgFtr1, 5⬘-ATGGCTGTTGATGTCTTTGCGG-3⬘ as forward primer, and 5⬘-CTATGCTGTTGTGGTCTTTTCA CTG-3⬘ as reverse primer ; for FgFtr2, 5⬘-ATGCTCG AATTGTTTTCGGTCC-3⬘ as forward primer, and 5⬘-CT AGTCTGTCCTGGCGTGCACTTC-3⬘ as reverse primer. The RT-PCR products ampliWed with each primer set were subcloned into pGEMT-easy vector (Promega, USA), and the sequences were conWrmed by DNA sequencing. For the expression vector in S. cerevisiae, the fragments which have ScFTR1 promoter regions were ampliWed by PCR using the following primers: 5⬘-AAAGAGCTCTCATTTTGCGCG CTTCTGTG-3⬘ for forward primer and 5⬘-TCTAGAGGC GGGAAGTATATGTGTGATGAC-3⬘ for reverse primer; and were then digested with SacI and XbaI. Ligation of the SacI/XbaI double-digested fragments of the ScFTR1 upstream region and SacI/XbaI double-digested pRS415 vector resulted in pFTR1-P. The FgFtr genes digested with SpeI and ApaI were introduced to XbaI/ApaI-digested pFTR1-P, resulting in pFgFtrs. For REXXE1 site-directed mutagenesis of FgFtr1, 5⬘-TGTCGTCTTTGGAGGGACT CTGGAGACGGTCATCA-3⬘, 5⬘-TGATGACCGTCTC CAGAGTCCCTCCAAAGACGACA-3⬘; for REXXE2 site-directed mutagenesis of FgFtr1, 5⬘-CACAGTACTAC CCGGGGGTATCGGGGCTGTTGTCTTC-3⬘, 5⬘-GAA GACAACAGCCCCGATACCCCCGGGTAGTACTGT G-3⬘; for REXXE1 site-directed mutagenesis of FgFtr2, 5⬘CGTTGTCTTTGGAGGGACTCTGGAGACTGCCAT CA-3⬘, 5⬘- TGATGGCAGTCTCCAGAGTCCCTCCAA AGACAACG-3⬘; for REXXE2 site-directed mutagenesis of FgFtr2, 5⬘-CACTGTTCTTCCCGGGGGTATTGGAG GCATCGTTTTC-3⬘, 5⬘-GAAAACGATGCCTCCAATA CCCCCGGGAAGAACAGTG-3⬘. The mutagenesis was performed using pfuUltra highWdelity DNA polymerase (Stratagene, USA) with pFgFtr plasmids. The PCR products were then treated with DpnI enzyme for 1 h. The DpnI-digested PCR products were transformed into XL-1 Blue strain. 2.3. Double-joint PCR, gene disruption, and fungal transformation PCR-mediated gene disruption was used to generate deletions of the FgFtr genes. For making the deletion strains of each FgFtr, a deletion cassette containing Geneticin marker was used. For FgFtr1/FgFtr2 double deletion, the strain was created from an FgFtr1-deleted strain using the FgFtr2 cassette containing Hygromycin B marker (Lee et al., 2003). A deletion cassette containing the selection marker was ampliWed using double-joint PCR (Yu et al., 2004) with the following primers: for the 5⬘ Xanking region of FgFtr1, 5⬘-CGGCATATTAACTATCCCTGTCTGC-3⬘ and 5⬘-CCTTCAATATCATCTTCTGTCGAAACAGG AAGCGGAAACGGAG-3⬘; for 3⬘ Xanking region of FgFtr1, 5⬘-GCACAGGTACACTTGTTTAGAGTCACC CACCACAAGAGGCATGG-3⬘ and 5⬘-ATACACCAAC

275

AACCTACGAAAACGTC-3⬘ ; for the 5⬘ Xanking region of FgFtr2, 5⬘-GTTGTCCAAAGTATTCTCTTTCGTGG3⬘ and 5⬘-CCTTCAATATCATCTTCTGTCGAATTCG AGCATCTTGGAGCAACC-3⬘; for 3⬘ Xanking region of FgFtr2, 5⬘-GCACAGGTACACTTGTTTAGAGAAGG AAGTGCACGCCAGGACAG-3⬘ and 5⬘- AGGTCAAT GCATCGGCAACACC-3⬘. For double deletion, the following primers were used: for the 5⬘ Xanking region of FgFtr2, 5⬘-AGAAGGTATGTCTTTCCGGGCTG-3⬘ and 5⬘-TACAGCAT CCAGGG TGACGGTGCAAATGAGT TTATTCCGAAGGCAC-3⬘; for 3⬘ Xanking region of FgFtr2, 5⬘-GAAGAAAAGCTGGCTGGCGGTGCGTC TACATGCGCGCCATCC-3⬘ and 5⬘- CCACGCTCCAA TTTGCGACC-3⬘; for Geneticin (G418) resistance cassette, 5⬘-CGACAGAAGATGATATTGAAGG-3⬘, 5⬘-CTCTA AACAAGTGTACCTGTGC-3⬘; for Hygromycin B resistance cassette, 5⬘-CACCGTCACCCTGGATGCTGTA-3⬘, 5⬘-CACCGCCAGCCAGCTTTTCTTC-3⬘. The Wnal PCR round was performed using the following primers: for FgFTR1, 5⬘-CGATAACTTTGATCTGAATCGGCAG-3⬘, 5⬘-AATCGTTGCCGATCGCTTCG-3⬘; for FgFtr2, 5⬘-TA AGAGCAGCTTGGGGAACTGG-3⬘, 5⬘-GGAGAAGG AACCAAAGCTCAACG-3⬘. The Wnal PCR product sizes of FgFtr1 and FgFtr2 were 4.2 and 4.4 kb, respectively. The PCR products were directly used for fungal transformation. Fungal transformation was carried out as described (Lee et al., 2003) and the transformants were selected on 50 g/ml of Hygromycin B and 200 g/ml of geneticin plates, respectively. Deletion strains were conWrmed by Southern blot analysis. 2.4. Southern and Northern blot analyses Genomic DNA was extracted from F. graminearum grown in potato dextrose broth (PDB) for 3 days at 25 °C. The mycelia were collected by Wltration, ground in liquid nitrogen using mortar, and then suspended in CTAB buVer (2% CTAB (cetyl trimethylammonium bromide), 20 mM EDTA, 0.1 M Tris–HCl, and 1.4 M NaCl). Genomic DNA was extracted with CH3Cl-isoamyl alcohol (24:1), precipitated with isopropyl alcohol, and then treated with RNase (20 g/ml) for 30 min at 37 °C, followed by repeated extraction with phenol:chloroform:isoamyl alcohol (25:24:1). The prepared DNA was used for Southern blot analysis. For the Southern blot of fgftr1, the extracted genomic DNA was digested with NcoI for 6 h, and for the Southern blot of fgftr2, it was digested with AvaI for 6 h. Ten micrograms of the digested DNA was separated on 1% TAE agarose gel for 4 h. The gel was submerged in denaturation solution (0.5 N NaOH, 1.5 M NaCl), and then in neutralization solution (0.5 M Tris–HCl, pH 7.5, 3 M NaCl) for 2 £ 15 min at room temperature. The DNA was blotted to Gene Screen plus membrane (Perkin-Elmer, USA) in 20£ SSC (3 M NaCl. 0.3 M sodium citrate, pH 7.0), as recommended by the supplier. Probes were prepared by PCR in the internal region of the reading frame: for FgFtr1, 5⬘-GCACAGGT AC ACTT GTTTAGAGTCACCCACCACAAGAGGCA

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TGG-3⬘ and 5⬘-ATACACCAACAACCTACGAAAACG TC-3⬘; for FgFtr2, 5⬘-GAAGAAAAGCTGGCTGGCGG TGCGTCTACATGCGCGCCATCC-3⬘ and 5⬘-CCACG CTCCAATTTGCGACC-3⬘. The PCR products were labeled with 32P using Ladderman labeling Kit (Takara, Japan). All hybridization reactions were performed at 53 °C using Rapid-Hyb buVer (Amersham, USA). The membrane was washed twice with 2£ SSC and 0.1% SDS at room temperature, and was subsequently washed with 0.1£ SSC and 0.1% SDS at 60 °C. For Northern analysis, the wild-type cells and FgFtr1-deleted cells of F. graminearum were grown at potato dextrose broth (PDB) containing a designated iron content and 100 M BPS for 3 days. After grinding in liquid nitrogen on mortar, total RNA was prepared using Trizol reagent (Life Technologies). The extracted total RNA was run at 1% formaldehyde agarose gel for 4 h. The procedures of blotting and hybridization were followed as described above. Probes of the internal region of the reading frame were prepared by PCR using the following primer: for FgFtr1, 5⬘-GTTTGGCTCGGAACGGGT ATG-3⬘ and 5⬘-GGCCGAAGCCGAGAAAGATACT-3⬘; for FgFtr2, 5⬘-ATACCTGGGCAGACCATGAGTA-3⬘ and 5⬘-CAACGAAAACGATGCCTTCAAT-3⬘; for FgAct, 5⬘-ACACGGTATTGTCACCAACTGGG-3⬘ and 5⬘-AGGACAAAACGGCTTGGATGG-3⬘. To quantify the Northern results the TINA 2.1 (Raytest, Straubenhardt, Germany) software was used. 2.5. Uptake assay, plate assay, and confocal microscopic analysis For iron uptake assay, S. cerevisiae cells cultured overnight in SD medium without Leucine were inoculated into fresh SD medium at optical density (600 nm) of 0.1 and cultured at 30 °C for 6 more hours. The cells were washed twice with citrate buVer and harvested with 2 £ 107/ml cells in citrate buVer. The 106 washed cells were incubated with 1 M 55Fe(II) at 30 °C for 1 h. The activated cells were applied to the Wltration plate wells equilibrated with distilled water for 30 min. After washing Wve times with 200 l of distilled water, the multi-screen Wltration plate (Millipore, USA) was dried at 60 °C for 20 min. The dried Wlter was punched into the scintillation cocktail solution, and the radioactivity was read with a liquid scintillation counter. Plate assay was performed as described using 10 M of ferrous ammonium sulfate and 100 M BPS. For confocal microscopic analysis, the cells were cultured on YPD medium overnight, and the slides mounted with the congenic S. cerevisiae cells were observed. A Zeiss Axiophot microscope equipped with Bio-Rad confocal optics was used to obtain confocal images. 2.6. The analysis of ICP-AES To analyze the iron contents of F. graminearum, the cells were grown in deWned minimal medium (sodium nitrate, 6 g; potassium chloride, 0.52 g; magnesium sulfate, 0.52 g;

potassium dihydrogen phosphate, 1.52 g; dextrose, 10 g; pH adjusted to 6.5 with sodium hydroxide) containing 1 M iron for three days. The mycellia were Wltrated, washed twice, and then dried at 80 °C for 12 h. After the mixed acid (HNO3:HF:HClO4, 4:4:1) was added to the dried cells, they were boiled on a hot plate at 158 °C, and then evaporated completely. The evaporated materials were dissolved with distilled water and then analyzed by inductively coupled plasma atomic emission spectrophotometer (ICP-AES). (Model:Jobin Yvon 138 Ultima 2C, Source: Argon plasma (6000K)) 2.7. Virulence test The virulence of fungal strains was determined on adult barley. Fungal macroconidia were harvested from strains grown on carrot agar plates for 2 weeks at 25 °C and were suspended in sterile water at a concentration of 1 £ 105 macroconidia/ml. This conidial suspension was sprayed onto heads of barley plants at the early anthesis stage; and the plants were left for 2 days in a growth chamber at 25 °C with 100% relative humidity and then transferred into a greenhouse. Head blight symptoms appeared 1 week after inoculation. 3. Results From a BLAST search, we identiWed two ScFTR1 homologous genes, FgFtr1 and FgFtr2, in the F. graminearum genome. From a genome database of F. graminearum, we found that FgFtr1 and FgFtr2 share 56 and 54% homology with ScFTR1, respectively. Interestingly, this means that F. graminearum has two diVerent genes with high homology, which may result from the gene duplication that occurs in many plants and fungi (Hansche, 1975). As indicated in Fig. 1A, FgFtr1 and FgFtr2 have a homology of 56% with each other and have a high homology with Candida albicans and human SFT (Gutierrez et al., 1997), a putative stimulator of iron transport. Two conserved REXXE motifs, which are thought to be iron-binding sites (Stearman et al., 1996), were found in both FgFtr1 and FgFtr2. As shown in Fig. 1A, the locations of REXXE motifs (Fang and Wang, 2002; Stearman et al., 1996) on the proteins in Ftr1p, caFtr1p, FgFtr1, and FgFtr2 were in the same region and were highly conserved. As shown in Fig. 1B, the secondary structures of FgFtr1 and FgFtr2 were postulated to be six transmembrane structures, similar to ScFtr1p. Northern blot analysis was performed to test the possibility of the involvement of FgFtr1 and FgFtr2 in iron metabolism. F. graminearum were cultured in iron-depleted (10 M FAS and 100 M BPS) and replete conditions (500 M FAS and 100 M BPS) to log phase, after which total RNA was extracted. A total of 5 g of RNA was separated by formaldehyde agarose gel electrophoresis. Northern blotting was performed with FgFtr1 and FgFtr2 probes in the internal regions of the ORF. Interestingly, transcript

Y.-S. Park et al. / Fungal Genetics and Biology 43 (2006) 273–282 Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

277

MPNKVFNVAVFFVVFRECLEAVIVISVLLSFLKQAIG--EHDRALYRKLRIQVWVGVLLG -MVDVFNVQIFFIVFRESLEAIIVVSVLLAFVKQSMGG-SSDPQLKKRLYRQIWLGAGLG MAVDVFAVPVFLVVFRETLETVIIVSVLLAFLKQTLDGPNGDVKVYKQLKRQVWLGTGIG -MLELFSVPVFVVVFRETLETAIIVSVLLAFLKQTLDGSHRDANLYKTLRTQVWAGTATG --MKEFNHWKVLNIY--CLSSPRKRSSLVPIQTDLILKLLHLACTVGSFAICRLCSFRIG

58 58 60 59 56

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

FIICLAIGAGF---------------IGAYYSLQKDIFG-SAEDLWEGIFCMIATIMISM VLVCL-YGVLS---------------IGASYGLGKDIFGVISEDLWEGIFCIIATVLITA FFICMVVAAALIGVFFFICMVVAAALIGVFYTVGSNSWE-KHEYYYEGAFCLFASLIISV FAVCAIASG---------------IIIGTFYILGSDTWA-DHEYYYEGVFSLISAIIITI FCKFLLEAGFC---------------IFDFCVFGYIFILCREIHELKNYFSLFYFCMNLS

102 102 119 103 101

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

MGIPMLRMNKMQSKWRVKIARSLVEIPHRK--------------RDYFKIGFLSRRYAMF MGIPMLRINKMKEKWRVKLAQALIKSPTNK--------------KDRFKLGYLGKKYALF MGAALLRIGKMQSKWRVKLAKALESPIKTGNVKLAKALESPIKTGNKGWFKQFVERYAMF MGGALLRVGKMEDKWRAKLSKAIEEPVVAG-------------HGKRAWLINLFEKYAMF HIDPVIDICVIIAIKYKKVEYIVLL--------------------DRCFLKYYFVCFYYI

148 148 179 150 141

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

LLPFITVLREGLEAVVFVAGAGITTQGSHASAYPLPVVVGLICGGLVG-----------ILPFLQVLREGLEAVVFVGGVGLN---SPATSFPIPVIVGLIAGIVVG-----------VLPFVTVLREGIEAVVFVAGVSFS---ASAKSIPLPTVVGLFAGCCVGYLPTVVGLFAGC VLPCITVLREGIEGIVFVAGVSFS---APASAVPLPVILGLTLGALVG-----------LFVYLQIQQTCKRMDSEICNVKIK------------------------------------

196 193 236 195 165

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

---YLLYYGASKSSLQIFLILS--TSILYLISAGLFSRGAWYFENYRFNLASG---------ALLYYFGSSMSMQIFLIIS--TCILYLIAAGLFSRGIWYFETNTYNKKTG------CVGYLLYKGGASTKLQFFLVLS--TCLLYLVGAGLFSRSVWSFEMAKWNEYIGGEADEFG ---YALYRGGSSAKLQYFLVAS--TCLLYLVAAGLFSRAIWLFEQQQWNKVVG---------NIFIYNQSTVKSRFFFNISSKLSTVYLFHCTMKPFDFYHFKCVSSKTKQT-------

244 241 294 243 215

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

--------GDASEGGDGNGSYNIRKAVYHVNCCNPELD--NGWDIFNALLGWQNTGYLSS --------GDASENGSGPGTYDISKSVWHVNCRNPETD--NGWDIFNAILGWQNSATYGS NGPGSYIGGEADEFGNGPGSYDIDQSVWHVNCCTSTDKIQNGWGIFNAILGWTNSATYGS --------GDAAELGDGPGSYDIDRSIWHVNCCNPQLNGGGGWAILNAVVGWNNSATYGS ---------SKNTLKTVMRAFIILYALRKTFIMVFKILGHLHLQLTRSTMQLKKQPNYFL

294 291 354 295 266

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

MLCYNIYWLVLIIVLSLMIFEERRGHLPFTKNLQLKHLNPGYWIKNKKKQELTEEQKRQL VISYNIYWLFIICVLLLMVYEEKHGHLPFTKNLTLVQLNPMYHIKGKKKLELNKAEKDEL VISYNLYWICVMTGFIVMRFKYWICVMTGFIVMRFKETH-GRYSFGKAKAPANAVDDAES VLAYNLYWVFVIA---------------QFTAMAYKEDH-GHWPLVKAK---------EV LKTRCFLHEKILYVCLRCQFYKS----VFRFHPLLAHFIICIFFLYRTKYILFTCMSTHY

354 351 413 330 322

Ftr1p caFtr1p FgFtr1 FgFtr2 SFT

FAKMENINFNEDGEINVQENYELPEQTTSHSSSQNVATDKEVLHVKADSL FTKLQQQNFGQAAEV---------DETSSN----------KWMDSQENSHATSSPRNVSS-------------EKTTTA-------------------HARTD--------------------------------------------FFPVNSIENPNRLIIK----------------------------------

404 381 430 335 338

Fig. 1. Sequence alignments of FgFtr1, FgFtr2, Ftr1p, CaFtr1, and SFT (Stearman et al., 1996). Bold areas indicate REXXE conserved regions. The sequence homology between ScFtr1p, CaFtr1p, AfFtrA, FgFtr1, and FgFtr2 is high and the two REXXE sequences are localized on the same N-terminus and C-terminus regions. SFT has one REXXE sequence and the position of REXXE on SFT is diVerent from Ftr1 protein homologues.

level of FgFtr1 increased dramatically in the iron-depleted condition and decreased with increasing iron concentration. However, even in an iron-depleted condition, FgFtr2 transcript was not detected (Fig. 3A). To test the redundancy, the fgftr1 strain was constructed as described under Section 2. Single and double-deletion strains were constructed (Fig. 2), and Northern blot analysis was then performed with the FgFtr2 probe in the internal region of the ORF. Interestingly, FgFtr2 transcript was detected in the fgftr1 strain, and the expression level increased dramatically in the iron-depleted condition and decreased with increasing iron concentration (Fig. 3B). These results indicate that F. graminearum has two genes homologous to ScFTR1, possibly resulting from gene duplication. More-

over, Northern blot results indicate that iron regulates the expressions of FgFtr1 and FgFtr2, giving a clue to the relationship between the FgFtrs and iron metabolism in F. graminearum. To identify the involvement of FgFtrs in iron metabolism, the Northern blot analysis was performed with a probe derived from FgSid, which has a high homology with SIDA of A. fumigatus. In A. fumigatus, SIDA encodes the intermediate L-ornithine-N5-monooxygenase in the siderophore biosynthesis pathway; when the SIDA was deleted, the growth of A. fumigatus became defected in an iron depletion. F. graminearum has one copy of the A. fumigatus SIDA homologue and, as shown in Fig. 3C, the expression level of FgSid was down-regulated by iron concentration in the medium.

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A

B

C

Fig. 2. Deletion of FgFtr1 and FgFtr2 from F. graminearum. (A) The Southern blot analysis to conWrm deletion of FgFtr1. PCR-mediated gene disruption was used to generate deletion of the FgFtr1 gene. For making the deletion strain of FgFtr1, a deletion cassette containing Geneticin marker was used. A deletion cassette containing the selection marker was ampliWed using double-joint PCR and transformation was performed with PCR product. The genomic DNA from transformants was digested with NcoI and hybridized with the probe of 3⬘-Xanking region of FgFtr1. (B) The Southern blot analysis to conWrm deletion of FgFtr2. To construct fgftr2 PCR-mediated deletion strategy was adapted using geneticin marker cassette described above. For the Southern blot of fgftr2, the genomic DNA of fgftr2 was digested with AvaI and hybridization was performed with probes of 5⬘-Xanking region of FgFtr2. (C) The Southern blot analysis of double-deletion stain of FgFtr1/FgFtr2. To construct double-deletion strain, the FgFtr2 of fgftr1 was deleted using PCR-mediated gene deletion with Hygromycin marker cassette. The genomic DNA from transformants was digested with EcoRI and hybridized with 5⬘-Xanking region of FgFtr2.

This result reXects the same pattern as A. fumigatus, and the eVects of deletions of FgFtr1 and FgFtr2 were tested by quantifying the FgSid transcript in the wild-type and deletion strains cultured on the Potato Dextrose media. As shown in Fig. 3D, the expression level of the FgSid in the single fgftr1 or fgftr2 strain was not detected in Northern blot analysis. However, the expression level of FgSid in a fgftr1/fgftr2 double-deletion strain was 2.2folds higher than in wild-type. These results indicate that the fgftr1/fgftr2 double-deletion strain may have a low iron concentration inside or outside of cell and points to the possibility that FgFtrs are involved in iron metabolism. To conWrm these results, cellular iron content was measured from wild-type cells and from FgFtr1/FgFtr2 doubledeletion strains with an ion absorption spectrometer in duplicated experiments. As shown in Fig. 4, iron content of the double-deletion strain cells was lower than the wildtype by about 30%, but copper and zinc concentrations were not. These results show that FgFtrs mediate iron uptake in F. graminearum speciWcally. To identify the functions of FgFtrs using biochemical and genetic approaches, iron uptake and growth assay were performed. Because of limitations of genetic and biochemical approaches such as quantitative iron uptake assay and making multiple gene deletion strain with F. graminearum, the S. cerevisiae system was adapted to identify the functions of FgFtrs. To carry out the experiment, the expression vector in S. cerevisiae was constructed as described in the Section 2. The promoter region of the ScFTR1 was subcloned into pRS415 vectors (Stratagene), and FgFtr1 and FgFtr2 cDNAs were subcloned into constructed S. cerevisiae expression vectors. The constructed FgFtrs in the S.

cerevisiae expression vector were transformed into the S. cerevisiae ftr1 strain, and plate assay was performed in the iron-depleted medium. The ftr1 strain with an empty vector was used as a negative control, and a wild-type cell with an empty vector was used as a positive control. As shown in Fig. 5A, even though there were diVerences in growth speed, FgFtr1 and FgFtr2 complemented the growth defect of the ftr1 strain in an iron-depleted condition. To identify the importance of the conserved REXXE motifs of the FgFtrs, as in S. cerevisiae, the REXXE motif amino acids were changed to diVerent amino acids, and plate assay was performed. In FgFtrs, there are two REXXE motifs on the amino terminus side and on the carboxy terminus side, as shown in Fig. 1, which are thought to be iron-binding motifs of Ftr1p in S. cerevisiae and SFT in humans (Gutierrez et al., 1997). As shown in Fig. 5B, RE residues of the amino terminus REXXE motif were changed to two glycine residues and to arginine, and two aspartic acids in the carboxy terminus REXXE motif were changed to proline, glycine, and glycine, respectively. The constructed FgFtr1 plasmids were then transformed to a ftr1-deletion strain, and plate assays were performed. As shown in Fig. 5B, the FgFtr1-deletion strains with modiWed REXXE motifs failed to grow on iron-depleted medium; these patterns were same for FgFtr2. These results indicate that REXXE motifs have an important function, as in S. cerevisiae. Moreover, a single amino acid change of conserved motif failed to complement the ftr1-deletion strain. These results indicate that FgFtrs encode the iron transporter protein in F. graminearum, as in S. cerevisiae. Iron uptake experiments were performed to identify the iron uptake activity of FgFtrs in S. cerevisiae. As shown in

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Fig. 3. The expressions of FgFtr1 and FgFtr2 were regulated by iron concentration. (A) Northern blot result from wild-type F. graminearum. The total RNAs were isolated from cells cultured in 10, 50, 500 M FAS plus 100 M BPS and subjected to Northern blot analysis. For FgFtr1 and FgFtr2, the same probes used in Southern blot analysis were used. (B) Northern blot analysis of FgFtr1 and FgFtr2 of fgftr1 strain. Total RNAs were isolated from fgftr1 strain cultured in 10, 50, and 500 M FAS plus 100 M BPS and Northern blot analysis was performed to identify expression level of FgFtr1 and FgFtr2. The probes used in this experiment were same with (A). In (A) and (B) the FgAct was used as a control. (C) Northern blot result to identify the expression level of FgSid by indicated FAS concentrations. The total RNAs were isolated from wild-type cells cultured in 0, 50, and 500 M FAS plus 100 M BPS and subjected to Northern blot analysis with probe of internal region of FgSid. (D) Northern blot result to identify the expression level of FgSid by deletion of FgFtr genes. The total RNAs were isolated from indicated deletion strains cultured in Potato Dextrose medium and Northern blot analysis was performed with probe of internal region of FgSid and quantiWed using TINA 2.1 (Raytest, Straubenhardt, Germany). The graph showed the relative amounts of transcripts of indicated deletion strains. The FgAct was used as a control in experiments (C) and (D).

Fig. 6, the iron uptake activity of the ftr1-deletion strain was not detected. However, ftr1-deletion strains with FgFtr1 or FgFtr2 showed high iron uptake activity, even though the activity was lower than the wild-type. Notably, FgFtr2 showed higher uptake activity than FgFtr1, even though FgFTR2 complemented the growth defect of the

Fig. 4. The cellular iron concentration in double-deletion strain was lower than wild-type. The wild-type and double-deletion strain of FgFtr1 and FgFtr2 were cultured in deWned iron-depleted minimal medium. The mycellia were Wltrated, washed twice, and then dried at 80 °C for 12 h. After the mixed acid (HNO3:HF:HClO4, 4:4:1) was added to the dried cells, they were boiled on a hot plate at 158 °C, and then evaporated completely. The evaporated materials were dissolved with distilled water and then analyzed by ICP-AES.

ftr1-deletion strain much less than FgFTR1 in the plate assay. Fet3p is localized on the plasma membrane (Stearman et al., 1996), and correct localization depends on the functional Ftr1p. In the ftr1 strain, Fet3p can be found on the ER in a round, ring form. To identify whether FgFtrs complement FTR1 or not in localization, the localization of Fet3p was investigated by the introduction of the FgFtrs to the ftr1-deletion strain. As shown in Fig. 7, Fet3p was detected in intracellular compartments in the ftr1 strain and found on the plasma membrane in the wild-type (Stearman et al., 1996). This result was same as that with the Scftr1 strain. However, Fet3p was found on the plasma membrane when the FgFtrs were introduced into the Scftr1 strain, which indicated that FgFtrs functionally interacted with Fet3p to localize it correctly. FgFtr1 complemented ScFTR1 better than FgFtr2, even though iron uptake activity was lower than that of FgFtr2. To conWrm the relationship between FgFTRs and virulence, we carried out an infection test with barley plants (Fig. 8). All of the deletion strains (FgFtr1, FgFtr2, and FgFtr1/2) examined still caused head blight symptoms on whole barley plants that were typical and similar to those of the wild-type strain. 4. Discussion From the genome sequence of F. graminearum, we identiWed two genes homologous to ScFTR1; this information provides a clue to the important function of the reductive iron uptake system in Wlamentous fungi. To identify the function of FTR1 homologous gene products, gene deletion was performed and an attempt was made to identify the functions of these gene products. We thus determined the relevance of iron as it relates to FgFtrps. Because of the

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Fig. 6. Requirements of FgFtr1 or FgFtr2 for 55Fe uptake. Congenic strains of the indicated genotype were grown in SD medium without leucine and assayed for the uptake of 55Fe(II) as described in Section 2. Assays were performed in duplicate, and the experiment was replicated three times. Data from a representative experiment are shown.

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Fig. 5. The FgFtrs complement ScFTR1 and REXXE regions of FgFtrs have important function in iron uptake. (A) The ability of complementation of FgFtr genes to ScFTR1. The FgFtr1 and FgFtr2 were cloned from F. graminearum cDNA and subcloned into S. cerevisiae expression vector constructed as stated in Section 2. The subcloned FgFtr1 and FgFtr2 were transformed into S. cerevisiae ftr1 strain and plate assay was carried out on deWned low iron medium and control SD plate. (B) The functions of REXXE conserved sequences in FgFtr1 and FgFtr2. The point mutations were performed in the REXXE sequences and the FgFtr genes which have mutated REXXE sequences were transformed into S. cerevisiae ftr1 strain. The transformants were subjected to plates assay on deWned irondepleted medium (see Section 2) and control SD plate. The amino acids indicate mutated amino acids.

diYculties of genetic and biochemical approaches with the Wlamentous fungi such as making multiple deletion strain and carrying out quantitative iron uptake assay, we then used the S. cerevisiae system to further examine these gene products. Most fungi produce a number of diVerent types of siderophores, and the reductive iron system may play a small part in iron metabolism. Complementation testing with the S. cerevisiae system has shown that FgFtrps function as ScFtr1p and also correctly complemented ScFtr1p, even though we observed a small diVerence in iron uptake activity, complementation of growth defect in iron-depleted medium, and complementation of Fet3p localization. Fusarium graminearum has two genes homologous with ScFTR1 likely arising from gene duplication. The expression level and redundancy of the FgFtrs are notable.

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Fig. 7. Localization of Fet3p depends on FgFtr1 and FgFtr2 in S. cerevisiae. The cells indicated were grown in iron-depleted medium and performed confocal microscopy. The strain FG101 and FG102 indicate that the GFP was tagged FET3 on chromosome DNA of wild-type and FTR1deletion strain, respectively. (A) The localization of Fet3p in the wild-type as a positive control and (B) Fet3p in FTR1-deletion strain as a negative control. (C) and (D) indicate the localization of Fet3p in the FTR1-deletion strains which have FgFtr1 and FgFtr2, respectively.

Expression of FgFtr2 is repressed in the normal condition and is even repressed in the iron-depleted condition. However, its expression is increased in the iron-depleted condition when fgftr1 is present. These results show that gene duplication has advantages in nutrient utilization and in the adaptation of fungi to new environments.

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Fig. 8. Virulence test in barley plants with deletion strains of F. graminearum. Fungal macroconidia were harvested from strains grown on carrot agar plates for 2 weeks at 25 °C and were suspended in sterile water at a concentration of 1 £ 105 macroconidia/ml. This conidial suspension was sprayed onto heads of barley plants at the early anthesis stage; and the plants were left for 2 days in a growth chamber at 25 °C with 100% relative humidity and then transferred into a greenhouse.

Candida albicans and S. cerevisiae (Fang and Wang, 2002; Urbanowski and Piper, 1999) also have three and two FTR1 homologous gene products, respectively,that are regulated by iron concentration. FTR1 in S. cerevisiae is down-regulated by iron concentration. Interestingly, in C. albicans, CaFTR1 is down-regulated and CaFTR2 is upregulated by iron (Ramanan and Wang, 2000). Moreover, there are two REXXE motifs in F. graminearum that are thought to be iron-binding domains in the iron uptake system of S. cerevisiae. When the amino acids of fungal REXXE motifs were changed to diVerent amino acids, FgFtrs lost their function in S. cerevisiae. These results indicated that the FgFtrs work as iron transporters in F. graminearum, as does Ftr1p in S. cerevisiae. Notably, FgFtr2p has higher iron uptake activity than FgFtr1p, although it resulted in more mis-localized proteins and slower growth in iron-depleted medium than did FgFtr1. These results indicate that there are other factors, such as cellular localization and iron aYnity that aVect the uptake activity and warrant further study. We also determined the expression of the FgFtrs in F. graminearum by Northern blot analysis. As shown in Fig. 4, FgSid mRNA was used as an indicator of cellular iron status (Eisendle et al., 2003). The expression level of FgSid was regulated by cellular iron concentration, and the expression level was high in the iron-depleted condition. We found a higher expression level of FgSid in the fgftr1/fgftr2 double-deletion strain and indications that the cellular iron concentration of the fgftr1/fgftr2 double-deletion strain was lower than that of the wild-type strain. From these results, we conclude that the FgFtrps mediate iron uptake in low-iron conditions. This result was conWrmed by the direct quantiWcation of cellular iron concentration using ICP-AES. The cellular iron concentration was low in the fgftr1/fgftr2 double-deletion strain, indicating that FgFtrs work as iron transporters in F. graminearum.

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