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Virus Research 144 (2009) 96–106

Contents lists available at ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

roteomic analysis of fungal host factors differentially expressed by Fusariumraminearum infected with Fusarium graminearum virus-DK21

un-Jung Kwon a, Sang-Yun Cho b, Kyung-Mi Lee b, Jisuk Yu b, Moonil Son b, Kook-Hyung Kim a,b,∗

Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of KoreaDepartment of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea

r t i c l e i n f o

rticle history:eceived 8 January 2009eceived in revised form 28 March 2009ccepted 5 April 2009vailable online 15 April 2009

eywords:-DESI-MS/MSusarium graminearum virus-DK21ypovirulence

a b s t r a c t

Fusarium graminearum virus–DK21 (FgV-DK21), which infects the plant pathogenic F. graminearum, per-turbs host developmental processes such as sporulation, morphology, pigmentation, and attenuates thevirulence (hypovirulence) of the host. To identify the differentially expressed F. graminearum proteinsby FgV-DK21 infection, we have used two-dimensional electrophoresis with mass spectrometry usingproteins extracted from virus-free and FgV-DK21-infected strains. A total of 148 spots showing an alteredexpression were identified by PDQuestTM program. Among these spots, 33 spots were exclusively analyzedincluding 14 spots from FgV-DK21-infected and 19 spots from virus-free strains by ESI-MS/MS analysesand successfully identified 23 proteins. Seven proteins including sporulation-specific gene SPS2, triosephosphate isomerase, nucleoside diphosphate kinase, and woronin body major protein precursor wereinduced or significantly up-regulated by FgV-DK21 infection. A significant decrease or down regulation

of 16 proteins including enolase, saccharopine dehydrogenase, flavohemoglobin, mannitol dehydroge-nase and malate dehydrogenase caused by FgV-DK21 infection was also identified. Variations of proteinexpression were also further investigated at the mRNA level by real-time RT-PCR analysis, which con-firmed the proteomic data for 9 out of the representative 11 selected proteins including 5 proteins fromup-regulated group and 6 proteins from down-regulated group. Further investigation of these differen-tially expressed proteins will provide novel insights into the molecular responses of F. graminearum to FgV-DK21 infection.

. Introduction

Fusarium graminearum is a filamentous fungal pathogen ofheat, maize, and other grains that causes blighting on heads and

eedlings and rotting on stalks and ears that directly affects eco-omic losses due to lower yields and poor grain quality (Windels,000). Also, it causes serious human and animal health problems

n several parts of the world especially in Korea, because it pro-uces mycotoxins that contaminate harvested grains (McMullen etl., 1997). We have isolated F. graminearum from diseased barleynd maize from Korea and occasionally found colonies with unusualorphology such as slow growth, ameboid mycelia, and increased

igmentation. These isolates contained mycoviruses with variousizes of double-stranded RNAs (dsRNAs) suggesting that these mor-hological changes might be associated with these various sizes ofsRNAs (Chu et al., 2002, 2004). Among these dsRNA mycoviruses,

∗ Corresponding author at: Department of Agricultural Biotechnology, Seoulational University, Seoul 151-921, Republic of Korea. Tel.: +82 2 880 4677;

ax: +82 2 873 2317.E-mail address: kookkim@snu.ac.kr (K.-H. Kim).

168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2009.04.004

© 2009 Elsevier B.V. All rights reserved.

especially Fusarium graminearum virus-DK21 (FgV-DK21) isolatedfrom the F. graminearum strain DK21 conferred the most strikingtrait associated with attenuation of virulence (hypovirulence) toF. graminearum. The FgV-DK21 infected isolate showed changes inmorphological and pathogenicity phenotypes as well as reducedlevel of mycotoxin productions (Chu et al., 2002, 2004).

The mechanism by which the FgV-DK21 causes the pheno-typic changes is not known. As previously described, however,loss of dsRNA of FgV-DK21 through conidial passage resultedin faster mycelial growth and less pigmentation, whereas itsacquisition through hyphal anastomosis restored the abnormaldsRNA-containing DK21 phenotype (Chu et al., 2002). Moreover, werecently reported that electron-dense and pleiomorphic particle-like structure in cytoplasm and intracellular degeneration werefrequently observed in FgV-DK21 infected cells but not in virus-freecells under EM observation of mycelia (Kwon et al., 2007). This sug-gests hypovirulence phenotype is caused by FgV-DK21 infection.

How the FgV-DK21 affects fungal virulence, however, is not knownyet. A hypovirulence phenotype associated with mycoviruses hasbeen reported for several plant pathogenic fungi such as Cryponec-tria parasitica and Ophiostoma novo-ulmi (Anagnostakis, 1987; Denget al., 2003; Hong et al., 1999; MacDonald and Fulbright, 1991;

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uss, 1992; van Alfen et al., 1975). The Cryponectria hypovirusesCHVs) have been reported to confer hypovirulence to the chestnutlight fungus C. parasitica. DsRNA mycovirus containing isolates of. parasitica showed reduced levels of virulence (hypovirulence),uppressed sporulation, altered colony morphology, and reducedigmentation (Choi and Nuss, 1992b). O. novo-ulmi, the causal agentf Dutch elm disease, may contain up to 12 uncapsidated mito-hondrial dsRNAs that cause dramatic changes in the fungal host,ncluding slow growth, formation of colonies with abnormal, irreg-lar margins, and a reduction in the number of variable asexualpores (Cole et al., 1998; Rogers et al., 1987). Other mycovirusesave been shown to interfere with their fungal host gene expressionathways related to fungal growth and virulence (Nuss, 2005).

It has been reported that various interactions between host andiral factors are involved for changes of phenotype and virulenceGhabrial and Suzuki, in press; Nuss, 2005). Much has been learnedrom the C. parasitica/hypovirus system in which multiple trans-ormation protocols for the host fungus (Faruk et al., 2008) andeverse genetics of some hypovirus strains (Choi and Nuss, 1992a)re available. Two multifunctional proteins, p29 and p48, encodedy the prototype hypovirus CHV1-EP713 are identified as symptometerminants involved in reduction in sporulation and pigmenta-ion (Choi and Nuss, 1992b; Deng and Nuss, 2008; Suzuki et al.,999). Powell and van Alfen (1987a,b) reported that expressions oflimited number of specific mRNAs and polypeptides are repressed

n virus-containing strains of C. parasitica. An expressed sequenceag (EST)-based microarray analysis has revealed that about 295enes (13.4% of the 2200 unique cDNAs) showed altered expressiont transcriptional level as a result of CHV1-EP713 infection (Allent al., 2003). In this regard, it is worth noting that only some genesncluding cpsp12 have proved to be required for virulence (Deng etl., 2007).

Proteins serve as the bridge between genetic informationncoded in genome and the phenotype and therefore the pro-ein analysis will lead to the characterization involved in certainiological functions, in specific organs, at different developmen-al stages or under particular condition. The aim of the presentuantitative proteomics study was to carry out a differential profil-

ng of F. graminearum proteins in response to FgV-DK21 infection,nd to identify differentially regulated proteins. A total of 148ifferentially displayed proteins were identified and 23 of themere further characterized by electrospray ionization tandem mass

pectrometry (ESI-MS/MS). Protein query results identified variousungal host proteins involved in diverse cellular function and sug-est that FgV-DK21 infection affected various steps in fungal lifeycles.

. Materials and methods

.1. Fungal strains and growth conditions

Virus-free and FgV-DK21-infected F. graminearum strains wereaintained on potato dextrose agar (PDA) at 25 ◦C with 12 h

ight–dark cycle. F. graminearum cultures used for protein and RNAxtractions were grown as described previously (Kwon et al., 2007).reshly grown mycelia from plate were inoculated in 100 ml of com-lete media (CM) broth and incubated for 5 days in an orbital shaker130 rpm). Hyphae were collected by filtering through 3 MM paper,ashed with distilled water, dried by blotting with paper towels

nd weighed (Chu et al., 2002).

.2. Protein sample preparation

The collected mycelia were ground to a fine powder and 10 gere suspended in 20 ml of extraction buffer (20 mM Tris-Cl, pH

ch 144 (2009) 96–106 97

7.5, 0.5% Triton X-100 (v/v), 0.5 M EDTA, pH 7.5, 0.5 M EGTA,pH 8.0, 10 mM �-mercaptoethanol, 25 �g/ml aprotinin, 25 �g/mlleupeptin, and 1 mM PMSF) and homogenized. The cells wereremoved by centrifugation at 10,000 × g for 15 min at 4 ◦C fol-lowed by filtering through a 0.45 �m pore-size membrane filter,and trichloroacetic acid was added to a filtrate at a final concentra-tion of 10% (w/v). The mixture was then centrifuged at 12,000 × gfor 30 min at 4 ◦C, and the pellets were washed several times withcold ethanol, dried, and stored at −80 ◦C. The protein concentrationof each sample was determined by Bradford assay using BSA as astandard (Bradford, 1976).

2.3. Isoeletric focusing (IEF) and 2D electrophoresis

Thirty �g of each protein extract were resuspended in 250 �l ofrehydration buffer (8 M urea, 2% CHAPS (w/v), 0.5% IPG buffer (v/v),25 mM DTT, and 0.002% bromophenol blue) and were passivelyrehydrated for 16 h. Rehydrated proteins were absorbed into 13 cmimmobilin pH gradient strip with pH ranges of 4–7 (GE healthcare,Buckinghamshire, UK) as described by the manufacturer’s manual.IEF was then conducted using an Ettan IPGphor (GE healthcare) ata constant temperature of 20 ◦C and was focused through 5 h at30 V, 1 h at 100 V, 1 h at 500 V, 3 h gradient to at 8000 V, and 7 hholding at 8000 V until a total of at least 56,000 V h was reached.After IEF, the strips were equilibrated in equilibration buffer A (6 Murea, 2% SDS (w/v), 75 mM Tris-Cl , pH8.8, 29.3% glycerol (v/v),1% dithiothreitol (w/v)) at room temperature for 15 min and thenin equilibration buffer B (6 M urea, 2% SDS (w/v), 75 mM Tris-Cl,pH8.8, 29.3% glycerol (v/v), 2.5% iodoacetamide (w/v)) for an addi-tional 5 min. After equilibration, the strips were positioned on topof the second dimension gel and sealed with 1% (w/v) agarose.SDS-PAGE was performed on 12.5% polyacrylamide gel using SE600Ruby complete system (GE healthcare). The 2-DE for each samplewas repeated three times using total proteins prepared from threeindependent experiments.

2.4. Silver staining and 2D analysis

Protein spots on 2-DE gels were visualized with a silver stain-ing kit (GE healthcare) according to the manufacturer’s instructionand were stored in a solution of 5% ethanol, 5% acetic acid at roomtemperature. Silver-stained gels were scanned with a flatbed scan-ner. The image analysis in this study was performed by either visualobservation or using PDQuestTM 2-D Analysis Software V 8.0 (Bio-Rad, Hercules, CA, USA).

2.5. In gel digestion and ESI-MS/MS

Proteins were identified by peptide mass finger printing meth-ods (Jensen et al., 1997) and ESI-MS/MS. Silver-stained protein spotswere manually excised individually from 2D-PAGE gels and driedby a freezer vacuum dryer (Sarvant, Holbrook, NY, USA). Excised gelspots were destained with 100 �l of destain solution (1:1 mixtureof 30 mM potassium ferricyanide and 100 mM sodium thiosulfate,v/v) with shaking for 5 min. After removal of the solution, gel spotswere incubated with 200 mM ammonium bicarbonate for 20 min.The gel pieces were dehydrated with 100 �l of acetonitrile and driedin a vacuum centrifuge. The dried gel pieces were then rehydratedwith 20 �l of 50 mM ammonium bicarbonate containing 0.2 �gmodified trypsin (Promega, Madison, WI, USA) for 45 min on ice.After removal of solution, 30 �l of 50 mM ammonium bicarbon-

ate was added. The digestion was performed overnight at 37 ◦C.The peptide solution was desalted using C18 nano column (porousC18). For MS/MS analyses, peptides were eluted with 1.5 �l 50%methanol/49% H2O/1% formic acid directly into a precoated borosil-icate nanoelectrospray needle (Micromass, Manchester, UK).

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Table 1Identification of proteins from Fusarium graminearum up-regulated by FgV-DK21 infection.

Spot no. Protein identification(locus/species)a

Sequence/site of peptideidentified by LC-MS/MS

MW (Da)/pIb Mascot score Sequence coverage (%) Fold changec GenBankaccession no.

Possiblefunctione

Mean SD p-Valued

s1606 Sporulation-specific geneSPS2(FG10089/Gibberella zeaePH-1)

1. LGELNFGTEGVTK/120-132 42529/4.66 67 3 4.865 0.705 0.0046 XP 390265 Differentiation

s3301 Ribosomal-associatedprotein S2(FG10905/Gibberella zeaePH-1)

1. FTPGSFTNYITR/105-116 31778/4.76 64 4 3.844 1.077 0.0177 XP 391081 Protein synthesis

s4101 Triose phosphateisomerase(FG06702/Gibberella zeaePH-1)

1. VIWCCGESLETR/122-133 27179/5.19 46 10 >100 0.0011 XP 386878 Metabolism

2. TIEFVSAQIESLK/138-150

s8005 FgV-DK21-ORF2(Fusarium graminearumdsRNA mycovirus)

1. DLQALLGNTPR/30-40 16844/5.91 122 29 >100 0.0084 YP 223921 –

2. GADGESSAACLPETSSNVSLDR/41-623. YTGVSEPDLTSR/75-86

s9002 Nucleoside diphosphatekinase(FG05972/Gibberella zeaePH-1)

1. SILLGATNPLASSPGTIR/173-189 26169/9.1 79 7 4.046 0.755 0.0007 XP 386148 Metabolism

s9103 Woronin body majorprotein precursor(FG08737/Gibberella zeaePH-1)

1. VLDMQEGQLVAMTETGDVK/101-119 24959/5.77 96 14 3.778 0.837 0.0013 XP 388913 Differentiation

2. QGLPVIDQSNLYSR/120-133

s9204 Hypothetical protein(FG10543/Gibberella zeaePH-1)

1. SIWSDTSSGVNEWWTAYR/54-71 25587/6.24 52 7 >100 0.0003 XP 390719 –

a From F. graminearum genome database (http://mips.gsf.de/genre/proj/fusarium/).b Theoretical molecular mass of pI value from the predicted protein available from the F. graminearum genome database.c The mean and SD of fold change was calculated by comparing spot intensities between virus-free and FgV-DK21 infected strain using the quantitative analysis by PDQuestTM 2-D Analysis Software V 8.0.d Student’s t-test.e Functional categories were classified by KEGG database (http://www.genome.jp/kegg/).

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Research144 (2009) 96–106

99Table 2Identification of proteins from Fusarium graminearum down-regulated by FgV-DK21 infection.

Spot no. Protein identification(locus/species)a

Sequence/site of peptideidentified by LC-MS/MS

MW (Da)/pIb Mascotscore

Sequencecoverage (%)

Fold changec GenBankaccession no.

Possiblefunctione

Mean SD p-Valued

s2111 Thiol-specific antioxidant I(TSAI)(FG03180/Gibberella zeaePH-1)

1. QGGLGPDLELPLIADK/92-107 22860/4.94 51 14 <0.01 0.0011 XP 383356 Defense response

2. NYGVLIEDEGIALR/114-127

s3007 Cofilin(FG06245/Gibberella zeaePH-1)

1. YAVYDFEYNLASGDGIR/66-82 16170/5.25 45 11 <0.01 0.0009 XP 386421 Cell biogenesis

s3102 Hypothetical protein(FG01228/Gibberella zeaePH-1)

1. FVTLDQVEDDFLK/111-123 20848/5.24 44 6 0.0329 0.005 0.0006 XP 381404

s3214 Transaldolase(FG08723/Gibberella zeaePH-1)

1. ATGTTVVSDSGDFASIGK/10-27 35532/5.10 56 5 <0.01 0.0011 XP 388899 Metabolism

s3303 Adenosine kinase(FG06932/Gibberella zeaePH-1)

1. LSIQELGPSYPFPK/409-422 46795/6.52 44 3 0.191 0.008 0.0008 XP 387108 Metabolism

s3513 Enolase(FG01346/Gibberella zeaePH-1)

1. AVENVNTVIAPALTEK/65-80 47538/5.01 203 11 0.263 0.014 0.0007 XP 381522 Metabolism

2. TQDIQVVGDDLTVTNPLR/312-3313. SGETEDVTIADIAVGLR/377-393

s4302 inorganic pyrophosphatase(FG00496/Gibberella zeaePH-1)

1. VLGVMALLDEEETDWK/142-157 32921/5.20 80 10 0.269 0.029 0.0023 XP 380672 Metabolism

2. VIVIDVNDPLASK/158-170

s4406 Saccharopinedehydrogenase(FG10925/Gibberella zeaePH-1)

1. TLVDAGYPVSVER/28-40 42643/5.09 153 14 <0.01 0.0004 XP 391101 Metabolism

2. LVNEGTWPNAEAGTLILGLK/63-823. GYYLNEEELVNQIR/170-183

s5103 Peroxiredoxin(FG07536/Gibberella zeaePH-1)

1. DIGEVTGGNVEFPIIGDK/87-104 25360/5.86 83 12 0.272 0.017 0.0001 XP 387712 Defense response

2. TIFSYPASTGR/146-156

s5307 Ser/Thr phosphatase(FG09532/Gibberella zeaePH-1)

1. VGVLGCPNLPVDDSAR/164-179 37957/5.20 43 4 0.184 0.025 0.0031 XP 389708 Signal transduction

s5405 Flavohemoglobin(FG04458/Gibberella zeaePH-1)

1. EGQLYDAAG EWNSWR/138-152 45991/5.38 49 14 0.253 0.016 0.0004 XP 384634 Defense response

2. LQG PEEEPSLEDLSAGK/223-2393. YNVGDEVELSPPAGEFFLNPADTSSK/252-278

s6206 Mannitol dehydrogenase(FG04826/Gibberella zeaePH-1)

1. GAAEMGADVAITYASR/40-55 28590/5.58 78 6 0.253 0.013 0.0001 XP 385002 Metabolism

s6409 Phosphoglycerate kinase(FG03992/Gibberella zeaePH-1)

1. AEDGSVILLENLR/111 -123 44882/5.91 223 13 0.267 0.023 0.0013 XP 384168 Metabolism

100 S.-J. Kwon et al. / Virus ResearTa

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MS/MS of peptides generated by in-gel digestion was performedby nano-ESI on a Q-TOF2 mass spectrometer (Micromass). Thesource temperature was 80 ◦C. A potential of 1 kV was applied to theprecoated borosilicate nanoelectrospray needles in the ion sourcecombined with a nitrogen back-pressure of 0–5 psi to produce astable flow rate (10–30 nl/min). The mass spectrometer operatedin an automatic data-dependent MS/MS to collect ion signals fromthe eluted peptides. In this mode, the most abundant peptide ionpeak with doubly or triply charged ion in a full scan mass spec-trum (m/z 400–1500) was selected as the precursor ion. Finally,an MS/MS spectrum was recorded to confirm the sequence of theprecursor ion using collision-induced dissociation with a relativecollision energy dependant on molecular weight. The cone voltagewas 40 V. The quadrupole analyzer was used to select precursor ionsfor fragmentation in the hexapole collision cell. The collision gaswas argon at a pressure of 6–7 × 10−5 mbar and the collision energywas 20–30 V. Product ions were analyzed using an orthogonal TOFanalyzer, fitted with a reflector, a micro-channel plate detector anda time-to-digital converter. The data were processed using a MassLynx Windows NT PC system.

2.6. Protein identification and sequence processing

To identify the protein, all MS/MS spectra recorded on tryp-tic peptides derived from spot were searched against proteinsequences from National Center for Biotechnology Information(NCBI) databases using the MASCOT search program (http://www.matrixscience.com).

2.7. Real-time RT-PCR analysis

Total RNAs for real-time RT-PCR were extracted by Trizol reagent(Invitrogen, Carlsbad, CA, USA) and further treated with Turbo DNA-free TM (Ambion, Austin, TX, USA) according to manufacturer’sprotocols. Real-time RT-PCR was performed using Power SYBR®

Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA)and a 7500 Real Time PCR System (Applied Biosystems) accordingto the manufacturer’s instructions. The thermal profile was 50 ◦C for2 min, 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s and60 ◦C for 1 min. All primers were designed using Primer Express3.0 software (Applied Biosystems) and listed in supplementaryTable S1. All real-time PCR reactions were technically repeatedthree times. The gene expression was calibrated using formula2−��Ct method (Livak and Schmittgen, 2001) where Ct refers tothe threshold cycle, sample strands for the gene of interest andcontrol indicates the endogenous housekeeping gene cyclophilin(CyP1; GenBank accession No. XP387615). The gene expressionratio was calibrated with the Ct value of the FgV-DK21 infectedsample with the Ct value of a virus-free sample, using formula2−��Ct−s–2−��Ct+s, where s is the strandard deviation of �Ct value.

3. Results

3.1. Comparison of proteomic patterns between virus-free andFgV-DK21-infected F. graminearum

To investigate the host proteins of F. graminearum responsiveto FgV-DK21 infection, the total proteins were extracted from 5day-old virus-free and FgV-DK21 infected F. graminearum myceliagrown in CM broth and were separated by 2-DE. Comparison of2-DE images indicated that there were both qualitative and quan-

titative differences between protein profiles from virus-free andFgV-DK21 infected F. graminearum. In protein extraction, indepen-dent culture mycelia were accounted as one biological replicate thatwere conducted in triplicate. The 2-DE for one biological replicatewas repeated technically at least three times. Thus, nine analytical

S.-J. Kwon et al. / Virus Research 144 (2009) 96–106 101

F -free (j

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ig. 1. 2-DE patterns of protein samples in the pH range of 4–7 extracted from virus) are enlarged in Fig. 2.

els were comparatively analyzed for virus-free and FgV-DK21-nfected F. graminearum strain. Fig. 1 shows a representative 2-DEel of total proteins from virus-free (A) and FgV-DK21-infected (B). graminearum strains in a pH gradient of 4–7. Numerous differ-ntly expressed protein spots were observed in these gels. About70 spots were detected on the map with PDQuestTM softwareBio-Rad). One hundred forty-eight spots that showed differentialxpression pattern in all analytical gels upon FgV-DK21 infectionere identified.

Among those, 33 proteins that consistently exhibited increasedr decreased in abundance, there were at least three fold differences

n spot intensity in gel for FgV-DK21-infected F. graminearum strain,ompared with those virus-free F. graminearum gel. Of these, 14 pro-ein spots (s1606, s2002, s2902, s3301, s4101, s5004, s6003, s8005,8511, s8610, s9002, s9103, s9204 and s9603) with an up-regulatedrend and 19 protein spots (s2111, s2507, 3007, s3102, s3214, s3303,3309, s3513, s4202, s4302, s4406, s5103, s5307, s5405, s6206,6409, s7306, s7310 and s7405) with a down-regulated trend bygV-DK21 infection were selected for MS analysis (Fig. 2). To exam-ne the reproducibility between gels, these proteins were confirmedy three biological replications and three independent technicaleplications of 2-DE before conducting protein spot isolation and

S analysis. The fold change value of expression level for eachrotein spots is listed in Tables 1 and 2.

.2. Identification of F. graminearum host proteins regulated bygV-DK21 infection

All the 33 differentially regulated proteins were subjected to in-el digestion and analyzed by ESI-MS/MS. However, 6 spots (s2507,3309, s4202, s5004, s6003 and s8610) showed keratin peaks fromhe mass list, presumably resulting from contamination during theandling of gel slices or in-gel digestion. And the other 4 spotss2002, s2902, s8511 and s9603) did not generate any signal that

ight be caused by insufficient spot intensity for analysis. Thus,nly the data of the residual 23 spots were used for further analysisnd summarized in Tables 1 and 2 as up- and down-regulated pro-eins by FgV-DK21 infection, respectively. Protein identification wasrst conducted by searching the EST database using the MASCOTequence query program. Protein database were searched in the F.

raminearum genome database (http://broad.mit.edu/annotation/enome/fusarium group/Multihome.html) and the Munich Infor-ation Center for Protein Sequences (MIPS) F. graminearum genome

atabase (http://mips.gsf.de/genre/proh/fusarium). All of the iden-ified protein spots, except s8005, were recognized as different

A) and FgV-DK21 infected (B) F. graminearum. Boxed region (a, d, c, d, e, f, g, h, i and

proteins originating from F. graminearum. Most (20/22) of theseprotein functions are known or can be predicted except for spotss3102 and s9204. Protein spot s8005 was annotated in the NCBI as aFgV-DK21 ORF2 sequence (GenBank accession No. YP 223921). Themolecular weights and pI values of most of the analyzed proteinsdetected by 2-DE were similar to those deduced from the genomesequence. However, some of the proteins, such as s1606, s3301and s3513, had similar pI values but different molecular weightthan their predicted protein. While s3102, s3303, s5103, s6206and s9002 were similar molecular weight but different pI values.These observations suggest that the proteins may be changed theircharacteristics by post-translational modification or the net chargealterations by phosphorylation.

3.3. Functional classification of up-regulated proteins inFgV-DK21 infected F. graminearum

To obtain the profile of up-regulated F. graminearum proteins byFgV-DK21 infection, we analyzed 14 spots as described above andfinally obtained information for only 7 protein spot by ESI-MS/MS(Table 1). These identified proteins were found to be involved indifferentiation (s1606 and s9103), metabolism (s4101 and s9002)and protein synthesis (s3301). Spot s1606 was highly similar tosporulation specific gene SPS2 and s9103 was matches to a woroninbody major protein precursor. Spot s4101 was identified as atriose phosphate isomereas (TPI) and spot s9002 was matched tonucleoside diphosphate kinase (NDK). TPI and NDK have impor-tant functions in carbohydrate (glycolysis) and nucleotide (purine)metabolism, respectively (Hasunuma et al., 2003; Janin and Deville-Bonne, 2002). Spot s3301 was matched to a ribosomal-associatedprotein S2 and s9204 showed identity with hypothetical proteinencoded FG10543 in F. graminearum genome database. Finally, spotnumber s8005 was identified as a putative ORF (ORF2) of FgV-DK21. ORF 2 encodes putative protein of 16.7 kDa. Since neitherthe nucleotide sequences of the ORF2 nor the sequence of theputative translation product show any similarities to sequencescurrently available in GenBank, it is not clear whether the ORF2encode real polypeptide that is significant for viral replication.Induction of this protein in FgV-DK21 infected F. graminearum,however, further confirmed the existence of ORF2 although we do

not know the possible role of this viral protein yet. Other puta-tive translation products from FgV-DK21 were not detected mainlybecause pI values of these putative proteins (ORF1, 8.34; ORF3, 9.28and ORF4, 9.07) were out of range on strip used in this experi-ment.

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gt(bscr

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.4. Functional classification of down-regulated proteins ingV-DK21 infected F. graminearum

A total of 19 proteins were selected as a down-regulated proteinroup on FgV-DK21 infected strain (Fig. 3). Out of these 19 pro-eins, 16 proteins were analyzed the protein identity by ESI-MS/MSTable 2). The analyzed proteins were found to be involved in diverseiological processes, including metabolism (s3214, s3303, s3513,4302, s4406, s6206, s6409, s7306, s7310 and s7405), biogenesis ofellular components (s3007), signal transduction (s5307) and cellescue, defense and virulence (s2111, s5103 and s5405).

Among the proteins responsible for metabolism, seven ofhem were related to carbohydrate metabolism: transaldolases3214) for pentose-phosphate pathway, enolase (s3513), manni-ol dehydrogenase (s6206), phosphoglycerate kinase (s6409), andlyceraldehydes 3-phosphate dehydrogenase (GAPDH, s7306) forlycolysis, malate dehydrogenase (MDH; s7310) and acetoacetyl-oA thiolase (s7405) for pyruvate metabolism. Adenosine kinases3303), inorganic pyrophosphatase and saccharopine dehydro-enase (SDH; s4406) have related to nucleotide (purine), energyoxidative phosphorylation) and amino-acid (lysine) metabolism,espectively. Cofilin (s3007) is related to biogenesis of cellularomponents. Cofilin binds to monomeric actin in 1:1 ratio, thusreventing the polymerization of actin into filaments (Bamburg,999; Carlier et al., 1999). Ser/Thr phosphatase (s5307) has impor-ant function in signal transduction for switching cellular pathways

n response to environmental situations. As a final group inown-regulated proteins, thio-specific antioxidant 1 (TSA1; s2111),eroxiredoxin (Prx; s5103) and flavohemoglobin (s5405) wereelated to cell rescue, defense and virulence. Both TSA1, isoform ofrx, and Prx have antioxidant function. Flavohemoglobin (s5405),

ig. 2. Enlarged comparisons of protein spots in boxed regions (A, D, C, D, E, F, G, H, I andgV-DK21 infection. Labeled numbers were analyzed with ESI-MS/MS identification in Ta

ch 144 (2009) 96–106

found in both prokaryotes and eukaryotes, metabolize nitric oxide(NO) to nitrate and protect bacteria and fungi from NO-mediateddamage, growth inhibition, and killing by NO-release immune cells(Cramm et al., 1994; Membrillo-Hernandez et al., 1996). However,it is unclear whether this protein has physiological roles and patho-logical meanings on fungus especially on fungus–virus interaction.

3.5. Correlation between mRNA expression and proteinabundance

To verify the up-regulated or down-regulated expression ofsome of the identified proteins in transcription level, total RNAswere extracted and were analyzed by real-time RT-PCR. Among 23identified proteins by 2-DE, F. graminearum EST sequences corre-sponding to the 11 identified proteins (5 proteins in up-regulatedgroup and 6 proteins in down-regulated group) were selectedfor comparing the protein and transcript expression level. Theprimer pairs used for real time RT-PCR are shown in supplementaryTable S1 and were designed based on nucleotide sequence in NCBIdatabases. Of five proteins identified as the up-regulated group,three genes (s4101, s8005 and s9103) showed a higher transcriptionlevel in the FgV-DK21 infected strain (Fig. 4A and B), consistent withproteome level observations. In contrast, two other genes (s1606and s9002) did not show correlation between transcription andtranslation expression levels (Fig. 4B; compare to Fig. 3A).

Of six proteins (s3513, s4406, s5103, s5307, s6206 and s7310)

identified as down-regulated group, all genes showed a signifi-cant decrease in transcription abundance in FgV-DK21 infectedstrain, although we did not observe completely consistent correla-tion between transcriptional and translational expression patterns.Thus, it is evident from the results presented in Fig. 4C that

J) marked in Fig. 1. Arrows indicated selected spots for regulated proteins followingbles 1 and 2.

S.-J. Kwon et al. / Virus Research 144 (2009) 96–106 103

F ins anT

tsIsaAb

4

tiftrgggb2ic

ig. 3. Expression histogram showing volume changes of (A) 14 up-regulated prote-ticks on the top of each bar indicated the standard error.

ranscript-level changes generally support the differential expres-ion of translation in virus-free strain following FgV-DK21 infection.n this regard, it is worth mention that we could not detect the tran-cript abundance for s4101and s8005 in virus-free strain and s4406nd s7310 in FgV-DK21 infected strain by real-time RT-PCR (Fig. 4A).ltogether, these data implied that expression of many genes mighte controlled directly through the transcriptional regulation.

. Discussion

In this study, proteins showing differential changes in pro-ein profiles on F. graminearum during FgV-DK21 infection weredentified by using a proteomic approach. Around 470 proteinsrom F. graminearum were visualized on 2-D gels for identifica-ion and quantitative analyses of differentially regulated proteins inesponse to FgV-DK21 infection were conducted. The genome of F.raminearum is around 36.45 Mb, and expected to contain ∼13,300enes (http://www.broad.mit.edu/annotation/genome/fusarium

raminearum/MultiHome.html). Our results showed that we haveeen observed less than 1% of the total protein expression following-DE. We identified 23 fungal proteins out of 148 spots show-

ng altered expression upon FgV-DK21 infection. These proteinsould be divided into three functional groups including metabolism

d (B) 19 down-regulated proteins for differentially displayed spots from 2-DE. The

(including protein synthesis and signal transduction), cellular mor-phology (differentiation and biogenesis of cellular components),and defense response.

Twelve proteins involved in metabolism showed a differentialprofile in response to FgV-DK21 infection. Relative abundance oftwo proteins, TPI and NDK, were up-regulated in FgV-DK21 infectedstrain (Table 1). NDK was reported as essential protein for perithe-cial polarity and conidiation in N. crassa and required for growth athigher temperature in Aspergillus nidulans (Hasunuma et al., 2003;Lin et al., 2003; Ogura et al., 2001). In Drosophila melanogaster,Caenorhabditis elegans, and Xenopus laevis, NDK was reported to bean essential gene for development (Chiadmi et al., 1993; Maedaet al., 2001; Ouatas et al., 1997). Crucial roles of mammalian NDKsinvolved in differentiation, cell survival, tumor metastasis, and pro-liferation was also reported (Lee and Lee, 1999; Nosaka et al., 1998;Postel et al., 2000). Assuming these diverse roles of NDK, it seemsthat the up-regulation of NDK in FgV-DK21 infected strain might berelated for survival of fungal host to overcome a strict environmentscaused by virus infection. In this regard, it is worth mentioning that

there was no significant difference in conidiation between virus-free strain and FgV-DK21 infected strain although mycelia growthwas inhibited in FgV-DK21 infected strain (Chu et al., 2002).

The other ten proteins including transaldolase (s3214), adeno-sine kinase (s3303), enolase (s3513), inorganic pyrophosphatase

104 S.-J. Kwon et al. / Virus Resear

Fig. 4. Transcription expression levels of selected genes. (A) Check on gel for quan-titative real-time PCR of mRNA level for s4101 and s8005 in up-regulated groupand s4406 and s7310 in down-regulated group. s4101 and s8005 were inducedonly in FgV-DK21 infected strain and s4406 and s7310 were induced only in virus-free strain. Lane M is 50 bp DNA ladder (New England Biolabs, Beverly, MA, USA).Comparison of differentially expressed mRNA patterns for selected up-regulatedproteins (B) and down-regulated proteins (C) by real time RT-PCR as described inSection 2. Each Value is showed as ratio of expression level of selected genes inFgV-DK21 infected strain to the corresponding genes in virus-free strain from threeindependent replicates.

ch 144 (2009) 96–106

(s4302), SDH (s4406), mannitol dehydrogenase (s6206), phos-phoglycerate kinase (s6409), GAPDH (s7306), MDH (s7310) andacetoacetyl-CoA thiolase (s7405) showed decreased expressionsupon FgV-DK21 infection (Table 2). These proteins have severalfunctions including carbohydrate, nucleotide, amino-acid or energymetabolism and have been reported as essential factors for fungalgrowth. Inorganic pyrophosphatase was characterized structurallyas soluble and membrane-bound proton pumping pyrophosphate-ses, sPPases and H+-PPases, respectively. In fungi, sPPases have beenidentified both in mitochondria and in cytosol (Maeshima, 2000)and were reported to play an important role in survival under dif-ferent biotic and abiotic stress conditions (Perez-Castineira et al.,2001, 2002). Mannitol dehydrogenase is a key enzyme in mannitolmetabolism. Important roles of mannitol for asexual sporulation(Solomon et al., 2006), for stress tolerance (Ruijter et al., 2003),and as a reservoir of reducing power (Hult et al., 1980) have beenreported. Moreover, mannitol is common storage carbon for mostfungi and it can also serve as a quencher of reactive oxygen species(ROS) such as H2O2 generated from the plant defense responsereaction, possibly aiding in pathogen colonization (Jennings et al.,1998). Down-regulation of these proteins causes slow mycelialgrowth and thus showing the typical hypovirulence phenotype of F.graminearum host following FgV-DK21 infection. Further identifica-tion and characterization of these proteins involved in metabolismwill provide important information for fungal cellular maintenanceunder physiological and nutritional stresses caused by FgV-DK21infection. Among the carbohydrate metabolism associated proteins,especially, enolase, phosphoglycerate kinase and GAPDH that cat-alyze the reversible reaction in glycolysis and regulation of theseproteins in most fungi is connected with the glycolysis of theirplant host (Zhou et al., 2006). Generally, fungus assimilates car-bon sources from its plant host. Therefore, it is reasonable tospeculate that the FgV-DK21 infected F. graminearum could notget enough carbon sources from plant host due to severe growthdefects and significant down-regulation of these proteins involvedin glysolysis by FgV-DK21 infection. Down-regulation of enolase,phosphoglycerate kinase and GAPDH can cause significant alter-nation of glycolysis in F. graminearum. Moreover, recent studiesindicated that many glycolytic proteins have additional functionsin addition to their roles in glycolysis. As for GAPDH, it is alsoinvolved in nuclear export of tRNA, regulation of actin filaments inmicrofilaments, DNA repair, and the regulation of calcium releasefrom the ER (Deveze-Alvarez et al., 2001). Therefore, variationsin GAPDH activity may also influence many other cellular activi-ties, facilitating a greater capacity for adaptation to environmentalchallenges or growth conditions (Deveze-Alvarez et al., 2001). Inaddition, recent studies show the importance of certain house-keeping enzymes including GAPDH as virulence factors for a vastvariety of microbial pathogens (Alderete et al., 2001; Pancholi andChhatwal, 2003). Reduced accumulation of GAPDH is, therefore, inagreement with our previous observation showing reduced patho-genecity of F. graminearum strain DK21 upon FgV-DK21 infections(Chu et al., 2002).

The relative abundance of proteins involved in cellular mor-phology, including differentiation (SPS2 and woronin body majorprotein precursor) and biogenesis of cellular components (cofilin)showed altered expression profiles of their relative abundancesupon FgV-DK21 infection. Two proteins (SPS2 and woronin bodymajor protein precursor, s1606 and s9103, respectively) involvedin differentiation showed increased accumulation levels whereascofilin (s3007) showed decreased accumulation levels (Table 2).

SPS2 and woronin body are known to be involved in the effi-ciency of spore and hyphae formation of mycelia (Collinge andMarkham, 1985; Percival-Smith and Segall, 1987). In filamentousascomycetes, they typically form mycelial colonies consisting ofinterconnected multinucleate hyphae. Loss of cytoplasm in injured

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yphae is restricted by a fast and efficient sealing of central poresf hyphal crosswalls by a specialized organelle called woronin bodyCollinge and Markham, 1985). Studies with N. crassa revealed thatlug formation in intact hyphae is fundamentally different fromeptal plugging in response to injury (Tenney et al., 2000). Increasednduction of this protein in FgV-DK21 infected strain might bexplained as a consequence of curing processes of damaged hyphaeaused by FgV-DK21 infection. Cofilin is actin depolymerisation fac-or that is important in vivo for the acceleration of actin filamenturnover during cell motility, cell growth, and cytokinesis (Huangnd Kolodner, 2005). With respect to the significant alteration inhe morphology of F. graminearum infected with FgV-DK21 strain,nd the reduction of cofilin on protein as well as mRNA level coulderhaps be inferred that this protein is important to phenotypichanges of F. graminearum.

Finally, three proteins including TSA1 (s2111), Prx (s5103) andavohemoglobin (s5405) were involved in defense responses. Allf these three proteins showed decreased accumulation of theirelative expressions in response to FgV-DK21 infection. The down-egulation of the relative abundances of these defense relatedroteins might be implied in rapid replication of FgV-DK21. Amonghese protein, the antioxidant proteins such as TSA1 and Prx haveeen known to eliminate ROS, including the superoxide anion,ydrogen peroxide and hydroxyl radical, that are generated asyproducts of normal metabolism and as a result of exposure toarious environmental agents (Newcomb and Loeb, 1998). ROS aresed as cellular signaling products during respiration, metabolismnd stress response pathways (Finkel, 2003). To survive against theamage of ROS, most organisms are provided with an antioxidantrotein for their defense (Halliwell and Gutterridge, 1989). In casef plant, several antioxidant proteins were up-regulated or inducedfter infection with pathogen in the resistance host (Zhou et al.,005). This report suggested that the antioxidant proteins were

nduced for defense activity against pathogen attack. Therefore,own-regulation of antioxidant proteins in hypovirulent FgV-DK21

nfected strain could be indirectly inferred that there might beigher oxidative burst in host in response to defects detoxificationf ROS.

Here, our proteome analysis revealed the change in the abun-ance of a number of proteins involved in diverse biological

unctions in both virus-free and FgV-DK21 infected strains. Inddition, the molecular responses of F. graminearum to FgV-DK21nfection obtained in this study further indicate the up regulation ofroteins with differentiation, and down regulation of proteins withntioxidant activities and glycolysis. Interestingly, most selectedroteins for mRNA expression study showed positive consistentegulation in both mRNA and protein expressions, although it haseen reported that the correlation between transcription and trans-

ation is known to be less than 50% (King and Sinha, 2001). Oureal-time RT-PCR results suggest that level of protein expression of

any genes is usually controlled at the gene expression level upongV-DK21 infection. In conclusion, this is a first report on the iden-ification of regulated proteins associated with virus infection in. graminearum utilizing a comparative proteomic analysis. Moreetailed analysis of the identified proteins is in progress to fur-her characterize their possible functional roles for hypovirulenceaused by FgV-DK21 infection.

cknowledgements

We thank Drs. Nobuhiro Suzuki and Miranda Gilda Jonson foreviewing this manuscript. This research was supported in party grants from the Agriculture Specific Research Project fundedy the Rural Development Administration; a Korea Researchoundation grant (MOEHRD, Basic Research Promotion Fund;

ch 144 (2009) 96–106 105

KRF-2005-217-F00001); and the Korea Science and EngineeringFoundation (KOSEF) grant (No. R11-2008-062-02003-0) fundedby the Korean Government (MEST). SYC, KML, JSY and MIS weresupported by the graduate research fellowships from the Ministryof Education through Brain Korea 21 Project.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.virusres.2009.04.004.

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