Functional analysis of the Fusarium graminearum phosphatome

Yingzi Yun1*, Zunyong Liu1*, Yanni Yin1, Jinhua Jiang2, Yun Chen1, Jin-Rong Xu3 and Zhonghua Ma1

1Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China; 2Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou

310021, Zhejiang, China; 3Purdue-NWAFU Joint Research Center and State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University,

Yangling, Shanxi, China

Author for correspondence:Zhonghua Ma

Tel: +86 571 88982268

Email: zhma@zju.edu.cn

Received: 27 January 2015

Accepted: 15 February 2015

New Phytologist (2015) 207: 119–134doi: 10.1111/nph.13374

Key words: Fusarium graminearum,mitogen-activated protein kinase (MAPK)pathways, mycotoxin, phosphatome,virulence.

Summary

� Phosphatases are known to play important roles in the regulation of various cellular pro-

cesses in eukaryotes. However, systematic characterization of the phosphatome has not been

reported in phytopathogenic fungi.� The wheat scab fungus Fusarium graminearum contains 82 putative phosphatases. The bio-

logical functions of each phosphatase were investigated in this study.� Although 11 phosphatase genes appeared to be essential, deletion mutants of the other 71

phosphatase genes were obtained and characterized for changes in 15 phenotypes, including

vegetative growth, nutrient response and virulence. Overall, the deletion of 63 phosphatase

genes resulted in changes in at least one of the phenotypes assayed. Interestingly, the deletion

of four genes (Fg06297, Fg03333, Fg03826 and Fg07932) did not dramatically affect hyphal

growth, but led to strongly reduced virulence. Western blot analyses showed that three phos-

phatases (Fg10516, Fg03333 and Fg12867) functioned as negative regulators of the

mitogen-activated protein kinase signaling pathways. In addition, we found, for the first time,

that FgCdc14 is dispensable for growth, but plays an important role in ribosome biogenesis.� Overall, in this first functional characterization of the fungal phosphatome, phosphatases

important for various aspects of hyphal growth, development, plant infection and secondary

metabolism were identified in the phytopathogenic fungus F. graminearum.

Introduction

Phosphorylation and dephosphorylation executed by kinases andphosphatases, respectively, regulate various cellular processes,including the cell cycle, gene transcription and metabolism. In atypical cell, the functions of approximately one-third of proteinsare regulated via phosphorylation/dephosphorylation (Cohen,2000). In eukaryotic cells, phosphorylation mainly occurs onthree hydroxyl-containing amino acids, serine (Ser), threonine(Thr) and tyrosine (Tyr), with Ser as the predominant target (Shi,2009). Proteomic analyses of 6600 phosphorylation sites on2244 human proteins have shown that phosphoserine (pSer),phosphothreonine (pThr) and phosphotyrosine (pTyr) accountfor 86.4%, 11.8% and 1.8%, respectively, of the phosphorylatedamino acids (Olsen et al., 2006). Based on sequence homology,structure and catalytic mechanism, protein phosphatases (PPs)can be divided into two major superfamilies: protein Tyr phos-phatases (PTPs) and Ser/Thr phosphatases (PSPs) (Shi, 2009).The PTP members have multiple functions, encountering pro-tein Tyr kinases by removing the phosphate group from Tyr resi-dues (Goberdhan et al., 1999). PTPs comprise the classical PTPs,the dual-specificity phosphatases (DSPs), the Cdc25-type phos-phatases and the low-molecular-weight phosphatases. PSPs

comprise three major families: phosphoprotein phosphatases(PPPs), metal-dependent protein phosphatases (PPMs) and theaspartate (Asp)-based protein phosphatases (APPs) (Son &Osmani, 2009).

In general, c. 0.6% of genes encode PPs in the genome of aeukaryote (Seshacharyulu et al., 2013). The human genome con-tains 140 putative PPs and Arabidopsis thaliana has c. 120 PPs(Brenchley et al., 2007; Kerk et al., 2008). In the budding yeastSaccharomyces cerevisiae, 33 PPs have been identified. Many playcritical roles in signal transduction, the cell cycle, sexual repro-duction and stress responses (Zolnierowicz & Bollen, 2000).More recently, 28 PP genes have been identified and character-ized in the filamentous fungus Aspergillus nidulans. Four arerequired for normal growth, and four have essential functionsrequired for mitosis (Son & Osmani, 2009).

Fusarium graminearum is an economically important plantpathogen that causes Fusarium head blight (FHB) on variouscereal crops (Starkey et al., 2007). Although yield loss caused bythe disease is a major concern, mycotoxins, such as deoxynivale-nol (DON) and its derivatives, produced by the fungus ininfested grains, pose a serious threat to human and animal health(Sutton, 1982). In addition to its economic importance,F. graminearum is a tractable genetic system amenable to molecu-lar and genomic studies (Dean et al., 2012). To date, three PPs inF. graminearum have been reported to be important for fungal*These authors contributed equally to this work.

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developmental and virulence (Jiang et al., 2011; Kim & Yun,2012). However, a systematic characterization of PPs inF. graminearum or other pathogenic fungi has not been reported.

In order to analyze the functions of phosphatases, we carriedout a systematic gene deletion analysis of all putative phosphatasegenes in F. graminearum. In silico analyses showed that theF. graminearum genome contains 82 putative phosphatase genes.Of these, 11 are likely to be essential. We generated deletionmutants for the other 71 genes and characterized their defects in15 phenotypic traits. Phosphatases that are important for variousaspects of hyphal growth and development, plant infection andsecondary metabolism were identified in this study. Our findingshelp to determine the functions of phosphatases in various cellarprocesses in F. graminearum, and such information could beextended to other pathogenic fungi.

Materials and Methods

Strains and culture conditions

The wild-type strain PH-1 of F. graminearum was used as aparental strain for transformation experiments. The wild-typestrain and resulting transformants were grown on potato dextroseagar (PDA) (200 g potato, 20 g glucose, 10 g agar and 1 l water),complete medium (CM) (10 g of glucose, 2 g peptone, 1 g yeastextract, 1 g casamino acids, nitrate salts, trace elements, 0.01% ofvitamins, 10 g agar and 1 l water, pH 6.5) (Klittich & Leslie,1987) or minimal medium (MM) (10 mM K2HPO4, 10 mMKH2PO4, 4 mM (NH4)2SO4, 2.5 mM NaCl, 2 mM MgSO4,0.45 mM CaCl2, 9 mM FeSO4, 10 mM glucose, 1% agar,pH 6.9) for mycelial growth tests, and in carboxymethylcellulose(CMC) medium (1 g NH4NO3, 1 g KH2PO3, 0.5 gMgSO4ˑ7H2O, 1 g yeast extract, 15 g CMC and 1 l water)(Cappellini & Peterson, 1965) for conidiation assays.

Generation of gene deletion mutants

The double-joint PCR approach was used to generate the genereplacement construct for each target gene (Yu et al., 2014). Theprimers used to amplify upstream and downstream of each geneare listed in Supporting Information Table S1. The resultingPCR products for each gene were transformed into protoplasts ofthe wild-type strain, as described previously (Hou et al., 2002).Hygromycin B (Calbiochem, La Jolla, CA, USA) was added to afinal concentration of 100 mg ml�1 for transformant selection.The putative targeted gene deletion mutants were identified byPCR assays with the primer pairs listed in Table S1, and furtherconfirmed by Southern hybridization assays.

Growth, conidiation and stress sensitivity assays

Colony morphology and mycelial growth were assayed on PDAand MM amended without/with various compounds, as indi-cated in the figure legends. For conidiation assays, fresh mycelia(50 mg) of each strain were inoculated in a 50-ml flask contain-ing 20 ml of CMC broth. The flasks were incubated at 25°C for

4 d in a shaker (180 rpm). Subsequently, the number of conidiain each flask was determined using a hemocytometer. In addition,conidial germination was examined after fresh conidia were incu-bated at 25°C in 2% sucrose water. Each experiment wasrepeated three times.

Pathogenicity and DON biosynthesis assays

Pathogenicity assays on wheat spikelets were conducted asdescribed previously (Jiang et al., 2011). To determine DONproduction, a 50-g aliquot of healthy wheat kernels was sterilizedand inoculated with fresh mycelia (100 mg) of each strain. Afterincubation at 25°C for 20 d, DON and fungal ergosterol wereextracted according to previously described protocols (Mirochaet al., 1998; Liu et al., 2011). The DON extracts were purifiedwith PuriToxSR DON column TC-T200 (Trilogy AnalyticalLaboratory, Washington, MO, USA), and the amounts of DONand ergosterol in each sample were determined by an Agilent1100 HPLC system (Palo Alto, CA, USA), as described previ-ously (Liu et al., 2013). The experiment was repeated three times.

Western blotting hybridization

Fresh mycelia (200 mg) of each strain were finely ground and sus-pended in 1 ml of extraction buffer (50 mM Tris-HCl, pH 7.5,100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 2 mM phen-ylmethylsulfonylfluoride (PMSF)) containing 10 ll of proteaseinhibitor cocktail (Sangon Co., Shanghai, China). After homoge-nization with a vortex shaker, the lysate was centrifuged at10 000 g for 20 min at 4°C. Then, 100 ll of supernatant wasmixed with an equal volume of 29 loading buffer and boiled for5 min. The resulting proteins were separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and transferred to Immobilon-P transfer membrane (Millipore,Billerica, MA, USA). The phosphorylated FgMgv1 andFgGmpk1 were detected with the antiphospho-p44/42 kit (CellSignaling Technology, Boston, MA, USA). The upper and lowerbands represent the phosphorylated FgMgv1 and FgGmpk1,respectively (Ramamoorthy et al., 2007). In addition, FgMgv1and FgGmpk1 were detected using the anti-Mpk1 antibody(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and p44/44antibody (Cell Signaling Technology Inc., Beverly, MA, USA),respectively. The phosphorylated FgHog1 and total FgHog1were detected by the antibody against dually phosphorylated p38(Thr180/Tyr182) (Cell Signaling Technology Inc.) and ananti-Hog1 antibody (Santa Cruz Biotechnology), respectively.Incubation with a secondary antibody and chemiluminescentdetection were performed as described previously (Yang et al.,2013b). The experiment was conducted three times.

Affinity purification and mass spectrometry (MS)

To identify putative FgCdc14-interacting proteins, FgCdc14was tagged with 39 FLAG and transferred in the FgCDC14(Fg00543) deletion mutant (DFgCDC14), and the resultingtransformant was used for protein extraction as described earlier.

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In addition, the strain transferred only with 39 FLAG was usedas a control. After centrifugation at 10 000 g for 20 min at 4°C,the supernatant was transferred into a sterilized Eppendorf tube.Approximately 50 ll of anti-FLAG agarose (Abmart, Shanghai,China) was added to capture FgCdc14-interacting proteins, fol-lowing the manufacturer’s instructions. After incubation at 4°Covernight, the agarose was washed three times with 500 ll ofTBS (20 mM Tris-HCl, 500 mM NaCl, pH 7.5). Proteinsbinding to the beads were immediately eluted with 60 ll of elu-tion buffer (0.2 M glycine, pH 2.5). Eluates were instantly neu-tralized with 3 ll of neutralization buffer (1.5 M Tris, pH 9.0)and digested with trypsin using a previously described protocol(Tao et al., 2005; Zhou et al., 2007). Tryptic peptides were ana-lyzed as described previously (Ding et al., 2010). In addition,the physical interaction of FgCdc14 with FgTfc7 was furtherconfirmed by the yeast two-hybrid assay according to the previ-ously published protocol (Ma et al., 1987). Three independentexperiments were performed to confirm yeast two-hybrid assayresults.

Results

Identification and classification of putative phosphatasegenes

The sequences of all the PPs from the budding yeast S. cerevisiae,A. nidulans and Neurospora crassa were used for BLASTP searchesto identify their orthologs in the genome of F. graminearum(http://www.broadinstitute.org/annotation/genome/fusari-um_graminearum/MultiHome.html). In addition, we also usedthe catalytic domains of various classes of phosphatases, whichencode enzymes removing a phosphate group from their sub-strates (Majerus et al., 1999; Brenchley et al., 2007; Sacco et al.,2012), to identify additional phosphatase genes. In total, 82putative phosphatase genes were thus identified (Table S2). Basedon the catalytic domains identified with the program InterPro(Sacco et al., 2012), these phosphatases were classified into sevenphosphatase families (Table S2), including four PP families(including PPP, PPM, PTP and APP), one lipid phosphatasefamily (LP), one histidine phosphatase family (HP) and one alka-line phosphatase family (AP).

Construction of phosphatase gene deletion mutants

To examine the biological functions of the 82 phosphatases, weattempted to generate deletion mutants for each of the phospha-tase genes. The resulting hygromycin-resistant transformants ofeach gene were screened by PCR with the gene-specific primerpairs listed in Table S1. For 71 genes, multiple knockout mutantswere identified (Fig. S1b; Table S2). Twenty-one randomlyselected knockout mutants were confirmed by Southern hybridiza-tion assays. For these 71 genes, we obtained at least three deletionmutants for each gene with similar phenotypes, as described later.

For the other 11 putative phosphatase genes (Table S3), wefailed to identify true gene knockout mutants after screening over70 ectopic transformants from at least three independent

transformation experiments, indicating that the deletion of thesegenes may be lethal because of the high homologous recombina-tion efficiency in F. graminearum (Son et al., 2011; Wang et al.,2011a). For four of these, their orthologs are also essential inS. cerevisiae. However, deletion of the orthologs of the otherseven essential F. graminearum phosphatase genes was not lethalin the budding yeast (Table S3). By contrast, SSU72 and CDC14are essential genes in S. cerevisiae, but FgSSU72 and FgCDC14were dispensable for viability in F. graminearum. Deletion ofFgSSU72 (Fg00930) showed no detectable phenotype, whereasthe FgCDC14 (Fg00543) deletion mutant exhibited severedefects in hyphal growth (Table S2).

All the resulting gene deletion mutants were characterized fortheir defects in mycelial growth, colony and hyphal morphology,pigmentation, conidiation, conidium morphology, conidiumgermination, virulence on wheat heads and responses to variousstresses and fungicides. The resulting data are summarized inTable S2.

Involvement of phosphatases in the regulation of hyphalgrowth and conidiation

Colony morphology analyses showed that the deletion of 22phosphatase genes led to reduced mycelial growth by > 20% incomparison with the wild-type strain (Fig. 1a). The growth ratesof eight mutants (including DFg09619, DFg01464, DFg05281,DFg05894, DFg10239, DFgCDC14, DFg12867 and DFg07926)were < 40% of the wild-type (Table S2). Morphology examina-tion showed that the mutants of 12 genes revealed reduced pig-mentation on PDA (Table S2; Fig. S2). In addition, the aerialhyphae of DFg07783 were fluffier than those of the wild-type. Bycontrast, DFg05432 formed a compacted colony with limitedhyphal growth (Fig. 1b). It is interesting that DFg07926 showedincreased hyphal branches. However, DFg01527 and DFg05894displayed fewer branches (Fig. 1c).

For the 71 phosphatase mutants, we also compared the colonymorphology of each mutant grown on PDA and on MM. Over-all, most phosphatase mutants displayed similar growth pheno-types on both media, except for DFg10302 and DFg09532. It isvery interesting that DFg10302 grew much more rapidly on MMthan on PDA (Fig. 2a). Furthermore, this mutant also exhibitedstrong growth defects on wheat-head (WA) and CM (Fig. 2b). Asshown in Fig. 2(a), DFg09532 was unable to grow on MM, butgrew on PDA. Fg09532 shares 44% identity with the yeastMet22 mutant, which dephosphorylates 30-phosphoadenosine-50-phosphate (PAP) and 30-phosphoadenosine-50-phosphosulfate(PAPS) (Patron et al., 2008; Hudson & York, 2012). The yeastMET22 mutant can use methionine (Met) or cysteine (Cys) as asulfur source to restore its growth defects on sulfur-free Bmedium, but is defective in the utilization of sulfate and sulfite(Murgu�ıa et al., 1996). In order to explore the function ofFg09532 in F. graminearum, DFg09532 was incubated on MMsupplemented with various compounds. As shown in Fig. 2(c),DFg09532 was able to grow on MM amended with 1 mM Met,but the addition of 1 mM Asp failed to restore the defect ofhyphal growth of DFg09532 on MM (Fig. 2c). These results

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indicate that Fg09532 may be involved in Met biosynthesis inF. graminearum.

Among the 71 non-essential genes, 10 gene deletion mutantsshowed reduced conidiation by > 40% in comparison with thewild-type (Fig. 3a). Among these 10 mutants, seven (DFg09619,DFg05281, DFg05894, DFg00543, DFg12867 DFg07926 andDFg09532) also exhibited strong defects in hyphal growth onPDA and MM (Table S2). By contrast, DFg10239 displayedincreased conidiation, but grew significantly more slowly thanPH-1 (Table S2). Microscopic examination showed that themutants of five phosphatase genes (Fg05894, Fg10516,Fg07926, Fg01527 and Fg12867) produced abnormal conidia(Fig. 3c). The conidia of these mutants were shorter and smallerwith fewer septa than those of PH-1 (Fig. 3d,e). Most (65%)conidia of the wild-type had five or six septa. However, 90% ofthe conidia of DFg05894 had only one or two septa (Fig. 3c,d).In addition, the conidia of DFg01527 showed less septation,although the mutant produced more conidia than PH-1(Fig. 3b,d). We also examined conidial germination for the 71phosphatase mutants and found that none was blocked inconidial germination, but DFg05894 showed a delay in conidialgermination (Table S2).

Involvement of phosphatase in the regulation of virulenceand DON biosynthesis

The virulence of each mutant was evaluated on flowering wheatheads. The wild-type strain caused scab symptoms in > 65% ofspikelets of inoculated wheat heads, and had a disease severity of5. Among the 71 mutants, 25 (35.2%) were significantly reducedin virulence and had a disease severity of < 3 (Fig. 4a). In general,the mutants that showed defects in mycelial growth were alsoimpaired in virulence (Fig. 4a). Although four mutants(DFg06297, DFg03333, DFg03826 and DFg07932) did not dis-play obvious defects in mycelial growth, they demonstrated sig-nificantly reduced virulence with a disease severity of < 3. Wefurther analyzed the distribution of orthologs of these four genesin 159 fungi and five oomycetes using the BLASTMatrix tool(CFGP, http://cfgp.riceblast.snu.ac.kr/). It is interesting that two(Fg03333 and Fg03826) were only distributed in Pezizomycotinapathogenic fungi, indicating that these genes may be stronglyrelated to virulence in these fungi.

Unexpectedly, DFg10516 was unable to infect the inoculatedspikelet at all (Fig. 4b). To analyze the virulence defect in detail,the penetration behavior of this mutant was also examined on

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Fig. 1 Phosphatase mutants of Fusariumgraminearum with vegetative defects.(a) Relative mycelial growth rate of thephosphatase mutants in comparison with thewild-type (WT) on potato dextrose agar(PDA) after 4 d of incubation at 25°C.(b) Comparison of colony morphology in WTand nine phosphatase mutants on PDA.(c) Hyphal tip growth and branchingpatterns of WT, DFg01527, DFg07926 andDFg05894 on complete medium (CM) slabagar. Bar, 100 lm.

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cellophane membranes and on wheat spikelets using previouslypublished protocols (Lopez-Berges et al., 2010). As shown inFig. 4(c), DFg10516 was unable to penetrate the cellophanesheet. In addition, the wild-type strain produced a number ofpenetration structures on inoculated spikelets, but it was difficultto find such structures for DFg10516 at 7 d post-inoculation(Fig. 4d), indicating that Fg10516 may be essential for the pene-tration of F. graminearum into host plant tissues.

DON has been identified as an important virulence factor ofF. graminearum (Seong et al., 2008). We thus determined theamount of DON production on wheat kernels for the mutantsthat displayed reduced virulence. Eleven mutants that wereimpaired in virulence also showed reduced DON biosynthesis(Table 1); in particular, DFg09532 produced an undetectableamount of DON. By contrast, DFg01464 did not show a signifi-cant change in DON production, and DF10302 exhibitedincreased DON biosynthesis. However, both mutants showedstrongly reduced virulence (Table 1), indicating that factors otherthan DON may be responsible for the reduced virulence in thesemutants.

Identification of negative regulators of mitogen-activatedprotein kinase (MAPK) pathways

MAPK cascades are activated by the dual phosphorylation of con-served Thr and Tyr residues within the activation loop (Hamelet al., 2012) by MAP kinases (MAPKs), which are, in turn,

activated by MAPK kinases (MAPKKs) to transduce extracellularsignals. In F. graminearum, three MAPK pathways, consisting ofGpmk1 MAPK, Hog1 MAPK and Mgv1 MAPK, have beencharacterized (Hou et al., 2002; Jenczmionka et al., 2003; Zhenget al., 2012). However, the phosphatases involved in these MAPKcascades as the negative regulators have rarely been identified.

The Mgv1 MAPK pathway, consisting of FgBck1, FgMmk1and FgMgv1, is homologous to the cell wall integrity (CWI)pathway in the budding yeast (Wang et al., 2011a). InS. cerevisiae, the PTP family members Msg5, Ptp2, Ptp3 andSdp1 function as negative regulators of the CWI pathway (Levin,2005). In this study, we tested the sensitivity of 71 phosphatasemutants to the cell wall stress agent Congo red (CR). Deletion ofthe orthologs of ScPTP2 (Fg06297), ScPTP3 (Fg11979) andScSDP1 (Fg04296) had no influence on the sensitivity to CR(Table S2), indicating that these PTPs may not be associated withthe CWI pathway or their functions may be redundant inF. graminearum. However, three mutants (DFg10239, DFg06977and DFg10516) showed significantly increased tolerance to CR(Fig. 5a; Table S2). FgMsg5 (Fg06977) has been found todephosphorylate FgMgv1 in a previous study (Yu et al., 2014);we therefore analyzed the phosphorylation level of FgMgv1 inthe other two mutants. Although DFg10239 did not show achange in the phosphorylation level of FgMgv1, deletion ofFg10516 resulted in an increased level of FgMgv1 phosphoryla-tion (Fig. 5b,c), indicating that Fg10516 functions as a negativeregulator of the CWI pathway in F. graminearum.

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Fig. 2 Involvement of phosphatases in response to nutrients in Fusarium graminearum. (a) Comparisons of mycelial growth in the wild-type (WT),DFg10302, and DFg05932 on potato dextrose agar (PDA) and minimal medium (MM). (b) DFg10302 grew much more rapidly on MM than on PDA,wheat-head medium (WA) and complete medium (CM). (c) Comparisons of mycelial growth betweenWT and DFg09532 on PDA and MMwithout orwith different agents as indicated in the figure. Asp, aspartate; Cys, cysteine; Met, methionine.

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The high osmolarity glycerol (HOG) MAPK signaling path-way is required for fungal growth under hyperosmotic conditions(Zheng et al., 2012). PPM family members (Ptc1, Ptc2 andPtc3), together with the PTP phosphatases Ptp2 and Ptp3,dephosphorylate the Hog1 kinase to inactivate the osmosensingHOG pathway in S. cerevisiae (Miermont et al., 2011). InF. graminearum, however, except for the mutant of FgPTC3,which displayed reduced sensitivity to osmotic stresses (Jiang

et al., 2011), the mutants of FgPTC1 (Fg04111), FgPTP2(Fg06297) and FgPTP3 (Fg11979) showed no detectable changesin response to osmotic stress mediated by 1M sorbitol or 0.7 MNaCl (Table S2). Unexpectedly, DFg12867 exhibited increasedtolerance to osmotic stress (Fig. 5d). Furthermore, the phosphor-ylation level of FgHog1 increased significantly in this mutant(Fig. 5e,f), indicating that the phosphatase Fg12867 acts as a neg-ative regulator of the HOG pathway in F. graminearum.

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Fig. 3 Phosphatase mutants of Fusarium graminearumwith defects in conidiation and conidium morphology. (a) Number of phosphatase mutants withdifferent conidiation abilities in comparison with the wild-type (WT). (b) The number of conidia produced by WT, DFg05894, DFg10516, DFg07926,DFg01527 or DFg12867. Line bars in each column denote + SE of three repeated experiments. Values on the bars followed by the same letter are notsignificantly different at P = 0.05. (c) Conidial morphology of WT, DFg05894, DFg10516, DFg07926, DFg01527 and DFg12867. Differential interferencecontrast (DIC) images of conidia stained with Calcofluor white were captured with an electronic microscope. Bar, 30 lm. (d) Percentages of conidia withdifferent numbers of septa in WT, DFg05894, DFg10516, DFg07926, DFg01527 and DFg1286. (e) Percentages of conidia with different lengths in WT,DFg05894, DFg10516, DFg07926, DFg01527 and DFg1286.

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In F. graminearum, the FgGpmk1 signaling pathway is knownto control a variety of virulence-related functions (Gu et al.,2015). When the 25 phosphatase deletion mutants with reducedvirulence were assayed for FgGpmk1 phosphorylation, we foundthat only DFg03333 exhibited increased phosphorylation ofFgGpmk1 (Fig. 5g,h). These results indicate that Fg03333 func-tions as a negative regulator of FgGpmk1.

FgCdc14 plays an important role in the regulation of celldivision

For each of the 71 gene deletion mutants, we also examined thedistribution of nuclei in conidia stained with 1 lg ml�1 of 40,6-diamidino-2-phenylindole (DAPI). In comparison with PH-1,70 of the 71 mutants exhibited no recognizable changes in

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Fig. 4 Phosphatase mutants of Fusarium graminearumwith defects in virulence. (a) The number of phosphatase mutants with different disease severities.Disease severity: 0, mutant was nonpathogenic on flowering wheat head; 1, infection existed only in the inoculated wheat spikelets; 2, 3 and 4,percentages of infected spikelets in the inoculated wheat heads range from 5% to 25%, 25% to 45% and 45% to 65%, respectively; 5, > 65% spikeletswere infected in the inoculated wheat heads. The wild-type (WT) caused typical scab symptoms with a disease severity of 5. (b) Comparison of virulencebetweenWT and DFg10516. (c) Penetration of WT and DFg10516 into cellophane membrane. Fungal colonies were grown for 2 d at 25°C on top of acellophane membrane placed on minimal medium (MM) (Before). The cellophane membranes with the fungal colonies were removed, and the plates wereincubated for an additional day to examine the presence of mycelial growth on the plate, indicating penetration of the cellophane (After). (d) Infectionassay on dissected wheat glumes. Wheat head spikelets were inoculated with conidia of WT or DFg10516. The infection structure (lobate appressoriumindicated by arrows) was observed easily fromWT, but not from DFg10516.

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nucleus distribution in conidia. However, DFgCDC14 containedmultiple nuclei in single conidial cells (Fig. S3). To analyze thisdefect in detail, the histone H1 fused with red fluorescent protein(RFP) was transferred into PH-1 and DFgCDC14. Again, theDFgCDC14 mutant contained multiple nuclei within singleconidial cells, but only one (rarely two) nucleus was observed ineach compartment in the wild-type conidia (Fig. 6c,e). In addi-tion, DFgCDC14 also showed strongly reduced conidiation, sep-tation and hyphal growth (Fig. 6a,b,d). Furthermore, it isinteresting that c. 20% of DFgCDC14 conidia were misshapenwith two conidia interconnected (Fig. 6c). However, such inter-connected conidia were not observed for PH-1.

FgCdc14 is homologous to S. cerevisiae Cdc14, a conservedDSP, which is present in a wide range of organisms from yeastto humans (Fig. 7a). Vertebrates contain three Cdc14 orthologs(Mocciaro & Schiebel, 2010), but both S. cerevisiae and the fil-amentous fungi (including Fusarium spp., Aspergillus spp. andNeurospora spp.) contain a single Cdc14 protein. Phylogenicanalyses showed that Cdc14 orthologs from fungi were clus-tered into a group (group I), which is separated from the ani-mal Cdc14 group (group II) (Fig. 7a). In S. cerevisiae, Cdc14 isessential and plays a critical role in late events of cell division,including the orchestration of several aspects of chromosomesegregation and triggering exit from mitosis (Amon, 2008). Tocharacterize its functions, the full-length FgCDC14 cDNA wascloned into the yeast expression vector pYES2 and introducedinto the yeast mutant BY4741CDC14 carrying a temperature-sensitive CDC14 allele. The mutant transformed with anempty pYES2 vector served as a negative control. As shown inFig. 7(b), the BY4741CDC14 mutant failed to grow on YPRG(yeast extract/peptone/raffinose/galactose) medium at 37°C,

and this growth defect was partially restored by genetic com-plementation of the yeast mutant with full-length FgCDC14.These results indicate that FgCdc14 and ScCdc14 may sharesimilar functions in the regulation of cell division.

FgCdc14 is involved in ribosome biogenesis inF. graminearum

Although S. cerevisiae CDC14 is an essential gene, DFgCDC14was viable, but with severe growth defects (Fig. 6a), indicatingthat the roles of Cdc14 orthologs in filamentous fungi may bepartially different from those in the budding yeast. Thus, to fur-ther elucidate the functions of FgCdc14, we performed serialanalysis of gene expression (SAGE) assays with DFgCDC14 andPH-1. For SAGE data, the analyses were usually limited to prede-fined tags showing at least five-fold difference in abundance withP ≤ 0.05 (Audic & Claverie, 1997). With this criterion, we iden-tified 194 genes upregulated and 310 genes downregulated inDFgCDC14. It was unexpected that, among the 194 upregulatedgenes, 68 encode ribosome-related proteins. These results indi-cate that FgCdc14 may be associated with ribosome biogenesis inF. graminearum. To verify this, we looked for ribosomes in thecells of DFgCDC14 by transmission electron microscopy. Asshown in Fig. 8(e), cells of PH-1 were filled with the small darkspheroid ribosomes, but ribosomes were strongly reduced inDFgCDC14 cells. These results indicate that FgCdc14 may playan important role in the regulation of ribosome biogenesis inF. graminearum.

To further explore the molecular mechanism of FgCdc14 inregulating ribosome biogenesis, we attempted to identifyFgCdc14-interacting proteins using the affinity purification andMS approach. Briefly, FgCdc14 was tagged with 39 FLAG, andproteins co-purified with FgCdc14-39 FLAG were analyzed byMS. In this assay, FgTfc7 (Fg09985), a transcription factor ofthe TFIIIC family, was co-purified with FgCdc14 (Table 2). Theinteraction of FgCdc14 with FgTfc7 was further confirmed byyeast two-hybrid assays (Fig. 8a). Furthermore, both FgCdc14and FgTfc7 localized to the nucleus (Fig. 8b).

Earlier studies have shown that Tfc7 is required for RNA poly-merase (Pol) III in transcribing 5S rRNA, which is a part of ribo-somes (Teichmann et al., 2010; Acker et al., 2013). InS. cerevisiae, Tfc7 is essential. Similarly, we failed to deleteFgTFC7 in F. graminearum, although we obtained 112 ectopictransformants from five independent transformation experi-ments. Thus, we silenced FgTFC7 using the double-strand RNA(dsRNA)-induced gene silencing strategy. A hairpin structurecontaining a 685-bp fragment of FgTFC7 was constructed andtransferred into PH-1. Subsequently, a transformant (DFgTFC7-PSli) with severe defects in mycelial growth was obtained(Fig. 8c). Real-time PCR assay showed that the expression levelof FgTFC7 was significantly reduced in DFgTFC7-PSli (Fig. 8d).Microscopic examination showed that DFgTFC7-PSli containedsignificantly fewer ribosomes in comparison with PH-1. Takentogether, these data indicate that FgCdc14 may play an impor-tant role in ribosome biosynthesis, probably via its interactingprotein FgTfc7, in F. graminearum.

Table 1 Phosphatase genes play important roles in virulence anddeoxynivalenol (DON) production in Fusarium graminearum

StrainDiseaseseveritya

DON productionb

(mgmg�1 ergosterol)

Wild-type 5 5.1� 0.12DFg09619 1 1.89� 0.56DFg05281 1 0.15� 0.13DFg05894 1 0.36� 0.28DFg10239 1 0.17� 0.05DFg00543 1 0.10� 0.23DFg10516 0 2.20� 0.89DFg05432 1 0.11� 0.01DFg12867 1 0.79� 0.22DFg01527 1 0.47� 0.07DFg07926 1 0.61� 0.04DFg09532 1 0DFg01464 1 4.98� 0.09DFg10302 1 14.29� 3.21

a

Virulence on flowering wheat heads was scored. Disease severity: 0,mutant was nonpathogenic on flowering wheat head; 1, infection existedonly in the inoculated wheat spikelets. The wild-type caused typical scabsymptoms on > 65% of spikelets in the inoculated wheat heads with adisease severity of 5.b

Percentage of DON production of each mutant in comparison with thatof the wild-type; � SE was calculated from three repeated experiments.

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An essential role of the HCX5R motif in FgCdc14

Clustalw2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) analysisshowed that the Cdc14 orthologs from various organisms containa highly conserved HCX5R motif in the N-terminus, whereas theC-terminus is highly variable (Fig. 7a). In order to identify itsfunctional motifs, we generated a series of FgCDC14 deletionconstructs (Fig. 9a). The truncated FgCDC14s were then trans-formed into DFgCdc14 for complementation assays, includingDFgCdc14-C1 (DFgCdc14 complemented with a fragment of 1–543 amino acids of FgCdc14 without the nucleus localization sig-nal (NLS) motif), DFgCdc14-C2 (complemented with 1–507amino acids of FgCdc14 without the nucleus export signal (NES)and NLS motifs), DFgCdc14-C3 (complemented with 1–405

amino acids of FgCdc14, which contain the HCX5R domain),DFgCdc14-C4 (DFgCdc14 bearing a FgCdc14 fragment lackingthe HCX5R motif) and DFgCdc14-C(R/A) (DFgCdc14 bearinga point-mutated FgCdc14, which has a site mutation from argi-nine (Arg) to alanine (Ala) at residue 348 in the HCX5R motif).As shown in Fig. 9(b), DFgCdc14-C1, DFgCdc14-C2 andDFgCdc14-C3 partially recovered the mycelial growth defects ofDFgCdc14. Furthermore, these three strains showed normal con-idiation and virulence (Fig. 9c,d). In addition, the growth defectof the BY4741CDC14 mutant was also restored partially bygenetic complementation of the yeast mutant with the truncatedFgCDC14 which contains 1–405 amino acids of FgCdc14(Fig. 7b). These results indicate that the C-terminus of FgCdc14containing NES and NLS motifs is dispensable for the important

(a) (b) (c)

(d) (e) (f)

(g) (h)

Fig. 5 Identification of Fg10516, Fg12867 and Fg03333 as negative regulators of mitogen-activated protein kinase (MAPK) pathways in Fusariumgraminearum. (a) Comparison of the sensitivity to Congo red between the wild-type (WT) and DFg10516. Bars denote � SE from three repeatedexperiments. Values on the bars followed by the same letter are not significantly different at P = 0.05. (b) Comparison of the phosphorylation level ofFgMgv1 and FgGmpk1 in WT and DFg10516. (c) The intensities of the Western blotting bands were quantified with the program IMAGE QUANT TL. Theintensity of the FgMgv1 band in each mutant is relative to the amount of FgMgv1 in the WT. Bars denote � SE from three repeated experiments. Valueson the bars followed by the same letter are not significantly different at P = 0.05. (d) Comparison of the sensitivity to sorbitol and NaCl betweenWT andDFg12867. Statistical analyses were performed as described in (a). (e) Comparison of the phosphorylation level of FgHog1 in WT and DFg12867. (f) Theintensities of the Western blotting bands were quantified and analyzed as described in (c). (g) Comparison of the phosphorylation level of FgGmpk1 andFgMgv1 in WT and DFg03333. (h) The intensities of the Western blotting bands were quantified and analyzed as described in (c).

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functions of FgCdc14. DFgCdc14-C4 and DFgCdc14-C(R/A),however, showed similar defects in mycelial growth, conidiationand virulence as DFgCdc14 (Fig. 9b,d), indicating that theHCX5R motif is essential for the biological functions ofFgCdc14.

Discussion

In this study, we targeted each of 82 putative phosphatases forgene deletion and characterized 71 gene deletion mutants ofF. graminearum. One advantage of large-scale genetic analysis isthat these mutants constructed from the same progenitor havethe potential to be phenotypically grouped as likely componentsof the same process when similar phenotypes are uncovered.Given that several phosphatases may function together in a sig-naling pathway, this phenotypic grouping provided experimental

evidence that previously uncharacterized phosphatases functionin the CWI pathway, as well as in the pathways regulating otherstress responses and virulence. In addition, the collection of phos-phatase mutants is also helpful in the identification of phosphata-ses which show species-specific functions. For example, it is notunexpected that phosphatases which are essential in one organismcan be nonessential in others. This also holds true within fungi.In this study, we found that Fg06103 and Fg02043 may beessential in F. graminearum (Table S3), but their orthologs arenonessential in A. nidulans (Son & Osmani, 2009), suggestingthat other unidentified phosphatases may share similar functionswith the Fg06103 and Fg02043 orthologs in A. nidulans. Simi-larly, the ortholog of Fg01918 is essential in N. crassa (Tanakaet al., 2007), but DFg01918 of F. graminearum grew as well asthe wild-type strain. Thus, it would be interesting to further char-acterize these phosphatases with species-specific functions in the

(a)

(c)

(b)

(d)

(e)

Fig. 6 Involvement of FgCdc14 in the regulation of hyphal growth, conidiation and cell cycle regulation in Fusarium graminearum. (a) Colony morphologyof the wild-type (WT), DFgCDC14 and the complemented strain DFgCDC14-C on potato dextrose agar (PDA) after 3 d of incubation at 25°C (upperpanel). Hyphae of WT, DFgCDC14 and DFgCDC14-C stained with Calcofluor white (CFW, lower panel). Bar, 100 lm. (b) Comparison of conidiation inWT, DFgCDC14 and DFgCDC14-C. Line bars in each column denote + SE of three repeated experiments. Values on the bars followed by the same letterare not significantly different at P = 0.05. (c) Conidial morphology of WT and DFgCDC14. The nuclei were observed by fusing histone H1 with redfluorescent protein (RFP). DIC, differential interference contrast image. Bar, 30 lm. (d) Percentages of conidia with different septum numbers in WT,DFgCDC14 and DFgCDC14-C. (e) The average number of nuclei in single conidial cells of WT, DFgCDC14 and DFgCDC14-C. Values on the bars followedby the same letter are not significantly different at P = 0.05.

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pathogenic fungi. In the long term, defining the function of spe-cies-specific phosphatases is important as they are potential candi-dates for the development of specific antifungal agents againstpathogenic fungi.

Phosphatases involved in virulence

Among the 71 phosphatase mutants of F. graminearum, 25 dis-played serious defects in virulence (disease severity of < 3).Twenty-one showed growth defects (Fig. 4a), which may bedirectly responsible for reduced virulence. However, the otherfour (including DFg06297, DFg03333, DFg03826 andDFg07932) showed no obvious changes in hyphal phenotype,but displayed dramatic defects in virulence. Fg06297 shared 32%identity with Ptp1 of S. cerevisiae, and its orthologs (BcPtpA andBcPtpB) in Botrytis cinerea are not only involved in the process ofplant infection, but also in the regulation of vegetative growth(Yang et al., 2013b). DFg03333 displayed defects in both viru-lence and the production of DON (Table S2), which is a critical

virulence factor for F. graminearum (Boenisch & Sch€afer, 2011).However, mutants of APP family member Fg03826 and LP fam-ily gene Fg07932 showed increased DON production, but, unex-pectedly, still displayed defects in plant infection, indicating thatthese phosphatases may play a critical role specifically in theinfection process.

PP2C members are well conserved in the filamentous fungi,including Aspergillus spp., B. cinerea, Magnaporthe oryzae,N. crassa and F. graminearum (Ari~no et al., 2011; Jiang et al.,2011; Yang et al., 2013a). A previous study has shown thatBcPtc1 and BcPtc3 of B. cinerea play an important role in plantinfection (Yang et al., 2013a). Similarly, in this study, we foundthat DFg10239 (DFgPTC3) of F. graminearum showed a seri-ous defect in virulence on wheat spikelets. The mutant ofFg07926 (FgTPS2), which encodes a trehalose 6-phosphatephosphatase involved in the biosynthesis of trehalose, also dis-played impaired virulence, which is consistent with a previousfinding (Song et al., 2014). In addition, FgTps2 orthologs inA. fumigatus and Candida albicans have been reported to beimportant in pathogenesis (Maidan et al., 2008; Puttikamonkulet al., 2010). These results highlight that the function of treha-lose 6-phosphate phosphatase in virulence may be conserved inpathogenic fungi.

The orthologs of the PP2A catalytic subunit are not essentialfor fungal growth, but display critical roles in pathogenesis in sev-eral pathogenic fungi, including B. cinerea, M. oryzae,F. verticillioides and Sclerotinia sclerotiorum (Erental et al., 2007;Choi & Shim, 2008; Giesbert et al., 2012; Du et al., 2013). Inthis study, we were unable to obtain a mutant of the PP2A cata-lytic subunit (Fg09825) in F. graminearum. In addition, for a sec-ond PPP member Ppz1, its orthologs have been focused onvirulence in several fungi. In C. albicans, the lack of PPZ1 led toimpaired virulence in a mouse model (�Ad�am et al., 2012). InA. fumigatus, deletion of PPZA also showed a defect in virulencein a murine model of corneal infection (Muszkieta et al., 2014).However, in this study, DFgPPZ1 (DFg00852) did not show anydetectable defects in virulence. These results indicate that thePPPs may show species-specific functions, although their struc-tures are conserved in pathogenic fungi.

Phosphatase involved in response to nutrient stresses

In this work, we found, unexpectedly, that DFg10302 grewmuch better on MM than on nutrient-rich media (includingPDA, wheat-head medium and CM). Fg10302 shares 43%identity to S. cerevisiae Nem1, which cooperates with its regula-tory subunit Spo7 to dephosphorylate Smp2. Smp2 is a keyregulator of nuclear membrane formation during the cell cycle(Karanasios et al., 2010; Dubots et al., 2014). The NEM1mutant showed enlarged and irregularly shaped nuclei, oftenconsisting of two or more interconnected lobes within a singlecell. Interestingly, despite these defects in nuclear structure, themutant grew normally at 30°C, and did not display defects innucleocytoplasmic transport (Santos-Rosa et al., 2005).Recently, the Nem1 ortholog has been characterized in thepathogenic fungus M. oryzae. In contrast with our observations

(a)

(b)

Fig. 7 FgCdc14 is homologous to the budding yeast Cdc14 (ScCdc14).(a) Phylogenetic analysis of Fusarium graminearum FgCdc14 with itsorthologs from other organisms, including Neurospora crassa (NcCdc14),Magnaporthe oryzae (MoCdc14), Aspergillus nidulans (AnCdc14),Saccharomyces cerevisiae (ScCdc14), Candida albicans (Clp1), Ustilagomaydis (UmCdc14), Caenorhabditis elegans (CeCdc14), Homo sapiens

(hCdc14A, hCdc14B and hCdc14C) and Xenopus laevis (XCdc14A andXCdc14B), using the neighbor-joining method with MEGA 5 software (leftpanel). Values on the branches of clusters represent the results ofbootstrap analysis. Schematic representations of the structures of Cdc14proteins (right panel). The conserved domain is shown in gray. Thecatalytic motif (HCX5R motif) and its amino acid sequence are highlightedin red, the nucleus localization signal (NLS) in green and the nucleus exportsignal (NES) in blue. (b) Fusarium graminearum FgCdc14 and thetruncated FgCdc14 (1–405 amino acids) could partially complement ayeast temperature-sensitive (ts) mutant of ScCDC14. The BY4741-derivedts mutant BY4741CDC14 was transferred with pYES2, pYES2-FgCdc14 orpYES2-FgCdc14 (1–405), respectively. Yeast cells as indicated in the figurewere spotted on yeast extract/peptone medium containing 2% galactosemedium (YPG) and incubated at 25 or 37°C for 3 d.

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in DFgNem1, DMoNem1 displayed dramatically slower growthon MM than on CM (Wang et al., 2011b). Thus, it would bevery interesting to further explore the species-specific functionsof FgNem1 in the regulation of the nutrient response inF. graminearum.

Phosphatases involved in the regulation of MAPKs

In plant and animal pathogens, MAPK cascades are known toregulate various fungal developmental and infection processes(Rispail et al., 2009). To date, five MAPK pathways have been

(a)

(c)

(d)

(e)

(b)

Fig. 8 Involvement of FgCdc14 in ribosome biogenesis of Fusarium graminearum. (a) Yeast two-hybrid analysis of the physical interaction betweenFgCdc14 and FgTfc7. The pair of plasmids pEXP32/Krev1 and pEXP22/RalGDS-wt served as a positive control. The pair of plasmids pEXP32/Krev1 andpEXP22/RalGDS-m2 was used as a negative control. Columns in each panel represent serial decimal dilution. (b) Subcellular localization of FgCdc14 (left)and FgTfc7 (right) in F. graminearum. FgCdc14-green fluorescent protein (GFP) and FgTfc7-GFP were both localized to the nucleus. Nuclei in hyphae werestained with 40,6-diamidino-2-phenylindole (DAPI). Bar, 10 lm. (c) Colony morphology of the wild-type (WT) and FgTFC7-silenced transformant(DFgTFC7-PSli) on potato dextrose agar (PDA) after 3 d of incubation at 25°C. (d) Transcription level of FgTFC7 in DFgTFC7-PSli in comparison with thatin the WT. Line bars in each column denote + SE of three repeated experiments. Values on the bars followed by the same letter are not significantlydifferent at P = 0.05. (e) Transmission electron microscopic examination of the hyphae of WT, DFgCDC14 and DFgTFC7-PSli. Cells of WT were filled withsmall dark spheroid ribosomes, but the numbers of ribosomes were reduced dramatically in DFgCDC14 and DFgTFC7-PSli.

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identified in S. cerevisiae; orthologs of core elements of MAPKcascades have also been characterized in several filamentousfungi, including M. oryzae, B. cinerea, Aspergillus spp. andFusarium spp. (Thomas et al., 1992; Yu et al., 2004; Rui &Hahn, 2007; Valiante et al., 2009; Hamel et al., 2012). How-ever, much attention has focused on the elements which trans-fer signals to activate the MAPK cascades; knowledge on thephosphatases that act as negative regulators to inactivate thephosphorylated elements is very limited. For the HOG cascadein B. cinerea, mutants of four phosphatases homologous to thenegative regulators of S. cerevisiae Hog1 showed drastic defectsin hyphal growth and conidiation, but only one (BcPTC3mutant) exhibited increased phosphorylated BcSak1 (the ortho-log of Hog1) (Yang et al., 2013a,b), indicating that the func-tions of the three other phosphatases may be redundant in theregulation of BcSak1 phosphorylation. In F. graminearum,FgPtc3 has also been found to be associated with the HOG

Table 2 Putative FgCdc14-interacting proteins identified by affinitycapture assays

Protein Putative function

Fg01158 Small heat shock protein (sHSP) with chaperone activityFg05216 Putative formin proteinFg05587 Inner plaque spindle pole body (SPB) componentFg08856 Putative rRNA-binding proteinFg09985 RNA Pol III transcription initiation factor complex (TFIIIC)

subunit, Tfc7Fg11563 Hypothetical Cdc28 substrateFg01241 Hypothetical proteinFg01414 Putative component involved in endoplasmic reticulum

(ER)-to-Golgi SNARE complexFg05972 Nucleoside diphosphate kinaseFg09974 Ribosomal 60S subunit proteinFg04213 GTPase-activating proteinFg01008 Translation elongation factor 1 beta

(a)

(c) (d)

(b)

Fig. 9 The essential role of the HCX5R motif in FgCdc14 of Fusarium graminearum. (a) Schematic representation of the structure of FgCdc14 and itstruncated proteins. The conserved protein tyrosine phosphatase (PTP) domain in Cdc14 orthologs is denoted in red, the N-terminus and C-terminusdomains in green and blue, respectively, the HCX5R motif in yellow, the nucleus export signal (NES) in purple and the nucleus localization signal (NLS) ingray. The truncated or mutated FgCdc14 (as indicated in the figure) was transferred into DFgCdc14. (b) The colony morphology of the wild-type(WT), DFgCDC14 and its complemented strains bearing truncated FgCdc14 (as shown in a) incubated on potato dextrose agar (PDA) at 25°C for 3 d.DFgCdc14-C was obtained from DFgCdc14 complemented with full-length FgCDC14. DFgCdc14-C1, DFgCdc14-C2, DFgCdc14-C3 and DFgCdc14-C4are the strains of DFgCdc14 complemented with fragments of 1–543, 1–507, 1–405 or 1–305 amino acids of FgCdc14, respectively. DFgCdc14-C(R/A)was obtained from DFgCdc14 transferred with full-length FgCDC14 containing a point mutation from arginine (Arg) to alanine (Ala) at residue 348 in theHCX5R motif. (c) Comparison of conidiation in WT, DFgCdc14 and the complemented strains. Line bars in each column denote + SE of three repeatedexperiments. Values on the bars followed by the same letter are not significantly different at P = 0.05. (d) Wheat heads were point inoculated with aconidial suspension of each strain.

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pathway (Jiang et al., 2011). In this study, we found that themutant of Fg12867 (a member of the APP family) showedincreased tolerance to osmotic stresses and elevated phosphory-lation level of FgHog1, indicating that Fg12867 is a secondnegative regulator of FgHog1. More recently, mutants of 24PP genes have been used to detect the phosphorylation level ofNcHog1 in N. crassa (Ghosh et al., 2014). Four showed an ele-vated basal phosphorylation level of NcHog1. These four phos-phatases belong to different families (PPP, PTP and APP).These results indicate that, in contrast with S. cerevisiae, diversephosphatases may function as the negative regulators of theHOG pathway in filamentous fungi.

In a previous study, we found that Fg06977 (FgMsg5) acts asa negative regulator of FgMgv1 in F. graminearum (Yu et al.,2014). In the current study, we further observed that deletion ofFg10516, which encodes a phosphatase sharing 32% identitywith S. cerevisiae ScYvh1, leads to increased phosphorylatedFgMgv1 (Fig. 5b). In S. cerevisiae, both Msg5 and Yvh1 belongto the VH1-like dual specific phosphatases (Doi et al., 1994;Sakumoto et al., 2001). Yvh1 orthologs are conserved from thebudding yeast to humans. The human ortholog can rescue theslow growth defect of the ScYVH1 mutant (Muda et al., 1999),indicating that the functions of the VH1-like dual specific phos-phatase may be highly conserved from fungi to humans.

The deletion of Fg03333, an APP gene, resulted in an elevatedphosphorylation level of FgGmpk1 in F. graminearum.Saccharomyces cerevisiae lacks a distinct ortholog of Fg03333.Although its orthologs exist in several filamentous ascomycetes,including Colletotrichum spp. and Metarhizium spp., to date, thefunction of this phosphatase has not been documented in anyfungal species. Interestingly, we observed that DFg03333 wasimpaired in virulence, although it did not show detectable defectsin hyphal growth and conidiation (Table S2), indicating that thisphosphatase is not required for fungal growth, but may play aspecific role in pathogenesis in filamentous fungi.

FgCdc14 is involved in the regulation of cell division andribosome biogenesis

In S. cerevisiae, ScCdc14 is essential and counteracts the activityof cyclin-dependent kinase Cdc28 at the end of mitosis, and thusplays an important role in the regulation of anaphase, mitotic exitand cytokinesis (Queralt & Uhlmann, 2008). In the currentstudy, we found that DFgCdc14 is viable, although it producedconidia with more than two nuclei in single conidial cells. Inaddition, the mutant also produced some interconnected conidia(Fig. 6c). Furthermore, FgCdc14 could partially restore thegrowth defect of the S. cerevisiae CDC14 mutant (Fig. 7b). Theseresults indicate that FgCdc14 is associated with cell division inF. graminearum. It is interesting that, although S. pombe also con-tains a single Cdc14 ortholog (named Clp1), Clp1 is not essentialand does not appear to be important for mitotic exit (Papado-poulou et al., 2010). In human cells, three Cdc14 orthologs(HCdc14A, HCdc14B and HCdc14C) have been identified (Liet al., 2000; Rosso et al., 2008). Analysis of the human Cdc14proteins by overexpression or depletion has suggested that their

functions are quite different from those of ScCdc14. ThehCdc14A protein is found in the cytoplasm and on centrosomesin interphase, but not on centrosomes in mitotic cells (Mailandet al., 2002). hCdc14B is predominantly localized in the nucleo-lus (Berdougo et al., 2008). It has been shown that overexpressionof hCdc14C fused to fluorescent reporters leads to its localizationon microtubules and at the endoplasmic reticulum (ER) (Rossoet al., 2008). Thus, the functions of Cdc14 orthologs vary signifi-cantly, although they are structurally conserved in a wide range oforganisms from yeasts to humans.

As Cdc14 phosphatases contribute to diverse functions in vari-ous organisms, we were interested in characterizing Cdc14 func-tions in a filamentous fungus. It was unexpected that FgCdc14was highly associated with ribosome biogenesis (Fig. 8e). Byaffinity purification and yeast two-hybrid assays, we identified anovel FgCdc14-interacting protein, Fg09985 (FgTfc7), which isrequired for RNA polymerase (Pol) III in transcribing 5S rRNA.To our knowledge, this is the first report of the regulation ofribosome biogenesis by a Cdc14 ortholog.

Acknowledgements

This research was supported by the National Key Basic Researchand Development Program (2013CB127802), National ScienceFoundation (31171881 and 31272000), Special Fund for Agro-scientific Research in the Public Interest (no. 201303016) andChina Agriculture Research System (CARS-3-1-15).

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Deletion of putative phosphatase genes in Fusariumgraminearum.

Fig. S2 Colony morphology of the wild-type (WT) and 71 phos-phatase gene deletion mutants of Fusarium graminearum onpotato dextrose agar (PDA) and minimal medium (MM) afterincubation at 25°C for 4 d.

Fig. S3 Conidia of the wild-type (WT) and DFgCDC14 ofFusarium graminearum were stained with 40,6-diamidino-2-phenylindole (DAPI) and observed under a microscope.

Table S1 Primers used in this study

Table S2 Collection of phenotypic analyses for 71 phosphatasedeletion mutants of Fusarium graminearum

Table S3 Comparisons of the putative essential phosphatasegenes in Fusarium graminearum with those in Saccharomycescerevisiae, Aspergillus nidulans and Neurospora crassa

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