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Biz1, a Zinc Finger Protein Required for Plant Invasion by Ustilago maydis, Regulates the Levels of a Mitotic Cyclin

Biz1, a Zinc Finger Protein Required for Plant Invasion by Ustilago maydis, Regulates the Levels of a Mitotic Cyclin
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  Biz1, a Zinc Finger Protein Required for Plant Invasion by  Ustilago maydis , Regulates the Levels of a Mitotic Cyclin W Ignacio Flor-Parra, a,1 Miroslav Vranes, b,1 Jo ¨ rg Ka ¨ mper, b and Jose´  Pe´ rez-Martı´ n a,2 a Departamento de Biotecnologı´ a Microbiana, Centro Nacional de Biotecnologı´ a, Consejo Superior de Investigaciones Cientı´ ficas, Campus de Cantoblanco, Universidad Autonoma de Madrid, 28049 Madrid, Spain b Max-Planck-Institut fu¨r Terrestrische Mikrobiologie, Abteilung Organismische Interaktionen, D-35043 Marburg, Germany Plant invasion by pathogenic fungi involves regulated growth and highly organized fungal morphological changes. Forinstance, when the smut fungus  Ustilago maydis  infects maize (   Zea mays  ), its dikaryotic infective filament is cell cycle ar-rested, and appressoria are differentiated prior to plant penetration. Once the filament enters the plant, the cell cycle block is released and fungal cells begin proliferation, suggesting a tight interaction between plant invasion and the cell cycle andmorphogenesis control systems. We describe a novel factor, Biz1 (   b -dependent zinc finger protein), which has two Cys 2 His 2 zinc finger domains and nuclear localization, suggesting a transcriptional regulatory function. The deletion of  biz1  shows nodetectable phenotypic alterations during axenic growth. However, mutant cells show a severe reduction in appressoriaformation and plant penetration, and those hyphae that invade the plant arrest their pathogenic development directly afterplant penetration.  biz1  is induced via the  b -mating–type locus, the key control instance for pathogenic development. Thegene is expressed at high levels throughout pathogenic development, which induces a G2 cell cycle arrest that is a directconsequence of the downregulation of the mitotic cyclin Clb1. Our data support a model in which Biz1 is involved in cellcycle arrest preceding plant penetration as well as in the induction of appressoria.INTRODUCTION Many phytopathogenic fungi are known to differentiate specificinfection structures, called appressoria, that facilitate the pene-tration of the plant epidermal cell layers (Emmett and Parbery,1975; Deising et al., 2000). In  Magnaporthe grisea ,  Colletotri-chum species,andmanyotherplantpathogens,appressoriaarevisible as discrete, lobed, or dome-shaped cells that are sepa-rated from the germ tube by a septum. This dome-shaped cellgenerates enormous turgor pressure and physical force, allow-ing the fungus to breach the host cuticle and to invade the planttissue (Mendgen et al., 1996; Talbot, 2003). In smut and rustfungi,appressoriaareinconspicuousswellingsofthegermtube;it is generally assumed that in these fungi cell wall–degradingenzymes, rather than force, aid the penetration of the invadinghypha (Gold and Mendgen, 1991). Appressorial developmentand the penetration step are still poorly understood processesthat require the integration of several environmental signals toproduce the appropriate differentiation (Dean, 1997). Like otherdevelopmental decisions in fungi, appressorium formation mustinvolve regulated growth, control of the cell cycle progression,and highly organized morphological changes.One of the model systems to investigate the connections be-tween cell cycle and morphogenesis in fungi during plant pen-etration is  Ustilago maydis , a basidiomycete causing smutdisease on maize (   Zea mays  ) plants (Basse and Steinberg,2004). Haploid cells (sporidia) of this fungus are unicellular andgrow saprophytically by budding. The pathogenic form, the fila-mentousdikaryon,isestablishedafterfusionoftwosporidiathathavetoharbordifferentallelesofthe  a- and  b -mating–typelociof  U. maydis . The  a  locus controls the cell fusion via a pheromonereceptor–based system. Upon pheromone stimulation, cells ar-restbuddinggrowthandstarttheformationofconjugationtubes(Spelligetal.,1994).Thesematingfilamentsundergodirectedtipgrowthtowardthepheromonesource(Snetselaaretal.,1996)fol-lowed by cell fusion and the formation of dikaryotic hyphae. Thesubsequent steps in filament formation and pathogenic devel-opment are controlled by the multiallelic  b -locus that encodestwo distinct homeodomain transcription factors, bE and bW. A heterodimeric complex of the two proteins is formed when theyare derived from different alleles, and the presence of this com-plex is sufficient to initiate pathogenic development (Kahmannand Ka ¨ mper, 2004).The dikaryon formed after the fusion of compatible sporidia isarrestedintheG2phaseofthecellcycle;forfurtherpropagation,it requires plant signals that have not been identified yet. On theplant surface, the filaments differentiate appressoria and pene-trate the cuticule (Snetselaar and Mims, 1992, 1993). In contrastwith appressoria from other phytopathogenic fungi, such as M. grisea  or  Colletotrichum  species (Bechinger et al., 1999;Talbot, 2003), appressoria of   U. maydis  are unmelanized, rathersmall swellings of the hyphal tip that form penetration structuresthatarelessconstricted(SnetselaarandMims,1993;Snetselaar 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. E-mail; fax 34-91-585-4506.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors ( is: Jose´  Pe´ rez-Martı´n( W Online version contains Web-only The Plant Cell, Vol. 18, 2369–2387, September 2006, ª 2006 American Society of Plant Biologists  Figure 1.  Expression of   biz1  Arrests the Cell Cycle in G2 Phase. 2370 The Plant Cell  et al., 2001). Since it is unlikely that entry of   U. maydis  occurs bymechanical force, it is believed that appressoria simply mark thepoint at which the growth direction changes. Once the filamententers the plant, the cell cycle is reactivated and the fungal cellsproliferate to a network of filaments with septated cell compart-ments each containing a pair of nuclei (Snetselaar and Mims,1992; Banuett and Herskowitz, 1996).Themorphologicalchangesof  U.maydis cellsduringthepath-ogenic process advocate for tight control of the cell cycle. Pre-viousresearcheffortshavedefinednetworksofregulatorygenesthat control cell cycle progression (Castillo-Lluva et al., 2004;Garcı´ a-Muse etal.,2004;Castillo-Lluva andPe´ rez-Martı´ n,2005;SgarlataandPe´ rez-Martı´n,2005a,2005b).Asinothereukaryoticorganisms,in U.maydis cyclin-dependentproteinkinases(Cdks)are key regulators of the cell division cycle. Two distinct Cdk-cyclin complexes are responsible for the different cell cycletransitionsin U.maydis .WhileCdk1-Clb1isrequiredfortheG1/Sand the G2/M transitions, the Cdk1-Clb2 complex is specific forthe G2/M transition (Garcı´a-Muse et al., 2004). A third Cdk1-cyclin complex composed of Cdk1 and the G1-like cyclin Cln1regulates the G1/S transition and has additional functions at themorphogenetic level (Castillo-Lluva and Pe´ rez-Martı´ n, 2005).Regulation of Cdk-cyclin activity throughout the different cellcyclephasesiscrucial.Inhibitoryphosphorylationandactivatingdephosphorylation of the Cdk1 catalytic subunit are one of themajor controls, and we have recently shown that this kind of regulation is crucial for cell cycle progression in  U. maydis (Sgarlata and Pe´ rez-Martı´ n, 2005a, 2005b). In addition, proteol-ysis of cyclins is pivotal for cell cycle control.  U. maydis  mitoticcyclinsaretargetedbytheanaphasepromotingcomplexinasso-ciation with different adaptor proteins, such as Cru1 (Castillo-Lluvaetal.,2004)andCdc20(J.TorreblancaandJ.Pe´ rez-Martı´n,unpublished data).Recently,theroleofcellcycleregulatorsforpathogenicdevel-opment of   U. maydis  has been addressed. Manipulation of thelevels of mitotic cyclins either by affecting their rate of degrada-tion or by alteration of their transcriptional regulation producefungalcellsthatareunabletoinfecttheplant(Castillo-Lluvaetal.,2004;Garcı´a-Museetal.,2004;Castillo-Lluva andPe´ rez-Martı´n,2005).Theseresultsstresstheimportanceofatightregulationof the Cdk-cyclin complexes through the pathogenic developmentof   U. maydis .Inthiswork,wedescribeanoveltranscriptionalregulator,Biz1(   b -dependent zinc finger protein), that functions as a repressorfor the mitotic cyclin Clb1.  biz1  is induced after initiation of path-ogenic development. The deletion of   biz1  does not lead to anydetectable phenotypic alterations during axenic growth. How-ever, strains deleted for  biz1  are completely apathogenic. Themutantcellsshowalowfrequencyofappressoriaformation,andthose that produce appressorium and invade the plant arresttheir pathogenic development directly after plant penetration. RESULTSHighLevelsof  biz1 ExpressionInduceaG2CellCycleArrest To isolate novel regulators for the control of cell cycle and mor-phogenesis during pathogenic development, we performed ageneticscreendevotedtotheisolationofmutantsaffectedincellcycle arrest and/or polarity induction in response to pheromonestimulation(seeSupplementalResultsforadescriptionofsuchagenetic screen). Serendipitously, in this screen we found that anopen reading frame (ORF) of 783 amino acids, which corre-spondstothepredictedhypotheticalproteinum02549annotatedattheMIPS Ustilagomaydis Database( proj/ustilago/),wasabletoinducecellcyclearrestandstrongpo-lar growth in cells expressing it (see below). We called the gene  biz1  (for  b -induced zinc finger; see below).To analyze the functions of Biz1, we examined the effects of increased expression of   biz1 . For this, we replaced the endog-enous promoter within the  biz1  locus with the carbon source–regulated  crg1  promoter (which is repressed by glucose andinduced by arabinose; Bottin et al., 1996). The resulting  biz1 crg1 allele shows a clear carbon source–dependent pattern of ex-pression (Figure 1A). Under repressing conditions (YPD), cellsharboring the  biz1 crg1 allele were morphologically indistinguish-ablefromwild-typestrains.However,aftertransfertoarabinose-containing medium (YPA), cells expressing high levels of   biz1 started to elongate (Figure 1B). We found that these elongatedcells contained a single nucleus and long microtubules that Figure 1.  (continued). (A)  Arabinose-inducedexpressionof   biz1. Wild-typeFB1andconditionalUMN53(FB1  biz1 crg1  )strainweregrownfor6hinnoninducingconditions(yeastextract-peptonemediumamendedwithglucose[YPD])orinducingconditions(yeastextract-peptonemediumamendedwitharabinose[YPA]).TheRNA was extracted and analyzed by RNA gel blotting, loading 10  m g total RNA per lane. A probe specific for 18s rRNA was used to control for loading. (B)  Micrographs showing the cell morphology of UMN53 cells after 12 h of growth in YPD (noninducing conditions) or YPA (inducing conditions) liquidcultures. Note the elongated shape and the presence of a single nucleus (4 9 ,6-diamidino-2-phenylindole [DAPI] staining). Bars ¼ 20  m m. (C)  FACS analysis of FB1 and UMN53 (FB1  biz1 crg1  ) to assess DNA content in noninducing (YPD) and inducing conditions (YPA). Samples were taken at0, 3, 6, and 9 h after transfer to inducing conditions. Cells expressing  biz1  arrest theirgrowth at G2 and thereby accumulate with a 2C DNA content. Theshift to DNA content higher than 2C observed in UMN53 cells incubated in YPA for 9 h was due to mitochondrial DNA staining. (D)  Microtubule network in UMN68 cells carrying an  a -tubulin-GFP fusion and expressing high levels of   biz1  after 8 h in YPA (Tub1-GFP;epifluorescence). Bar ¼ 20  m m. DIC, differential interference contrast. (E)  UMN53 cells incubated for 24 h in inducing conditions display a distinct phenotype characterized by an extensive polarized growth (top panel; DIC)and single nuclei content (DAPI; arrow points to the nucleus), and empty sections behind that are separated by septa (stained with wheat germagglutinin [WGA]; arrows) generated by formation of basal vacuoles as result of a permanent G2 cell cycle arrest. The vertical panel shows a detailedview of the basal area of the hypha where the septa-separated empty sections can be observed. Bars ¼ 50  m m in horizontal panels and 15  m m in thevertical panel. Biz1 Controls Virulence in Smut Fungus 2371  Figure 2.  biz1  Encodes a Zinc Finger–Containing Protein. 2372 The Plant Cell  reached to the tip of the growing pole (Figure 1D). This polargrowth phenotype has been described for  U. maydis  cellsarrested in the G2 phase (Steinberg et al., 2001; Banuett andHerskowitz, 2002). Consistently, fluorescence-activated cellsorter (FACS) analysis revealed that the cells have a 2C DNA content (Figure 1C). We did not observe cell divisions afterinduction of   biz1 crg1 . Prolonged incubation (>24 h) under induc-ing conditions resulted in long filaments in which only the tip cellwas filled with cytoplasm and the remaining part of the hyphaeconsisting of empty sections separated by septa (Figure 1E).Strikingly, these hyphae are reminiscent of the growth mode of the dikaryotic hyphae produced after mating. Under these con-ditions, cell division is arrested and cells show permanentpolarized growth (Steinberg et al., 1998). We believe that thisgrowth mode of   U. maydis  could be a default response to an ar-restedcelldivision.Forinstance,theconditionalremovalof  clb1 ,encoding a mitotic cyclin, led to the same phenotype (Garcı´ a-Muse et al., 2004). Biz1,aPutativeZincFingerTranscriptionFactorin U.maydis The predicted Biz1 protein contains two putative zinc finger do-mainsoftheCys 2 His 2 class,oneofthemostcommonDNAbind-ing motifs found in Eukaryota. The two zinc fingers weredesignatedZf1(fromresidues159to183)andZf2(fromresidues186 to 211). Both are predicted to be composed of two short b -strands (  b 1 and  b 2) separated by a Cys-containing short loop(L1),followedbyasecondloop(L2)andan a -helix(H)(Figure2A).WhileZf2comprisestheCysX 2-4 CysX 12 HisX 3-5 HisconsensusforCys 2 His 2 zincfingers(Jacobs,1992),thetwoCysresiduesinZf1are separated only by a single amino acid (Figure 2A). Both zincfingers contain a conserved hydrophobic residue, a Met, atposition4oftheproposed a -helix.ThePheresidueatposition3inthesecond b -strand(  b 2)isafrequentbutnotessentialfeatureof Cys 2 His 2  zinc fingers (Suzuki et al., 1994). We have been unableto find any significant sequence similarity between the Biz1protein and other entries inthe databases outside the zinc fingerregion. Analysis of the amino acid sequence using PROSITEindicated a Gln-rich region located between residues 507 and526, a Ser-rich region located between residues 655 and 702,andaputativenuclearlocalizationsignal(NLS)locatedupstreamof the first zinc finger (residues 145 to 150). PSORT (Nakai andHorton, 1999) predicts for Biz1 a high probability for nuclearlocalization (P  ¼  94.1). To verify the subcellular location, weexpressed a Biz1–green fluorescent protein (GFP) fusion underthecontrolofthearabinose-inducible crg1 promoter(Bottinetal.,1996) in the  U. maydis  haploid wild-type strain FB1 (   a1b1  ).Fluorescence microscopy revealed bright signals in the nucleusupon induction of the fusion protein (Figure 2B). Inductionresulted in filament formation comparable to that of the cellsexpressing wild-type Biz1, indicating that the GFP fusion did notrender the protein inactive.The presence of two conserved Cys 2 His 2  class zinc fingerdomains in the predicted sequence of Biz1 suggests a role astranscriptionalfactor.However,wecannotruleoutotherfunctions,asithas beenshownthatzinc-centereddomainscan beinvolvedinprotein–protein interaction in other proteinsthan transcriptionalregulators(MatthewsandSunde,2002).TogainsupportforaroleofBiz1asatranscriptionfactor,firstwetriedtoshowthattheNLSand zinc fingers are required for the function of Biz1. For this, weexpressed an N-terminal truncated protein lacking the NLS andzinc finger–containing region fused to a triple HA tag at the Cterminus(Figure 2C). Ascontrol, wealso expressed a C-terminal-taggedfull-lengthprotein.Bothproteinsweredetectedbyproteingelblotassayusinganti-HAantibodies(Figure2C).Theadditionof aC-terminaltripleHAtagdidnotaffecttheactivityofBiz1,ashighlevels of the full-length protein induced the already observed cellcycle arrestand hyperpolarized growth (Figure 2D). Strikingly, theN-terminaldeletedmutantproducednoeffect,supportingaroleof the N-terminal end carrying the NLS and the zinc fingers in theactivity of the Biz1 protein.Takentogether, thepresence oftwoCys 2 His 2 zincfingers anda Gln-rich region, two features that frequently associate withtranscription factors (Escher et al., 2000), and the nuclear local-ization suggest that Biz1 probably functions as a transcriptionfactor. Biz1 Downregulated the Expression of the MitoticCyclin  clb1 In  U. maydis  cells, entry into mitosis from G2 phase requires theactivityoftwoB-typecyclins,Clb1andClb2,thatformacomplexwith the catalytic subunit Cdk1 (Garcı´a-Muse et al., 2004). Since  biz1  overexpression inhibits the G2/M transition, we wonderedwhether Biz1 could control the expression of these cell cycle Figure 2.  (continued). (A) DomainstructureofBiz1.Domainsindicated wereidentified usingPROSITE.Biz1containsanNLS,aGln-richregion,aSer-richregion,andtwozincfingers. The bottom part shows the sequence of the two putative Cys 2 His 2  zinc finger domains; amino acidspredicted to be involved in the formation of the  b -strands (  b 1 and  b 2), the loops (L1 and L2), and the  a -helix (H) are indicated. Residues corresponding to the Cys 2 His 2  zinc finger consensussequence are in gray. (B)  The Biz1-GFP fusion protein localizes to the nucleus. Cells harboring an ectopic copy of a  biz1-gfp  fusion under the control of the arabinose-inducible  crg1  promoter (UMN68 strain) were grown for 4 h under inducing conditions (complete medium supplemented with 1% arabinose as carbonsource). Bars ¼ 15  m m. (C)  In the top part, a scheme of the C-terminal-tagged Biz1 protein and its derivative lacking the NLS and zinc fingers, Biz1 D 218-HA, is shown. In thebottom part,a protein gelblot assayto detect Biz1proteins isshown. Extractswere prepared from FB1 Pcrg:Biz1HA andFB1 Pcrg:Biz1 D 218HAgrownininducingconditions (YPA)orrepressive conditions (YPD) atanOD 600 of0.5. Anequalamount oftotal protein(50 m g)wasloadedintothegel.Anti-HA-peroxidase (Roche) antibodies were used to detect the fusion proteins. The bars at right indicate the molecular masses of the protein marker ladder.Biz1-HA and Biz1 D 218HA have an estimated molecular mass of 88 and 64 kD, respectively. (D)  Morphology of cells producing the Biz1-HA or the Biz1 D 218HA protein, growing in inducing conditions (YPA). Bars ¼ 20  m m. Biz1 Controls Virulence in Smut Fungus 2373
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