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The RNA-Binding Protein Rrm4 is Essential for Efficient Secretion of Endochitinase Cts1

2011; Elsevier BV; Volume: 10; Issue: 12 Linguagem: Inglês

10.1074/mcp.m111.011213

1535-9484

Janine Koepke, Florian Kaffarnik, Carl Haag, Kathi Zarnack, Nicholas M. Luscombe, Julian König, Jernej Ule, Ronny Kellner, Dominik Begerow, Michael Feldbrügge,

Protein Tyrosine Phosphatases

Long-distance transport of mRNAs is crucial in determining spatio-temporal gene expression in eukaryotes. The RNA-binding protein Rrm4 constitutes a key component of microtubule-dependent mRNA transport in filaments of Ustilago maydis. Although a number of potential target mRNAs could be identified, cellular processes that depend on Rrm4-mediated transport remain largely unknown. Here, we used differential proteomics to show that ribosomal, mitochondrial, and cell wall-remodeling proteins, including the bacterial-type endochitinase Cts1, are differentially regulated in rrm4Δ filaments. In vivo UV crosslinking and immunoprecipitation and fluorescence in situ hybridization revealed that cts1 mRNA represents a direct target of Rrm4. Filaments of cts1Δ mutants aggregate in liquid culture suggesting an altered cell surface. In wild type cells Cts1 localizes predominantly at the growth cone, whereas it accumulates at both poles in rrm4Δ filaments. The endochitinase is secreted and associates most likely with the cell wall of filaments. Secretion is drastically impaired in filaments lacking Rrm4 or conventional kinesin Kin1 as well as in filaments with disrupted microtubules. Thus, Rrm4-mediated mRNA transport appears to be essential for efficient export of active Cts1, uncovering a novel molecular link between mRNA transport and the mechanism of secretion. Long-distance transport of mRNAs is crucial in determining spatio-temporal gene expression in eukaryotes. The RNA-binding protein Rrm4 constitutes a key component of microtubule-dependent mRNA transport in filaments of Ustilago maydis. Although a number of potential target mRNAs could be identified, cellular processes that depend on Rrm4-mediated transport remain largely unknown. Here, we used differential proteomics to show that ribosomal, mitochondrial, and cell wall-remodeling proteins, including the bacterial-type endochitinase Cts1, are differentially regulated in rrm4Δ filaments. In vivo UV crosslinking and immunoprecipitation and fluorescence in situ hybridization revealed that cts1 mRNA represents a direct target of Rrm4. Filaments of cts1Δ mutants aggregate in liquid culture suggesting an altered cell surface. In wild type cells Cts1 localizes predominantly at the growth cone, whereas it accumulates at both poles in rrm4Δ filaments. The endochitinase is secreted and associates most likely with the cell wall of filaments. Secretion is drastically impaired in filaments lacking Rrm4 or conventional kinesin Kin1 as well as in filaments with disrupted microtubules. Thus, Rrm4-mediated mRNA transport appears to be essential for efficient export of active Cts1, uncovering a novel molecular link between mRNA transport and the mechanism of secretion. Fungal filaments are highly polarized cellular structures that expand at their apical pole. A key feature of this growth mode is the polarized secretion of cell wall material and cell wall-remodeling enzymes at the hyphal tip. Macromolecular structures like the Spitzenkörper (apical body) as well as the adjacent polarisome, which contains polarity factors to organize the fungal cytoskeleton, have been implicated in this specialized growth form. The Spitzenkörper is proposed to function as a vesicle supply center (1Gierz G. Bartnicki-Garcia S. A three-dimensional model of fungal morphogenesis based on the vesicle supply center concept.J. Theor. Biol. 2001; 208: 151-164Crossref PubMed Scopus (80) Google Scholar), mediating secretion of enzymes such as chitin synthases for cell wall synthesis. Important for the function of the Spitzenkörper and thus polar growth is the continuous supply of vesicles such as chitosomes that are transported along microtubules to the region of active growth (2Fischer R. Zekert N. Takeshita N. Polarized growth in fungi - interplay between the cytoskeleton, positional markers and membrane domains.Mol. Microbiol. 2008; 68: 813-826Crossref PubMed Scopus (154) Google Scholar, 3Harris S.D. Cell polarity in filamentous fungi: shaping the mold.Int. Rev. Cytol. 2006; 251: 41-77Crossref PubMed Scopus (96) Google Scholar, 4Steinberg G. Hyphal growth: a tale of motors, lipids, and the Spitzenkörper.Euk. Cell. 2007; 6: 351-360Crossref PubMed Scopus (235) Google Scholar). A well-studied model for fungal filamentous growth is the corn pathogen Ustilago maydis (5Brefort T. Doehlemann G. Mendoza-Mendoza A. Reissmann S. Djamei A. Kahmann R. Ustilago maydis as a Pathogen.Annu. Rev. 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Cell Sci. 1998; 111: 2235-2246PubMed Google Scholar). This developmental program is intimately coupled to mating of two compatible partners, which recognize each other using pheromones (9Bölker M. Urban M. Kahmann R. The a mating type locus of U. maydis specifies cell signaling components.Cell. 1992; 68: 441-450Abstract Full Text PDF PubMed Scopus (298) Google Scholar). Stimulated cells form conjugation tubes that fuse at their tips. This activates the heterodimeric homeodomain transcription factor bW/bE that is necessary and sufficient for filamentous growth. Its activity is dependent on cell fusion, because it is only functional as heterodimer with subunits derived from compatible mating partners (10Kämper J. Reichmann M. Romeis T. Bölker M. Kahmann R. Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis.Cell. 1995; 81: 73-83Abstract Full Text PDF PubMed Scopus (200) Google Scholar). The infectious filament penetrates the plant surface and reinitiates proliferation to form a multicellular mycelium within the plant (11Feldbrügge M. Bölker M. Steinberg G. Kämper J. Kahmann R. Regulatory and structural netwoks orchestrating mating, dimorphism, cell shape, and pathogenesis in Ustilago maydis.in: Kües U. Fischer R. The Mycota I. Springer-Verlag, Berlin Heidelberg2006: 375-391Google Scholar, 12Vollmeister E. Schipper K. Baumann S. Haag C. Pohlmann T. Stock J. Feldbrügge M. Fungal development of the plant pathogen Ustilago maydis.FEMS Microbiol. Rev. 2011; 10.1111/j.1574–6976.2011.00296.xPubMed Google Scholar). Important for infection is the secretion of effector proteins that are thought, for example, to suppress defense mechanisms (13Kämper J. Kahmann R. Bölker M. Ma L.J. Brefort T. Saville B.J. Banuett F. Kronstad J.W. Gold S.E. Müller O. Perlin M.H. Wösten H.A. de Vries R. Ruiz-Herrera J. Reynaga-Peña C.G. Snetselaar K. McCann M. Pérez-Martín J. Feldbrügge M. Basse C.W. Steinberg G. Ibeas J.I. Holloman W. Guzman P. Farman M. Stajich J.E. Sentandreu R. González-Prieto J.M. Kennell J.C. Molina L. Schirawski J. Mendoza-Mendoza A. Greilinger D. Münch K. Rössel N. Scherer M. Vranes M. Ladendorf O. Vincon V. Fuchs U. Sandrock B. Meng S. Ho E.C. Cahill M.J. Boyce K.J. Klose J. Klosterman S.J. Deelstra H.J. Ortiz-Castellanos L. Li W. Sanchez-Alonso P. Schreier P.H. Häuser-Hahn I. Vaupel M. Koopmann E. Friedrich G. Voss H. Schlüter T. Margolis J. Platt D. Swimmer C. Gnirke A. Chen F. Vysotskaia V. Mannhaupt G. Güldener U. Münsterkötter M. Haase D. Oesterheld M. Mewes H.W. Mauceli E.W. DeCaprio D. Wade C.M. Butler J. Young S. Jaffe D.B. Calvo S. Nusbaum C. Galagan J. Birren B.W. Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.Nature. 2006; 444: 97-101Crossref PubMed Scopus (879) Google Scholar, 14Doehlemann G. van der Linde K. Assmann D. Schwammbach D. Hof A. Mohanty A. Jackson D. Kahmann R. Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells.PLoS Pathog. 2009; 5: e1000290Crossref PubMed Scopus (247) Google Scholar, 15Schirawski J. Mannhaupt G. Münch K. Brefort T. Schipper K. Doehlemann G. Di Stasio M. Rössel N. Mendoza-Mendoza A. Pester D. Müller O. Winterberg B. Meyer E. Ghareeb H. Wollenberg T. Münsterkötter M. Wong P. Walter M. Stukenbrock E. Güldener U. Kahmann R. Pathogenicity determinants in smut fungi revealed by genome comparison.Science. 2010; 330: 1546-1548Crossref PubMed Scopus (247) Google Scholar). In recent years, it has been shown that post-transcriptional control is important for filament formation (16Feldbrügge M. Zarnack K. Vollmeister E. Baumann S. Koepke J. König J. Münsterkötter M. Mannhaupt G. The posttranscriptional machinery of Ustilago maydis.Fungal Genet. Biol. 2008; 45: S40-S46Crossref PubMed Scopus (24) Google Scholar, 17Zarnack K. Feldbrügge M. Microtubule-dependent mRNA transport in fungi.Eukaryot. Cell. 2010; 9: 982-990Crossref PubMed Scopus (28) Google Scholar). In particular, microtubule-dependent transport of mRNAs is essential for fast polar growth (18König J. Baumann S. Koepke J. Pohlmann T. Zarnack K. Feldbrügge M. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs.EMBO J. 2009; 28: 1855-1866Crossref PubMed Scopus (74) Google Scholar, 19Vollmeister E. Feldbrügge M. Posttranscriptional control of growth and development in Ustilago maydis.Curr. Opin. Microbiol. 2010; 13: 693-699Crossref PubMed Scopus (21) Google Scholar). The RNA-binding protein Rrm4 is a key player in this transport process. In vivo UV-crosslinking revealed that Rrm4 binds more than 50 different mRNAs encoding cytotopically related proteins such as polarity or translation factors. RNA live imaging demonstrated that target mRNAs colocalize with Rrm4 in ribonucleoprotein particles, so-called mRNPs, that shuttle along microtubules (18König J. Baumann S. Koepke J. Pohlmann T. Zarnack K. Feldbrügge M. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs.EMBO J. 2009; 28: 1855-1866Crossref PubMed Scopus (74) Google Scholar). Rrm4 contains three N-terminal RNA recognition motifs (RRMs) 1The abbreviations used are:AAAprotease ATPase associated with diverse cellular activitiesBLASTBasic Local Alignment Search ToolCLIPUV crosslinking and immunoprecipitationERendoplasmic reticulumDICdifferential interference contrastFISHfluorescence in situ hybridizationGfpgreen fluorescence proteinGPIglycosyl-phosphatidyl-inositolHygRhygromycin resistance cassetteIPGimmobilized pH gradientMLmaximum likelihoodMLLEMademoiseLLE domainmRNPribonucleoprotein particleMUMDBMIPS Ustilago maydis DatabaseNatRnourseothricin resistance cassettePIApeak-identifying algorithmppipeptidyl prolyl isomeraseRFUrelative fluorescence unitsRRMRNA recognition motifSMARTSimple Modular Architecture Research ToolTGFtransforming growth factorXcorrcross-correlation. and a C-terminal MLLE domain (MademoiseLLE domain forming a defined peptide binding pocket involved in protein-protein interactions; found at the C terminus of poly[A]-binding protein PABPC, 20Kozlov G. De Crescenzo G. Lim N.S. Siddiqui N. Fantus D. Kahvejian A. Trempe J.F. Elias D. Ekiel I. Sonenberg N. O'Connor-McCourt M. Gehring K. Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase.EMBO J. 2004; 23: 272-281Crossref PubMed Scopus (90) Google Scholar, 21Kozlov G. Ménade M. Rosenauer A. Nguyen L. Gehring K. Molecular determinants of PAM2 recognition by the MLLE domain of poly(A)-binding protein.J. Mol. Biol. 2010; 397: 397-407Crossref PubMed Scopus (51) Google Scholar). The latter domain is essential for the formation of shuttling particles (22Becht P. König J. Feldbrügge M. The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules.J. Cell Sci. 2006; 119: 4964-4973Crossref PubMed Scopus (97) Google Scholar). Loss of the conventional kinesin Kin1 interferes with mRNP shuttling, suggesting that active transport by molecular motors is important for function (22Becht P. König J. Feldbrügge M. The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules.J. Cell Sci. 2006; 119: 4964-4973Crossref PubMed Scopus (97) Google Scholar). Removal of the RNA-binding domain causes loss of transported mRNPs although Rrm4 is still shuttling along microtubules. Thus, Rrm4 constitutes an integral component of the main transport unit of microtubule-dependent mRNA transport (17Zarnack K. Feldbrügge M. Microtubule-dependent mRNA transport in fungi.Eukaryot. Cell. 2010; 9: 982-990Crossref PubMed Scopus (28) Google Scholar, 18König J. Baumann S. Koepke J. Pohlmann T. Zarnack K. Feldbrügge M. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs.EMBO J. 2009; 28: 1855-1866Crossref PubMed Scopus (74) Google Scholar). protease ATPase associated with diverse cellular activities Basic Local Alignment Search Tool UV crosslinking and immunoprecipitation endoplasmic reticulum differential interference contrast fluorescence in situ hybridization green fluorescence protein glycosyl-phosphatidyl-inositol hygromycin resistance cassette immobilized pH gradient maximum likelihood MademoiseLLE domain ribonucleoprotein particle MIPS Ustilago maydis Database nourseothricin resistance cassette peak-identifying algorithm peptidyl prolyl isomerase relative fluorescence units RNA recognition motif Simple Modular Architecture Research Tool transforming growth factor cross-correlation. Loss of Rrm4 leads to impaired virulence and filamentous growth (23Becht P. Vollmeister E. Feldbrügge M. Role for RNA-binding proteins implicated in pathogenic development of Ustilago maydis.Eukaryot. Cell. 2005; 4: 121-133Crossref PubMed Scopus (50) Google Scholar). A significantly increased number of filaments grow bipolar. Deletion strains fail to insert retraction septa at the basal pole resulting in the formation of shorter filaments (22Becht P. König J. Feldbrügge M. The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules.J. Cell Sci. 2006; 119: 4964-4973Crossref PubMed Scopus (97) Google Scholar; Fig. 1A). Although substantial progress has been made in elucidating the function of Rrm4 during microtubule-dependent mRNA transport, the molecular consequences of this transport process are still unclear. To identify proteins with altered abundance in rrm4Δ strains we applied differential proteomics comparing wild type and rrm4Δ filaments. We found that the amount of endochitinase Cts1 was significantly increased. Consistently, we could show that cts1 mRNA is a direct target of Rrm4 and that secretion of Cts1 is almost abolished in the absence of Rrm4. Thus, posttranscriptional control at the level of mRNA transport is crucial for secretion of this cell wall-remodeling enzyme. Escherichia coli K-12 derivates DH5α (Invitrogen, Carlsbad, CA) and Top10 (Invitrogen) were used for cloning. Growth conditions for U. maydis strains and source of antibiotics are described elsewhere (23Becht P. Vollmeister E. Feldbrügge M. Role for RNA-binding proteins implicated in pathogenic development of Ustilago maydis.Eukaryot. Cell. 2005; 4: 121-133Crossref PubMed Scopus (50) Google Scholar, 24Brachmann A. König J. Julius C. Feldbrügge M. A reverse genetic approach for generating gene replacement mutants in Ustilago maydis.Mol. Genet. Genomics. 2004; 272: 216-226Crossref PubMed Scopus (184) Google Scholar). U. maydis strains were constructed by transformation of progenitor strains with linearized plasmids (supplemental Table S1). Homologous integration events at the cts1 and rrm4 locus were verified by Southern blot analysis (24Brachmann A. König J. Julius C. Feldbrügge M. A reverse genetic approach for generating gene replacement mutants in Ustilago maydis.Mol. Genet. Genomics. 2004; 272: 216-226Crossref PubMed Scopus (184) Google Scholar). Filamentous growth of AB33 derivates for 8 h was induced by shifting cells of an exponential growing culture (OD600 = 0.5) from liquid complete medium to nitrate minimal medium each supplemented with 1% glucose. Cells were incubated at 28 °C shaking with 200 r.p.m. Standard molecular techniques were followed. Plasmids pCR2.1-Topo (Invitrogen) and pBluescriptSKII (Stratagene, La Jolla, CA) were used as cloning vehicles. Genomic DNA of wild type strain UM521 (a1b1) was used as template for PCR amplifications unless otherwise noted. Detailed plasmid description, constructs, and oligonucleotide sequences (supplemental Table S2) are given in supplemental data. All constructs were confirmed by sequencing. Plasmid sequences are available upon request. Microscopy was carried out as described previously. Epifluorescence was observed using filter sets for detection of Gfp (ET470/40BP, ET495LP, BP525/50), and TexasRed (HC562/40BP, HC593LP, HC624/40BP). To detect Cy3 either TexasRed or TRITC (HC543/22BP, HC562LP, HC593/40BP) filter sets were used (18König J. Baumann S. Koepke J. Pohlmann T. Zarnack K. Feldbrügge M. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs.EMBO J. 2009; 28: 1855-1866Crossref PubMed Scopus (74) Google Scholar). Fluorescence in situ hybridization (FISH) analysis was performed as described (18König J. Baumann S. Koepke J. Pohlmann T. Zarnack K. Feldbrügge M. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs.EMBO J. 2009; 28: 1855-1866Crossref PubMed Scopus (74) Google Scholar). Four Cy3-labeled probes complementary to gfp were used (oSL387–390, 4 pmol each; see supplemental Table S2). Peaks in the resulting fluorescence intensity graphs were identified using PIA (peak-identifying algorithm by K. Zarnack, J. König, and M. Feldbrügge to be published elsewhere). This algorithm detects all positions within the graph that surmount their local environment (to a maximum distance w to either side) by at least height h. Peak identification was performed using the parameters h = 130 and w = 8 (values were optimized for varying imaging techniques and signal intensities). Whole-cell extracts were prepared from 50 ml (OD600 = 0.5) budding cells or filaments growing for 8 h under inducing conditions. After centrifugation (860 × g for 5 min at 4 °C) cells or filaments were resuspended in 2 ml lysis buffer (100 mm sodium phosphate buffer, pH 8; 10 mm Tris/HCl, pH 8; 8 m urea; 2 × complete protease inhibitor mixture (Roche)) frozen in liquid nitrogen and ground in a pebble mill (Retsch; shaking for 10 min, 30 times/sec). Following a second round of centrifugation (860 × g for 5 min at 4 °C), an aliquot of the supernatant was removed (total cell fraction). The supernatant of a third centrifugation step (51,590 × g for 30 min at 4 °C) constituted the soluble protein fraction. Protein yield was determined by Bradford assay (Bio-Rad). The pellet was washed twice with lysis buffer, resuspended in 800 μl membrane protein buffer (10 mm Tris acetate, pH 7.6; 1 mm Mg acetate; 0,1 mm EDTA; 8% glycerine (v/v); 0,1% TritonX-100 (v/v); 4 mm dodecyl-β-d-maltoside; 0,7 mm cholesteryl hemisuccinate; 2 × complete protease inhibitor mixture) and incubated for 5 h on a turning wheel at 4 °C. The supernatant of a fourth centrifugation step (51,590 × g for 30 min at 4 °C) represented the membrane-associated protein fraction. For Western blot experiments 20 μg of total and soluble protein fractions as well as 20 μl of the membrane-associated protein fractions were analyzed. Gfp fusion protein and α-tubulin were detected using an α-Gfp antibody (Roche; mixture of two mouse monoclonal antibodies directed against Gfp) and an α-Tub antibody (Merck4Biosciences; Anti-α-Tubulin Mouse IgG), respectively. Proteins were labeled with DIGE-specific Cy3, Cy2, or Cy5 according to the manufacturer's instructions (GE Healthcare) with the following modifications: 200 μl of the membrane-associated protein fraction was precipitated with 10% trichloroacetic acid and washed five times with acetone (−20 °C) according to published procedures (25Görg A. Obermaier C. Boguth G. Csordas A. Diaz J.J. Madjar J.J. Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins.Electrophoresis. 1997; 18: 328-337Crossref PubMed Scopus (207) Google Scholar). For the internal standard, a mixture of 600 μl was used (100 μl of each sample). Samples were resuspended in labeling buffer and tagged with the CyDyes as described (26Westermeier R. Scheibe B. Difference gel electrophoresis based on lys/cys tagging.Methods Mol. Biol. 2008; 424: 73-85Crossref PubMed Scopus (28) Google Scholar). Cy2-, Cy3-, and Cy5-tagged protein samples were mixed (50 μl of each sample, including 25 μl labeling buffer with labeled probe and 25 μl 2 × lysis buffer). DIGE sample buffer (7 m urea; 2 m thiourea; 4% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; 20 mm dithiothreitol and 0.5% IPG buffer (GE Healthcare)) was added to adjust the volume to 550 μl. Samples were applied to 24 cm Immobiline Drystrips IPG pH 3–11 (GE Healthcare). IEF (isoelectric focusing) was carried out after 12 h rehydration using an Ettan IPGphor II (GE Healthcare) at 20 °C with maximum 50 μA/strip and the following settings: gradient increase to 500 V in 4 h, continued 500 V for 4 h, gradient increase to 3500 V in 5 h followed by continued 3500 V for 13 h reaching the desired total Vh of 59,000. Subsequently, IPG strips were incubated in equilibration buffer (6 m urea; 30% (w/v) glycerol; 2% SDS; 50 mm Tris/HCl, pH 8.0) first with 0.5% dithiothreitol and then with 2% iodoacetamide each for 15 min. Strips were transferred to 10% SDS-PAGE gels (Ettan Dalt Six gel system, GE Healthcare; 1 W/gel for 2 h and 3 W/gel for 12–16 h). Images were acquired with the multifluorescent point laser scanner Typhoon 9410 (GE Healthcare) and analyzed by the image analysis software (DeCyderTM software, GE Healthcare). Only those protein spots were analyzed further that were present in all nine gels and passed standard protein filter criteria (area ≤ 200; peak height ≤ 200; volume ≤ 10,000). 94% of the ∼600 spots did not change in abundance. Applying a threshold of 2.5-fold difference in abundance, ten protein spots were detected as differentially expressed (p value < 0.01 by Student's t test; DeCyder software, GE Healthcare; Table I).Table IProtein variants identified by DIGESpotRelative fold differencearrm4Δ versus wild type.Student's t-testUnique peptidesSequence coverage %Scoreum numberbMUMDB, http://mips.gsf.de/genre/proj/ustilago/ (13).GenebMUMDB, http://mips.gsf.de/genre/proj/ustilago/ (13).Predicted gene functionbMUMDB, http://mips.gsf.de/genre/proj/ustilago/ (13).114.70.00054377004662rps19Probable RPS19B - ribosomal protein S1925.30.0019113312010419cts1Chitinase34.50.0047354000898afg3Probable AFG3 - protease of the SEC18/CDC48/PAS1 family of ATPases (AAA)44.10.00289269010419cts1Chitinase53.30.0047n. i.cnot identified.n. i.n. i.n. i.n. i.n. i.63.20.017197010419cts1Chitinase730.0013n. i.n. i.n. i.n. i.n. i.n. i.8−3.10.00529489010548atp4Probable H+-transporting two-sector ATPase chain b precursor, mitochondrial9−3.60.0061n. i.n. i.n. i.n. i.n. i.n. i.10−5.80.00074375011495nuo2Related to NADH-ubiquinone oxidoreductase 21.3 kDa subunita rrm4Δ versus wild type.b MUMDB, http://mips.gsf.de/genre/proj/ustilago/ (13Kämper J. Kahmann R. Bölker M. Ma L.J. Brefort T. Saville B.J. Banuett F. Kronstad J.W. Gold S.E. Müller O. Perlin M.H. Wösten H.A. de Vries R. Ruiz-Herrera J. Reynaga-Peña C.G. Snetselaar K. McCann M. Pérez-Martín J. Feldbrügge M. Basse C.W. Steinberg G. Ibeas J.I. Holloman W. Guzman P. Farman M. Stajich J.E. Sentandreu R. González-Prieto J.M. Kennell J.C. Molina L. Schirawski J. Mendoza-Mendoza A. Greilinger D. Münch K. Rössel N. Scherer M. Vranes M. Ladendorf O. Vincon V. Fuchs U. Sandrock B. Meng S. Ho E.C. Cahill M.J. Boyce K.J. Klose J. Klosterman S.J. Deelstra H.J. Ortiz-Castellanos L. Li W. Sanchez-Alonso P. Schreier P.H. Häuser-Hahn I. Vaupel M. Koopmann E. Friedrich G. Voss H. Schlüter T. Margolis J. Platt D. Swimmer C. Gnirke A. Chen F. Vysotskaia V. Mannhaupt G. Güldener U. Münsterkötter M. Haase D. Oesterheld M. Mewes H.W. Mauceli E.W. DeCaprio D. Wade C.M. Butler J. Young S. Jaffe D.B. Calvo S. Nusbaum C. Galagan J. Birren B.W. Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.Nature. 2006; 444: 97-101Crossref PubMed Scopus (879) Google Scholar).c not identified. Open table in a new tab Total protein post staining using Deep Purple dye was applied according to the manufacturer's instructions (GE Healthcare). The spot picking list was generated with the DeCyder software (GE Healthcare) and picked automatically with an Ettan Spot Picker. Gel plugs were subjected to in-gel tryptic digest (Trypsin, Promega). Peptides were eluted and analyzed by LC-electrospray ionization (ESI)-MS/MS using an ion trap mass spectrometer (LTQ, Thermo Scientific, Hemel Hempstead, UK) with automated data-dependent acquisition. A nanoflow-HPLC system (Surveyor, Thermo Scientific) was used to deliver a flow rate of ∼250 nl/min to the mass spectrometer. Desalting was performed by using a precolumn (C18 material) in line to an analytical self-packed C18, 8-cm column (Picotip, 75 μm inner diameter, 15 μm tip; New Objective). Peptides were eluted by a gradient of 2–60% acetonitrile over 35 min. The mass spectrometer was operated in positive ion mode controlled by the Excalibur software package. It was equipped with a nanospray source and run at a capillary temperature of 200 °C; no sheath gas was used, and the source voltage and the focusing voltage were optimized for the transmission of angiotensin. Data-dependent analysis consisted of six most-abundant ions in each cycle: MS range of mass-to-charge ration (m/z) 300-2000, minimum signal 1000, normalized collision energy 30 and five repeated hits. Isolation width for MS2 analysis was 2 m/z. Data analysis was performed using the software package BioWorks 3.2 (Thermo Scientific) with the SEQUEST protein identification algorithm using default settings (mass tolerance for parent and fragment ions was 2 and 1 amu, respectively). Fragment ion spectra were searched against the U. maydis protein database MUMDB (http://mips.gsf.de/genre/proj/ustilago/, release March 2006, 6892 predicted protein-coding genes 13; supplemental Table S3). Data analysis revealed typical contaminations (trypsin, keratin, BSA, casein, angiotensin). Search criteria included oxidation of methionine (+16) as variable modification and alkylation of cysteine (+57) as fixed modification. Furthermore, two tryptic termini and up to one missing cleavage site were allowed. Peptides with Xcorr values of 2.0, 2.3, and 3.5 for charges states 1+, 2+, and 3+, respectively, were considered identified. To allow high-throughput sequencing of the two independent CLIP libraries that were previously prepared for standard sequencing (18König J. Baumann S. Koepke J. Pohlmann T. Zarnack K. Feldbrügge M. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs.EMBO J. 2009; 28: 1855-1866Crossref PubMed Scopus (74) Google Scholar, 27Ule J. Jensen K.B. Ruggiu M. Mele A. Ule A. Darnell R.B. CLIP identifies Nova-regulated RNA networks in the brain.Science. 2003; 302: 1212-1215Crossref PubMed Scopus (833) Google Scholar), adapter regions were introduced by five cycles of re-amplification with the oligonucleotides oMFP51 or oMFP52 and oMFP3 (supplemental Table S2). In addition to the adapter regions, oMFP51 and oMFP52 introduced a 3-nt barcode (CCC and GGG, respectively) to each of the two libraries to mark them for computational separation after sequencing. Both libraries were mixed and sequenced on an Illumina GA2 flow cell. The 1,290,949 and 7,594,055 sequence reads from the two CLIP libraries were mapped to the U. maydis genome (MUMDB; ftp://ftpmips.gsf.de/ustilago/Umaydis_MIPS_nuc, version April 2008) using Bowtie allowing only unique hits with no more than one mismatch and one single reportable alignment (bowtie -m 1 -v 1; 28Langmead B. Trapnell C. Pop M. Salzberg S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol. 2009; 10: R25Crossref PubMed Scopus (15287) Google Scholar), yielding 616,664 and 5,585,696 mapping reads originating from 1565 and 1521 unique transcript locations (referred to as CLIP tags), respectively. Combining reads from the two libraries resulted in a total of 2551 unique CLIP tags. Associated genes were identified based on current gene annotations (MUMDB; ftp://ftpmips.gsf.de/ustilago/Umaydis_chromosomal/p3_t237631_Ust_maydi2.gff3, version March 2010; 6786 annotated mRNA genes). To include 5′ and 3′ UTRs, each annotation was extended by 300 nt on either side. 1657 of the CLIP tags overlapped in sense orientation with 948 annotated genes. Three genes were omitted, because corresponding CLIP tags mapped to rRNA regions. Detailed annotation on individual genes will be published elsewhere. Plant infections of corn variety Early Golden Bantam (Olds Seeds) were performed as previously described (29Brachmann A. Schirawski J. Müller P. Kahmann R. An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis.EMBO J. 2003; 22: 2199-2210Cro