Genome And Secretome Analysis Of The Hemibiotrophic Fungal Pathogen, Moniliophthora Roreri, Which Causes Frosty Pod Rot Disease Of Cacao: Mechanisms Of The Biotrophic And Necrotrophic Phases

Background: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp.Results: We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase.Conclusions: Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease. © 2014 Meinhardt et al.; licensee BioMed Central Ltd.151USDA; U.S. Department of AgricultureLatunde-Dada, A.O., Colletotrichum: tales of forcible entry, stealth, transient confinement and breakout (2001) Mol Plant Pathol, 2 (4), pp. 187-198. , 10.1046/j.1464-6722.2001.00069.x, 20573006Oliver, R.P., Ipcho, S.V.S., Arabidopsis pathology breathes new life into the necrotrophs-vs.-biotrophs classification of fungal pathogens (2004) Mol Plant Pathol, 5 (4), pp. 347-352. , 10.1111/j.1364-3703.2004.00228.x, 20565602Catanzariti, A.M., Dodds, P.N., Lawrence, G.J., Ayliffe, M.A., Ellis, J.G., Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors (2006) Plant Cell, 18 (1), pp. 243-256. , 10.1105/tpc.105.035980, 1323496, 16326930Link, T.I., Voegele, R.T., Secreted proteins of Uromyces fabae: similarities and stage specificity (2008) Mol Plant Pathol, 9 (1), pp. 59-66Brown, N.A., Antoniw, J., Hammond-Kosack, K.E., The predicted secretome of the plant pathogenic fungus Fusarium graminearum: a refined comparative analysis (2012) Plos One, 7 (4), pp. e33731. , 10.1371/journal.pone.0033731, 3320895, 22493673Thomma, B.P., Alternaria spp.: from general saprophyte to specific parasite (2003) Mol Plant Pathol, 4 (4), pp. 225-236. , 10.1046/j.1364-3703.2003.00173.x, 20569383Evans, H.C., Stalpers, J.A., Samson, R.A., Benny, G.L., Taxonomy of Monilia-Roreri, an important pathogen of theobroma-cacao in South-America (1978) Can J Bot, 56 (20), pp. 2528-2532Aime, M.C., Phillips-Mora, W., The causal agents of witches' broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae (2005) Mycologia, 97 (5), pp. 1012-1022. , 10.3852/mycologia.97.5.1012, 16596953Phillips-Mora, W., Wilkinson, M.J., Frosty pod of cacao: a disease with a limited geographic range but unlimited potential for damage (2007) Phytopathology, 97 (12), pp. 1644-1647. , 10.1094/PHYTO-97-12-1644, 18943726Meinhardt, L.W., Rincones, J., Bailey, B.A., Aime, M.C., Griffith, G.W., Zhang, D.P., Pereira, G.A.G., Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao: what's new from this old foe? (2008) Mol Plant Pathol, 9 (5), pp. 577-588. , 10.1111/j.1364-3703.2008.00496.x, 19018989Ferreira, L.F.R., Duarte, K.M.R., Gomes, L.H., Carvalho, R.S., Leal, G.A., Aguiar, M.M., Armas, R.D., Tavares, F.C.A., Genetic diversity of polysporic isolates of Moniliophthora perniciosa (Tricholomataceae) (2012) Genet Mol Res, 11 (3), pp. 2559-2568. , 10.4238/2012.July.10.11, 22869076Phillips-Mora, W., Wilkinson, M.J., Frosty pod: a disease of limited geographic distribution but unlimited potential for damage (2006) Phytopathology, 96 (6), pp. S138-S138Evans, H.C., (1981) Pod Rot of Cacao caused by Moniliophthora (Monilia) roreri, , London: Commonwealth Agricultural Bureau, 24Joosten, M., de Wit, P., THE TOMATO-CLADOSPORIUM FULVUM INTERACTION: a versatile experimental system to study plant-pathogen interactions (1999) Annu Rev Phytopathol, 37, pp. 335-367. , 10.1146/annurev.phyto.37.1.335, 11701827Perfect, S.E., Green, J.R., Infection structures of biotrophic and hemibiotrophic fungal plant pathogens (2001) Mol Plant Pathol, 2 (2), pp. 101-108. , 10.1046/j.1364-3703.2001.00055.x, 20572997Scarpari, L.M., Meinhardt, L.W., Mazzafera, P., Pomella, A.W.V., Schiavinato, M.A., Cascardo, J.C.M., Pereira, G.A.G., Biochemical changes during the development of witches' broom: the most important disease of cocoa in Brazil caused by Crinipellis perniciosa (2005) J Exp Bot, 56 (413), pp. 865-877. , 10.1093/jxb/eri079, 15642708Melnick, R.L., Marelli, J., Bailey, B.A., The molecular interaction of Theobroma cacao and Moniliophthora perniciosa, causal agent of witches' broom, during infection of young pods (2011) Phytopathology, 101 (6), pp. S274-S274Melnick, R.L., Marelli, J.P., Sicher, R.C., Strem, M.D., Bailey, B.A., The interaction of Theobroma cacao and Moniliophthora perniciosa, the causal agent of witches' broom disease, during parthenocarpy (2012) Tree Genet Genomes, 8 (6), pp. 1261-1279Thomazella, D.P., Teixeira, P.J., Oliveira, H.C., Saviani, E.E., Rincones, J., Toni, I.M., Reis, O., Pereira, G.A., The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development (2012) New Phytol, 194 (4), pp. 1025-1034. , 10.1111/j.1469-8137.2012.04119.x, 3415677, 22443281Mondego, J.M., Carazzolle, M.F., Costa, G.G., Formighieri, E.F., Parizzi, L.P., Rincones, J., Cotomacci, C., Pereira, G.A.G., A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom disease of cacao (2008) Bmc Genomics, 9, p. 548. , 10.1186/1471-2164-9-548, 2644716, 19019209Bailey, B.A., Crozier, J., Sicher, R.C., Strem, M.D., Melnick, R., Carazzolle, M.F., Costa, G.G.L., Meinhardt, L., Dynamic changes in pod and fungal physiology associated with the shift from biotrophy to necrotrophy during the infection of Theobroma cacao by Moniliophthora roreri (2013) Physiol Mol Plant P, 81, pp. 84-96Henrissat, B., A classification of glycosyl hydrolases based on amino acid sequence similarities (1991) Biochem J, 280 (PART 2), pp. 309-316. , 1130547, 1747104Dias, F.M., Vincent, F., Pell, G., Prates, J.A., Centeno, M.S., Tailford, L.E., Ferreira, L.M., Gilbert, H.J., Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A (2004) J Biol Chem, 279 (24), pp. 25517-25526. , 10.1074/jbc.M401647200, 15014076Fibriansah, G., Masuda, S., Koizumi, N., Nakamura, S., Kumasaka, T., The 1.3 A crystal structure of a novel endo-beta-1,3-glucanase of glycoside hydrolase family 16 from alkaliphilic Nocardiopsis sp. strain F96 (2007) Proteins, 69 (3), pp. 683-690. , 10.1002/prot.21589, 17879342Markovic, O., Janecek, S., Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution (2001) Protein Eng, 14 (9), pp. 615-631. , 10.1093/protein/14.9.615, 11707607Vandermarliere, E., Bourgois, T.M., Winn, M.D., van Campenhout, S., Volckaert, G., Delcour, J.A., Strelkov, S.V., Courtin, C.M., Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family (2009) Biochem J, 418 (1), pp. 39-47. , 10.1042/BJ20081256, 18980579Tiels, P., Baranova, E., Piens, K., De Visscher, C., Pynaert, G., Nerinckx, W., Stout, J., Callewaert, N., A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes (2012) Nat Biotechnol, 30 (12), pp. 1225-1231. , 10.1038/nbt.2427, 23159880Ferreira, P., Hernandez-Ortega, A., Herguedas, B., Martinez, A.T., Medina, M., Aryl-alcohol oxidase involved in lignin degradation: a mechanistic study based on steady and pre-steady state kinetics and primary and solvent isotope effects with two alcohol substrates (2009) J Biol Chem, 284 (37), pp. 24840-24847. , 10.1074/jbc.M109.011593, 2757187, 19574215Mayer, A.M., Staples, R.C., Laccase: new functions for an old enzyme (2002) Phytochemistry, 60 (6), pp. 551-565. , 10.1016/S0031-9422(02)00171-1, 12126701Kersten, P.J., Glyoxal oxidase of Phanerochaete chrysosporium: its characterization and activation by lignin peroxidase (1990) Proc Natl Acad Sci U S A, 87 (8), pp. 2936-2940. , 10.1073/pnas.87.8.2936, 53808, 11607073Henrissat, B., Callebaut, I., Fabrega, S., Lehn, P., Mornon, J.P., Davies, G., Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases (1995) Proc Natl Acad Sci U S A, 92 (15), pp. 7090-7094. , 10.1073/pnas.92.15.7090, 41477, 7624375Wostemeyer, J., Kreibich, A., Repetitive DNA elements in fungi (Mycota): impact on genomic architecture and evolution (2002) Curr Genet, 41 (4), pp. 189-198. , 10.1007/s00294-002-0306-y, 12172959Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., Galibert, F., Oliver, S.G., Life with 6000 genes (1996) Science, 274 (5287), pp. 546-563. , 547, 10.1126/science.274.5287.546, 8849441Dean, R.A., Talbot, N.J., Ebbole, D.J., Farman, M.L., Mitchell, T.K., Orbach, M.J., Thon, M., Nicol, R., The genome sequence of the rice blast fungus Magnaporthe grisea (2005) Nature, 434 (7036), pp. 980-986. , 10.1038/nature03449, 15846337Labbe, J., Murat, C., Morin, E., Tuskan, G.A., Le Tacon, F., Martin, F., Characterization of transposable elements in the ectomycorrhizal fungus Laccaria bicolor (2012) Plos One, 7 (8), pp. e40197. , 10.1371/journal.pone.0040197, 3411680, 22870194Adomako, D., Cocoa pod husk pectin (1972) Phytochemistry, 11 (3), p. 1145Gan, P., Ikeda, K., Irieda, H., Narusaka, M., O'Connell, R.J., Narusaka, Y., Takano, Y., Shirasu, K., Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi (2013) New Phytol, 197 (4), pp. 1236-1249. , 10.1111/nph.12085, 23252678Garcia, O., Macedo, J.A.N., Tiburcio, R., Zaparoli, G., Rincones, J., Bittencourt, L.M.C., Ceita, G.O., Cascardo, J.C., Characterization of necrosis and ethylene-inducing proteins (NEP) in the basidiomycete Moniliophthora perniciosa, the causal agent of witches' broom in Theobroma cacao (2007) Mycol Res, 111, pp. 443-455. , 10.1016/j.mycres.2007.01.017, 17512713Pemberton, C.L., Salmond, G.P., The Nep1-like proteins-a growing family of microbial elicitors of plant necrosis (2004) Mol Plant Pathol, 5 (4), pp. 353-359. , 10.1111/j.1364-3703.2004.00235.x, 20565603Zaparoli, G., Barsottini, M.R., de Oliveira, J.F., Dyszy, F., Teixeira, P.J., Barau, J.G., Garcia, O., Dias, S.M., The crystal structure of necrosis-and ethylene-inducing protein 2 from the causal agent of cacao's Witches' Broom disease reveals key elements for its activity (2011) Biochemistry-Us, 50 (45), pp. 9901-9910Cabral, A., Oome, S., Sander, N., Kufner, I., Nurnberger, T., Van den Ackerveken, G., Nontoxic Nep1-like proteins of the downy mildew pathogen Hyaloperonospora arabidopsidis: repression of necrosis-inducing activity by a surface-exposed region (2012) Mol Plant Microbe Interact, 25 (5), pp. 697-708. , 10.1094/MPMI-10-11-0269, 22235872Mosquera, G., Giraldo, M.C., Khang, C.H., Coughlan, S., Valent, B., Interaction transcriptome analysis identifies magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast disease (2009) Plant Cell, 21 (4), pp. 1273-1290. , 10.1105/tpc.107.055228, 2685627, 19357089Paper, J.M., Scott-Craig, J.S., Adhikari, N.D., Cuomo, C.A., Walton, J.D., Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum (2007) Proteomics, 7 (17), pp. 3171-3183. , 10.1002/pmic.200700184, 17676664van den Burg, H.A., Harrison, S.J., Joosten, M.H., Vervoort, J., de Wit, P.J., Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection (2006) Mol Plant Microbe Interact, 19 (12), pp. 1420-1430. , 10.1094/MPMI-19-1420, 17153926Roby, D., Gadelle, A., Toppan, A., Chitin oligosaccharides as elicitors of chitinase activity in melon plants (1987) Biochem Biophys Res Commun, 143 (3), pp. 885-892. , 10.1016/0006-291X(87)90332-9, 3566760Deising, H., Siegrist, J., Chitin deacetylase activity of the rust uromyces-viciae-fabae is controlled by fungal morphogenesis (1995) Fems Microbiol Lett, 127 (3), pp. 207-211Teixeira, P.J.P.L., Thomazella, D.P.T., Vidal, R.O., Do Prado, P.F.V., Reis, O., Baroni, R.M., Franco, S.F., Mondego, J.M.C., The fungal pathogen moniliophthora perniciosa has genes similar to plant PR-1 that are highly expressed during its interaction with cacao (2012) Plos One, 7 (9)Riviere, M.P., Marais, A., Ponchet, M., Willats, W., Galiana, E., Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(1→ 3)-glucanase activity at the onset of tobacco defence reactions (2008) J Exp Bot, 59 (6), pp. 1225-1239. , 10.1093/jxb/ern044, 18390849Levy, A., Guenoune-Gelbart, D., Epel, B.L., Beta-1,3-Glucanases: plasmodesmal gate keepers for intercellular communication (2007) Plant Signal Behav, 2 (5), pp. 404-407. , 10.4161/psb.2.5.4334, 2634228, 19704615Prados-Rosales, R.C., Roldan-Rodriguez, R., Serena, C., Lopez-Berges, M.S., Guarro, J., Martinez-del-Pozo, A., Di Pietro, A., A PR-1-like protein of fusarium oxysporum functions in virulence on mammalian hosts (2012) J Biol Chem, 287 (26), pp. 21970-21979. , 10.1074/jbc.M112.364034, 3381157, 22553200Kershaw, M.J., Talbot, N.J., Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis (1998) Fungal Genet Biol, 23 (1), pp. 18-33. , 10.1006/fgbi.1997.1022, 9501475Zelena, K., Takenberg, M., Lunkenbein, S., Woche, S.K., Nimtz, M., Berger, R.G., PfaH2: a novel hydrophobin from the ascomycete Paecilomyces farinosus (2013) Biotechnol Appl Biochem, 60 (2), pp. 147-154. , 10.1002/bab.1077, 23600571Wosten, H.A., Hydrophobins: multipurpose proteins (2001) Annu Rev Microbiol, 55, pp. 625-646. , 10.1146/annurev.micro.55.1.625, 11544369Bayry, J., Aimanianda, V., Guijarro, J.I., Sunde, M., Latge, J.P., Hydrophobins-unique fungal proteins (2012) PLoS Pathog, 8 (5), pp. e1002700. , 10.1371/journal.ppat.1002700, 3364958, 22693445De Oliveira, A.L., Gallo, M., Pazzagli, L., Benedetti, C.E., Cappugi, G., Scala, A., Pantera, B., Cicero, D.O., The structure of the elicitor cerato-platanin (CP), the first member of the CP fungal protein family, reveals a double psi beta-barrel fold and carbohydrate binding (2011) J Biol Chem, 286 (20), pp. 17560-17568. , 10.1074/jbc.M111.223644, 3093830, 21454637Baccelli, I., Comparini, C., Bettini, P.P., Martellini, F., Ruocco, M., Pazzagli, L., Bernardi, R., Scala, A., The expression of the cerato-platanin gene is related to hyphal growth and chlamydospores formation in Ceratocystis platani (2012) Fems Microbiol Lett, 327 (2), pp. 155-163. , 10.1111/j.1574-6968.2011.02475.x, 22136757Zaparoli, G., Cabrera, O.G., Medrano, F.J., Tiburcio, R., Lacerda, G., Pereira, G.G., Identification of a second family of genes in Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, encoding necrosis-inducing proteins similar to cerato-platanins (2009) Mycol Res, 113, pp. 61-72. , 10.1016/j.mycres.2008.08.004, 18796332Lombardi, L., Faoro, F., Luti, S., Baccelli, I., Martellini, F., Bernardi, R., Picciarelli, P., Pazzagli, L., Differential timing of defense-related responses induced by cerato-platanin and cerato-populin, two non-catalytic fungal elicitors (2013) Physiol Plant, 149, pp. 408-421Yang, Y., Zhang, H., Li, G., Li, W., Wang, X., Song, F., Ectopic expression of MgSM1, a Cerato-platanin family protein from Magnaporthe grisea, confers broad-spectrum disease resistance in Arabidopsis (2009) Plant Biotechnol J, 7 (8), pp. 763-777. , 10.1111/j.1467-7652.2009.00442.x, 19754836Bhadauria, V., Banniza, S., Vandenberg, A., Selvaraj, G., Wei, Y., EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum (2011) Bmc Genomics, 12, p. 327. , 10.1186/1471-2164-12-327, 3149586, 21699715Frischmann, A., Neudl, S., Gaderer, R., Bonazza, K., Zach, S., Gruber, S., Spadiut, O., Seidl-Seiboth, V., Self-assembly at air/water interfaces and carbohydrate binding properties of the small secreted protein EPL1 from the fungus trichoderma atroviride (2013) J Biol Chem, 288 (6), pp. 4278-4287. , 10.1074/jbc.M112.427633, 3567679, 23250741Jeong, J.S., Mitchell, T.K., Dean, R.A., The magnaporthe grisea snodprot1 homolog, MSP1, is required for virulence (2007) Fems Microbiol Lett, 273 (2), pp. 157-165. , 10.1111/j.1574-6968.2007.00796.x, 17590228Peter, M., Courty, P.E., Kohler, A., Delaruelle, C., Martin, D., Tagu, D., Frey-Klett, P., Martin, F., Analysis of expressed sequence tags from the ectomycorrhizal basidiomycetes Laccaria bicolor and Pisolithus microcarpus (2003) New Phytol, 159 (1), pp. 117-129Cosgrove, D.J., Loosening of plant cell walls by expansins (2000) Nature, 407 (6802), pp. 321-326. , 10.1038/35030000, 11014181Quiroz-Castaneda, R.E., Martinez-Anaya, C., Cuervo-Soto, L.I., Segovia, L., Folch-Mallol, J.L., Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta (2011) Microb Cell Fact, 10, p. 8. , 10.1186/1475-2859-10-8, 3050684, 21314954Brotman, Y., Briff, E., Viterbo, A., Chet, I., Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization (2008) Plant Physiol, 147 (2), pp. 779-789. , 10.1104/pp.108.116293, 2409044, 18400936Yamada, M., Sakuraba, S., Shibata, K., Taguchi, G., Inatomi, S., Okazaki, M., Shimosaka, M., Isolation and analysis of genes specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes by fluorescence differential display (2006) Fems Microbiol Lett, 254 (1), pp. 165-172. , 10.1111/j.1574-6968.2005.00023.x, 16451195Rincones, J., Scarpari, L.M., Carazzolle, M.F., Mondego, J.M.C., Formighieri, E.F., Barau, J.G., Costa, G.G.L., Pereira, G.A., Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches' broom pathogen Moniliophthora perniciosa (2008) Mol Plant Microbe In, 21 (7), pp. 891-908Zerbino, D.R., Birney, E., Velvet: algorithms for de novo short read assembly using de Bruijn graphs (2008) Genome Res, 18 (5), pp. 821-829. , 10.1101/gr.074492.107, 2336801, 18349386Sommer, D.D., Delcher, A.L., Salzberg, S.L., Pop, M., Minimus: a fast, lightweight genome assembler (2007) BMC Bioinforma, 8, p. 64Ter-Hovhannisyan, V., Lomsadze, A., Chernoff, Y.O., Borodovsky, M., Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training (2008) Genome Res, 18 (12), pp. 1979-1990. , 10.1101/gr.081612.108, 2593577, 18757608Stanke, M., Keller, O., Gunduz, I., Hayes, A., Waack, S., Morgenstern, B., AUGUSTUS: ab initio prediction of alternative transcripts (2006) Nucleic Acids Res, 34, pp. W435-W439. , Web Server issue, 1538822, 16845043Stanke, M., Tzvetkova, A., Morgenstern, B., AUGUSTUS at EGASP: using EST, protein and genomic alignments for improved gene prediction in the human genome (2006) Genome Biol, 7 (SUPPL. 1), pp. S11 11-18Slater, G.S., Birney, E., Automated generation of heuristics for biological sequence comparison (2005) BMC Bioinforma, 6, p. 31Borodovsky, M., Lomsadze, A., Ivanov, N., Mills, R., Eukaryotic gene prediction using GeneMark.hmm (2003) Curr Protoc Bioinformatics, , Chapter 4, Unit4 6Haas, B.J., Salzberg, S.L., Zhu, W., Pertea, M., Allen, J.E., Orvis, J., White, O., Wortman, J.R., Automated eukaryotic gene structure annotation using EVidenceModeler and the program to assemble spliced alignments (2008) Genome Biol, 9 (1), pp. R7. , 10.1186/gb-2008-9-1-r7, 2395244, 18190707Koski, L.B., Gray, M.W., Lang, B.F., Burger, G., AutoFACT: an automatic functional annotation and classification tool (2005) BMC Bioinforma, 6, p. 151Suzek, B.E., Huang, H., McGarvey, P., Mazumder, R., Wu, C.H., UniRef: comprehensive and non-redundant UniProt reference clusters (2007) Bioinformatics, 23 (10), pp. 1282-1288. , 10.1093/bioinformatics/btm098, 17379688Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Eddy, S.R., Griffiths-Jones, S., Sonnhammer, E.L., The Pfam protein families database (2002) Nucleic Acids Res, 30 (1), pp. 276-280. , 10.1093/nar/30.1.276, 99071,

Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree

It is increasingly recognized that macro-organisms (corals, insects, plants, vertebrates) consist of both host tissues and multiple microbial symbionts that play essential roles in their host’s ecological and evolutionary success. Consequently, identifying benefits and costs of symbioses, as well as mechanisms underlying them are research priorities. All plants surveyed under natural conditions harbor foliar endophytic fungi (FEF) in their leaf tissues, often at high densities. Despite producing no visible effects on their hosts, experiments have nonetheless shown that FEF reduce pathogen and herbivore damage. Here, combining results from three genomic, and two physiological experiments, we demonstrate pervasive genetic and phenotypic effects of the apparently asymptomatic endophytes on their hosts. Specifically, inoculation of endophyte-free (E−) Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant FEF species in healthy T. cacao, induces consistent changes in the expression of hundreds of host genes, including many with known defensive functions. Further, E+ plants exhibited increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes. These phenotypic changes observed in E+ plants correspond to changes in expression of specific functional genes in related pathways. Moreover, a cacao gene (Tc00g04254) highly up-regulated by C. tropicale also confers resistance to pathogen damage in the absence of endophytes or their products in host tissues. Thus, the benefits of increased pathogen resistance in E+ plants are derived in part from up-regulation of intrinsic host defense responses, and appear to be offset by potential costs including reduced photosynthesis, altered host nitrogen metabolism, and endophyte heterotrophy of host tissues. Similar effects are likely in most plant-endophyte interactions, and should be recognized in the design and interpretation of genetic and phenotypic studies of plantsIt is increasingly recognized that macro-organisms (corals, insects, plants, vertebrates) consist of both host tissues and multiple microbial symbionts that play essential roles in their host’s ecological and evolutionary success. Consequently, identifying benefits and costs of symbioses, as well as mechanisms underlying them are research priorities. All plants surveyed under natural conditions harbor foliar endophytic fungi (FEF) in their leaf tissues, often at high densities. Despite producing no visible effects on their hosts, experiments have nonetheless shown that FEF reduce pathogen and herbivore damage. Here, combining results from three genomic, and two physiological experiments, we demonstrate pervasive genetic and phenotypic effects of the apparently asymptomatic endophytes on their hosts. Specifically, inoculation of endophyte-free (E−) Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant FEF species in healthy T. cacao, induces consistent changes in the expression of hundreds of host genes, including many with known defensive functions. Further, E+ plants exhibited increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes. These phenotypic changes observed in E+ plants correspond to changes in expression of specific functional genes in related pathways. Moreover, a cacao gene (Tc00g04254) highly up-regulated by C. tropicale also confers resistance to pathogen damage in the absence of endophytes or their products in host tissues. Thus, the benefits of increased pathogen resistance in E+ plants are derived in part from up-regulation of intrinsic host defense responses, and appear to be offset by potential costs including reduced photosynthesis, altered host nitrogen metabolism, and endophyte heterotrophy of host tissues. Similar effects are likely in most plant-endophyte interactions, and should be recognized in the design and interpretation of genetic and phenotypic studies of plant

A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao

<p>Abstract</p> <p>Background</p> <p>The basidiomycete fungus <it>Moniliophthora perniciosa </it>is the causal agent of Witches' Broom Disease (WBD) in cacao (<it>Theobroma cacao</it>). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. <it>M. perniciosa</it>, together with the related species <it>M. roreri</it>, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage) of <it>M. perniciosa </it>was analyzed to evaluate the overall gene content of this phytopathogen.</p> <p>Results</p> <p>Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that <it>M. perniciosa </it>has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that <it>M. perniciosa </it>have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in <it>M. perniciosa </it>genome survey.</p> <p>Conclusion</p> <p>This genome survey gives an overview of the <it>M. perniciosa </it>genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the <it>M. perniciosa</it>/cacao pathosystem.</p

Development time and new product sales: A contingency analysis of product innovativeness and price

Opposing theories and conflicting empirical results with regard to the effect of development time on new product sales suggest the need for a contingency analysis into factors affecting this relationship. This study uses a unique combination of accounting and perceptual data from 129 product development projects to test the combined contingency effect of product innovativeness and new product price on the relationship between development time and new product sales. The results show that for radically new products with short development times, price has no effect on new product sales. When the development time is long, price has a negative effect on the sales of radical new products. The findings additionally show that price has no effect on sales for incremental new products with short development times and a negative effect for incremental new products with long development times. Together, these findings shed new light on the relationship between development time and new product sales

Two cold inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens

The barley genesHvLtp4.2 andHvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reportedLtp4 cDNA (nowLtp4.1). Southern blot analysis indicated the existence of three or moreLtp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genesHvLtp4.2 andHvLtp4.3 following transformation by particle bombardment, using promoter fusions to the-glucuronidase reporter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ss1) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except thatLtp4.2 was more active thanLtp4.3 in endosperm, andLtp4.3 was active in roots, whileLtp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using theLtp4-specific probe, indicated thatXanthomonas campestris pv.translucens induced an increase over basal levels ofLtp4 mRNA, whilePseudomonas syringae pv.japonica caused a decrease. TheLtp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas theLtp4.2-Gus construction did not respond to infectio

Systematic identification of functional modules and cis-regulatory elements in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>Several large-scale gene co-expression networks have been constructed successfully for predicting gene functional modules and cis-regulatory elements in Arabidopsis (<it>Arabidopsis thaliana</it>)<it>.</it> However, these networks are usually constructed and analyzed in an <it>ad hoc</it> manner. In this study, we propose a completely parameter-free and systematic method for constructing gene co-expression networks and predicting functional modules as well as cis-regulatory elements.</p> <p>Results</p> <p>Our novel method consists of an automated network construction algorithm, a parameter-free procedure to predict functional modules, and a strategy for finding known cis-regulatory elements that is suitable for consensus scanning without prior knowledge of the allowed extent of degeneracy of the motif. We apply the method to study a large collection of gene expression microarray data in Arabidopsis. We estimate that our co-expression network has ~94% of accuracy, and has topological properties similar to other biological networks, such as being scale-free and having a high clustering coefficient. Remarkably, among the ~300 predicted modules whose sizes are at least 20, 88% have at least one significantly enriched functions, including a few extremely significant ones (ribosome, <it>p</it> < 1E-300, photosynthetic membrane, <it>p</it> < 1.3E-137, proteasome complex, <it>p</it> < 5.9E-126). In addition, we are able to predict cis-regulatory elements for 66.7% of the modules, and the association between the enriched cis-regulatory elements and the enriched functional terms can often be confirmed by the literature. Overall, our results are much more significant than those reported by several previous studies on similar data sets. Finally, we utilize the co-expression network to dissect the promoters of 19 Arabidopsis genes involved in the metabolism and signaling of the important plant hormone gibberellin, and achieved promising results that reveal interesting insight into the biosynthesis and signaling of gibberellin.</p> <p>Conclusions</p> <p>The results show that our method is highly effective in finding functional modules from real microarray data. Our application on Arabidopsis leads to the discovery of the largest number of annotated Arabidopsis functional modules in the literature. Given the high statistical significance of functional enrichment and the agreement between cis-regulatory and functional annotations, we believe our Arabidopsis gene modules can be used to predict the functions of unknown genes in Arabidopsis, and to understand the regulatory mechanisms of many genes.</p

Characterization of miRNAs in Response to Short-Term Waterlogging in Three Inbred Lines of Zea mays

Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the microRNA-mediated gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic stress responses. To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and their 92 predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17). Based on our studies, miR159, miR164, miR167, miR393, miR408 and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress and development related cis-regulatory elements on the promoters of these miRNAs; and a probable miRNA-mediated gene regulatory network in response to short-term waterlogging stress was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a gene regulatory network; and crosstalk occurs between different biochemical pathways

Conference highlights of the 15th international conference on human retrovirology: HTLV and related retroviruses, 4-8 june 2011, Leuven, Gembloux, Belgium

The June 2011 15th International Conference on Human Retrovirology: HTLV and Related Viruses marks approximately 30 years since the discovery of HTLV-1. As anticipated, a large number of abstracts were submitted and presented by scientists, new and old to the field of retrovirology, from all five continents. The aim of this review is to distribute the scientific highlights of the presentations as analysed and represented by experts in specific fields of epidemiology, clinical research, immunology, animal models, molecular and cellular biology, and virology