Expression analysis of transcription factors from the interaction between cacao and Moniliophthora perniciosa (Tricholomataceae)

Cacao (Theobroma cacao) is one of the most important tropical crops; however, production is threatened by numerous pathogens, including the hemibiotrophic fungus Moniliophthora perniciosa, which causes witches' broom disease. To understand the mechanisms that lead to the development of this disease in cacao, we focused our attention on cacao transcription factors (TFs), which act as master regulators of cellular processes and are important for the fine-tuning of plant defense responses. We developed a macroarray with 88 TF cDNA from previously obtained cacao-M. perniciosa interaction libraries. Seventy-two TFs were found differentially expressed between the susceptible (Catongo) and resistant (TSH1188) genotypes and/or during the disease time course - from 24 h to 30 days after infection. Most of the differentially expressed TFs belonged to the bZIP, MYB and WRKY families and presented opposite expression patterns in susceptible and resistant cacao-M. perniciosa interactions (i.e., up-regulated in Catongo and down-regulated in TSH1188). The results of the macroarray were confirmed for bZIP and WRKY TFs by real-time PCR. These differentially expressed TFs are good candidates for subsequent functional analysis as well as for plant engineering. Some of these TFs could also be localized on the cacao reference map related to witches' broom resistance, facilitating the breeding and selection of resistant cacao trees. (Résumé d'auteur

First Microsatellite Markers Developed From Cupuassu Ests: Application In Diversity Analysis And Cross-species Transferability To Cacao

The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit's pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta-And trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches' broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively. The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit's pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta-And trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches' broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively. © 2016 Ferraz dos Santos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.11

Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches’ broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed.ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.374683693Aime, M.C., Phillips-Mora, W., The causal agents of witches’ broom and frost pod rot of cacao (chocolate, Theobroma cacao) from a new lineage of Marasmiaceae (2005) Mycologia, 97, pp. 1012-1022Albertini, C., Thebaud, G., Fournier, E., Leroux, P., Eburicol 14α-demethylase gene (CYP51) polymorphism and speciation in Botrytis cinerea (2002) Mycol Res, 106, pp. 1171-1178Altschul, S.F., Gish, W., Miller, W., Myersewand Lipman, D.J., Basic local alignment search tool (1990) J Mol Biol, 215, pp. 403-410Bak, S., Kahn, R.A., Oisen, C.E., Halkier, B.A., Cloning and expression in Escherichia coli of the obtusifoliol 14α-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α demethylases (CYP51) from fungi and mammals (1997) Plant J, 11, pp. 191-201Barrett-Bee, K., Dixon, G., Ergosterol biosynthesis inhibition: A target for antifungal agents (1995) Acta Biochim Pol, 42, pp. 465-480Bellamine, A., Mangla, A.T., Nes, W.D., Waterman, M.R., Characterization and catalytic properties of the sterol 14α-demethylase from Mycobacterium tuberculosis (1999) Proc Natl Acad Sci USA, 96, pp. 8937-8942Butler, G., Rasmussen, M.D., Lin, M.F., Santos, M.A., Sakthikumar, S., Munro, C.A., Rheinbay, E., Reedy, J.L., Evolution of pathogenicity and sexual reproduction in eight Candida genomes (2009) Nature, 459, pp. 657-662Carrillo-Muñoz, A.J., Giusiano, G., Ezkurra, P.A., Quindós, G., Antifungal agents: Mode of action in yeast cells (2006) Rev Esp Quim, 19, pp. 130-139Ceita, G.O., Macedo, J.N., Santos, T.B., Alemanno, L., Gesteira, A.S., Micheli, F., Mariano, A.C., Meinhardt, L.W., Involvement of calcium oxalate degradation during programmed cell death in Theobroma cacao tissues triggered by the hemibiotrophic fungus Moniliophthora perniciosa (2007) Plant Sci, 173, pp. 106-117D’souza, C.A., Kronstad, J.W., Taylor, G., Warren, R., Yuen, M., Hu, G., Jung, W.H., Tangen, K., Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts (2011) MBio, 2, pp. e00342-e00410Délye, C., Laigret, F., Corio-Costet, M.F., Cloning and sequence analysis of the eburicol 14α-demethylase gene of the obligate biotrophic grape powdery mildew fungus (1997) Gene, 195, pp. 29-33Dujon, B., Sherman, D., Fischer, G., Durrens, P., Casaregola, S., Lafontaine, I., De Montigny, J., Talla, E., Genome evolution in yeasts (2004) Nature, 430, pp. 35-44Evans, H.C., Cacao diseases - The trilogy revisited (2007) Phytopathology, 97, pp. 1640-1643Felsenstein, J., Confidence limits on phylogenies: An approach using the bootstrap (1985) Evolution, 39, pp. 783-791Formighieri, E.F., Tiburcio, R.A., Armas, E.D., Medrano, F.J., Shimo, H., Carels, N., GóEs Neto, A., Sardinha-Pinto, N., The mitochondrial genome of the phytopathogenic basidiomycete Moniliophthora perniciosa is 109 kb in size and contains a stable integrated plasmid (2008) Mycol Res, 112, pp. 1136-1152Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., Bairoch, A., Protein identification and analysis tools on the ExPASy Server (2005) The Proteomics and Protocols Handbook, pp. 571-607. , In: Walker JM, Humana Press, TotowaGoffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., Galibert, F., Johnston, M., Life with 6000 genes (1996) Science, 265, pp. 2077-2082Griffith, G.W., Bravo-Velasquez, E., Wilson, F.J., Lewis, D.M., Hedger, J.N., Autecology and evolution of the witches’ broom pathogen (Crinipellis perniciosa) of cocoa (1994) The Ecology of Plant Pathogens, pp. 245-265. , In: Blakeman JP and Williamson B, CAB International, WallingfordGriffith, G.W., Nicholson, J., Neinninger, A., Birch, R., Witches’ brooms and frosty pods: Two major pathogens of cacao (2003) New Zeal J Bot, 41, pp. 423-435Hall, T.A., BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT (1999) Nucleic Acids Res, 41, pp. 95-98Hof, H., Critical annotations to the use of azole antifungals for plant protection (2001) Antimicrob Agents Chemother, 45, pp. 2987-2990Jackson, C.J., Lamb, D.C., Marczylo, T.H., Parker, J.E., Manning, N.L., Kelly, D.E., Kelly, S.L., Conservation and cloning of CYP51: A sterol 14α-demethylase from Mycobacterium smegmatis (2003) Biochem Biophys Res Commun, 301, pp. 558-563James, T.Y., Kauff, F., Schoch, C.L., Matheny, P.B., Hofstetter, V., Cox, C.J., Celio, G., Miadlikowska, J., Reconstructing the early evolution of Fungi using a six-gene phylogeny (2006) Nature, 19, pp. 818-822Jones, T., Federspiel, N.A., Chibana, H., Dungan, J., Kalman, S., Magee, B.B., Newport, G., Magee, P.T., The diploid genome sequence of Candida albicans (2004) Proc Natl Acad Sci USA, 101, pp. 7329-7334Kairuz, P.B., Zuber, J.P., Jaunin, P., Buchman, T.G., Bille, J., Rossier, M., Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-α-demethylase (L1A1) gene fragment (1994) J Clin Microbiol, 32, pp. 1902-1907Kalb, V.F., Woods, C.W., Turi, T.G., Dey, C.R., Sutter, T.R., Loper, J.C., Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae (1987) DNA, 6, pp. 529-537Kall, L., Krogh, A., Sonnhammer, E.L., Advantages of combined transmembrane topology and signal peptide prediction- the Phobius web server (2007) Nucleic Acids Res 35:429-, p. 432Kim, D., Lim, Y.R., Ohk, S.O., Kim, B.J., Chun, Y.J., Functional expression and characterization of CYP51 from dandruffcausing Malassezia globosa (2011) FEMS Yeast Res, 11, pp. 80-87Lai, M.H., Kirsch, D.R., Nucleotide sequence of cytochrome P450 L1A1 (lanosterol 14α-demethylase) from Candida albicans (1989) Nucleic Acids Res, 17, p. 804Lamb, D.C., Kelly, D.E., Manning, N.M., Hollomon, D.W., Kelly, S.L., Expression, purification, reconstitution and inhibition of Ustilago maydis sterol 14α-demethylase (CYP 51P450) (1998) FEMS Microbiol Lett, 169, pp. 369-373Lee, C.H., Hsu, K.H., Wang, S.Y., Chang, T.T., Chu, F.H., Shaw, J.F., Cloning and characterization of the lanosterol 14α-demethylase gene from Antrodia cinnamomea (2010) J Agr Food Chem, 58, pp. 4800-4807Lees, N.D., Skaggs, B., Kirsch DR and BirdM(1995) Cloning of the late genes in the ergosterol biosynthetic pathway of Saccharomyces cerevisiae - A review Lipids, 30, pp. 221-226Lepesheva, G.I., Waterman, M.R., Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms (2007) Biochim Biophys Acta, 3, pp. 467-477Luo, C.X., Schnabel, G., The cytochrome P450 lanosterol 14α-demethylase gene is a demethylation inhibitor fungicide resistance determinant in Monilia fructicola field isolates from Georgia (2008) Appl Environ Microb, 74, pp. 359-366Martin, F., Aerts, A., Ahrén, D., Brun, A., Danchin, E.G., Duchaussoy, F., Gibon, J., Pereda, V., The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis (2008) Nature, 452, pp. 88-92McQuilken, M.P., Rudgard, S.A., Sensitivity of Crinipellis periciosa to two triazole fungicides in vitro and their effect on development of the fungus in cocoa (1988) Plant Pathol, 37, pp. 499-506Meinhardt, L.W., Bellato, C.M., Rincones, J., Azevedo, R.A., Cascardo, J.C.M., Pereira, G.A.G., In vitro production of biotrophic- like cultures of Crinipellis perniciosa, the causal agent of Witches’ broom disease of Theobroma cacao (2006) Curr Microbiol, 52, pp. 191-196Mellado, E., Guerra, T.M.D., Estrela, M.C., Tudela, J.L.R., Identification of two different 14α-sterol demethylase related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species (2001) J Clin Microbiol 39:2431-, p. 2438Mondego, J.M.C., Carazzolle, M.F., Costa, G.G.L., Formighieri, E.F., Parizzi, L.P., Rincones, J., Cotomacci, C., Carrer, H., A genome survey of Moniliophthora perniciosa gives new insights into Witches’ broom disease of cacao (2008) BMC Genomics, 9, pp. 1-25Morales, I.J., Vohra, P.K., Puri, V., Kottom, T.J., Limper, A.H., Thomas, C.F., Characterization of a lanosterol 14α demethylase from Pneumocystis carinii (2003) Am J Resp Cell Mol, 29, pp. 232-238Mota, S.G.R., Barros, T.F., Castilho, M.S., In vitro screening and chemometrics analysis on a series of azole derivativeswith fungicide activity against Moniliophthora perniciosa (2010) J Braz Chem Soc, 21, pp. 510-519Nierman, W.C., Pain, A., Anderson, M.J., Wortman, J.R., Kim, H.S., Arroyo, J., Berriman, M., Bermejo, C., Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus (2005) Nature, 438, pp. 1151-1156Page, R.D.M., TREEVIEW: An application to display phylogenetic trees on personal computers (1996) Comput Appl Biosci, 12, pp. 357-358Park, H.G., Lee, I.S., Chun, Y.J., Yun, C.H., Johnston, J.B., Montellano, P.R.O., Kim, D., Heterologous expression and characterization of the sterol 14α-demethylase CYP51F from Candida albicans (2011) Arch Biochem Biophys, 509, pp. 9-15Pereira, J.L., Ram, A., Figueiredo, J.M., Almeida, L.C.C., Primeira ocorrência de vassoura-de-bruxa na principal região produtora de cacau do Brasil (1989) Agrotrópica, 1, pp. 79-81Petersen, T.N., Brunak, S., Heijne, G., Nielsen, H., SignalIP 4.0: Discriminating signal peptides from transmembrane regions (2011) Nat Methods, 10, pp. 785-786Pietila, M.P., Vohra, P.K., Sanyat, B., Wengenack, N.L., Raghavakaimal, S., Thomas, C.F., Cloning and characterization of CYP51 from Mycobacterium avium (2006) Am J Resp Cell Mol, 35, pp. 236-240Pires, A.B.L., Gramacho, K.P., Silva, D.C., Góes-Neto, A., Silva, M.M., Muniz-Sobrinho, J.S., Porto, R.F., Cascardo, J.C.M., Early development of Moniliophthora perniciosa basidiomata and developmentally regulated genes (2009) BMC Microbiol, 9, p. e158Purdy, L.H., Schimidt, R.A., Status of cacao witches’ broom: Biology, epidemiology, and management (1996) Annu Rev Phytopathol, 34, pp. 573-594Raeder, U., Broda, P., Rapid preparation of DNA from filamentous fungi (1985) Lett Appl Microbiol, 1, pp. 17-20Revankar, S.G., Fu, J., Rinaldi, M.G., Kelly, S.L., Kelly, D.E., Lamb, D.C., Keller, S.M., Wickes, B.L., Cloning and characterization of the lanosterol 14α-demethylase (ERG11) gene in Cryptococcus neoformans (2004) Biochem Biophys Res Commun, 324, pp. 719-728Rincones, J., Scarpari, L.M., Carazzolle, M.F., Mondego, J.M.C., Formighieri, E.F., Barau, J.G., Costa, G.G.L., Vilas-Boas, L.A., Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches’ broom pathogen Moniliophthora perniciosa (2008) Mol Plant Microbe Int, 21, pp. 891-908Rio, M.C.S., Oliveira, B.V., Tomazella, D.P.T., Silva, J.A.F., Pereira, G.A.G., Production of calcium oxalate crystals by the basidiomycete Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao (2008) Curr Microbiol, 56, pp. 363-370Rozman, D., Stromstedt, M., Tsui, L.C., Scherer, S.W., Waterman, M.R., Structure and mapping of the human lanosterol 14α-demethylase gene (CYP51) encoding the cytochrome P450 involved in cholesterol biosynthesis: Comparison of exon/intron organization with other mammalian and fungal CYP genes (1996) Genomics, 38, pp. 371-381Sheng, C., Miao, Z., Ji, H., Yao, J., Wang, W., Che, X., Dong, G., Zhang, W., Three-dimensional model of lanosterol 14α-demethylase from Cryptococcus neoformans: Active- site characterization and insights into azole binding (2009) Antimicrob Agents Chemother, 53, pp. 3487-3495Sigrist, C.J.A., Cerutti, L., Castro, E., Langendijk-Genevaux, P.S., Bulliard, V., Bairoch, A., Hulo, N., PROSITE, a protein domain database for functional characterization and annotation (2009) Nucleic Acids Res, 38, pp. 161-166Stajich, J.E., Wilke, S.K., Ahrén, D., Au, C.H., Birren, B.W., Borodovsky, M., Burns, C., Cheng, C.K., Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus) (2010) Proc Natl Acad Sci USA, 107, pp. 11889-11894Stanke, M., Keller, O., Gunduz, I., Hayes, A., Waack, S., Morgenstern, B., AUGUSTUS: Ab initio prediction of alternative transcripts (2006) Nucleic Acids Res, 34, pp. 435-439Swofford, D.L., PAUP - Phylogenetic Analysis Using Parsimony (and other methods). Version 4.0b10 (2002) Sinauer Associates, , Sunderland, MATer-Hovhannisyan, V., Lomsadze, L., Chernoff, Y.O., Borodovsky, M., Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training (2008) Genome Res, 18, pp. 1979-1990Thompson, J.D., Higgins, D.G., Gibson, T.J., CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weigh matrix choice (1994) Nucleic Acids Res, 22, pp. 4673-4680Veen, M., Lang, C., Interactions of the ergosterol biosynthetic pathway with other lipid pathways (2005) Biochem Soc Trans, 33, pp. 1178-1181Warrilow, A.G.S., Melo, N., Martel, C.M., Parker, J.E., Nes, W.D., Kelly, S.L., Kelly, D.E., Expression, purification and characterization of Aspergillus fumigatus sterol 14α demethylase (CYP51) isoenzymes A and B (2010) Antimicrob Agents Chemother, 54, pp. 4225-4234Waterman, M.R., Lepesheva, G.I., Sterol 14 _-demethylase, an abundant and essential mixed-function oxidase (2005) Biochem Biophys Res Commun, 338, pp. 418-422Wood, H.M., Dickinson, M.J., Lucas, J.A., Dyer, P.S., Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme (2001) FEMS Microbiol Lett, 196, pp. 183-187Zhao, L., Liu, D., Zhang, Q., Zhang, S., Wan J and XiaoW(2007) Expression and homology modeling of sterol 14α-demethylase from Penicillium digitatium FEMS Microbiol Lett, 277, pp. 37-4

Genome sequence and effectorome of Moniliophthora perniciosa and Moniliophthora roreri subpopulations

Background: The hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity. Results: Candidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection. Conclusions: The present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity. © 2018 The Author(s).This work was done in the frame of the International Consortium in Advanced Biology (CIBA; https://www.ciba-network.org). The authors thank the Molecular Plant Pathology Laboratory and the Plant Pathology Laboratory at INIAP personnel for their assistance in obtaining the DNAs, Dr Carmen Suarez Capello for her kind assistance in Ecuador, and the Núcleo de Biologia Computacional e Gestão de Informações Biotecnológicas - UESC (NBCGIB), and Copenhague University for providing bioinformatics facility. Data sets were processed in sagarana HPC cluster, CPAD-ICB-UFMG. The authors would also like to thank Dr. Claudia Fortes Ferreira (Embrapa CNPMF, Brazil) and Dr. Raul Renné Valle (CEPLAC/CEPEC, Brazil) for English language revision. We are also grateful to Ivanna Michelle Meraz Pérez for helping translating an early version of this manuscript and to the anonymous reviewers who provided helpful comments to our work. KPG, FM and CPP were supported by research fellowship Pq-1 from CNPq. National Council for Scientific Development (CNPq) n° 311759/2014–9. CSB acknowledges FAPESB (Foundation for Research Support of the State of Bahia) for supporting her with a research assistantship during her Master’s Programme

Boto, A Class Ii Transposon In Moniliophthora Perniciosa, Is The First Representative Of The Pif/ Harbinger Superfamily In A Phytopathogenic Fungus

Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus. © 2013 SGM.1591112125Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., Gapped BLAST and PSI-BLAST: A new generation of protein database search programs (1997) Nucleic Acids Res, 25, pp. 3389-3402Anaya, N., Roncero, M.I.G., Stress-induced rearrangement of Fusarium retrotransposon sequences (1996) Mol Gen Genet, 253, pp. 89-94Andebrhan, T., Furtek, D.B., Random amplified polymorphic DNA (RAPD) analysis of Crinipellis perniciosa isolates from different hosts (1994) Plant Pathol, 43, pp. 1020-1027Andebrhan, T., Figueira, A., Yamada, M.M., Cascardo, J., Furtek, D.B., Molecular fingerprinting suggests two primary outbreaks of witches' broom disease (Crinipellis perniciosa) of Theobroma cacao in Bahia, Brazil (1999) Eur J Plant Pathol, 105, pp. 167-175Bastos, C.N., Evans, H.C., A new pathotype of Crinipellis perniciosa (Witches' broom disease) on solanaceous hosts (1985) Plant Pathol, 34, pp. 306-312Bastos, C.N., Andebrhan, T., de Almeida, L.C., Comparação morfológica de isolados de Crinipellis perniciosa (1988) Fitopatol Bras, 13, pp. 202-205Benton, W.D., Davis, R.W., Screening lambdagt recombinant clones by hybridization to single plaques in situ (1977) Science, 196, pp. 180-182Bergemann, M., Lespinet, O., M'Barek, S.B., Daboussi, M.J., Dufresne, M., Genome-wide analysis of the Fusarium oxysporum mimp family of MITEs and mobilization of both native and de novo created mimps (2008) J Mol Evol, 67, pp. 631-642Daboussi, M.J., Capy, P., Transposable elements in filamentous fungi (2003) Annu Rev Microbiol, 57, pp. 275-299Daboussi, M.J., Langin, T., Transposable elements in the fungal plant pathogen Fusarium oxysporum (1994) Genetica, 93, pp. 49-59Daboussi, M.J., Langin, T., Brygoo, Y., Fot1, a new family of fungal transposable elements (1992) Mol Gen Genet, 232, pp. 12-16de Arruda, M.C., Ferreira, M.A., Miller, R.N., Resende, M.L., Felipe, M.S., Nuclear and mitochondrial rDNA variability in Crinipellis perniciosa from different geographic origins and hosts (2003) Mycol Res, 107, pp. 25-37de Arruda, M.C., Miller, R.N., Ferreira, M.A., Felipe, M.S., Comparison of Crinipellis perniciosa isolates from Brazil by ERIC repetitive element sequence-based PCR genomic fingerprint (2003) Plant Pathol, 52, pp. 236-244Dobinson, K.F., Harris, R.E., Hamer, J.E., Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea (1993) Mol Plant Microbe Interact, 6, pp. 114-126Dufresne, M., Hua-Van, A., El Wahab, H.A., Ben M'Barek, S., Vasnier, C., Teysset, L., Kema, G.H.J., Daboussi, M.J., Transposition of a fungal miniature inverted-repeat transposable element through the action of a Tc1-like transposase (2007) Genetics, 175, pp. 441-452Evans, H.C., Witches' broom disease of cocoa (Crinipellis perniciosa) in Ecuador (1978) Ann Appl Biol, 89, pp. 185-192Felipe, M.S.S., Azevedo, M.A., Vainstein, M.H., Schrank, A., Biologia molecular de fungos filamentosos: Construção de banco genômico e de cDNA (1992) Manual Técnico, p. 99. , In, Piracicaba, SP, Brazil: Escola Superior de Agricultura 'Luiz de Queiroz'Fleetwood, D.J., Scott, B., Lane, G.A., Tanaka, A., Johnson, R.D., A complex ergovaline gene cluster in epichloe endophytes of grasses (2007) Appl Environ Microbiol, 73, pp. 2571-2579Fleetwood, D.J., Khan, A.K., Johnson, R.D., Young, C.A., Mittal, S., Wrenn, R.E., Hesse, U., Scott, B., Abundant degenerate miniature inverted-repeat transposable elements in genomes of epichloid fungal endophytes of grasses (2011) Genome Biol Evol, 3, pp. 1253-1264Gómez-Gómez, E., Anaya, N., Roncero, M.I.G., Hera, C., Folyt1, a new member of the hAT family, is active in the genome of the plant pathogen Fusarium oxysporum (1999) Fungal Genet Biol, 27, pp. 67-76Goodwin, T.J., Poulter, R.T., The DIRS1 group of retrotransposons (2001) Mol Biol Evol, 18, pp. 2067-2082Goodwin, T.J., Butler, M.I., Poulter, R.T., Cryptons: A group of tyrosine-recombinase-encoding DNA transposons from pathogenic fungi (2003) Microbiology, 149, pp. 3099-3109Griffith, G.W., Hedger, J.N., A novel method for producing basidiocarps of the cocoa pathogen Crinipellis perniciosa using a branvermiculite medium (1993) Eur J Plant Pathol, 99, pp. 227-230Grzebelus, D., Yau, Y.Y., Simon, P.W., Master: A novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.) (2006) Mol Genet Genomics, 275, pp. 450-459Hancock, C.N., Zhang, F., Wessler, S.R., Transposition of the Tourist-MITE mPing in yeast: An assay that retains key features of catalysis by the class 2 PIF/Harbinger superfamily (2010) Mob DNA, 1, p. 5He, C., Nourse, J.P., Irwin, J.A.G., Manners, J.M., Kelemu, S., CgT1: A non-LTR retrotransposon with restricted distribution in the fungal phytopathogen Colletotrichum gloeosporioides (1996) Mol Gen Genet, 252, pp. 320-331Hedger, J.N., Pickering, V., Aragundi, J., Variability of populations of the witches' broom disease of cocoa (Crinipellis perniciosa) (1987) Trans Br Mycol Soc, 88, pp. 533-546Hua-Van, A., Davière, J.M., Kaper, F., Langin, T., Daboussi, M.J., Genome organization in Fusarium oxysporum: Clusters of class II transposons (2000) Curr Genet, 37, pp. 339-347Ignacchiti, M.D.C., Santana, M.F., Araújo, E.F., Queiroz, M.V., The distribution of a transposase sequence in Moniliophthora perniciosa confirms the occurrence of two genotypes in Bahia, Brazil (2011) Trop Plant Pathol, 36, pp. 276-286Jiang, N., Bao, Z., Zhang, X., Hirochika, H., Eddy, S.R., McCouch, S.R., Wessler, S.R., An active DNA transposon family in rice (2003) Nature, 421, pp. 163-167Jurka, J., Kapitonov, V.V., PIFs meet Tourists and Harbingers: A superfamily reunion (2001) Proc Natl Acad Sci U S A, 98, pp. 12315-12316Kaneko, I., Tanaka, A., Tsuge, T., REAL, an LTR retrotransposon from the plant pathogenic fungus Alternaria alternata (2000) Mol Gen Genet, 263, pp. 625-634Kapitonov, V.V., Jurka, J., Molecular paleontology of transposable elements from Arabidopsis thaliana (1999) Genetica, 107, pp. 27-37Kapitonov, V.V., Jurka, J., Harbinger transposons and an ancient HARBI1 gene derived from a transposase (2004) DNA Cell Biol, 23, pp. 311-324Keyhani, N.O., Fungal genomes and beyond (2011) Fungal Genom Biol, 1, pp. e101Kito, H., Takahashi, Y., Sato, J., Fukiya, S., Sone, T., Tomita, F., Occan, a novel transposon in the Fot1 family, is ubiquitously found in several Magnaporthe grisea isolates (2003) Curr Genet, 42, pp. 322-331Langin, T., Capy, P., Daboussi, M.J., The transposable element impala, a fungal member of the Tc1-mariner superfamily (1995) Mol Gen Genet, 246, pp. 19-28Marchler-Bauer, A., Bryant, S.H., CD-Search: Protein domain annotations on the fly (2004) Nucleic Acids Res, 32, pp. W327-W331. , WEB SERVER ISSUEMaurer, P., Réjasse, A., Capy, P., Langin, T., Riba, G., Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene (1997) Mol Gen Genet, 256, pp. 195-202McClintock, B., The significance of responses of the genome to challenge (1984) Science, 226, pp. 792-801Mes, J.J., Haring, M.A., Cornelissen, B.J.C., Foxy: An active family of short interspersed nuclear elements from Fusarium oxysporum (2000) Mol Gen Genet, 263, pp. 271-280Mondego, J.M.C., Carazzolle, M.F., Costa, G.G.L., Formighieri, E.F., Parizzi, L.P., Rincones, J., Cotomacci, C., Cunha, A.F., A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao (2008) BMC Genomics, 9, p. 548Nakayashiki, H., Nishimoto, N., Ikeda, K., Tosa, Y., Mayama, S., Degenerate MAGGY elements in a subgroup of Pyricularia grisea: A possible example of successful capture of a genetic invader by a fungal genome (1999) Mol Gen Genet, 261, pp. 958-966Niella, G., Resende, M.L., Castro, H.A., de Carvalho, G.A., Silva, L.H.C.P., Aperfeiçoamento da metodologia de produção artificial de basidiocarpos de Crinipellis perniciosa (1999) Fitopatol Bras, 24, pp. 523-527Ogasawara, H., Obata, H., Hata, Y., Takahashi, S., Gomi, K., Crawler, a novel Tc1/mariner-type transposable element in Aspergillus oryzae transposes under stress conditions (2009) Fungal Genet Biol, 46, pp. 441-449Okuda, M., Ikeda, K., Namiki, F., Nishi, K., Tsuge, T., Tfo1: An Ac-like transposon from the plant pathogenic fungus Fusarium oxysporum (1998) Mol Gen Genet, 258, pp. 599-607Pereira, J.L., de Almeida, L.C.C., Santos, S.M., Witches' broom disease of cocoa in Bahia: Attempts at eradication and containment (1996) Crop Prot, 15, pp. 743-752Pereira, J.F., Araújo, E.F., Brommonschenkel, S.H., Queiroz, M.V., Elementos transponíveis em fungos fitopatogênicos (2006) Rev Anual Patol Plant, 14, pp. 303-362Pereira, J.F., Ignacchiti, M.D.C., Araújo, E.F., Brommonschenkel, S.H., Cascardo, J.C.M., Pereira, G.A.G., Queiroz, M.V., PCR amplification and sequence analyses of reverse transcriptaselike genes in Crinipellis perniciosa isolates (2007) Fitopatol Bras, 32, pp. 373-380Ploetz, R.C., Schnell, R.J., Ying, Z.T., Zheng, Q., Olano, C.T., Motamayor, J.C., Johnson, E.S., Analysis of molecular diversity in Crinipellis perniciosa with AFLP markers (2005) Eur J Plant Pathol, 111, pp. 317-326Pontecorvo, G., Roper, J.A.L., Hemmons, L.M., McDonald, K.D., Bufton, A.W.J., The genetics of Aspergillus nidulans (1953) Adv Genet, 5, pp. 141-238Rep, M., van der Does, H.C., Cornelissen, B.J., Drifter, a novel, low copy hAT-like transposon in Fusarium oxysporum is activated during starvation (2005) Fungal Genet Biol, 42, pp. 546-553Rincones, J., Meinhardt, L.W., Vidal, B.C., Pereira, G.A.G., Electrophoretic karyotype analysis of Crinipellis perniciosa, the causal agent of witches' broom disease of Theobroma cacao (2003) Mycol Res, 107, pp. 452-458Rincones, J., Mazotti, G.D., Griffith, G.W., Pomela, A., Figueira, A., Leal Jr., G.A., Queiroz, M.V., Azevedo, R.A., Genetic variability and chromosome-length polymorphisms of the witches' broom pathogen Crinipellis perniciosa from various plant hosts in South America (2006) Mycol Res, 110, pp. 821-832Rincones, J., Scarpari, L.M., Carazzolle, M.F., Mondego, J.M.C., Formighieri, E.F., Barau, J.G., Costa, G.G.L., Brentani, H.P., Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches' broom pathogen Moniliophthora perniciosa (2008) Mol Plant Microbe Interact, 21, pp. 891-908Saitou, N., Nei, M., The neighbor-joining method: A new method for reconstructing phylogenetic trees (1987) Mol Biol Evol, 4, pp. 406-425Sambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual, , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor LaboratorySanger, F., Nicklen, S., Coulson, A.R., DNA sequencing with chain-terminating inhibitors (1977) Proc Natl Acad Sci U S A, 74, pp. 5463-5467Schmidt, S.M., Panstruga, R., Pathogenomics of fungal plant parasites: What have we learnt about pathogenesis? (2011) Curr Opin Plant Biol, 14, pp. 392-399Shim, W.B., Dunkle, L.D., Malazy, a degenerate, speciesspecific transposable element in Cercospora zeae-maydis (2005) Mycologia, 97, pp. 349-355Shnyreva, A.V., Transposable elements are the factors involved in various rearrangements and modifications of fungal genomes (2003) Russ J Genet, 39, pp. 505-518Shull, V., Hamer, J.E., Genetic differentiation in the rice blast fungus revealed by the distribution of the Fosbury retrotransposon (1996) Fungal Genet Biol, 20, pp. 59-69Sinzelle, L., Kapitonov, V.V., Grzela, D.P., Jursch, T., Jurka, J., Izsvák, Z., Ivics, Z., Transposition of a reconstructed Harbinger element in human cells and functional homology with two transposon-derived cellular genes (2008) Proc Natl Acad Sci U S A, 105, pp. 4715-4720Specht, C.A., DiRusso, C.C., Novotny, C.P., Ullrich, R.C., A method for extracting high-molecular-weight deoxyribonucleic acid from fungi (1982) Anal Biochem, 119, pp. 158-163Stanke, M., Morgenstern, B., AUGUSTUS: A web server for gene prediction in eukaryotes that allows user-defined constraints (2005) Nucleic Acids Res, 33, pp. W465-W467. , WEB SERVER ISSUEThompson, J.D., Higgins, D.G., Gibson, T.J., CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice (1994) Nucleic Acids Res, 22, pp. 4673-4680Walker, E.L., Eggleston, W.B., Demopulos, D., Kermicle, J., Dellaporta, S.L., Insertions of a novel class of transposable elements with a strong target site preference at the r locus of maize (1997) Genetics, 146, pp. 681-693Wessler, S.R., Bureau, T.E., White, S.E., LTRretrotransposons and MITEs: Important players in the evolution of plant genomes (1995) Curr Opin Genet Dev, 5, pp. 814-821Wicker, T., Sabot, F., Hua-Van, A., Bennetzen, J.L., Capy, P., Chalhoub, B., Flavell, A., Morgante, M., A unified classification system for eukaryotic transposable elements (2007) Nat Rev Genet, 8, pp. 973-982Wöstemeyer, J., Kreibich, A., Repetitive DNA elements in fungi (Mycota): Impact on genomic architecture and evolution (2002) Curr Genet, 41, pp. 189-198Yang, G.J., Zhang, F., Hancock, C.N., Wessler, S.R., Transposition of the rice miniature inverted repeat transposable element mPing in Arabidopsis thaliana (2007) Proc Natl Acad Sci U S A, 104, pp. 10962-10967Yeadon, P.J., Catcheside, D.E., Guest: A 98 bp inverted repeat transposable element in Neurospora crassa (1995) Mol Gen Genet, 247, pp. 105-109Zhang, X., Feschotte, C., Zhang, Q., Jiang, N., Eggleston, W.B., Wessler, S.R., P instability factor: An active maize transposon system associated with the amplification of Tourist-like MITEs and a new superfamily of transposases (2001) Proc Natl Acad Sci U S A, 98, pp. 12572-12577Zhang, X., Jiang, N., Feschotte, C., Wessler, S.R., PIF-and Pong-like transposable elements: Distribution, evolution and relationship with Tourist-like miniature inverted-repeat transposable elements (2004) Genetics, 166, pp. 971-986Zhou, M.B., Lu, J.-J., Zhong, H., Liu, X.-M., Tang, D.-Q., Distribution and diversity of PIF-like transposable elements in the Bambusoideae subfamily (2010) Plant Sci, 179, pp. 257-266Zhou, M.B., Liu, X.M., Tang, D.Q., PpPIF-1: First isolated full-length PIF-like element from the bamboo Phyllostachys pubescens (2012) Genet Mol Res, 11, pp. 810-82

Protein Extraction For Proteome Analysis From Cacao Leaves And Meristems, Organs Infected By Moniliophthora Perniciosa, The Causal Agent Of The Witches' Broom Disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction - under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF - using infiltration-centfifugation procedure - that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.291123912401Kris-Etherton, P., Keen, C., (2002) Curr. Opin. Lipidol, 13, pp. 41-49(1985) Cocoa, , Wood, G. A. R, Lass, R. A, Eds, Longman, New YorkAime, M.C., Phillips-Mora, W., (2005) Mycologia, 97, pp. 1012-1022Purdy, L.H., Schmidt, R.A., (1996) Annu. Rev. Phytopathol, 34, pp. 573-594Andebrhan, T., Figueira, A., Yamada, M.M., Cascardo, J., Furtek, D.B., (1999) Eur. J. Plant Pathol, 105, pp. 167-175Gesteira, A.S., Micheli, F., Carels, N., Da Silva, A.C., (2007) Ann. Bot, 100, pp. 129-140Ceita, G.O., Macêdo, J.N.A., Santos, T.B., Alemanno, L., (2007) Plant Sci, 173, pp. 106-117Garcia, O., Macedo, J.N., Tiburcio, R., Zaparoli, G., (2007) Mycol. Res, 111, pp. 443-455Leal, G.A., Albuquerque, P.S.B., Figueira, A., (2007) Mol. Plant Pathol, 8, pp. 279-292Zak, D.L., Keeney, P.G.J., (1976) Agric. Food Chem, 24, pp. 479-483Zak, D.L., Keeney, P.G.J., (1976) Agric. Food Chem, 24, pp. 483-486Gesteira, A.S., Micheli, F., Ferreira, C.F., Cascardo, J.C.M., (2003) BioTechniques, 35, pp. 494-500Figueira, A., Janick, J., BeMiller, J.N., (1994) Carbohydr. Polym, 24, pp. 133-138Gorg, A., Weiss, W., Dunn, M.J., (2004) Proteomics, 4, pp. 3665-3685Carpentier, S.C., Witters, E., Laukens, K., Deckers, P., (2005) Proteomics, 5, pp. 2497-2507Saravanan, R.S., Rose, J.K., (2004) Proteomics, 4, pp. 2522-2532Dani, V., Simon, W.J., Duranti, M., Croy, R.R.D., (2005) Proteomics, 5, pp. 737-745Fecht-Christoffers, M.M., Braun, H.P., Lemaitre-Guillier, C., VanDorsselaer, A., Horst, W.J., (2003) Plant Physiol, 133, pp. 1935-1946Bolwell, P.P., Page, A., Pislewska, M., Wojtaszek, P., (2001) Protoplasma, 217, pp. 20-32Diaz-Vivancos, P., Rubio, M., Mesonero, V., Periago, P.M., (2006) J. Exp. Bot, 57, pp. 3813-3824Frias, G.A., Purdy, L.H., Schmidt, R.A., (1991) Plant Dis, 75, pp. 552-556Wang, W., Scali, M., Vignani, R., Spadafora, A., (2003) Electrophoresis, 24, pp. 2369-2375Carpentier, S.C., Witters, E., Laukens, K., Deckers, P., (2005) Proteomics, 5, pp. 2497-2507Silva, S.D., Luz, E.D.M.N., Almeida, O.C., Gramacho, K., Bezerra, J.L., (2002) Agrotropica, 1, pp. 1-23Dannel, F., Pfeffer, H., Marschner, H., (1995) J. Plant Physiol, 146, pp. 273-278Micheli, F., Sundberg, B., Goldberg, R., Richard, L., (2000) Plant Physiol, 124, pp. 191-199Laemmli, U.K., (1970) Nature, 227, pp. 680-485Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W., (1988) Electrophoresis, 9, pp. 255-262Ramírez, M.G., Avelizapa, L.I.R., Avelizapa, N.G.R., Camarillo, R.C., (2004) J. Microbiol. Methods, 56, pp. 213-219Lopes, M.A., Gomes, D.S., Koblitz, M.G.B., Pirovani, C.P., (2008) Mycol. Res, 112, pp. 399-406Eletroforese de Isoenzimas a Proteinas Afins (1998) Fundamentos a Aplicações em Plantas a Microrganismos, Editora UFV, , Alfenas, A. C, Dusi, A, Zerbini, F. M, Jr, Robinson, I. P. et al, Eds, Universidade Federal de ViçosaWang, Y.T., Yang, C.Y., Chen, Y.T., Lin, Y., Shaw, J.F., (2004) Plant Physiol. Biochem, 42, pp. 663-670Distefano, S., Palma, J.M., Gomez, M., Del Rio, L.A., (1997) Biochem. J, 327, pp. 399-405Braren, O., Liberei, R., Müller, M.W., Machado, R.C.R., (1993) Proceedings of the 11th International Cocoa Research Conference, pp. 859-863. , 18-24 July, Yamoussoukro, Côte d'lvoire. Cocoa Producers' Alliance, Lagos, NigeriaHartung, W.J., Radin, W., Hendrix, D.L., (1988) Plant Physiol, 86, pp. 908-913Bersntein, L., (1971) Plant Physiol, 47, pp. 361-365Long, J.M., Widders, I.E., (1990) Plant Physiol, 94, pp. 1040-1047Behling, J.P., Gabelman, W.H., Gerloff, G.C., (1989) Plant Soil, 113, pp. 189-196Rohringer, R., Ebrahim-Nesbat, F., Wolf, G., (1983) J. Exp. Bot, 34, pp. 1589-1605Pfanz, H., Oppman,. B., in: Lobarzewski, J., Greppin, H., Penel, C., Gaspar, T (Eds.), Biochemical, Molecular and Physiological Aspects of Plant Peroxidases, University of Geneva, Switzerland 1991, pp. 400-417Mühling, K.H., Sattelmacher, B., (1995) J. Plant Physiol, 147, pp. 81-86Lohaus, G., Pennewiss, K., Sattelmacher, B., Hussmann, M., Muehling, K.H., (2001) Physiol. Plantarum, 111, pp. 457-465Abo-Hamed, S., Collin, H.A., Hardwick, K., (2001) New Phytol, 95, pp. 9-17Terry, M.E., Bonner, B.A., (1980) Plant Physiol, 66, pp. 321-325Li, Z.C., McClure, J.W., Hagerman, A.E., (1989) Plant Physiol, 90, pp. 185-190Nikolic, M., Römheld, V., (2002) Plant Soil, 274, pp. 67-74Tang, S., Hettiarachchy, N.S., Shellhammer, T.H., (2002) J. Agric. Food Chem, 50, pp. 7444-7448Schuster, A.M., Davies, E., (1983) Plant Physiol, 73, pp. 809-816Kasprzewska, A., (2003) Cell Mol. Biol. Lett, 8, pp. 809-824Passardi, F., Cosio, C., Penel, C., Dunand, C., (2005) Plant Cell Rep, 24, pp. 255-265van der Hoorn, R.A.L., Jones, J.D.G., (2004) Curr. Opin. Plant Biol, 7, pp. 400-407Barett, A.J., (2004) Methods Enzymol, 244, pp. 1-1