Orientadores: Anete Pereira de Souza, Antonio Augusto Franco Garcia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:06:27Z (GMT). No. of bitstreams: 1 Mantello_CamilaCampos_D.pdf: 14575904 bytes, checksum: 3588e88f982780fe09718e2b837d7824 (MD5) Previous issue date: 2014 / Resumo: Aproximadamente 2.500 espécies são conhecidas por produzirem borracha natural e a seringueira, [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg.], espécie nativa da Amazônia e pertencente ao gênero Hevea, é a maior fonte de borracha natural do mundo. A borracha natural é matéria prima para fabricação de mais de 40.000 produtos tendo importância fundamental na indústria de pneus. Apesar de a região Amazônica oferecer condições climáticas adequadas para seu crescimento e desenvolvimento, esta área também possui condições favoráveis à ocorrência do mal-das-folhas (Microcyclus ulei P. Henn v. Arx), doença também conhecida como mal sulamericano das folhas (South American Leaf Bligth ¿ SALB). Dessa maneira, a heveicultura se expandiu para áreas de escape que propiciam novas condições de estresse, limitando o seu crescimento e a produção de látex. O melhoramento genético vem buscando cultivares adaptados a estas regiões de escape, porém o ciclo de melhoramento da seringueira é longo e não permite o rápido desenvolvimento de novos cultivares. O desenvolvimento de ferramentas na biologia molecular permite o melhor entendimento da espécie e pode diminuir o tempo gasto nos ensaios de campo. O presente trabalho desenvolveu novos marcadores microssatélites (SSRs) a partir de bibliotecas enriquecidas em microssatélites. A caracterização destes marcadores mostrou a alta variabilidade alélica dentro de H. brasiliensis e o teste de transferibilidade dos SSRs em outras seis espécies do gênero Hevea mostrou alelos exclusivos para as mesmas e taxas de amplificação superior a 80%. Com o objetivo desenvolver novos marcadores SSRs e single nucleotide polymorphisms (SNPs) em larga escala, foi sequenciado na plataforma Illumina GAIIx o transcriptoma de painel de dois cultivares importantes para a heveicultura (GT1 e PR255). A montagem e a caracterização do transcriptoma permitiu o melhor entendimento da dinâmica do transcriptoma em H. brasiliensis e identificou novos transcritos para a espécie. As sequências do transcriptoma foram submetidas à busca de SSRs e SNPs. No transcriptoma, foram identificados 1.709 novas sequências contendo SSRs para seringueira, a uma frequência de um SSR a cada 2,8 kb. Já a busca de SNPs identificou 404.114 SNPs com frequência de um SNP a cada 125 pb. Através da anotação no Kyoto Encyclopedia of Genes and Genomes (KEGG), foram identificadas sequências anotadas a todas as enzimas referentes às duas vias de síntese de látex (mevalonato - MVA e C-metileritritol 4-fosfato -MEP). Apesar de as vias MVA e MEP serem muitos estudadas, esta foi a primeira vez que SNPs foram identificados e validados. Os marcadores SSRs e SNPs foram então mapeados em uma população segregante F1. O mapa genético obtido contém 383 marcadores mapeados em 20 grupos de ligação. Neste trabalho foram desenvolvidos 52 SSRs e 51 SNPs do total de marcadores mapeados. Como o número de grupos de ligação esperado é 18 (2n=36), conclui-se que o mapa genético obtido mostra que ainda há uma cobertura incompleta do genoma. Devido à alta frequência de SNPs no genoma, o desenvolvimento de novos marcadores poderá saturar o mapa de forma homogênea, permitindo o agrupamento dos marcadores nos 18 grupos de ligação esperados / Abstract: Approximately 2.500 species are known to produce natural rubber. Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg. also known as rubber tree is a species native to the Amazon rainforest and is the largest source of natural rubber in the world. Natural rubber has been used in more than 40,000 products and has great importance in the tire industry. Although the Amazon rainforest offers optimal conditions for growth and rubber yields due to its warm and humid climate, this region also provides optimal conditions for the fungus Microcyclus ulei P. Henn v. Arx which causes the South American Leaf Blight (SALB) disease. Thus, rubber tree plantations have expanded to escape areas that provides new stress conditions limiting their growth and latex production. The rubber tree breeding is trying to create a new cultivar that is resistant to these new conditions but the rubber tree cycle breeding is long and does not allow a rapid cultivar development. Thus, molecular biology techniques could provide a greater knowledge of H. brasiliensis genetic and could optimize field evaluation and, thus, reduce the time and area required for experiments. The present work developed new microsatellites (SSRs) markers for rubber tree from genomic enriched libraries. The new microsatellites were characterized and demonstrated a high allelic variability within H. brasiliensis genotypes. The transferability rate in other six species of the genus Hevea was greater than 80%. To develop new SSRs and single nucleotide polymorphisms (SNPs) markers, the panel transcriptome from two important cultivars (GT1 and PR255) was sequenced in Illumina GAIIx platform. The transcriptome obtained allowed a better knowledge about H. brasiliensis transcriptome and identified new transcripts for rubber tree public database. The sequences were submitted to a SSR and SNP search. The SSR frequency was one SSR each 2.8 kb and it was identified 1.709 new sequences with new SSRs for rubber tree database. A total of 404.114 putative SNPs were detected with a frequency of one SNP every 125 bp. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, it was identified contigs corresponding to all the enzymes of mevalonate (MVA) and -C-methyl-D-erythritol 4-phosphate (MEP). Despite MVA and MEP pathways are being very well studied, since they are directly involved to rubber biosynthesis, this is the first time that molecular markers have been developed for such important pathways. The SSRs and SNPs developed were mapped in a full-sib population. The genetic linkage map has 383 molecular markers distributed in 20 linkage groups. This project contributed with 52 SSRs and 51 SNPs of the total mapped markers. Although the expected number of linkage groups are 18 (2n=36), the new genetic linkage map still has an incomplete coverage of the genome. Due to the high frequency of the SNPs in the genome, the development of new markers can saturate this map homogeneously / Doutorado / Genetica Vegetal e Melhoramento / Doutora em Genética e Biologia Molecular

Orientadores: Gonçalo Amarante Guimarães Pereira, Jorge Maurício Costa Mondego / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T22:21:16Z (GMT). No. of bitstreams: 1 Teixeira_PauloJosePereiraLima_D.pdf: 96794209 bytes, checksum: ecb31f47caf999afd03d8b9da31315f5 (MD5) Previous issue date: 2013 / Resumo: O cacaueiro se destaca como uma das principais culturas perenes na agricultura, sendo economicamente relevante por fornecer a matéria prima para a fabricação do chocolate, um produto que movimenta bilhões de dólares no mercado mundial a cada ano. Apesar de sua importância, o cacaueiro é drasticamente atacado por diversas doenças que diminuem sua produtividade e reduzem a qualidade das amêndoas do cacau. Dentre estas, a vassoura de bruxa, causada pelo basidiomiceto Moniliophthora perniciosa, é um importante fator limitante da produção cacaueira nas Américas. Utilizando tecnologias de sequenciamento de DNA de nova geração, realizamos uma abrangente análise transcriptômica da vassoura de bruxa neste trabalho. Um banco de dados denominado Atlas Transcriptômico da Vassoura de Bruxa foi construído, o qual compreende aproximadamente 60 bibliotecas de RNA-seq representativas dos mais variados estágios de desenvolvimento, condições de crescimento e respostas a estresse do fungo M. perniciosa sob condições in vitro e in planta. O primeiro capítulo desta tese apresenta uma análise global do Atlas Transcriptômico da vassoura de bruxa. Este conjunto de dados tem suportado uma série de estudos específicos relacionados a variados aspectos da doença, os quais são apresentados e detalhados nos demais capítulos da tese. Notavelmente, uma análise detalhada da interação biotrófica entre o cacaueiro e o fungo M. perniciosa (Capítulo II) revelou a ocorrência de intensa reprogramação transcricional e importantes alterações fisiológicas em plantas infectadas, incluindo a ativação de respostas de defesa ineficientes e a ocorrência de privação de carbono. Curiosamente, um processo de senescência prematura se estabelece no tecido infectado e parece ser um evento central no desenvolvimento da doença, possivelmente disparando o início da fase necrotrófica desta interação planta-patógeno. Ainda, nossos dados também permitiram a identificação de potenciais efetores de virulência em M. perniciosa, como também a caracterização do status metabólico do fungo durante a infecção do cacaueiro. Um modelo detalhado que sumariza os aspectos moleculares da vassoura de bruxa foi elaborado. De maneira geral, o Atlas Transcriptômico da Vassoura de Bruxa representa um importante avanço no estudo desta doença e tem servido como ponto de partida para uma série de estudos adicionais. A utilização destes dados na identificação e caracterização de potenciais fatores de patogenicidade de M. perniciosa (Capítulos III, IV e VI) e de mecanismos de defesa do cacaueiro (Capítulo V) também é apresentada nesta tese / Abstract: Cacao stands out as one of the major perennial crops in the world, being economically relevant as the source of chocolate, a multi-billion dollar product appreciated worldwide. Despite its importance, cacao is seriously affected by several diseases that reduce crop yield and decrease the quality of cocoa beans. Among them, the witches' broom disease (WBD), caused by the basidiomycete Moniliophthora perniciosa, is a major constraint for cacao production in the Americas. Using next generation sequencing technologies, a comprehensive transcriptomic analysis of WBD was performed. We developed a database named "WBD Transcriptome Atlas", which comprises approximately 60 RNA-seq libraries that represent a wide range of developmental stages, growth conditions and stress responses of the fungus, either under in vitro or in planta conditions. The first chapter of this thesis presents a global analysis of the WBD Transcriptome Atlas. This data set has supported a number of specific analyses related to several aspects of WBD, which are presented and detailed in the other chapters of the thesis. Strikingly, a detailed analysis of the biotrophic interaction between M. perniciosa and cacao (Chapter I) revealed the occurrence of intense transcriptional reprogramming and remarkable physiological alterations in infected plants, including the activation of ineffective defense responses and the occurrence of carbon deprivation. Curiously, a premature senescence process is established in infected tissues and appears to be a central event in WBD, possibly triggering the onset of the necrotrophic stage of this plant-pathogen interaction. Additionally, our data also allowed the identification of potential virulence effectors in M. perniciosa, as well as the characterization of the metabolic status of the fungus during cacao infection. A detailed model summarizing the molecular aspects of WBD is presented. Overall, the WBD Transcriptome Atlas represents an important advance in the study of this disease and constitutes a starting point for a number of additional studies. The use of these data in the identification and characterization of potential pathogenicity factors of M. perniciosa (Chapters III, IV and VI) and defense mechanisms of cacao (Chapter V) will also be presented in this thesis / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Orientador: Gonçalo Amarante Guimarães Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:25:00Z (GMT). No. of bitstreams: 1 Alvarez_Javier_D.pdf: 13138837 bytes, checksum: 410e11bd8ba7df37c003fae0919eec2e (MD5) Previous issue date: 2013 / Resumo: A doença vassoura de bruxa do cacaueiro, causada pelo fungo Moniliophthora perniciosa tem sido um dos maiores problemas fitopatológicos do hemisfério sul. O fungo tem uma fase biotrófica longa, pouco comum entre fungos com o estilo de vida hemibiotrófico. Este fato sugere que M. perniciosa deve dispor de importantes mecanismos de evasão do sistema de defesa da planta e que estes teriam um papel fundamental para o sucesso da infecção. Assim sendo, o objetivo deste trabalho é entender os mecanismos de supressão envolvidos na resposta de defesa basal da planta. Especificamente, aqueles mecanismos mediados por hormônios que poderiam influenciar na suscetibilidade da planta ao patógeno durante a invasão do M. perniciosa. Com este fim, foram construídas bibliotecas de RNAseq da interação cacau-M. perniciosa durante o inicio da fase assintomática da doença. As bibliotecas representantes desta interação mostraram expressão diferencial de genes relacionados com a sinalização de auxina, mas não foi observado indução de genes da biossíntese planta. Estes dados sugerem a presença de auxina "exógena" produzida provavelmente pelo patógeno. Adicionalmente, foram encontrados genes relacionados com o ácido jasmônico e etileno. Com o intuito de identificar se M. perniciosa era capaz de secretar um composto com função de auxina, sementes de Arabidopsis thaliana foram crescidas em amostras de sobrenadante do fungo. Estes experimentos mostraram alteração da morfologia das raízes semelhantes à adição de auxina exógena. Amostras de sobrenadantes do fungo foram submetidas às técnicas de RMN, LC MS/MS e GC-MS e mostraram a presença de um composto com estrutura semelhante ao indol-3-acetoácido sendo produzido pelo fungo. Estudos do time course de produção deste composto mostraram que a auxina produzida é rapidamente metabolizada formando diferentes derivados indólicos com provável função na patogênese do fungo. Finalmente, encontramos um novo composto com estrutura parecida à auxina e capacidade de indução de genes de resposta a este hormônio. Este trabalho é o primeiro relato a mostrar o time course da síntese de auxina por M. perniciosa e a indução de respostas de sinalização de auxina nos tecidos de cacau infectados pelo fungo. A intervenção nas vias de síntese de auxina pelo fungo seria um caminho plausível na tentativa por conter a doença vassoura de bruxa / Abstract: The witches' broom disease in Cacao is caused by the fungus Moniliophthora perniciosa has been one of the most important problems phytopathological in the southern hemisphere. The fungus has an unusual long biotrophic phase with the lifestyle hemibiotrofic suggesting that M. perniciosa must have important evasion mechanisms for breaking of the plant defense system and it would have a key role to success in the infection. Therefore, the aim of this work is to understand some of the mechanisms involved in the suppression of basal defense response of the plant. Specifically, hormones-mediated mechanisms that could influence plant susceptibility to pathogens during the infection of M. perniciosa. For this purpose, RNAseq libraries were constructed of the interaction cacao - M. perniciosa during the asymptomatic phase of the disease. Genes related with the auxin and jasmonic acid signaling and ethylene production were differential expressed during the initial stage of the disease. Furthermore, auxin-responsive genes were induced, but not observed induction of plant biosynthesis genes for this hormone. These data suggest presence of auxin "exogenous" probably produced by the pathogen. In order to identify whether M. perniciosa was able to secrete a compound like an auxin, Arabidopsis thaliana seedlings were grown in mediums with supernatant of the fungus. These experiments showed alterations on the roots morphology, similar to the addition of exogenous auxin. Samples of supernatants were subjected to the techniques of NMR, LC-MS/MS and GCMS and showed the presence of a compound with a similar structure of Indole-3- Acetic Acid (IAA) being produced by the fungus. A time course of the production of this compound was examinated by LC-MS/MS and showed that the auxin is produced by M. perniciosa, but is rapidly metabolized forming different indole derivatives, some of them with a probable role in the pathogenesis. Finally, we found a new compound with a structure similar to auxin and with the ability to induce gene response to this hormone / Doutorado / Genetica Vegetal / Doutor em Genetica e Biologia Molecular

Propionibacterium freudenreichii est une bactérie très utilisée par l’industrie laitière. Elle appartient aux Actinomycètes connus pour leur survie pendant de longues périodes, dans des conditions environnementales défavorables. Pour mieux comprendre ce phénomène, la caractérisation phénotypique de 8 souches de P. freudenreichii a été réalisée sur 11 jours dans un milieu en carence nutritionnelle. Le taux de survie bactérienne a été mesuré par densité optique, par énumération et évaluation de la viabilité cellulaire. En outre, l’absence de lyse cellulaire a été évaluée par PCR quantitative. La croissance de P. freudenreichii a été décrite en phases exponentielle, stationnaire, stationnaire tardive et survie à long terme.Dans nos conditions expérimentales pendant la période de survie à long terme, les bactéries sont restées viables. La caractérisation phénotypique a montré que P. freudenreichii CIRM-BIA138 était la plus résistante à la carence nutritionnelle et entrait dans un état viable mais non-cultivable. Cette souche a été utilisée pour une étude fonctionnelle par RNA-Seq ainsi que pour des analyses biochimiques sur les surnageants de culture, en phases exponentielle et stationnaire. L’association de ces données transcriptomiques et métabolomiques a permis de déduire les stratégies impliquées dans la survie de cette bactérie. La préparation à l’état de dormance, la diminution du métabolisme et l’utilisation de sources alternatives d’énergie semblent impliquées dans l’adaptation et la persistence de P. freudenreichii CIRM-BIA138 en carence nutritionnelle durant de long / Propionibacterium freudenreichii is a dairy bacterium belonging to the Actinobacteria group, which is known to survive for long periods in harsh environmental conditions. In order to investigate the long-term survival phenomenon in P. freudenreichii, 8 strains were phenotypically characterized for a period of 11 days in nutrient shortage condition. Bacterial survival rate was assessed by optical density, CFU counting and live-dead cellular viability. In addition, the absence of cell lysis was evaluated by quantitative PCR. P. freudenreichii growth phases were classified as exponential, stationary, late stationary and long-term survival. Moreover, it was observed that bacterial viability was maintained during long-term survival.Phenotypical characterization indicated that P. freudenreichii CIRM-BIA138 was more resistant to nutrient shortage being able to enter into a viable but nonculturable dormant state. In addition, functional studies of this strain were conducted by RNA-Seq on cultures sampled in exponential and stationary growth phases. Concomitantly, several biochemical analyses were carried out on the culture supernatant. An integrative approach of metabolomic and transcriptomic data allowed us to infer strategies associated with the survival of this bacterium, such as preparation for the dormant state, slow down of metabolic activity and utilization of alternative sources of energy, which altogether might allow P. freudenreichii CIRM-BIA 138 to adapt and persist through nutrient shortage for long periods.

Nous proposons de modéliser les données issues des technologies de séquençage du transcriptome (RNA-Seq) à l'aide de la loi binomiale négative, et nous construisons des modèles de segmentation adaptés à leur étude à différentes échelles biologiques, dans le contexte où ces technologies sont devenues un outil précieux pour l'annotation de génome, l'analyse de l'expression des gènes, et la détection de nouveaux transcrits. Nous développons un algorithme de segmentation rapide pour analyser des séries à l'échelle du chromosome, et nous proposons deux méthodes pour l'estimation du nombre de segments, directement lié au nombre de gènes exprimés dans la cellule, qu'ils soient précédemment annotés ou détectés à cette même occasion. L'objectif d'annotation précise des gènes, et plus particulièrement de comparaison des sites de début et fin de transcription entre individus, nous amène naturellement à nous intéresser à la comparaison des localisations de ruptures dans des séries indépendantes. Nous construisons ainsi dans un cadre de segmentation bayésienne des outils de réponse à nos questions pour lesquels nous sommes capable de fournir des mesures d'incertitude. Nous illustrons nos modèles, tous implémentés dans des packages R, sur des données RNA-Seq provenant d'expériences sur la levure, et montrons par exemple que les frontières des introns sont conservées entre conditions tandis que les débuts et fin de transcriptions sont soumis à l'épissage différentiel. / We propose to model the output of transcriptome sequencing technologies (RNA-Seq) using the negative binomial distribution, as well as build segmentation models suited to their study at different biological scales, in the context of these technologies becoming a valuable tool for genome annotation, gene expression analysis, and new-transcript discovery. We develop a fast segmentation algorithm to analyze whole chromosomes series, and we propose two methods for estimating the number of segments, a key feature related to the number of genes expressed in the cell, should they be identified from previous experiments or discovered at this occasion. Research on precise gene annotation, and in particular comparison of transcription boundaries for individuals, naturally leads us to the statistical comparison of change-points in independent series. To address our questions, we build tools, in a Bayesian segmentation framework, for which we are able to provide uncertainty measures. We illustrate our models, all implemented in R packages, on an RNA-Seq dataset from a study on yeast, and show for instance that the intron boundaries are conserved across conditions while the beginning and end of transcripts are subject to differential splicing.

Dans le domaine des plantes, la formation de la fleur a été un pas crucial dans la capacité des végétaux à coloniser une grande diversité de niches écologiques sur notre planète. Les deux espèces Petunia x hybrida et Arabidopsis thaliana représentent deux groupes majeurs des plantes à fleur. Nous avons montré que les gènes à homéodomaine d’une famille appelée WOX (Wuschel homeobOX) sont fortement impliqués dans le développement des organes dotés de polarité (dont les feuilles et des organes de la fleur : sépales, pétales, carpelles). Un double mutant (maw mawb), chez le Pétunia, développe des pétales en forme de filament, avec disparition du tube floral. De plus, nous avons découvert que ces mêmes gènes interagissent au niveau génétique avec d’autres gènes (appelés gènes à boite MADS) dans la formation des ovules, structures à partir desquelles les graines se forment. Nous avons aussi montré que des gènes de la même famille sont impliqués dans la formation d’autres structures chez le Pétunia : les trichomes ou poils aériens de surface. Ces derniers sont impliqués dans plusieurs taches, qui vont de la protection contre les pathogènes à celles contre les stress abiotiques. Grâce à des études de génétique fonctionnelle nous avons pu montrer un recrutement différentiel des gènes WOX ici étudiés, dépendant de l’organe et de l’espèce. Ces travaux de thèse montrent l’importance de cette famille génique pour les études d’evo-devo (Biologie Evolutionniste du Développement). Finalement, une analyse de RNA-Seq (séquençage du transcriptome), dévoile les réseaux génétiques contrôlés par ces gènes WOX. / In the Kingdom of Plants, the emergence of flowers was a crucial step in their ability to colonize a large variety of ecological niches on our planet. The two species Petunia x hybrida and Arabidopsis thaliana represent two major groups of flowering plants. In this work, we have shown that HOMEOBOX genes from the WOX family (Wuschel homeoboxes) are heavily involved in polar organ development (such as leaves and sepals, petals, and carpels at the flower level). The maw mawb double mutant in Petunia displays string-like petals, with consequent disappearance of the floral tube. Moreover, we found that these two genes genetically interact with genes from a different family (the MADS family) in ovule identity (ovules are the structures from which seeds develop). We have also shown that other genes from the WOX family are involved in development of a different kind of structures in Petunia: the trichomes. Trichomes are involved in different tasks, protecting the plant from pathogens or abiotic stress. Thanks to functional genetics studies, we have shown functional genetic recruitment of these WOX genes among different plant organs and among different species. This PhD thesis provides evidence for the importance of the WOX family in Evo-Devo studies. Eventually, we unravelled genetic networks controlled by MAW and MAWB trough RNA-Seq analysis.

Orientadores: Gustavo Henrique Goldman, Juliana Velasco de Castro Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:49:45Z (GMT). No. of bitstreams: 1 Borin_GustavoPagotto_M.pdf: 4025117 bytes, checksum: 5d652481786ac0f6cf4259069d181514 (MD5) Previous issue date: 2015 / Resumo: A parede celular vegetal é uma estrutura recalcitrante, composta por polissacarídeos complexos que podem ser quebrados em açúcares fermentáveis. A desconstrução desse material complexo pode ser feita por diversos tipos de enzimas hidrolíticas, que são produzidas naturalmente por uma variedade de microrganismos. Entre eles, o fungo Trichoderma reesei se destaca pela capacidade de produzir e secretar estas enzimas em grandes quantidades. Embora alguns trabalhos utilizando abordagens de proteômica e transcriptômica já tenham sido realizados com esse fungo, ainda não são conhecidos em detalhes os mecanismos moleculares responsáveis pela degradação da parede e a regulação gênica envolvida nesse sistema lignocelulolítico. O presente trabalho tem como objetivo principal a análise da expressão gênica global de T. reesei, crescido por 6, 12 e 24 horas em bagaço e colmo de cana-de-açúcar como fontes únicas de carbono, pela técnica de sequenciamento high-throughput de RNA (RNA-Seq). No transcriptoma de T. reesei foram identificadas sendo hiper-expressas as principais celulases, hemicelulases e proteínas acessórias relacionadas direta ou indiretamente com a desconstrução da parede vegetal. De modo geral, as celulases e hemicelulases apresentaram uma expressão maior do que outras enzimas, e o nível dos seus transcritos foi crescente ao longo do tempo tanto em colmo quanto no bagaço. A grande maioria dos genes de CAZymes e proteínas acessórias hiper-expressos foram compartilhados pelos dois substratos, o que demonstra que a estratégia usada por T. reesei para degradar a parede celular do colmo e do bagaço é similar. Adicionalmente, vários fatores de transcrição, proteínas de função desconhecida e transportadores supostamente envolvidos na assimilação dos açúcares liberados também foram hiper-expressos nas condições amostradas. Para validação do RNA-Seq, foi realizado PCR em tempo real de diversos genes hiper-expressos que codificam para enzimas hidrolíticas, reguladores transcricionais, proteínas acessórias e genes ainda não caracterizados. Para isso, a análise temporal foi ampliada para 30 minutos, 2, 4, 6, 12 e 24 horas de crescimento após o inóculo, o que permitiu uma análise mais detalhada da expressão desses genes. Como objetivo secundário, foi analisado o secretoma deste fungo e os açúcares concomitantemente liberados no sobrenadante. Estas análises indicaram que a desconstrução da parede celular já se inicia dentro de 6 horas pós inoculo, com a liberação de monômeros (principalmente xilose e glicose) dos polissacarídeos e secreção de diversas CAZymes. Ensaios enzimáticos também foram realizados, mostrando atividades celulo e hemicelulolíticas. Assim, descrevemos pela primeira vez o arsenal de enzimas transcritas e secretadas por T. reesei RUT C30, desde pontos inicias de crescimento, em bagaço explodido e colmo de cana-de-açúcar. Por fim, este trabalho também permitiu a identificação de vários genes, com função predita ou não, que podem abrir caminho para a descoberta de novos atuantes na resposta do fungo ao substrato lignocelulósico / Abstract: Plant cell wall is a recalcitrant structure composed of complex polysaccharides which can be broken down into fermentable sugars. The deconstruction of this complex material can be made by a variety of hydrolytic enzymes which are naturally produced by a variety of microorganisms. Among them, stands out the fungus Trichoderma reesei, able to produce and secrete those enzymes in large quantities. Although some studies using transcriptomics and proteomics approaches have been performed with this fungus, the molecular mechanisms responsible for the degradation of the cell wall and gene regulation involved in this lignocellulosic system are not well known. This work has as main objective the analysis of global gene expression of T. reesei grown at 6, 12 and 24 hours in sugarcane bagasse and culm as sole carbon sources by high-throughput RNA sequencing technology (RNA-Seq). In the T. reesei transcriptome, it was identified the major cellulases, hemicellulases and accessory proteins directly or indirectly related to the deconstruction of plant cell wall. In general, cellulases and hemicellulases exhibited higher expression than other enzymes, and the level of their transcripts was increased over the time in both culm and bagasse cultures. Most of up-regulated CAZymes and accessory proteins were shared between the two substrates, which demonstrates the strategy used by T. reesei to degrade the bagasse and culm cell wall is similar. Furthermore, several transcription factors, proteins of unknown function and transporters supposedly involved in the assimilation of sugars were also up-regulated in the sampled conditions. To validate the RNA-Seq data, real-time PCR of several up-regulated genes encoding hydrolytic enzymes, transcriptional regulators, accessory proteins and proteins not yet characterized was carried out. The time points was extended to 30 min, 2, 4, 6, 12 and 24 hours of growth after inoculation, allowing a more detailed analysis of the expression of these genes. As a secondary objective, T. reesei secretome and the sugars released in the supernatant were analyzed. It was shown that the sugarcane cell wall deconstruction begins within the first 6 hours post inoculation, releasing sugar monomers (mainly xylose and glucose) from polysaccharides due to the secretion of several hydrolytic enzymes. Enzymatic assays were also performed, showing cellulosic and hemicellulosic activities. Finally, this is the first study showing the arsenal of enzymes transcribed and secreted by T. reesei grown on steam exploded sugarcane bagasse and culm, at early time points. It was possible identify several genes, with predicted function or not, that can open new paths to discover novel players on the fungus response to lignocellulosic substrate / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Les nématodes à galles (RKN) du genre Meloidogyne spp. sont des parasites obligatoires des plantes qui induisent la formation d’un site nourricier spécialisé au sein des racines. Mon projet de thèse a pour objectif d’identifier des gènes spécifiques de ces nématodes qui sont impliqués dans le parasitisme en se focalisant sur des protéines sécrétées ou effecteurs. La technologie de séquençage Illumina a été utilisée pour comparer les transcriptomes de M. incognita au cours de son cycle de vie. A partir de 307 gènes surexprimés dans -au moins- un stade du cycle de vie, nous avons sélectionné 14 candidats d’effecteurs. Des expériences de RT-qPCR, d’hybridation in situ et d’ARN interférence ont permis de confirmer le profil d’expression, de localiser l’expression des effecteurs et d’étudier leur rôle dans la pathogénicité. Ce travail a permis de démontrer le rôle important d’une petite protéine, MiSCR1, dans les stades précoces du parasitisme. Parallèlement, nous avons réalisé l’assemblage de novo du transcriptome de M. enterolobii, qui représente une nouvelle menace pour l’agriculture mondiale du fait de sa capacité à se reproduire sur la majorité des plantes résistantes aux autres RKN. Les premières comparaisons avec d'autres RKN nous ont permis d'identifier, non seulement des effecteurs en commun, mais aussi ceux qui sont spécifiques à certaines espèces de RKN et qui pourraient expliquer des différences de gamme d'hôtes. En conclusion, les analyses de transcriptomes de RKN ont permis de caractériser des nouveaux effecteurs candidats impliqués dans la pathogénicité, et d’apporter de nouvelles connaissances pour le développement de méthodes de lutte contre ces bioagresseurs / Root-knot nematodes (RKN) are obligate endoparasites that maintain a biotrophic relationship with their hosts inducing specialized feeding cells. My PhD project aims to identify RKN genes specifically involved in plant parasitism with an emphasis on genes encoding new secreted proteins, named effectors. Using Illumina RNA-seq technologies, we compared transcriptomes of Meloidogyne incognita during its life cycle. From 307 genes over-expressed at -at least- one stage of the life cycle, we selected 14 effector candidates. RT-qPCR, in situ hybridisation and siRNA soaking experiments were carried out to confirm their expression profile, localize the spatial expression of these candidates in the nematode and to study their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 gene and its paralogues resulted in a significant, reproducible decrease in the number of egg masses, demonstrating a potentially important role for the small cysteine-rich effector, MiSCR1, it encodes in early stages of giant cell formation. In parallel, we perform a de novo assembly of M. enterolobii transcriptome. This RKN species represents a new threat for the agriculture worldwide because of its ability to reproduce on the majority of known RKN-resistant plants. First comparisons with others RKN allowed us to identify, not only the common set of effectors, but also those specific to some RKN species and possibly involved in host range differences. In conclusion, the transcriptome profiling of RKNs allowed the characterisation of new candidate effectors involved in the plant pathogenicity, and provided a better knowledge for the development of new methods to control these pests

The scope and application of high throughput techniques has expanded from studying a single genome, transcriptome or proteome to understanding complex environments at a greater resolution with the help of novel computational frameworks. Comprehensive structural annotation i.e. description of all functional elements in the genome, is required for measuring genome response accurately, using high throughput methods. Annotation of genome sequences using high throughput data from RNA-seq and proteomics experiments complement computational methods for identifying functional elements and can help validate existing in silico annotation, correct annotation errors, and could potentially identify novel functional elements. Re-annotation studies in recent times have revealed shortcomings of automated methods and the necessity to validate existing annotations using experimental data. This dissertation elucidates re-annotation of Mannheimia haemolytica, Pasteurella multocida and Histophilus somni, bacterial pathogens associated with bovine respiratory disease in cattle. Experimental re-annotation of these bacterial genomes using RNA-seq and proteomics enabled the validation of existing annotation and discovery of novel functional elements that can be utilized in future functional genomics studies. We also addressed the need for developing an automated bioinformatics workflow that is broadly applicable for bacterial genome re-annotation, by developing open source Perl pipeline that can use RNA-seq and proteomics data as input. Simultaneous analysis of host and pathogen gene expression profiling using metatranscriptomics approaches is necessary to improve our understanding of infectious diseases. Traditional methods for analysis of RNA-seq data do not address the impact of cross-mapping of reads to multiple genomes for data originating from a metatranscriptomic study. Analysis of sequence conservation between species can help determine a metric for cross mapping to correct for signal vs. noise. We generated artificial RNA-seq data and evaluated the impact of read length and sequence conservation on cross-mapping. Comparative genomics was used to identify a core and pan-genome for quantifying gene expression. Our results show that cross mapping between genomes can directly be related to evolutionary distance between these genomes and that an increase in RNA-seq read length tends to negate cross mapping.

A biomassa do bagaço da cana-de-açúcar é composta de material lignocelulósico, principalmente celulose e hemicelulose, os quais são constituídos por açúcares de alta energia que podem ser convertidos a etanol. No entanto, a associação recalcitrante dessa biomassa impõe um grande desafio para a produção de biocombustíveis de segunda geração, devido à dificuldade em recuperar esses açúcares sob a forma de monômeros com elevado grau de pureza. Na natureza, muitos microrganismos realizam a degradação da biomassa de plantas através da ação de múltiplas CAZymes. Dentre esses, os fungos filamentosos se destacam devido a sua capacidade de produzir misturas enzimáticas altamente específicas para os substratos com que se deparam e, por essa razão, são a principal fonte das enzimas utilizadas nos coquetéis enzimáticos comercializados, em especial a espécie T. reesei. Contudo, esses coquetéis precisam ser otimizados e, para tanto, é necessário investir no estudo de outros fungos, como o A. fumigatus. Apesar de patogênico, o mesmo é considerado um importante produtor de enzimas lignocelulolíticas, cujas eficiências são aumentadas devido ao efeito sinérgico entre elas. Dessa forma, uma melhor compreensão dos mecanismos utilizados por esse fungo durante a exposição a biomassas vegetais é necessária. Nesse sentido, a determinação das atividades de celulases e xilanases após diferentes tempos de incubação foi realizada nos sobrenadantes das culturas do A. fumigatus crescido em SEB e em frutose, resultando em valores 21, 59 e 271 vezes maiores para as celulases e 150, 541 e 74 vezes para as xilanases, após 24, 48 e 72 horas de cultivo, respectivamente, na presença do bagaço. Para verificar se as enzimas estavam efetivamente hidrolisando a biomassa, foi realizada a dosagem dos açúcares redutores nos sobrenadantes das culturas em SEB, tendo sido observado um aumento na concentração desses açúcares à medida que o tempo de exposição ao bagaço aumentava. Com o propósito de compreender o mecanismo envolvido na hidrólise do bagaço, foi realizada a análise transcricional do A. fumigatus por RNA-seq, quando esse fungo foi crescido na presença desse substrato, assim como em frutose. A análise dos dados revelou 144 CAZymes induzidas em SEB, frente a 65 reprimidas nessa biomassa. Dentre os genes induzidos, foram identificados muitos potencialmente envolvidos na desconstrução da lignocelulose, como aqueles que codificam endoglucanases, celobiohidrolases, glucosidades, xilanases, xilosidases, manosidases, LPMOs, pectinases, entre outros. Para verificar se essas proteínas estavam sendo secretadas pelo fungo, o secretoma do mesmo foi analisado por espectrometria de massas LC/MS. Com isso, identificou-se uma gama muito maior de proteínas na presença de SEB (130) em comparação ao crescimento em frutose (44), sendo a maioria CAZymes (59%). Todos esses resultados evidenciam o potencial do A. fumigatus na hidrólise do bagaço de cana-de-açúcar, podendo suas enzimas contribuir para a produção de coquetéis enzimáticos mais eficientes e, consequentemente, para a produção de etanol de segunda geração / Sugarcane bagasse biomass is composed of lignocellulosic material, mainly cellulose and hemicellulose, which are composed of high energy sugars that can be converted into ethanol. However, the recalcitrant association of this biomass imposes a major challenge for the production of second-generation biofuels, due to the difficulty in recovering these sugars in the form of monomers with high purity. In nature, many microorganisms perform the degradation of plant biomass through the action of multiple CAZymes. Among them, filamentous fungi stand out for their ability to produce highly specific enzymatic mixtures for the substrates they are in the presence of and, therefore, they are the main source of enzymes used in commercialized enzymatic cocktails, especially T. reesei. However, these cocktails need to be optimized and, for this reason, it is necessary to invest in the study of other fungi, such as the A. fumigatus. Although pathogenic, it is considered an important producer of lignocellulolytic enzymes, whose efficiencies are increased due to the synergistic effect between them. Thus, a better understanding about the mechanisms used by this fungus during exposure to plant biomass is necessary. In this sense, the determination of cellulases and xylanases activities after different incubation times was performed after collection of supernatants from A. fumigatus grown in SEB and fructose cultures, resulting in 21, 59 and 271-fold higher values for cellulases, and 150, 541 and 74-fold higher values for xylanases, after 24, 48 and 72 hours of cultivation, respectively, in presence of the bagasse. To verify if the enzymes were effectively hydrolyzing the biomass, the supernatants from SEB cultures were collected and the reducing sugars were quantified. An increase in the concentration of these sugars was observed as the exposure time to the bagasse increased. In order to understand the mechanism involved in bagasse hydrolysis, the transcriptional analysis of A. fumigatus grown in the presence of this substrate and in fructose was performed by RNA-seq. Data analysis revealed 144 CAZymes induced by SEB, compared to 65 repressed by this biomass. Among the induced genes, many potentially involved in the lignocellulose deconstruction, such as those encoding for endoglucanases, cellobiohydrolases, glucosidases, xylanases, xylosidases, mannosidases, LPMOs, pectinases, among others, were identified. To verify if these proteins were being secreted, the secretome of the fungus was analyzed by LC/MS mass spectrometry. Hence, a much larger range of proteins was identified in the presence of SEB (130) as compared to growth in fructose (44), most of them being CAZymes (59%). All these results show the potential of A. fumigatus in the hydrolysis of sugarcane bagasse and its enzymes can contribute to the production of more efficient enzymatic cocktails and, consequently, to the production of second-generation ethanol