Phytopathogenic Dickeya species inflict large economic losses on a variety of crops. A lack of effective chemical control methods has generated interest in the use of bacteriophages (phages) as a novel tool for biocontrol. In the last decade, six phages have been isolated in Belgium and Poland using Dickeya solani as the host. Previous work in this laboratory has isolated ninety phages capable of infecting D. solani. The majority have been morphologically classified as members of the Ackermannviridae family. In agreement with findings in Salmonella and Klebsiella species, the capsule of D. solani is a likely receptor of Ackermannviridae family phages. Analysis of D. solani strains carrying reporter fusions suggested that the capsule genes are expressed in response to nutritional stress, however disruption of the capsular polysaccharide cluster did not significantly impact virulence. Experiments assessing capsular polysaccharide as a putative receptor for Ackermannviridae family phages in nosocomial pathogen Serratia produced inconclusive results. Phageresistance due to random transposon mutagenesis identified genes encoding transcription factors and regulators, but none directly linked to capsular polysaccharide production. Thirteen phages were capable of infecting a wider host range of Dickeya species. Morphological and genomic analysis showed that six were Podoviridae family members, whilst the other seven were Myoviridae family members. These are part of the recently defined 'hairy Myoviridae', characterised by a distinct morphology. Another member of this grouping was isolated during this study, but is more closely related to phages of Erwinia amylovora. A subset of the Ackermannviridae family phages were shown to be capable of facilitating transduction. This makes them unsuitable for use in the environment due to the risk of deleterious horizontal gene transfer. This is also true for the Myoviridae family members, but not for one of the Podoviridae family members. This phage could therefore be a promising candidate for therapeutic use.

Les cytochalasines, métabolites secondaires issus de PKS-NRPS fongiques, représentent une large famille de composés bioactifs intéressants de par leur système tricyclique. Ce document expose les stratégies de synthèse vers l’obtention de cytochalasines de petite taille, les périconiasines A-C, isolées de Periconia sp. F-31, qui partagent un système 9/6/5 et des cytotoxicités intéressantes envers l’adénocarcinome du colon, et la synthèse totale de la périconiasine G qui possède le plus petit macrocycle rencontré jusqu’à présent dans cette famille avec un système 7/6/5. Plusieurs analogues du produit naturel dont l’bis-iso-périconiasine C qui se caractérise par une isomérisation des alcènes initiaux sont décrits, ainsi que leurs tests sur des cellules HeLa et MDA ce qui a permis l’identification d’un composé hautement cytotoxique. D’autre part, la synthèse totale de la périconiasine G a conduit à réviser la structure initialement attribuée par Dai et al. et réaliser des tests biologiques pertinents sur les bactéries gram-négatives Micrococcus luteus et Staphylococcus aureus ainsi que sur le phytopathogène Botrytis cinerea responsable de la « pourriture grise » des cultures à travers le monde. Une spécificité de la périconiasine G contre le phytopathogène a pu être découverte ce qui amène à considérer le rôle protecteur des endophytes dans le mutualisme plante-champignon. / Cytochalasins are secondary metabolites born from fungal PKS-NRPS which possess a characteristic tricyclic core and display a large variety of biological properties. This document exposes the strategies elaborated toward total syntheses of small cytochalasins periconiasins A-C, isolated from Periconia sp. F-31 and sharing a 9/6/5 backbone, and periconiasin G which possesses the smallest macrocyclic ring so far in this family with a 7/6/5 tricyclic ring. Several analogues of natural product periconiasin C, including isomer bis-iso-périconiasin C, are described, as well as their bioassays on HeLa and MDA cell lines which allowed identification of a highly toxic lead. Moreover, the total synthesis of periconiasin G led us to the revise the structure published by Dai et al. and carry out pertinent bioassays on gram-positives bacteria Micrococcus luteus and Staphylococcus aureus, and on the phytopathogen Botrytis cinerea responsible of the gray mold disease throughout the world. A specificity of periconiasin G against the phytopathogen was discovered, leading us to consider the protective role of endophytes in the plant-fungus mutualism.

Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes the disease Stewart's wilt in corn. The insect vector Chaetocnema pulicaria, the corn flea beetle, transmits P. stewartii into corn plants through wounds in the leaves. The bacteria can then move to the xylem of the plant where they form a biofilm that inhibits the flow of water. A previous in planta RNA-Seq study resulted in the selection of lrp as a gene of interest for further analyses. A reverse genetics approach was used for the creation of a strain containing the in-frame deletion of lrp, as well as a revertant strain. The strain with the deletion of the lrp gene showed reduced motility and capsule formation when in vitro assays were conducted. It has previously been demonstrated that these characteristics are both important for the bacteria's ability to form a biofilm in the xylem of corn plants and produce disease symptoms. The in planta virulence and competition assays demonstrated that the lrp gene deletion also results in reduced disease symptoms in infected corn plants, as well as an inability to outcompete wildtype P. stewartii in xylem colonization. In a bioinformatics approach, the transcriptional regulator Lrp of P. stewartii was present in the same node of the phylogeny as homologues from other closely related phytopathogens. This demonstrates that Lrp from P. stewartii and such homologues have evolved from a recent common ancestral gene. Examining the genomic islands present in P. stewartii, it is possible to begin to predict where some of the genes which have functions involved in plant colonization may have originated. Overall, the results collected from the studies in this thesis contribute to improving understanding of how P. stewartii is successful at colonizing the xylem of corn plants and cause disease. This research could result in the development of methods to decrease crop susceptibility to infection with P. stewartii. / Master of Science / Stewart's wilt is a disease of corn plants caused by the bacterium Pantoea stewartii subsp. stewartii via the insect vector Chaetocnema pulicaria, the corn flea beetle. This infection has proven to be costly as it impacts the health of corn crops and impedes the export of corn seeds from varieties that are susceptible to infection by P. stewartii. The focus of the research conducted for this thesis has been on learning more about how specific P. stewartii genes impact the ability of the bacterium to colonize corn plants and cause Stewart's wilt disease symptoms. The information collected from this study is important for developing a better understanding of how wilt disease-causing pathogens are able to successfully infect plants, as well as for developing future treatments to prevent further infection of corn plants. In addition, preliminary bioinformatics work has shown that some of the P. stewartii genes of interest share a common ancestor with select genes from other known plant pathogens. Additional preliminary bioinformatics work on regions of the DNA called genomic islands has revealed where some genes of importance to the bacterium's ability to colonize plants may have originated. Overall, the work presented in this thesis contributes to improving our understanding of the roles that different parts of the P. stewartii genome have in allowing the bacterium to successfully colonize and cause disease in corn plants.

A cultura do eucalipto apresenta grande importância no setor florestal no mundo. No Brasil, 70% da área florestal plantada é destinada ao eucalipto. Entretanto, a ferrugem das mirtáceas, também conhecida como ferrugem do eucalipto, causada pelo fungo Puccinia psidii Winter, afeta o enorme potencial produtivo das plantações de eucalipto. A biologia, mecanismos de patogenicidade e genética desse patógeno são pouco conhecidos, apesar de sua importância para o setor florestal. Os elementos transponíveis (TEs) são sequências de DNA com a capacidade de migrar e influenciar a organização, integridade e evolução do genoma hospedeiro. O presente trabalho teve como principal objetivo estudar os TEs presentes no genoma de P. psidii, combinando ferramentas in silico e moleculares. A classificação dos elementos transponíveis no genoma de P. psidii MF-1 foi realizada utilizando contigs previamente minerados e remontados, bem como sem seleção prévia dos contigs, por meio do programa RepeatMasker. Ambas estratégias apontaram o predomínio de elementos da Classe I - LTR Retrotransposons no genoma de P. psidii MF-1. O resultado condiz com a composição de TEs em fungos fitopatogênicos descrita na literatura. Algumas análises in silico, como verificação de integridade e anotação manual de sequências proteicas foram também realizadas para alguns contigs classificados como TEs. Assim, foi possível observar a presença de sequências conservadas pertencentes à região pol em LTR Retrotransposons. Além disso, as análises permitiram inferir sobre a existência de TEs híbridos no genoma parcialmente sequenciado de P. psidii MF-1. Paralelamente foi também realizada uma análise comparativa entre os TEs presentes nos genomas de P. graminis, P. striiformis, P. triticina e P. psidii. Observou-se que P. graminis, P. striiformis e P. triticina apresentam maior frequência de elementos da Classe II, do tipo DNA Transposons ao contrário de P. psidii, com maior frequência de elementos da Classe I. Interessantemente, a quantidade de elementos desconhecidos foi similarmente alta para todos os quatro genomas avaliados. Este tipo de análise é muito importante, pois evidencia a grande quantidade de famílias de TEs novas a serem descobertas. Elas podem estar potencialmente relacionadas ao silenciamento de genes importantes à virulência destes patógenos. A utilização de TEs no estudo de diversidade genética entre populações é bastante comum. A técnica molecular IRAP foi utilizada para acessar a diversidade entre populações de P. psidii originárias de três híbridos de Eucalyptus spp., goiabeira, jambeiro e jabuticabeira. No entanto, esta técnica não se mostrou eficiente para detectar polimorfismos existentes entre estas populações. A anotação de TEs foi difícil devido à observação de sequências de elementos sobrepostas, o que podem representar híbridos de TEs, entretanto, visando a confirmação desta hipótese por meio da PCR, alguns contigs serão sequenciados e mais estudos devem ser realizados para a continuação desta confirmação. Os resultados apresentados neste trabalho são inéditos e representam uma etapa crucial no entendimento de TEs em fungos do gênero Puccinia, em especial do patógeno P. psidii para o desenvolvimento de melhores mecanismos de controle de ferrugem. / The culture of eucalyptus has great importance worldwide in forestry sector. In Brazil, 70% of cultivated forest area is intended for Eucalyptus. However, the eucalyptus potential productive has been affected by rust disease, caused by the fungus Puccinia psidii Winter. Despite its importance to brazilian and world forest sector, the knowledge of biology, genetic and pathogenic mechanisms of this pathogen is scarce. Transposable elements (TEs) are mobile DNA fragments that influence the organization and development of the host genome. These elements have the ability to move within host genome, and their insertion can cause a wide spectrum of mutations in their hosts. This study aims to decipher the TEs in P. psidii genome by combining in silico and molecular tools. P. psidii MF-1 TEs classification was performed automatically, through RepeatMasker software, being observed a predominance of Class I - LTR Retrotransposons in P. psidii MF-1 genome. This result is consistent with the TEs composition described in phytopathogenic fungi. Some in silico analysis, as integrity and manual annotation of conserved protein sequences from TEs were carried out with P. psidii MF-1 contigs classified as transposable elements. The presence of conserved sequences belonging to pol region in LTR Retrotransposons was observed. Furthermore, these analysis allowed the inference of hybrid TEs in P. psidii MF-1. At the same time, a comparative analysis of TEs present in other Puccinia genomes and P. psidii MF-1 was also performed. The P. graminis, P. striiformis and P. triticina genomes have higher frequency of Class II - DNA Transposons unlike the results found for P. psidii. Interestingly, the number of unknown elements was similarly high for all genomes. This type of analysis is very importante because it shows a great number of potential new TEs families to be discovered. They may be potentially related to the virulence gene silencing of these pathogens. Using TEs for study the fungal genetic diversity is quite common. The IRAP technique was used to access the diversity among P. psidii populations originated from three Eucalyptus spp. hybrids, guava, syzigium and jabuticaba. However, this technique was not efficient to detect existing polymorphisms between these populations. TEs annotation was labored due to the existence of overlapping elements, which may represent hybrids TEs. PCR tool was used to confirm some sequences annotated as hybrids and more studies are needed to confirm this hyphotesis. The results presented in this study are novel and is a crucial step in understanding the genetic of P. psidii pathogen for further improvements of rust control mechanisms.

Gramíneas cultivadas, como trigo, cevada e milho são produtos agrícolas de fundamental importância no Brasil. Entre os fatores causadores de perdas na produção de grãos dessas espécies estão os estresses causados por fitopatógenos como Fusarium graminearum Schwabe (teleomorfo Gibberella zeae Schw.), agente causador da fusariose e de difícil controle químico, biológico ou mesmo genético. Uma estratégia que tem se mostrado eficiente no controle de doenças é a utilização de microrganismos antagonistas a diferentes fitopatógenos, dentre os quais destaca-se a bactéria P. agglomerans. O presente trabalho teve como objetivo identificar genes diferencialmente expressos em interações fungo fitopatogênico-microrganismo antagonista, considerando como modelo o sistema F. graminearum-P. agglomerans. A construção de uma biblioteca de cDNA de F. graminearum cultivado in vitro proporcionou a geração de 1.983 seqüências válidas, resultando em 1.283 unigenes. As categorias de maior representatividade desta biblioteca foram aquelas constituídas por proteínas envolvidas em vias da informação genética - DNA-RNA-proteína (26 %); proteínas hipotéticas (24 %) e proteínas do metabolismo (16 %). Tanto a categoria de proteínas envolvidas nos processos de desenvolvimento como as envolvidas na percepção a estímulos externos constituíram 10 % dos unigenes. Dentre os genes presumivelmente anotados, foram identificados aqueles codificadores de enzimas de importantes rotas metabólicas como gliceraldeído-3-fosfato-desidrogenase, fosfoglicerato quinases e fosfoenolpiruvato carboxilases, como também componentes produzidos pelo metabolismo secundário como micotoxinas e outras proteínas associadas a estresse e patogenicidade de fungos. Neste trabalho também foi verificado o potencial de antagonismo in vitro da bactéria P. agglomerans frente a três fitopatógenos de trigo: Drechslera tritici-repentis (Died.) Shoem e Bipolaris sorokiniana (Sacc. in Sorok.) e F. graminearum. Foi verificado que a inibição do crescimento destes fungos está associada à liberação de compostos solúveis e voláteis pela bactéria, que foram responsáveis por cerca de 50 % e 40 % de inibição, respectivamente. O perfil da expressão gênica de F. graminearum na interação com a bactéria P. agglomerans foi avaliado via macroarranjo. Dos 1.014 genes avaliados, 29 genes de F. graminearum foram diferencialmente expressos (p < 0,05) durante a interação com a bactéria antagonista, sendo 19 genes induzidos e 10 genes reprimidos. Entre os transcritos induzidos foram identificadas proteínas envolvidas nos processos de defesa e/ou virulência de fungos, cuja expressão foi induzida em resposta a estresses tanto abióticos como bióticos. Dos genes que foram reprimidos, destacaram-se: um transcrito com similaridade a uma proteína com um domínio do tipo dedo de zinco ?zinc finger? que é um fator de transcrição importante no processo de divisão celular, bem como proteínas envolvidas na cadeia respiratória, na modulação protéica e sinalização celular. Os dados do macroarranjo foram validados via transcrição reversa seguida de PCR quantitativo em tempo real (RT-PCRq), metodologia que se mostrou adequada para complementar a análise transcricional obtida por macroarranjo. As informações geradas na análise de antagonismo in vitro, bem como a análise e seqüenciamento dos transcritos, juntamente com a quantificação do nível de expressão na interação, foram fundamentais para compreender o padrão de resposta do fungo F. graminearum na interação com a bactéria P. agglomerans. / Cultivated grasses such as wheat, barley and maize are agricultural products of fundamental economic and social importance in Brazil. Among causing factors of important grain production losses in these species are diseases caused by phytopathogenic fungi such as Fusarium graminearum Schwabe (teleomorfo Gibberella zeae Schw.), the causal agent of fusariosis, a disease of difficult chemical, biological or even genetic control. An efficient and promising strategy to be adopted in order to protect cultivated plants against such diseases is the selection of antagonist microorganisms, amongst them the bacteria Pantoea agglomerans. This microbiota might have an important impact in scab control, isolated or in an integrated management program with chemical treatment. The present work aimed at identifying differentially expressed sequences in pathogenic fungi-antagonistic microorganisms interactions, considering the F. graminearum ? P. agglomerans model. The construction of a cDNA library for F. graminearum grown in PDA medium generated 1,983 valid sequences and provided 1,283 unigenes. The most representative categories in this library were proteins involved in genetic information pathways, DNA-RNA-protein (26 %); hypothetical proteins (24 %); and proteins involved in metabolism (16 %). The protein category involved in developmental processes as well as those related to external stimuli perception comprised 10 % of the obtained unigenes. Among putatively annotated genes, some coding for enzymes of important metabolic routes were identified, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and phophoenolpyruvate carboxylase. Also secondary metabolism compounds, specially micotoxins and proteins related to fungi stresses and pathogenicity were identified. In the present work, the control of three wheat phytopathogens, Drechslera tritici-repentis (Died.) Shoem, Bipolaris sorokiniana (Sacc.in Sorok.) and F. graminearum, using specific isolates of P. agglomerans was demonstrated. It was observed that the 50 % and 40 % growth inhibition of these fungi is associated to the bacteria release of soluble and volatile compounds, respectively. The gene expression profile of F. graminearum during interaction with the bacteria P. agglomerans was evaluated via macroarray. Among the 1,014 analysed genes, 29 F. graminearum genes were differentially expressed (p < 0,05) during its interaction with the antagonist bacteria: 19 genes were induced while 10 genes were repressed. Among the induced transcripts, proteins involved in fungi defense and/or virulence processes were identified, whose expression was induced in reponse to abiotic or biotic stresses. Among the identified repressed genes, a transcript similar to a protein containing a zinc finger-type domain, a transcription factor relevant in cell division, deserves special attention, as well as proteins involved in respiratory chain, in protein modulation and in cell signaling. Additionally, the macroarray data were validated by reverse transcription followed by real-time quantitative PCR (RT-PCRq), a suitable method for complementing transcriptional analysis through macroarray. Finally, the information generated in in vitro pathogenic fungi-antagonistic microorganisms interactions analysis, as well as in the analysis and sequencing of the obtained transcripts, together with the determination of the level of expression during the evaluated interactions were essential for better understanding the response pattern of the fungus F. graminearum in interaction with the bacteria P. agglomerans

A biodiversidade microbiana de diferentes ecossistemas tem incentivado estudos químicos e biológicos com micro-organismos dos mais variados habitats, os quais têm conduzido à obtenção de moléculas bioativas com aplicações na medicina, indústria química e agricultura, proporcionando melhorias na qualidade de vida ao homem. O presente trabalho teve como objetivos a bioprospecção por actinobactérias endofíticas e seus metabólitos, além do estudo da via biossintética dos sesquiterpenos aristoloquenos produzidos pelo fungo fitopatogênico Botrytis cinerea. No estudo de bioprospecção foram isoladas 41 linhagens de actinobactérias endofíticas de duas espécies de Asteraceae (Thitonia diversifolia e Lychnophora ericoides). A identificação através do sequenciamento de DNAr indicou predominância do gênero Streptomyces. As linhagens foram cultivadas em meio de arroz e os extratos etanólicos submetidos aos ensaios de citotoxicidade frente a células tumorais e antimicrobiano. Um total de 58,5% dos extratos apresentou atividade em pelo menos um dos ensaios realizados. Foram selecionadas as linhagens Streptomyces cattleya RLe 4 e Streptomyces sp. RLe 8 para cultivo em escala ampliada, isolamento e identificação de metabólitos bioativos. O isolamento dos compostos foi realizado através de diferentes técnicas cromatográficas e a identificação estrutural foi baseada em dados de ressonância magnética nuclear de 1H e 13C e espectrometria de massas. De S. cattleya RLe 4 foram isolados quatro compostos: 2-hidroxibenzamida, desferrioxamina E, 1-(3\',4\'-dimetoxifenil)-1-propanona e 1-(3\',4\'-dimetoxifenil)-1-etanona. Dos extratos de Streptomyces sp. RLe 8 foram isolados dez compostos: benzamida, 3- hidroxibenzamida, 3-hidróxi-4-metoxibenzamida, 4-hidróxi-3-metoxibenzamida, 3,4- dimetoxibenzamida, 2-fenilacetamida, dois isômeros de 3,4-diidro-3,4,6,8-tetraidróxi-1(2H)- naftalenona, 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona e desferrioxamina B. O composto 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona apresentou elevada atividade frente as células de câncer de cólon (HCT-8) e glioblastoma (SF295), com 93,9 % e 87.0 % de inibição, respectivamente. O outro enfoque da tese envolveu a otimização da produção de sesquiterpenos aristoloquenos por linhagens de B. cinerea, seguido de estudo biossintético destes produtos naturais através de experimentos de incorporação de precursores isotopicamente enriquecidos com 2H (deutério) e 13C (carbono treze). As análises dos dados obtidos de RMN de 2H e de 13C do sesquiterpeno majoritário indicaram que a biossíntese desta substância ocorre pela via do mevalonato (MVA). Os resultados também sugeriram o possível envolvimento da via do metil-eritritolfosfato ou 1-desoxi-D-xilulose-fosfato (MEP/DPX) na biossíntese deste sequiterpeno. Estes resultados podem contribuir para o planejamento racional de fungicidas seletivos com aplicação na agricultura. O trabalho desenvolvido mostrou o grande potencial de actinobactérias endofíticas para a obtenção de moléculas bioativas e que estudos usando precursores isotopicamente marcados fornecem informações precisas acerca da origem biossintética de produtos naturais. / The microbial biodiversity from different ecosystems has incited chemical and biological studies with microorganisms from several habitats, leading to the isolation of bioactive natural products with applications in medicine, chemical industry and agriculture, and thus contributing to a better quality of life. This thesis aimed the biopropecting on endophytic actinobacteria and their natural products, and also the biosynthetic study of aristolochene sesquiterpenes in the phytopathogenic fungus Botrytis cinerea. A total of 41 actinobacterial strains were isolated of two Asteraceae species (Thitonia diversifolia and Lychnophora ericoides) for the bioprospecting study. The rDNA sequencing showed predominancy of Streptomyces genus. All the strains were cultured on rice medium, and the ethanolic extracts were screened in cytotoxity and antimicrobial assays. As a result, 58.5% of the extracts showed activity in al least one bioassay. The strains Streptomyces cattleya RLe 4 and Streptomyces sp. RLe 8 were selected for scale up cultures, isolation and identification of bioactive compounds. Different chromatographic methods were applied for the isolation of compounds, and structural analysis were based on 1H and 13C nuclear magnetic resonance and mass spectrometry data. Four compounds were isolated from S. cattleya RLe 4: 2- hydroxybenzamide, desferrioxamine E, 1-(3\',4\'-dimethoxyphenyl)-1-propanone, and 1-(3\',4\'- dimethoxyphenyl)-1-etanone. Ten compounds were isolated from Streptomyces sp. Rle 8: benzamide, 3-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-hydroxy-3- methoxybenzamide, 2-phenylacetamide, two isomers of 3,4-dihydro-3,4,6,8-tetrahydroxy- 1(2H)-naphthalenone, 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone, and desferrioxamine B. Compound 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone showed high antiproliferative activity against colon cancer cells (HCT-8) and glioblastoma cells (SF295), with 93.9 and 87.0% of inhibition, respectively. The second focus of the thesis involved the optimization of aristolochene sesquiterpenes production by two strains of B. cinerea, followed by the biosynthetic study through feeding experiments with 2H (deuterium) and 13C isotopically labeled precursors. The 2H and 13C NMR obtained data showed that the biosynthesis of the sesquiterpene proceeds by the mevalonate pathway (MVA). The results also suggested the possible participation of the non mevalonate pathway, methylerytritol phosphate ou 1-deoxy- D-xylulose phosphate (MEP/DXP), in the biosynthesis. These results might contribute to the rational design of selective fungides with application in agriculture. This thesis showed the endophytic actinobacteria as promising sources of bioactive natural products, and also showed that the isotopically labeled feeding experiments give reliable information about the natural products biosynthetic pathways.

Les champignons endophytes sont des microorganismes non-pathogènes impliqués dans des associations mutualistes avec les plantes. Les endophytes foliaires, en particulier, représentent un groupe très divers, mais leurs interactions avec la plante hôte et ses micro-organismes associés sont peu connues. Des travaux préliminaires initiés par notre équipe, explorant la diversité microbienne foliaire du conifère Cephalotaxus harringtonia, ont permis d’isoler la souche fongique Paraconiothyrium variabile (Ascomycota), un antagoniste du phytopathogène Fusarium oxysporum. Au cours de leur interaction, on détecte des quantités moindres de beauvéricine, une mycotoxine synthétisée par F. oxysporum. En parallèle, on observe une augmentation de la synthèse de deux oxylipines, l’acide 13-hydroperoxyoctadécadiénoïque (13-HPODE) et l’acide 13-oxo-octadécadiénoïque (13-oxo-ODE), dans la zone de confrontation. L'objectif de ce travail était de comprendre les mécanismes conduisant à la diminution de la beauvéricine au cours de l'interaction et d'explorer le rôle des oxylipines dans la régulation de cette dernière. Dans mon travail de thèse, je montre la présence de deux gènes lox chez P. variabile (pvlox1 et pvlox2) codant tous deux pour des manganèse lipoxygénases, potentiellement à l'origine des acides 13-HPODE et 13-oxo-ODE. Pvlox2 est spécifiquement induit pendant l'interaction, et ces résultats sont corroborés par une synthèse accrue de 13-HPODE chez P. variabile. Par ailleurs, l'interaction avec l’endophyte, ainsi que l'ajout de l’oxylipine 13-HPODE, régulent positivement la voie de biosynthèse de la beauvéricine, comme l’indiquent les teneurs plus élevées en mycotoxines observées chez F. oxysporum. Enfin, nous avons montré que la beauvéricine inhibait la croissance de l’endophyte, mais que ce dernier était capable de dégrader la mycotoxine, expliquant ainsi les faibles quantités de beauvericine observées initialement dans la zone de compétition. Ce travail contribue à la compréhension du rôle des oxylipines dans la communication inter-microbienne. / Endophytic fungi are non-pathogenic microorganisms involved in mutualistic associations with their host. Foliar endophytes, in particular, represent a very diverse group but little is known about their interactions with the host and its associated micro-organisms. In preliminary work, exploring the leaf microbial diversity of the conifer Cephalotaxus harringtonia, our team isolated the fungal strain Paraconiothyrium variabile (Ascomycota), an antagonist of the phytopathogen Fusarium oxysporum. During their interaction, decreased amounts of the F. oxysporum mycotoxin beauvericin, and higher amounts of the two oxylipins, 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-oxo-octadecadienoic acid (13-oxo-ODE), were observed in the confrontation zone. The objective of the present work was to understand the mechanisms leading to beauvericin decrease during the interaction and to explore the role of oxylipins in beauvericin regulation. In my thesis work I show the presence of two lox genes in P. variabile (pvlox1 and pvlox2) coding both for manganese lipoxygenases, potentially at the origin of 13-HPODE and 13-oxo-ODE. Pvlox2 is specifically induced during the interaction, which lead to an increased synthesis of 13-HPODE in P. variabile. The endophyte itself, as well as the oxylipin 13-HPODE, up-regulated the beauvericin biosynthesis gene beas, which was paralleled by higher mycotoxin content in the mycelium of F. oxysporum. Finally, we showed that beauvericin inhibited the endophyte’s growth, but the latter was capable to degrade the mycotoxin, which explains the lower amounts of beauvericin found in the competition zone. This work presents pioneer undertaking to elucidate the role of oxylipins in inter-microbial crosstalk.

Gramíneas cultivadas, como trigo, cevada e milho são produtos agrícolas de fundamental importância no Brasil. Entre os fatores causadores de perdas na produção de grãos dessas espécies estão os estresses causados por fitopatógenos como Fusarium graminearum Schwabe (teleomorfo Gibberella zeae Schw.), agente causador da fusariose e de difícil controle químico, biológico ou mesmo genético. Uma estratégia que tem se mostrado eficiente no controle de doenças é a utilização de microrganismos antagonistas a diferentes fitopatógenos, dentre os quais destaca-se a bactéria P. agglomerans. O presente trabalho teve como objetivo identificar genes diferencialmente expressos em interações fungo fitopatogênico-microrganismo antagonista, considerando como modelo o sistema F. graminearum-P. agglomerans. A construção de uma biblioteca de cDNA de F. graminearum cultivado in vitro proporcionou a geração de 1.983 seqüências válidas, resultando em 1.283 unigenes. As categorias de maior representatividade desta biblioteca foram aquelas constituídas por proteínas envolvidas em vias da informação genética - DNA-RNA-proteína (26 %); proteínas hipotéticas (24 %) e proteínas do metabolismo (16 %). Tanto a categoria de proteínas envolvidas nos processos de desenvolvimento como as envolvidas na percepção a estímulos externos constituíram 10 % dos unigenes. Dentre os genes presumivelmente anotados, foram identificados aqueles codificadores de enzimas de importantes rotas metabólicas como gliceraldeído-3-fosfato-desidrogenase, fosfoglicerato quinases e fosfoenolpiruvato carboxilases, como também componentes produzidos pelo metabolismo secundário como micotoxinas e outras proteínas associadas a estresse e patogenicidade de fungos. Neste trabalho também foi verificado o potencial de antagonismo in vitro da bactéria P. agglomerans frente a três fitopatógenos de trigo: Drechslera tritici-repentis (Died.) Shoem e Bipolaris sorokiniana (Sacc. in Sorok.) e F. graminearum. Foi verificado que a inibição do crescimento destes fungos está associada à liberação de compostos solúveis e voláteis pela bactéria, que foram responsáveis por cerca de 50 % e 40 % de inibição, respectivamente. O perfil da expressão gênica de F. graminearum na interação com a bactéria P. agglomerans foi avaliado via macroarranjo. Dos 1.014 genes avaliados, 29 genes de F. graminearum foram diferencialmente expressos (p < 0,05) durante a interação com a bactéria antagonista, sendo 19 genes induzidos e 10 genes reprimidos. Entre os transcritos induzidos foram identificadas proteínas envolvidas nos processos de defesa e/ou virulência de fungos, cuja expressão foi induzida em resposta a estresses tanto abióticos como bióticos. Dos genes que foram reprimidos, destacaram-se: um transcrito com similaridade a uma proteína com um domínio do tipo dedo de zinco ?zinc finger? que é um fator de transcrição importante no processo de divisão celular, bem como proteínas envolvidas na cadeia respiratória, na modulação protéica e sinalização celular. Os dados do macroarranjo foram validados via transcrição reversa seguida de PCR quantitativo em tempo real (RT-PCRq), metodologia que se mostrou adequada para complementar a análise transcricional obtida por macroarranjo. As informações geradas na análise de antagonismo in vitro, bem como a análise e seqüenciamento dos transcritos, juntamente com a quantificação do nível de expressão na interação, foram fundamentais para compreender o padrão de resposta do fungo F. graminearum na interação com a bactéria P. agglomerans. / Cultivated grasses such as wheat, barley and maize are agricultural products of fundamental economic and social importance in Brazil. Among causing factors of important grain production losses in these species are diseases caused by phytopathogenic fungi such as Fusarium graminearum Schwabe (teleomorfo Gibberella zeae Schw.), the causal agent of fusariosis, a disease of difficult chemical, biological or even genetic control. An efficient and promising strategy to be adopted in order to protect cultivated plants against such diseases is the selection of antagonist microorganisms, amongst them the bacteria Pantoea agglomerans. This microbiota might have an important impact in scab control, isolated or in an integrated management program with chemical treatment. The present work aimed at identifying differentially expressed sequences in pathogenic fungi-antagonistic microorganisms interactions, considering the F. graminearum ? P. agglomerans model. The construction of a cDNA library for F. graminearum grown in PDA medium generated 1,983 valid sequences and provided 1,283 unigenes. The most representative categories in this library were proteins involved in genetic information pathways, DNA-RNA-protein (26 %); hypothetical proteins (24 %); and proteins involved in metabolism (16 %). The protein category involved in developmental processes as well as those related to external stimuli perception comprised 10 % of the obtained unigenes. Among putatively annotated genes, some coding for enzymes of important metabolic routes were identified, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and phophoenolpyruvate carboxylase. Also secondary metabolism compounds, specially micotoxins and proteins related to fungi stresses and pathogenicity were identified. In the present work, the control of three wheat phytopathogens, Drechslera tritici-repentis (Died.) Shoem, Bipolaris sorokiniana (Sacc.in Sorok.) and F. graminearum, using specific isolates of P. agglomerans was demonstrated. It was observed that the 50 % and 40 % growth inhibition of these fungi is associated to the bacteria release of soluble and volatile compounds, respectively. The gene expression profile of F. graminearum during interaction with the bacteria P. agglomerans was evaluated via macroarray. Among the 1,014 analysed genes, 29 F. graminearum genes were differentially expressed (p < 0,05) during its interaction with the antagonist bacteria: 19 genes were induced while 10 genes were repressed. Among the induced transcripts, proteins involved in fungi defense and/or virulence processes were identified, whose expression was induced in reponse to abiotic or biotic stresses. Among the identified repressed genes, a transcript similar to a protein containing a zinc finger-type domain, a transcription factor relevant in cell division, deserves special attention, as well as proteins involved in respiratory chain, in protein modulation and in cell signaling. Additionally, the macroarray data were validated by reverse transcription followed by real-time quantitative PCR (RT-PCRq), a suitable method for complementing transcriptional analysis through macroarray. Finally, the information generated in in vitro pathogenic fungi-antagonistic microorganisms interactions analysis, as well as in the analysis and sequencing of the obtained transcripts, together with the determination of the level of expression during the evaluated interactions were essential for better understanding the response pattern of the fungus F. graminearum in interaction with the bacteria P. agglomerans

A biodiversidade microbiana de diferentes ecossistemas tem incentivado estudos químicos e biológicos com micro-organismos dos mais variados habitats, os quais têm conduzido à obtenção de moléculas bioativas com aplicações na medicina, indústria química e agricultura, proporcionando melhorias na qualidade de vida ao homem. O presente trabalho teve como objetivos a bioprospecção por actinobactérias endofíticas e seus metabólitos, além do estudo da via biossintética dos sesquiterpenos aristoloquenos produzidos pelo fungo fitopatogênico Botrytis cinerea. No estudo de bioprospecção foram isoladas 41 linhagens de actinobactérias endofíticas de duas espécies de Asteraceae (Thitonia diversifolia e Lychnophora ericoides). A identificação através do sequenciamento de DNAr indicou predominância do gênero Streptomyces. As linhagens foram cultivadas em meio de arroz e os extratos etanólicos submetidos aos ensaios de citotoxicidade frente a células tumorais e antimicrobiano. Um total de 58,5% dos extratos apresentou atividade em pelo menos um dos ensaios realizados. Foram selecionadas as linhagens Streptomyces cattleya RLe 4 e Streptomyces sp. RLe 8 para cultivo em escala ampliada, isolamento e identificação de metabólitos bioativos. O isolamento dos compostos foi realizado através de diferentes técnicas cromatográficas e a identificação estrutural foi baseada em dados de ressonância magnética nuclear de 1H e 13C e espectrometria de massas. De S. cattleya RLe 4 foram isolados quatro compostos: 2-hidroxibenzamida, desferrioxamina E, 1-(3\',4\'-dimetoxifenil)-1-propanona e 1-(3\',4\'-dimetoxifenil)-1-etanona. Dos extratos de Streptomyces sp. RLe 8 foram isolados dez compostos: benzamida, 3- hidroxibenzamida, 3-hidróxi-4-metoxibenzamida, 4-hidróxi-3-metoxibenzamida, 3,4- dimetoxibenzamida, 2-fenilacetamida, dois isômeros de 3,4-diidro-3,4,6,8-tetraidróxi-1(2H)- naftalenona, 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona e desferrioxamina B. O composto 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona apresentou elevada atividade frente as células de câncer de cólon (HCT-8) e glioblastoma (SF295), com 93,9 % e 87.0 % de inibição, respectivamente. O outro enfoque da tese envolveu a otimização da produção de sesquiterpenos aristoloquenos por linhagens de B. cinerea, seguido de estudo biossintético destes produtos naturais através de experimentos de incorporação de precursores isotopicamente enriquecidos com 2H (deutério) e 13C (carbono treze). As análises dos dados obtidos de RMN de 2H e de 13C do sesquiterpeno majoritário indicaram que a biossíntese desta substância ocorre pela via do mevalonato (MVA). Os resultados também sugeriram o possível envolvimento da via do metil-eritritolfosfato ou 1-desoxi-D-xilulose-fosfato (MEP/DPX) na biossíntese deste sequiterpeno. Estes resultados podem contribuir para o planejamento racional de fungicidas seletivos com aplicação na agricultura. O trabalho desenvolvido mostrou o grande potencial de actinobactérias endofíticas para a obtenção de moléculas bioativas e que estudos usando precursores isotopicamente marcados fornecem informações precisas acerca da origem biossintética de produtos naturais. / The microbial biodiversity from different ecosystems has incited chemical and biological studies with microorganisms from several habitats, leading to the isolation of bioactive natural products with applications in medicine, chemical industry and agriculture, and thus contributing to a better quality of life. This thesis aimed the biopropecting on endophytic actinobacteria and their natural products, and also the biosynthetic study of aristolochene sesquiterpenes in the phytopathogenic fungus Botrytis cinerea. A total of 41 actinobacterial strains were isolated of two Asteraceae species (Thitonia diversifolia and Lychnophora ericoides) for the bioprospecting study. The rDNA sequencing showed predominancy of Streptomyces genus. All the strains were cultured on rice medium, and the ethanolic extracts were screened in cytotoxity and antimicrobial assays. As a result, 58.5% of the extracts showed activity in al least one bioassay. The strains Streptomyces cattleya RLe 4 and Streptomyces sp. RLe 8 were selected for scale up cultures, isolation and identification of bioactive compounds. Different chromatographic methods were applied for the isolation of compounds, and structural analysis were based on 1H and 13C nuclear magnetic resonance and mass spectrometry data. Four compounds were isolated from S. cattleya RLe 4: 2- hydroxybenzamide, desferrioxamine E, 1-(3\',4\'-dimethoxyphenyl)-1-propanone, and 1-(3\',4\'- dimethoxyphenyl)-1-etanone. Ten compounds were isolated from Streptomyces sp. Rle 8: benzamide, 3-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-hydroxy-3- methoxybenzamide, 2-phenylacetamide, two isomers of 3,4-dihydro-3,4,6,8-tetrahydroxy- 1(2H)-naphthalenone, 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone, and desferrioxamine B. Compound 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone showed high antiproliferative activity against colon cancer cells (HCT-8) and glioblastoma cells (SF295), with 93.9 and 87.0% of inhibition, respectively. The second focus of the thesis involved the optimization of aristolochene sesquiterpenes production by two strains of B. cinerea, followed by the biosynthetic study through feeding experiments with 2H (deuterium) and 13C isotopically labeled precursors. The 2H and 13C NMR obtained data showed that the biosynthesis of the sesquiterpene proceeds by the mevalonate pathway (MVA). The results also suggested the possible participation of the non mevalonate pathway, methylerytritol phosphate ou 1-deoxy- D-xylulose phosphate (MEP/DXP), in the biosynthesis. These results might contribute to the rational design of selective fungides with application in agriculture. This thesis showed the endophytic actinobacteria as promising sources of bioactive natural products, and also showed that the isotopically labeled feeding experiments give reliable information about the natural products biosynthetic pathways.

A cultura do eucalipto apresenta grande importância no setor florestal no mundo. No Brasil, 70% da área florestal plantada é destinada ao eucalipto. Entretanto, a ferrugem das mirtáceas, também conhecida como ferrugem do eucalipto, causada pelo fungo Puccinia psidii Winter, afeta o enorme potencial produtivo das plantações de eucalipto. A biologia, mecanismos de patogenicidade e genética desse patógeno são pouco conhecidos, apesar de sua importância para o setor florestal. Os elementos transponíveis (TEs) são sequências de DNA com a capacidade de migrar e influenciar a organização, integridade e evolução do genoma hospedeiro. O presente trabalho teve como principal objetivo estudar os TEs presentes no genoma de P. psidii, combinando ferramentas in silico e moleculares. A classificação dos elementos transponíveis no genoma de P. psidii MF-1 foi realizada utilizando contigs previamente minerados e remontados, bem como sem seleção prévia dos contigs, por meio do programa RepeatMasker. Ambas estratégias apontaram o predomínio de elementos da Classe I - LTR Retrotransposons no genoma de P. psidii MF-1. O resultado condiz com a composição de TEs em fungos fitopatogênicos descrita na literatura. Algumas análises in silico, como verificação de integridade e anotação manual de sequências proteicas foram também realizadas para alguns contigs classificados como TEs. Assim, foi possível observar a presença de sequências conservadas pertencentes à região pol em LTR Retrotransposons. Além disso, as análises permitiram inferir sobre a existência de TEs híbridos no genoma parcialmente sequenciado de P. psidii MF-1. Paralelamente foi também realizada uma análise comparativa entre os TEs presentes nos genomas de P. graminis, P. striiformis, P. triticina e P. psidii. Observou-se que P. graminis, P. striiformis e P. triticina apresentam maior frequência de elementos da Classe II, do tipo DNA Transposons ao contrário de P. psidii, com maior frequência de elementos da Classe I. Interessantemente, a quantidade de elementos desconhecidos foi similarmente alta para todos os quatro genomas avaliados. Este tipo de análise é muito importante, pois evidencia a grande quantidade de famílias de TEs novas a serem descobertas. Elas podem estar potencialmente relacionadas ao silenciamento de genes importantes à virulência destes patógenos. A utilização de TEs no estudo de diversidade genética entre populações é bastante comum. A técnica molecular IRAP foi utilizada para acessar a diversidade entre populações de P. psidii originárias de três híbridos de Eucalyptus spp., goiabeira, jambeiro e jabuticabeira. No entanto, esta técnica não se mostrou eficiente para detectar polimorfismos existentes entre estas populações. A anotação de TEs foi difícil devido à observação de sequências de elementos sobrepostas, o que podem representar híbridos de TEs, entretanto, visando a confirmação desta hipótese por meio da PCR, alguns contigs serão sequenciados e mais estudos devem ser realizados para a continuação desta confirmação. Os resultados apresentados neste trabalho são inéditos e representam uma etapa crucial no entendimento de TEs em fungos do gênero Puccinia, em especial do patógeno P. psidii para o desenvolvimento de melhores mecanismos de controle de ferrugem. / The culture of eucalyptus has great importance worldwide in forestry sector. In Brazil, 70% of cultivated forest area is intended for Eucalyptus. However, the eucalyptus potential productive has been affected by rust disease, caused by the fungus Puccinia psidii Winter. Despite its importance to brazilian and world forest sector, the knowledge of biology, genetic and pathogenic mechanisms of this pathogen is scarce. Transposable elements (TEs) are mobile DNA fragments that influence the organization and development of the host genome. These elements have the ability to move within host genome, and their insertion can cause a wide spectrum of mutations in their hosts. This study aims to decipher the TEs in P. psidii genome by combining in silico and molecular tools. P. psidii MF-1 TEs classification was performed automatically, through RepeatMasker software, being observed a predominance of Class I - LTR Retrotransposons in P. psidii MF-1 genome. This result is consistent with the TEs composition described in phytopathogenic fungi. Some in silico analysis, as integrity and manual annotation of conserved protein sequences from TEs were carried out with P. psidii MF-1 contigs classified as transposable elements. The presence of conserved sequences belonging to pol region in LTR Retrotransposons was observed. Furthermore, these analysis allowed the inference of hybrid TEs in P. psidii MF-1. At the same time, a comparative analysis of TEs present in other Puccinia genomes and P. psidii MF-1 was also performed. The P. graminis, P. striiformis and P. triticina genomes have higher frequency of Class II - DNA Transposons unlike the results found for P. psidii. Interestingly, the number of unknown elements was similarly high for all genomes. This type of analysis is very importante because it shows a great number of potential new TEs families to be discovered. They may be potentially related to the virulence gene silencing of these pathogens. Using TEs for study the fungal genetic diversity is quite common. The IRAP technique was used to access the diversity among P. psidii populations originated from three Eucalyptus spp. hybrids, guava, syzigium and jabuticaba. However, this technique was not efficient to detect existing polymorphisms between these populations. TEs annotation was labored due to the existence of overlapping elements, which may represent hybrids TEs. PCR tool was used to confirm some sequences annotated as hybrids and more studies are needed to confirm this hyphotesis. The results presented in this study are novel and is a crucial step in understanding the genetic of P. psidii pathogen for further improvements of rust control mechanisms.