A citricultura destaca-se dentro do agronegócio brasileiro gerando dividendos diretos com exportações e empregos, e indiretos com arrecadação de impostos. No entanto, ainda padece com problemas fitossanitários, com destaque para o HLB (Huanglongbing, HLB, ex-greening), que nos últimos dez anos tem dizimado pomares e provocado o abandono da cultura por muitos produtores. Causada no Brasil pelas bactérias Candidatus Liberibacter asiaticus e Ca. Liberibacter americanus, é uma doença sistêmica, restrita aos vasos do floema, e induz ao aparecimento de sintomas semelhantes àqueles causados por deficiência de nutrientes em folhas e ramos, frutos com tamanhos reduzidos e assimétricos e coloração amarelada das plantas, tornando-as economicamente inviáveis. A transmissão da doença acontece naturalmente pelo inseto Diaphorina citri, e artificialmente por meio de borbulhas contaminadas. Todas as variedades de citros e rutáceas próximas são comprovadamente suscetíveis ao HLB; entretanto, há diferenças marcantes entre níveis de suscetibilidade entre os genótipos, como Poncirus trifoliata, que apresenta certa resistência ou tolerância à doença. Os mecanismos de patogenicidade de Ca. Liberibacter spp. e a resposta molecular da planta à infecção ainda não estão bem definidos e o estudo da ativação dos mecanismos de defesa da planta induzidos por hormônios vegetais é um importante foco na elucidação deste complexo patossistema. Diante deste panorama, este estudo propôs avaliar o perfil de expressão de genes associados às vias do jasmonato e do etileno em plantas de Citrus sinensis L. Osb. (suscetível) e Poncirus trifoliata (L.) Raf. (resistente ou tolerante) em resposta à infecção pelas duas espécies de bactérias causadoras do HLB no Brasil, separadamente. Os perfis transcricionais dos genes avaliados não foram estatisticamente distintos, mas mereceram destaque os genes de biossíntese e metabolismo de jasmonato LOX2, AOC3 e JMT, os genes sinalizadores da via de jasmonato JAZ2 e MYC2, o gene de biossíntese de etileno SAM1, os receptores de etileno ETR1, ERS1 e EIN4, o regulador negativo de etileno CTR1, o regulador positivo da sinalização de etileno EIN2, os fatores de transcrição EIN3, EIL1, ERF1, ERF2, ERF/AP2, uma MAPkinase MAPK6 e uma proteína PR PR-4. Estes resultados mostram que as vias sinalizadas por jasmonato e etileno são modificadas pelas bactérias Ca. Liberibacter spp. No entanto, não foi possível comprovar a importância destas vias na resposta diferencial de genótipos suscetível e resistente/ tolerante de citros ao HLB. / The citrus industry stands out in the Brazilian agribusiness generating direct and indirect jobs and significant revenues. However, the citrus crop faces several diseases, especially the HLB (Huanglongbing, ex-greening), which, in the last ten years, have caused severe losses to growers. In Brazil, HLB is caused by the bacteria Candidatus Liberibacter asiaticus and Ca. Liberibacter americanus, which are restricted to the phloem and induce symptoms similar to those caused by nutrient deficiency in citrus leaves and branches. Fruits are of reduced size and asymmetrical, and the plants tend to show overall yellowing. These symptoms altogether lead to the economical death of the plant. Transmission of the pathogens occurs naturally by the Asian citrus psyllid Diaphorina citri, or by contaminated buds. All citrus varieties and relatives are considered susceptible to HLB; however, there are clear differences in levels of susceptibility among genotypes. Poncirus trifoliata, for instance, exhibits some resistance or tolerance to the disease. The mechanisms involved in the pathogenesis of Ca. Liberibacter spp. and the molecular responses of the hosts to the infection are still unknown, but emphasis has been given to the defense mechanisms induced by plant hormones in order to try to elucidate the interactions of such complex pathosystem. Based on this scenario, this study aimed to evaluate the expression profile of Citrus sinensis (L.) Osb. (susceptible) and Poncirus trifoliata (L.) Raf. (resistant or tolerant) genes associated with the jasmonate and ethylene pathways in response to infection by both species of Ca. Liberibacter spp. causing HLB in Brazil, separately. The transcriptional profiles did not differ statistically, for the evaluated genes, including those in the jasmonate biosynthesis and metabolism (LOX2, AOC3 and JMT), signaling pathway (JAZ2 and MYC2), biosynthesis of ethylene (SAM1), ethylene receptors (ETR1, ERS1 and EIN4), negative regulator of ethylene (CTR1), positive regulator of ethylene signaling (EIN2), transcription factors (EIN3, EIL1, ERF1, ERF2, ERF/AP2), a MAPkinase (MAPK6) and a PR protein (PR-4). These results suggest that the jasmonate and ethylene pathways exhibit some modulation caused by the bacteria Ca Liberibacter spp. However, it does not seem that these pathways are relevant for the differential response of susceptibility or resistance/ tolerance of the citrus genotypes to HLB.

Pyrenophora tritici-repentis (Ptr) is the necrotrophic fungus responsible for tan spot of wheat (Triticum aestivum). Ptr causes disease on susceptible wheat cultivars through the production and secretion of host-selective toxins (HSTs). HSTs are compounds that are only known to be produced by fungi and considered to be primary determinants of pathogenicity. Infiltration of these toxins into sensitive wheat elicits the same symptoms as the pathogen, which simplifies investigations of host- pathogen interactions due to exclusion of the pathogen. These characteristics make HSTs ideal molecules to dissect molecular plant-microbe interactions. Known HSTs of Ptr include Ptr ToxA (ToxA), Ptr ToxB (ToxB) and Ptr ToxC (ToxC). ToxA is the most characterized toxin of Ptr, as well as the first proteinaceous HST identified. The proposed mode-of-action for ToxA includes internalization into sensitive wheat mesophyll cells, localization to the chloroplast, photosystem perturbations and elicitation of high amounts of reactive oxygen species (ROS), all of which lead to necrosis. However, it is still unknown how ToxA is transported to the chloroplast. To identify additional interacting components involved in ToxA symptom development, genes were silenced in tobacco plants (Nicotiana benthamiana) using the tobacco rattle virus (TRV) virus-induced gene-silencing (VIGS) system. Four genes were identified that potentially could play a role in ToxA-induced cell death: a 40S ribosomal subunit, peroxisomal glycolate oxidase (GOX), a thiamine biosynthetic enzyme (Thi1), and the R-gene mediator, Sgt1. Ptr exhibits a complex race structure determined by the HST(s) produced and the symptom(s) elicited on sensitive wheat cultivars. Currently, there are eight characterized races and other HSTs and races have been proposed. Isolate SO3 was discovered in southern Oregon and elicits ToxA-like symptoms on a wheat differential set, yet lacks the ToxA gene. The transcriptome of SO3 was sequenced, assembled, and aligned to a ToxA-producing isolate, Pt-1C-BFP, which will aid in the identification of the protein(s) that may be responsible for these ToxA-like symptoms. SO3 contains a set of 497 sequences that were not found in the ToxA-producing isolate Pt-1C-BFP (BFP). These sequences should be further investigated to identify those that encode small secreted proteins (SSPs) and could potentially serve as HSTs and pathogenicity factors of SO3. / Graduation date: 2013

A citricultura destaca-se dentro do agronegócio brasileiro gerando dividendos diretos com exportações e empregos, e indiretos com arrecadação de impostos. No entanto, ainda padece com problemas fitossanitários, com destaque para o HLB (Huanglongbing, HLB, ex-greening), que nos últimos dez anos tem dizimado pomares e provocado o abandono da cultura por muitos produtores. Causada no Brasil pelas bactérias Candidatus Liberibacter asiaticus e Ca. Liberibacter americanus, é uma doença sistêmica, restrita aos vasos do floema, e induz ao aparecimento de sintomas semelhantes àqueles causados por deficiência de nutrientes em folhas e ramos, frutos com tamanhos reduzidos e assimétricos e coloração amarelada das plantas, tornando-as economicamente inviáveis. A transmissão da doença acontece naturalmente pelo inseto Diaphorina citri, e artificialmente por meio de borbulhas contaminadas. Todas as variedades de citros e rutáceas próximas são comprovadamente suscetíveis ao HLB; entretanto, há diferenças marcantes entre níveis de suscetibilidade entre os genótipos, como Poncirus trifoliata, que apresenta certa resistência ou tolerância à doença. Os mecanismos de patogenicidade de Ca. Liberibacter spp. e a resposta molecular da planta à infecção ainda não estão bem definidos e o estudo da ativação dos mecanismos de defesa da planta induzidos por hormônios vegetais é um importante foco na elucidação deste complexo patossistema. Diante deste panorama, este estudo propôs avaliar o perfil de expressão de genes associados às vias do jasmonato e do etileno em plantas de Citrus sinensis L. Osb. (suscetível) e Poncirus trifoliata (L.) Raf. (resistente ou tolerante) em resposta à infecção pelas duas espécies de bactérias causadoras do HLB no Brasil, separadamente. Os perfis transcricionais dos genes avaliados não foram estatisticamente distintos, mas mereceram destaque os genes de biossíntese e metabolismo de jasmonato LOX2, AOC3 e JMT, os genes sinalizadores da via de jasmonato JAZ2 e MYC2, o gene de biossíntese de etileno SAM1, os receptores de etileno ETR1, ERS1 e EIN4, o regulador negativo de etileno CTR1, o regulador positivo da sinalização de etileno EIN2, os fatores de transcrição EIN3, EIL1, ERF1, ERF2, ERF/AP2, uma MAPkinase MAPK6 e uma proteína PR PR-4. Estes resultados mostram que as vias sinalizadas por jasmonato e etileno são modificadas pelas bactérias Ca. Liberibacter spp. No entanto, não foi possível comprovar a importância destas vias na resposta diferencial de genótipos suscetível e resistente/ tolerante de citros ao HLB. / The citrus industry stands out in the Brazilian agribusiness generating direct and indirect jobs and significant revenues. However, the citrus crop faces several diseases, especially the HLB (Huanglongbing, ex-greening), which, in the last ten years, have caused severe losses to growers. In Brazil, HLB is caused by the bacteria Candidatus Liberibacter asiaticus and Ca. Liberibacter americanus, which are restricted to the phloem and induce symptoms similar to those caused by nutrient deficiency in citrus leaves and branches. Fruits are of reduced size and asymmetrical, and the plants tend to show overall yellowing. These symptoms altogether lead to the economical death of the plant. Transmission of the pathogens occurs naturally by the Asian citrus psyllid Diaphorina citri, or by contaminated buds. All citrus varieties and relatives are considered susceptible to HLB; however, there are clear differences in levels of susceptibility among genotypes. Poncirus trifoliata, for instance, exhibits some resistance or tolerance to the disease. The mechanisms involved in the pathogenesis of Ca. Liberibacter spp. and the molecular responses of the hosts to the infection are still unknown, but emphasis has been given to the defense mechanisms induced by plant hormones in order to try to elucidate the interactions of such complex pathosystem. Based on this scenario, this study aimed to evaluate the expression profile of Citrus sinensis (L.) Osb. (susceptible) and Poncirus trifoliata (L.) Raf. (resistant or tolerant) genes associated with the jasmonate and ethylene pathways in response to infection by both species of Ca. Liberibacter spp. causing HLB in Brazil, separately. The transcriptional profiles did not differ statistically, for the evaluated genes, including those in the jasmonate biosynthesis and metabolism (LOX2, AOC3 and JMT), signaling pathway (JAZ2 and MYC2), biosynthesis of ethylene (SAM1), ethylene receptors (ETR1, ERS1 and EIN4), negative regulator of ethylene (CTR1), positive regulator of ethylene signaling (EIN2), transcription factors (EIN3, EIL1, ERF1, ERF2, ERF/AP2), a MAPkinase (MAPK6) and a PR protein (PR-4). These results suggest that the jasmonate and ethylene pathways exhibit some modulation caused by the bacteria Ca Liberibacter spp. However, it does not seem that these pathways are relevant for the differential response of susceptibility or resistance/ tolerance of the citrus genotypes to HLB.

O Brasil é o maior produtor mundial de maracujá azedo (Passiflora edulis f. flavicarpa) sendo esta a espécie de maior expressão comercial dentre as passifloras cultivadas. A bacteriose do maracujazeiro, causada por Xanthomonas axonopodis pv. passiflorae (Xap), é uma das doenças mais severas da cultura, acarretando grandes prejuízos aos produtores. Atualmente, é incipiente o conhecimento sobre a interação maracujá azedo-Xap. Diante disso, a identificação e a caracterização dos genes envolvidos no processo de defesa são passos importantes para dar suporte ao desenvolvimento de variedades resistentes. Assim, o objetivo deste trabalho foi identificar e caracterizar genes de maracujá azedo diferencialmente expressos durante a resposta de defesa à Xap, bem como mensurar a sua expressão. Para isso, foram construídas duas bibliotecas subtrativas de cDNA (forward e reverse) usando o método SSH a partir de transcritos de folhas, que foram inoculadas com o patógeno ou solução salina (controle). Após o sequenciamento dos clones, o processamento e a montagem das sequências, as unisequências foram anotadas através da Plataforma PLAZA e do programa computacional Blast2GO. Genes envolvidos em diversos processos biológicos foram selecionados para a validação das bibliotecas por PCR quantitativo. Usando a Plataforma PLAZA, 78 % (764) das unisequências mostraram similaridade com proteínas de Arabidopsis thaliana, enquanto 87 % (866) delas apresentaram similaridade com proteínas putativas de diversas espécies vegetais, quando se utilizou Blast2GO. Na biblioteca forward, foram identificadas 73 proteínas relacionadas à resposta de defesa, dentre as quais estão proteínas envolvidas na sinalização intracelular, na ativação da transcrição e regulação da expressão de genes de defesa, bem como proteínas de defesa, de resistência e relacionadas à patogênese (PRs). Dentre os 22 transcritos validados, 95 % foram diferencialmente expressos em pelo menos um dos três períodos avaliados; os genes mais expressos em resposta à infecção pelo patógeno são os que codificam as enzimas lipoxigenase, (+)-neomentol desidrogenase e quitinase, as quais participam diretamente nas respostas de defesa vegetal. Dos genes cuja expressão foi mais reprimida, dois codificam proteínas relacionadas à fotossíntese e dois codificam proteínas envolvidas na detoxificação da amônia e do H2O2. Nossos resultados sugerem que a planta utiliza um arsenal de transcritos para responder à infecção; entretanto, este arsenal não é eficiente para impedir a ação do patógeno e, consequentemente, o desenvolvimento da bacteriose nas condições estudadas. Nosso estudo é inédito e gerou informações sobre a reprogramação transcricional durante a interação maracujá azedo-Xap, o que constitui um importante passo para o melhor entendimento sobre este patossistema. / Brazil is the main producer of yellow passion fruit (Passiflora edulis f. flavicarpa) worldwide, which is the most widely commercialized crop among the cultivated passifloras. The bacterial leaf spot induced by Xanthomonas axonopodis pv. passiflorae (Xap) is one of the most severe diseases of the crop, causing great losses to producers. Currently, we understand very little about the yellow passion fruit-Xap interaction. Therefore, the identification and characterization of genes involved in the defense process are important steps to support the development of resistant varieties. Thus, the objective of this study was identify and characterize differentially expressed genes during the defense response to Xap, as well as to measure their expression. For that, we constructed two subtractive cDNA libraries (the forward and the reverse) by performing the SSH method from leaf transcripts, which were inoculated with the pathogen or saline solution (control). After sequencing the clones and sequence data processing, sequences were assembled into unique sequences, which were annotated using the PLAZA Platform and the computational program Blast2GO. Genes involved in several biological processes were selected to validate the libraries by quantitative PCR. When PLAZA was used for sequence similarity searches, 78 % (764) of the yellow passion fruit unique sequences showed similarity to proteins of Arabidopsis thaliana; when Blast2GO was used, 87 % (866) of the unique sequences showed similarities to putative proteins of several plant species. For the forward library, 73 proteins related to defense response were identified, such as those involved in intracellular signaling, transcription activation and regulation of defense gene expression, as well as defense and resistance proteins, and pathogenesis-related proteins (PRs). Of the 22 validated transcripts, 95 % were differentially expressed during at least one of the three periods evaluated; the genes up-regulated in response to the pathogen infection were those that code for the enzymes lipoxygenase, (+)-neomenthol dehydrogenase and chitinase, which participate directly in plant-defense responses. Out of down-regulated genes, two code for photosynthesis-related proteins, and two for ammonia and H2O2 detoxification. Our results suggest the plant uses an arsenal of transcripts to respond to infection; however, this arsenal is not effective to prevent pathogen action and consequently the occurrence of bacterial leaf spot under the evaluated conditions. The present study is the first to produce information on the transcriptional reprogramming during the passion fruit-Xap interaction, which represents an important step for a better understanding of this pathosystem.

Abstract Medicinal plants have been for long remedies for human diseases because they contain components of therapeutic value. The growing problem of antibiotic resistance by organisms demands the search for novel compounds from plant based sources. The present study was aimed at evaluating the bioactivity and phytochemical analysis of Hydnora africana on clinical and standard strains of Helicobacter pylori (PE 252C and ATCC 43526), Aeromonas hydrophila ATCC 35654, and Staphylococcus aureus NCT 6571 in an effort to identify potential sources of cheap starting materials for the synthesis of new drugs against these strains. Ethyl acetate, acetone, ethanol, methanol, and water crude extracts of H. africana were screened for activity against the test organisms using the agar well diffusion assay. The Minimum Inhibitory Concentration (MIC50) and Minimum Bactericidal Concentration (MBC) of the most potent extracts were determined by the microdilution method, followed by qualitative phytochemical analysis. Results were analyzed statistically by ANOVA one - way test. Different concentrations (200,100, 50mg/mL) of the methanol, acetone, ethanol and ethyl acetate extracts showed activity against S. aureus and A. hydrophila while for H. pylori, only methanol and ethyl acetate extracts were active; water showed no activity for all studied bacterial pathogens. Mean zone diameter of inhibition which ranged from 0-22mm were observed for all test bacterial pathogens and 14-17mm for ciprofloxacin. The activity of methanol and ethyl acetate extracts were statistically significant (P< 0.05) compared to all the other extracts. MIC50 and MBC ranged from 0.078 – 2.5mg/mL, 0.78-25mg/mL respectively for all tested bacterial pathogens. For ciprofloxacin, the MIC50 and MBC ranged from 0.00976 – 0.078mg/mL and 0.098– 0.78mg/mL respectively. There was no statistically significant difference between extracts (methanol, acetone, ethanol, ethyl acetate) and the control antibiotic (ciprofloxacin) (P> 0.05). Qualitative phytochemical analysis confirmed the presence of alkaloids, saponins, steroids, tannins and flavonoids in the methanol, acetone,ethanol and ethyl acetate extracts. The results demonstrate that H. africana may contain compounds with therapeutic potentials which can be lead molecules for semi-synthesis of new drugs.

Les pratiques culturales modifient l'architecture des couverts de manière à augmenter ou diminuer le développement des épidémies mais les processus mis en jeu sont complexes ; des modèles mécanistes simulant l'interaction entre plante et pathogène devraient aider à les clarifier. Les modèles de Plantes Virtuelles, qui permettent de décrire explicitement la structure tridimensionnelle de la plante, semblent particulièrement prometteurs pour exprimer les effets de l'architecture de la plante sur le développement des épidémies. L'objectif de cette étude est d'examiner la possibilité de simuler l'effet de l'architecture des plantes sur le développement de la maladie en utilisant un modèle Plante Virtuelle. Dans ce travail, nous nous intéressons au pathosystème blé-Septoria tritici, dans lequel l'architecture joue un rôle important. En effet, les spores de Septoria tritici sont propagées par les éclaboussures de pluie depuis les feuilles infectées du bas du couvert vers les nouvelles feuilles saines. Notre travail s'est appuyé sur un modèle pré-existant d'épidémie de la septoriose, Septo3D. L'architecture du blé a été étudiée pour une gamme de densités et de date de semis. Les différences de phyllochrone entre traitements ont été dans une gamme susceptible de modifier le développement de la septoriose. Ces variations ont été représentées par un modèle descriptif qui tient compte du nombre de feuilles final et de la photopériode. Une description détaillée des variables d'architecture à l'échelle des organes et du couvert a fourni une documentation originale et complète sur la plasticité de l'architecture du blé. Ces données ont été utilisées pour paramétrer la description du blé dans Septo3D. Globalement, les traitements étudiés ont conduit à de fortes différences de la densité de végétation au cours du temps. Les dynamiques de développement de la septoriose ont été suivies pour trois traitements de densités contrastées. Les cinétiques de la maladie simulées par le modèle étaient conformes aux mesures expérimentales. Bien que, l'approche nécessite davantage de validation, les résultats confirment que l'approche Plante Virtuelle apporte un nouvel éclairage sur les processus et les caractéristiques des plantes qui impactent les épidémies. En conclusion, nous proposons quelques perspectives en vue de nouvelles applications et améliorations de l'approche. / Agronomic practices modify crop architecture in ways that may facilitate or hamper disease development. The processes involved are complex and mechanistic models simulating plant-pathogen interaction should help clarifying them. Virtual Plants, i.e. models in which the three-dimensional structure of the plant is explicitly described, appear specially promising to express the effects of the plant architecture on the epidemic development. The objective of this study is to examine the ability to simulate the effect of plant architecture on disease development using a Virtual Plant model.The work focuses on the pathosystem wheat-Septoria tritici, in which architecture plays an important role because spores of Septoria are propagated from infected leaves to upper healthy leaves by rain splash. We build on a pre-existing model of Septoria epidemics, Septo3D. Wheat architecture was examined for a range of sowing date and density treatments. Differences of phyllochron between treatments were in a range sufficient to likely modify epidemic development; they were well represented by a descriptive model depending on photoperiod and final leaf number. A detailed description of architectural variables at the organ and canopy scale provided an original and comprehensive documentation of the plastic response of wheat, which was used for parameterising the wheat description in Septo3D. Overall, the investigated treatments resulted in strong differences in the time course of vegetation density. Septoria dynamics were monitored in a subset of three treatments of contrasted densities. Simulated disease kinetics were consistent with field measurements. Although, the approach needs further validation, results support that virtual plant modelling provides new insights into the processes and plant traits that impact epidemics. We conclude with prospects for further improvements and applications.

Lily leaf spot is an emerging disease of the globally rare Lilium grayi S. Wats., a species endemic to the southern Appalachian Blue Ridge, USA. The species is considered Threatened or Endangered in the three states where it naturally occurs (North Carolina, Tennessee, Virginia). Disease leads to premature senescence of aboveground tissues and curtailment of sexual reproduction. Spore morphology, completion of Koch’s postulates, and DNA sequence data showed the causative agent of lily leaf spot to be Pseudocercosporella inconspicua (G. Winter) U. Braun, a Lilium-specific basidiomycete. Diagnostic disease symptomatology includes amphigenous necrotic lesions with tan to green margins encircling a white to grey powdery mass of conidia. Studies conducted in the field at Roan Mountain, NC/TN, in the largest known population, showed that a visual disease diagnosis based on morphology can be highly accurate in predicting P. inconspicua infection, and that high concentrations of conidia of P. inconspicua are strongly associated with infected L. grayi, but are largely absent or few on uninfected L. grayi and other species. Field inoculation trials using infected L. grayi leaf tissue as inocula resulted in transmission of disease and induction of premature senescence under natural conditions.

Xylella fastidiosa é uma bactéria gram-negativa, não flagelada, agente causal de doenças de importância econômica como a doença de Pierce nas videiras e a clorose variegada dos citros (CVC) nas laranjeiras. O objetivo do presente trabalho foi realizar análises in silico das sequências promotoras dos genes deste fitopatógeno em uma tentativa de arrecadar novas evidências para o melhor entendimento da dinâmica de regulação transcricional de seus genes, incluindo aqueles envolvidos em mecanismos de patogenicidade e virulência. Para tanto, duas estratégias foram utilizadas para predição de elementos cis-regulatórios em regiões promotoras do genoma da cepa referência 9a5c, comprovadamente associada à CVC. A primeira, conhecida como phylogenetic footprinting, foi empregada para identificação de elementos regulatórios conservados em promotores de unidades transcricionais ortólogas, levando em consideração o conjunto de genes de X. fastidiosa e 7 espécies comparativas. O critério para identificação de unidades transcricionais ortólogas, isto é, unidades trancricionais oriundas de espécies distintas e cujos promotores compartilham elementos cis-regulatórios, foi paralelamente estudado utilizando-se informações regulatórias das bactérias modelos: Pseudomonas aeruginosa, Bacillus subtilis e Escherichia coli. Os resultados obtidos com análise de phylogenetic footprinting nos permitiu acessar a rede regulatória transcricional da espécie de forma compreensiva (global). Foram estabelecidas 2990 interações regulatórias, compreendendo 80 motivos distribuídos nos promotores de 56.8% das unidades transcricionais do genoma de X. fastidiosa. Na segunda estratégia recuperamos informações regulatórias experimentalmente validadas em E. coli e complementamos o conhecimento de dez regulons de X. fastidiosa, através de uma metodologia de scanning (varredura), dos quais algumas interações regulatórias já haviam sido previamente descritas por outros trabalhos. Destacamos os regulons de Fur e CRP, reguladores transcricionais globais, que se mostraram responsáveis pela modulação de genes relacionados a mecanismos de invasão e colonização do hospedeiro vegetal entre outros. Por fim, análises comparativas em regiões regulatórias correspondentes entre cepas foram realizadas e diferenças possivelmente associadas a particularidades fenotípicas foram identificadas entre 9a5c e J1a12, um isolado de citros não virulento, e 9a5c e Temecula1, um isolado de videira causador da doença de Pierce. / Xylella fastidiosa is a gram-negative, non-flagellated bacterium responsible for causing economically important diseases such as Pierce\'s disease in grapevines and Citrus Variegated Clorosis (CVC) in sweet orange trees. In the present work we performed in silico analysis on promoter sequences of protein-coding genes from this phytopathogen, including those involved in virulence and pathogenic mechanisms, in an attempt to better understand the underlying transcriptional regulatory dynamics. Two strategies for cis-regulatory elements prediction were applied on promoter sequences from 9a5c strain genome, a proven causal agent of CVC. The first one, known as phylogenetic footprinting, involved the prediction of regulatory motifs conserved on promoter sequences of orthologous transcription units from X. fastidiosa and a set of 7 comparatives species. The criteria to identify orthologous transcription units, i. e., those from different species and whose promoter sequences share at least one common regulatory motif, was studied based on regulatory information available for model organisms: Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli. The results obtained with the phylogenetic footprinting analysis permitted us to access the underlying transcriptional regulatory network from the species in a comprehensive manner (genome-wide), with a total of 2990 regulatory interactions corresponding to 80 predicted motifs distributed on promoter sequences of 56.8% of all transcription units. In the second strategy regulatory information from E. coli was recovered and used to expand the knowledge of ten regulons in X. fastidiosa, through a scanning process, of which some regulatory interactions were previously described by independent studies. We emphasize some genes related to host invasion and colonization present in the Fur and CRP regulons, two global transcription regulators. Lastly, comparative analysis on corresponding regulatory regions among strains were performed and differences possibly associated to phenotypic variation were identified between 9a5c and J1a12, a non-virulent strain isolated from orange trees, and between 9a5c and Temecula1, a strain associated to Pierce\'s disease on grapevines.

No description available.

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.