In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...

In the United States corn is one of the leading agricultural products and one of the top exports. The majority of U.S corn is grown in the Midwestern region of the U.S. known as the Corn Belt where the bacterial disease Stewart's Wilt reduces crop yield. Pantoea stewartii subsp. stewartii (Pss) is transmitted into corn via the corn flea beetle insect vector, Chaetocnema pulicaria. As the beetle feeds on the corn plant leaves, Pss deposited in beetle feces enter the leaf through lesions. The early stage of Pss infection begins in the mesophyll apoplast of the corn leaf where a type III secretion system (T3SS) and its associated effectors induce water soaking (WS) and nutrient release. Ultimately, Pss will enter the plant xylem apoplast (will be referred to as the xylem) and use quorum sensing (QS) to initiate a lifestyle shift. Within the xylem, Pss grows to high cell density and secretes exopolysaccharide (EPS), forming a biofilm which eventually obstructs water transport, leading to wilting and necrosis. Previous Tn-Seq experiments provided insights into genes that are essential for in planta survival, including the master transcriptional regulator, Leucine-responsive Regulatory Protein (Lrp). To better understand the role of Lrp when Pss inhabits the xylem, RNA-Seq experiments comparing Pss wild-type and ∆lrp strains grown in planta were conducted to ascertain differential gene expression. The RNA-Seq data was further analyzed using DESeq2 and validated using qRT-PCR methods. Following validation, the Pss genome was annotated using Blast2GO software and genes upregulated and downregulated by Lrp were linked with biological processes. Lrp was found to be involved in regulating capsule biosynthesis and nitrogen-associated assimilation and metabolism during Pss survival in the xylem. This provides further insight into how Pss contends with harmful host defense compounds and extracts scarce nutrients present in the in planta xylem environment. A corn xylem fluid extraction method was developed that has enabled more physiologically relevant growth experiments to be conducted in vitro. Extracted xylem fluid was used to grow Pss wild-type and ∆lrp mutant strains as monocultures to observe any differences in growth patterns in different growth media. When grown separately in xylem fluid or Luria-Bertani (LB) medium, the Pss wild-type and ∆lrp mutant strains grew at similar rates and to final cell densities . The Pss ∆lrp mutant strain greatly outcompeted the wild type when grown together in LB medium. However, when the two Pss strains were growth together in xylem fluid, a shift in relative competition was observed, providing evidence of the wild type slightly outcompeting the ∆lrp mutant. Analysis of the composition of extracted xylem fluid through metabolomics will help define the nutrients specifically utilized by Pss in planta. Altogether, the outcome of these research projects was to provide pertinent discoveries to contribute to understanding the mechanisms used by Pss to survive in the corn xylem environment. Broadly, increased understanding of Pss pathogenesis may translate to understanding pathogenesis mechanisms in other bacterial wilt-disease causing plant pathogens. / Master of Science / Corn is a significant agricultural product and export in the United States. This important crop is used as a food source for humans, a primary nutrient source of livestock, and a major ingredient for corn-based industries manufacturing commodities such as culinary additives, biofuels, and preservatives. Certain bacteria are greatly beneficial to plants, able to increase their overall health and growth, while other bacteria share a more insidious relationship with plants and cause disease. The research discussed in this thesis focuses on the bacterial pathogen Pantoea stewartii subspecies stewartii (Pss), the causal agent of Stewart's wilt disease in corn. Pss grows inside the plant xylem (vascular tissues which distribute water throughout the plant) and forms a biofilm that causes plant wilt leading to lower crop yield and even plant death. Previous research on Pss identified important genes for successful Pss survival inside the corn plant xylem. One of those genes codes for the Leucine-responsive Regulatory Protein (Lrp). Using a combination of experimental (RNA-Seq) and computational (bioinformatics) analyses, Lrp was found to control other genes related in biological process important for living inside the plant, necessary for the metabolism of available nutrients and production the protect slime layer within biofilm. By better understanding the key bacterial genes needed for Pss to grow inside the xylem, new disease intervention strategies can be developed to disrupt these genes and impede the ability of the bacterium to infect the plant. A second part of this research project was to develop a method for extracting corn xylem fluid from the plant. Using this extracted xylem fluid, experiments could be conducted in the laboratory to study Pss growth in more detail. The original strain of Pss (wild type) was grown separately and in combination with a Pss mutant lacking the Lrp gene in the extracted xylem fluid. Both strains grew similarly in the xylem fluid, but the wild type slightly outcompeted the mutant strain when they were grown in competition. Future work in the lab will use extracted xylem fluid to determine its precise nutrient composition and the development of synthetic xylem fluid that will enable a more detailed analysis of mechanisms used by Pss to grow in the xylem. Work on Pss serves as a model for the study of other bacterial wilt-disease causing pathogens.

Pantoea stewartii subsp. stewartii is a bacterium that causes Stewart’s wilt disease in corn plants. The bacteria are transmitted to the plants via an insect vector, the corn flea beetle Chaetocnema pulicaria. Once in the plant, the bacteria migrate to the xylem and grow to high cell densities, forming a biofilm by secreting excess capsular exopolysaccharide, which blocks water transport and causes wilting. The timing of virulence factor synthesis is regulated by the cell-density dependent quorum sensing (QS) system. Such temporal regulation is crucial in establishing infection and is orchestrated by the QS-dependent transcriptional regulator EsaR. EsaR represses expression of capsular exopolysaccharide at low cell densities. At high cell densities, an acylated homoserine lactone (AHL) molecule produced during growth by the cognate AHL-synthase EsaI accumulates. The AHL binds to and inactivates EsaR, causing derepression of capsule production. EsaR is a member of the LuxR family of QS-dependent transcriptional factors. Most LuxR homologs are unstable and/or insoluble in the absence of AHL which has hindered structural studies. Chapter Two describes the changes in the structure of EsaR due to binding of AHL ligand as determined through biochemical methods. EsaR was found to be stable and retain its multimeric state in the absence or presence of AHL, but intra- and inter-domain changes occurred that affect its DNA-binding capacity. Apart from repressing expression of capsule at low cell-densities, EsaR represses its own expression and activates production of a small RNA, EsaS, with unknown function. In Chapter Three a proteomic approach was used to identify an additional 30 QS-controlled proteins. Genes encoding three of these proteins are directly regulated by EsaR and the EsaR binding sites in the respective promoters were defined. In Chapter Four, a high-throughput RNA-Seq method identified even more genes in the QS regulon that the proteomic approach overlooked. RNA-Seq analysis of rRNA-depleted RNA from two strains of P. stewartii was used as a screen to help identify 11 promoters, subsequently shown to be directly regulated by EsaR in vitro. Most of the genes controlled by QS grouped into three major physiological responses, capsule & cell wall production, surface motility & adhesion and stress response. In Chapter Five, the role of two QS regulated genes, dkgA (encoding 2, 5-diketo-D-gluconate) and lrhA (encoding a repressor of chemotaxis, adhesion and motility), in plant virulence were examined. These studies have better characterized the QS regulator EsaR and its interaction with the AHL ligand, and shown that QS has a more global response in P. stewartii than previously recognized. Further characterization of the genes identified in this study could facilitate identification of factors crucial in plant pathogenesis or insect-vector symbiosis and aid in the development of molecular-based approaches for possible disease intervention. / Ph. D.

Corn is a valuable agricultural commodity in the United States and in the world. The causal agent of Stewart's wilt disease in corn, Pantoea stewartii subsp. stewartii, is a bacterial phytopathogen that is vectored into the plant by the corn flea beetle, Chaetocnema pulicaria. After entering the apoplast of the leaf, the bacteria cause water soaking symptoms before traveling to the plant xylem to form a dense biofilm, thereby blocking water transport and inducing necrosis and wilt. This results in reduced crop yield and may even lead to death of the corn plant. To better understand the in planta requirements of this pathogen, a whole transcriptome study was performed via RNA-Seq to determine genes differentially expressed in the bacteria while inside the corn. It was found that nutrient transporters and stress response genes were upregulated specifically when the bacteria are in their host plant, suggesting a response to nutrient availability and host defense in the xylem. Further elucidation of the genes required for the P. stewartii in planta lifestyle was performed via a reverse genetics approach where in-frame gene deletions and the corresponding complementation strains were constructed for genes that had shown a fitness defect in corn based on a previously published Tn-Seq study: genes encoding seven transcription factors, nsrR, iscR, lrp, nac, DSJ_00125, DSJ_03645, and DSJ_18135, as well as a hypothetical protein DSJ_21690. Investigation of the physiological role of these genes was performed using in planta virulence and competition assays for all strains. An in planta qRT-PCR analysis of bacterial gene transcription was also completed for the strains with deletions in nsrR and iscR. In vitro assays were performed on all strains to determine their capsule production and motility phenotypes. Taken together, it was seen that iscR is important for colonization capabilities in planta, both NsrR and IscR act as regulators, and lrp is important for full disease capabilities, perhaps due to reduced capsule and motility phenotypes. These findings lay the groundwork for finding potential disease intervention strategies not only against P. stewartii, but also other xylem-dwelling bacterial phytopathogens. In addition to exploring ways to enhance crop yield, an additional research area was on repurposing a byproduct of corn ethanol production, syrup. It was hypothesized that this corn-based syrup could be utilized as a carbon source to grown bacteria. In turn, the resulting bacterial biomass could then be added as a fish feed supplement in aquaculture. Syrup was tested as a growth medium for individual soil bacterial isolates as well as a full mixed bacterial community consortium to determine which bacteria could grow most efficiently, both in rate and yield. It was found that the highest growth rate and yield was from Bacillus species, some of which may have probiotic benefits to fish. Ultimately, the collective outcomes from these projects in basic research about a bacterial corn pathogen and applied research about beneficial microbes grown on a corn-based substrate are expected to improve scientific endeavors as well as agricultural practices. / Doctor of Philosophy / Corn is a top agricultural commodity in the United States, as a food for human consumption, a primary nutrient source used in animal feed, and a substrate consumed during biofuel production. These various corn-based industries are impacted by bacteria in multiple ways; in some cases, bacteria may cause disease that reduces crop yield, but other bacteria serve beneficial roles that enhance health. This dissertation research describes studies about the bacterium that causes Stewart's wilt disease in corn, Panteoa stewartii subsp. stewartii. In an initial experiment, the genes that P. stewartii expresses at the highest levels when it grows inside the corn plant were identified. These genes were deduced to be important for the ability of the bacterium to live successfully in this environment. This work was followed up with a more specific approach that examined the role of certain genes that were predicted to be master regulators of the expression of other genes in the ability of the P. stewartii to colonize the plant and/or cause disease. By identifying key bacterial genes, disease intervention strategies to combat Stewart's wilt and other similar bacterial plant pathogen diseases might become possible. Protecting corn yields is important for ethanol production. The final study of this dissertation examined the ability of bacteria to grow on a byproduct of ethanol production called syrup. The goal was to then use the biomass of these beneficial microbes as a food source for animals being produced in aquaculture facilities. Among the species tested, the highest growth rate and yield was from Bacillus subtilis, a safe-to-eat bacterium that has known beneficial health properties when consumed by fish. Overall, the research studies that were completed for this dissertation have the potential to improve agricultural practices by decreasing corn disease leading to increased corn yield and developing new downstream corn-based animal feed products.

O fungo Moniliophthora perniciosa, fitopatógeno responsável pela doença vassoura-de-bruxa no cacaueiro, é responsável por reduzir a produtividade de frutos de cacau nas Américas. Visto que os métodos atuais de controle desta doença não são eficientes, faz-se necessário a busca por compostos para seu controle químico. Os produtos naturais têm contribuído na síntese de novos pesticidas e a família Solanaceae é reconhecida como uma fonte promissora de compostos antifúngicos. Neste contexto, este trabalho teve por objetivo explorar o potencial biológico e químico de metabólitos secundários produzidos por plantas da família Solanaceae com atividade antifúngica à M. perniciosa. Para tanto, extratos aquosos de folhas de dez plantas foram avaliados quanto sua atividade inibitória a M. perniciosa e, observou-se maior atividade antifúngica pelos extratos de Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Destas, as plantas S. seaforthianum e B. uniflora mostram-se como promissoras e foram selecionadas para isolamento de seus compostos ativos. A partir do extrato aquoso de S. seaforthianum obteve-se duas frações constituídas por saponinas furostanas, a mistura (25R)-karatavioside C / (25R)-purpureagitoside, com CI50 de 40,9 &mu;g mL-1, e a mistura (25R)-timosaponin H1/ (25R)-uttroside B, com CI50 de 22,3 &mu;g mL-1 ao crescimento micelial. E, a partir do extrato aquoso de B. uniflora obteve-se três compostos da classe das saponinas espirostanas, o composto karatavioside A e os compostos parcialmente identificados BuM8i4, com aglicona identificada como (25R)-yucagenina, e BuM8i6, com aglicona identificada como (25R)-diosgenina, todos com CI50.< 15,63 &mu;g mL-1 ao crescimento micelial de M. perniciosa. Todos os compostos caracterizados neste trabalho estão sendo relatados pela primeira vez a partir das plantas estudas e também para atividade antifúngica. / The fungal phytopathogen Moniliophthora perniciosa is the causal agent of Witches\' Broom disease and the main responsible by limiting cacao production in Americas. The current disease control methods are not efficient and new antifungal compounds are necessary to the chemical management. Natural products has contributed to the development of natural product-based pesticides and the Solanaceae plants are known as a promising source of antifungal compounds. In this context, this work aimed to explore the biological and chemical potential of secondary metabolites produced by Solanaceae plants with antifungal activity against M. perniciosa. For this, antifungal activity of water extracts from leaves of ten plants was evaluated against M. perniciosa and the best results were observed by using extracts from Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Among these plants, S. seaforthianum and B. uniflora were selected for active compounds isolation due to their promising characteristics. From water extract of S. seaforthianum two furostan saponin fractions were obtained, the mixture (25R)-karatavioside C / (25R)- purpureagitoside, with IC50 of 40,9 &mu;g mL-1, and the mixture (25R)-timosaponin H1/ (25R)-uttroside B, with IC50 of 22,3 &mu;g mL-1 to the mycelial growth. From water extract of B. uniflora three spirostan saponin compounds were obtained, the compound karatavioside A and the compounds partially identified BuM8i4, with aglycone identified as (25R)-yucagenina, and BuM8i6, with aglycone identified as (25R)- diosgenina, all these compounds showed IC50< 15,63 &mu;g mL-1 to the mycelial growth of M. perniciosa. All compounds characterized in this study were obtained for the first time from these plants and also described about their antifungal activity.

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante &#916;xadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes &#916;xadA1 e &#916;xadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa. / Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce\'s disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the &#916;xadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both &#916;xadA1 and &#916;xadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce\'s disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence.

O fungo Moniliophthora perniciosa, fitopatógeno responsável pela doença vassoura-de-bruxa no cacaueiro, é responsável por reduzir a produtividade de frutos de cacau nas Américas. Visto que os métodos atuais de controle desta doença não são eficientes, faz-se necessário a busca por compostos para seu controle químico. Os produtos naturais têm contribuído na síntese de novos pesticidas e a família Solanaceae é reconhecida como uma fonte promissora de compostos antifúngicos. Neste contexto, este trabalho teve por objetivo explorar o potencial biológico e químico de metabólitos secundários produzidos por plantas da família Solanaceae com atividade antifúngica à M. perniciosa. Para tanto, extratos aquosos de folhas de dez plantas foram avaliados quanto sua atividade inibitória a M. perniciosa e, observou-se maior atividade antifúngica pelos extratos de Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Destas, as plantas S. seaforthianum e B. uniflora mostram-se como promissoras e foram selecionadas para isolamento de seus compostos ativos. A partir do extrato aquoso de S. seaforthianum obteve-se duas frações constituídas por saponinas furostanas, a mistura (25R)-karatavioside C / (25R)-purpureagitoside, com CI50 de 40,9 &mu;g mL-1, e a mistura (25R)-timosaponin H1/ (25R)-uttroside B, com CI50 de 22,3 &mu;g mL-1 ao crescimento micelial. E, a partir do extrato aquoso de B. uniflora obteve-se três compostos da classe das saponinas espirostanas, o composto karatavioside A e os compostos parcialmente identificados BuM8i4, com aglicona identificada como (25R)-yucagenina, e BuM8i6, com aglicona identificada como (25R)-diosgenina, todos com CI50.< 15,63 &mu;g mL-1 ao crescimento micelial de M. perniciosa. Todos os compostos caracterizados neste trabalho estão sendo relatados pela primeira vez a partir das plantas estudas e também para atividade antifúngica. / The fungal phytopathogen Moniliophthora perniciosa is the causal agent of Witches\' Broom disease and the main responsible by limiting cacao production in Americas. The current disease control methods are not efficient and new antifungal compounds are necessary to the chemical management. Natural products has contributed to the development of natural product-based pesticides and the Solanaceae plants are known as a promising source of antifungal compounds. In this context, this work aimed to explore the biological and chemical potential of secondary metabolites produced by Solanaceae plants with antifungal activity against M. perniciosa. For this, antifungal activity of water extracts from leaves of ten plants was evaluated against M. perniciosa and the best results were observed by using extracts from Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Among these plants, S. seaforthianum and B. uniflora were selected for active compounds isolation due to their promising characteristics. From water extract of S. seaforthianum two furostan saponin fractions were obtained, the mixture (25R)-karatavioside C / (25R)- purpureagitoside, with IC50 of 40,9 &mu;g mL-1, and the mixture (25R)-timosaponin H1/ (25R)-uttroside B, with IC50 of 22,3 &mu;g mL-1 to the mycelial growth. From water extract of B. uniflora three spirostan saponin compounds were obtained, the compound karatavioside A and the compounds partially identified BuM8i4, with aglycone identified as (25R)-yucagenina, and BuM8i6, with aglycone identified as (25R)- diosgenina, all these compounds showed IC50< 15,63 &mu;g mL-1 to the mycelial growth of M. perniciosa. All compounds characterized in this study were obtained for the first time from these plants and also described about their antifungal activity.

Submitted by JELENA PURIC (jelena0puric@gmail.com) on 2018-04-16T12:39:06Z No. of bitstreams: 1 Jelena Puric Dissertação de Mestrado.pdf: 2830343 bytes, checksum: 555cfd034f96e02e12c50107d2077d5f (MD5) / Rejected by Adriana Aparecida Puerta null (dripuerta@rc.unesp.br), reason: Prezada Jelena, Solicitamos que realize uma nova submissão seguindo as orientações abaixo: 1) A folha de aprovação está em local errado. No arquivo enviado, está em primeiro lugar, antes da capa. O correto de acordo com as normas vigentes é que a folha de aprovação venha após a ficha catalográfica (que o seu arquivo também não tem) e venha antes do Resumo. 2) Falta a ficha catalográfica, que deve ser solicitada pelo site da biblioteca da Unesp de Rio Claro e inserida após a folha de rosto no arquivo pdf e no verso da folha de rosto na versão impressa. O documento enviado não foi excluído. Para revisá-lo e realizar uma nova tentativa de envio, acesse: https://repositorio.unesp.br/mydspace Agradecemos a compreensão e aguardamos o envio do novo arquivo. Atenciosamente, Biblioteca Campus Rio Claro Repositório Institucional UNESP on 2018-04-16T14:20:27Z (GMT) / Submitted by JELENA PURIC (jelena0puric@gmail.com) on 2018-04-16T14:31:14Z No. of bitstreams: 1 Dissertação Jelena Puric.pdf: 2938364 bytes, checksum: b7379106bbe1a2cdf5ce930d5337096b (MD5) / Approved for entry into archive by Adriana Aparecida Puerta null (dripuerta@rc.unesp.br) on 2018-04-16T18:28:58Z (GMT) No. of bitstreams: 1 puric_j_me_rcla.pdf: 2383486 bytes, checksum: e2cb2e6c5d3d71426040a339c65c55ce (MD5) / Made available in DSpace on 2018-04-16T18:28:58Z (GMT). No. of bitstreams: 1 puric_j_me_rcla.pdf: 2383486 bytes, checksum: e2cb2e6c5d3d71426040a339c65c55ce (MD5) Previous issue date: 2018-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho teve como objetivo obter extratos contendo metabólitos secundários de fungos filamentosos isolados de sedimentos marinhos da Antártica e avaliar sua potencial atividade antibacteriana em Xanthomonas citri subsp. citri, Xanthomonas euvesicatoria e Xanthomonas axonopodis pv. passiflorae (bactérias fitopatogênicas que causam doenças em cítricos, pimenta e tomate, e maracujá, respectivamente). Entre os 90 extratos brutos intracelulares e extracelulares obtidos a partir de fungos, 19 mostraram a capacidade de impedir o crescimento de X. citri subsp. citri e X. euvesicatoria in vitro e 22 mostraram a capacidade de dificultar o crescimento de X. axonopodis pv. passiflorae in vitro. Os extratos extracelulares inibiram em média 97,12% de X. citri subsp. citri, 95,94% de X. euvesicatoria e 96,58% de X. axonopodis pv. passiflorae a 3,0 mg / mL. / This work aimed to obtain extracts containing secondary metabolites from filamentous fungi isolated from marine sediments from Antarctic and assess their potential antibacterial activity on Xanthomonas citri subsp. citri, Xanthomonas euvesicatoria and Xanthomonas axonopodis pv. passiflorae (phytopathogenic bacteria causing diseases in citrus, pepper and tomato, and passionfruit, respectively). Among the 90 raw intracellular and extracellular extracts obtained from fungi, 19 showed the ability to hamper the growth of Xanthomonas citri subsp. citri and X. euvesicatoria in vitro and 22 showed the ability to hamper the growth of X. axonopodis pv. passiflorae in vitro. The extracellular extracts inhibited on average 97,12% of Xanthomonas citri subsp, 95,94% of X. euvesicatoria and 96,58% of X. axonopodis pv. passiflorae at 3,0 mg/mL.

O fitopatógeno Xylella fastidiosa coloniza o lúmen dos vasos do xilema de seus hospedeiros e o aparelho bucal do inseto-vetor. É responsável por doenças de extrema gravidade em videira, laranjeira, e oliveira, entre outras plantas de relevância econômica. Há evidência de especificidade entre cepas de X. fastidiosa e as diferentes espécies de plantas que colonizam, mas as bases moleculares desta interação são desconhecidas. O objetivo central deste trabalho foi elucidar o repertório completo de genes expressos e/ou diferencialmente expressos por diferentes cepas X. fastidiosa em meios e tempos de cultivo distintos e relacionar as respostas transcricionais a mecanismos de virulência, patogenicidade e especificidade ao hospedeiro. Foram sequenciados, analisados e comparados os transcritomas de cepas de laranjeiras (9a5c, J1a12, U24d e Fb7), de cafeeiro (3124), de hibisco (Hib4), ameixeira (Pr8x) e de videira (Temecula1), no início e fim da fase a exponencial de crescimento populacional em meio rico PWG e em meio mínimo PIM6, que mimetiza a seiva do xilema. Foi observado que a maioria dos genes de X. fastidiosa é expressa, ainda que, dependendo da cepa e da condição experimental, 40-80% dos transcritos sejam pouco abundantes. Por outro lado, foi verificado um conjunto de transcritos muito abundantes, uma parte deles comuns a todas as cepas, e que incluem os ncRNAs 6S e RNAse P, além de transcritos de microcinas, proteases, lipases, proteínas de resposta a estresse e proteínas de função desconhecida. Além da definição de perfis transcricionais, foram descritas as regiões 5\' e 3\' não-traduzidas dos transcritos. As estruturas de 545 e 386 operons expressos, respectivamente pelas cepas 9a5c e Temecula1, também foram mapeadas, e pela primeira vez foi obtido o perfil de sRNAs expressos por X. fastidiosa. As análises de expressão diferencial entre transcritomas das duas fases de crescimento no mesmo meio indicam que o estresse gerado pela limitação nutricional do meio PIM6 exigiu mudanças mais drásticas na expressão gênica do que no meio PWG. Foi também observado que diferentes cepas respondem de maneiras distintas a uma mesma condição, indicando que genes ortólogos são regulados de formas diferentes. Além disso, a transcritômica comparativa revelou diferenças relevantes na regulação gênica de cepas de hospedeiros vegetais distintos que podem estar relacionadas à especificidade ao hospedeiro. Por fim, as análises dos transcritomas evidenciaram vários genes candidatos que poderão ser futuramente investigados quanto ao seu papel na biologia e na virulência de X. fastidiosa. / The phytopathogenXylella fastidiosa colonizes the lumen of xylem vessels from its hosts and the mouth apparatus of the insect-vector. It is responsible for severe diseases in grapevine, orange and olive trees, among other plants of economic relevance. There is evidence for specificity between X. fastidiosa strains and the different plant species they colonize, but the molecular bases of this interaction are unknown. The main objective of this work was elucidate the complete repertoire of expressed and/or differentially expressed genes by different X. fastidiosa strains in distinct media and growth times and associate transcriptional responses to virulence mechanisms, pathogenicity and host specificity. Transcriptomes of orange strains (9a5c, J1a12, U24d and Fb7), coffee (3124), hibiscus (Hib4), plum (Pr8x) and grapevine (Temecula1), were sequenced, analyzed and compared from cells at the beginning and end stages of exponential growth phase in rich medium PWG and in minimum medium PIM6, which mimics xylem sap. It was observed that the majority of X. fastidiosa genes is expressed, although, depending of the strain and experimental condition, 40-80% of transcripts are less abundant. On the other hand, it was verified a set of more abundant transcripts, some of them shared by all strains, including 6S and RNAse P ncRNAs as well as transcripts for microcins, proteases, lipases, stress response proteins and proteins of unknown function. Besides the definition of transcriptional profiles, 5\' and 3\' untranslated regions of transcripts were described. The structure of 545 and 386 expressed operons, respectively for 9a5c and Temecula1 strains, were also mapped, and for the first time the expressed profile of sRNAs in X. fastidiosa was obtained. The differential expression analyzes between transcriptomes of two growth phases in the same medium indicate that the stress generated by nutritional limitation of PIM6 medium required more drastic changes in gene expression than PWG medium. It was also observed that different strains respond in distinct manners to a same condition, indicating that orthologous genes are regulated in different ways. Moreover, comparative transcriptomics revealed relevant differences in gene regulation of strain of distinct plant hosts that can be related to host specificity. Lastly, transcriptomic analyzes pointed to several gene candidates that could be further investigated for their roles in X. fastidiosa biology and virulence.

Xylella fastidiosa é uma bactéria Gram-negativa, do subgrupo das Gama-Proteobactérias, não-flagelada, que coloniza o xilema de diversas plantas cultivadas e silvestres, podendo ser causadora de doenças. Sua disseminação é feita por insetos conhecidos como cigarrinhas. Genomas de cepas de X. fastidiosa isoladas de distintos hospedeiros já foram sequenciados completa ou parcialmente: 9a5c de citros; Temecula-1 e GB514 de videira; Dixon, M12 e M23 de amendoeira; Ann-1 de espirradeira e EB92-1, isolada de sabugueiro e utilizada como bio-controle para Doença de Pierce de videiras. Estudos de genômica comparativa associados a abordagens de genômica funcional e de genética molecular têm possibilitado o estudo detalhado de mecanismos potencialmente relevantes tanto para a colonização de plantas e insetos por este fitopatógeno como para o desenvolvimento de sintomas associados a doenças específicas em seus respectivos hospedeiros vegetais. Exceto o genoma de 9a5c, todos os demais genomas conhecidos são de cepas isoladas na América do Norte. Neste trabalho descrevemos o pirossequenciamento dos genomas da cepa J1a12, que exibe fenótipo não-virulento em citros, e das cepas Pr8x e Hib4, isoladas, respectivamente, de ameixeira e hibisco. A cepa J1a12 possui além de seu cromossomo principal de 2.788.789 pb dois plasmídeos, pXF51 e pXF27, respectivamente de 51.180 pb e 27.268 pb. pXF51 já foi descrito também na cepa de citros 9a5c e pXF27 tem similaridade com outros plasmídeos de cepas de X. fastidiosa norte-americanas isoladas de amoreira e videira. A cepa Pr8x possui além de seu cromossomo principal de 2.666.242 pb um plasmídeo, pXF39, de 39.580 pb, o qual contém a maioria das CDS presentes no pXF51. A cepa Hib4, isolada de hibisco, tem o maior cromossomo (2.813.297 pb) e também o maior plasmídeo (pXF64 com 64.251 pb) já descritos para X. fastidiosa. pXF64 apresenta extensa similaridade com o plasmídeo pBVIE04 de Burkholderia vietnamensis cepa G4, sendo descrito pela primeira vez em cepas de X. fastidiosa. Análises comparativas destes genomas possibilitaram a identificação de alterações que podem ser correlacionadas com os fenótipos exibidos por estas cepas, além da variedade e diversidade de regiões relacionadas a bacteriófagos e de plasmídeos que co-existem nas diferentes cepas deste fitopatógeno. / Xylella fastidiosa is a Gram-negative bacteria, of the Gamma-proteobacterium subgroup, non-flagellated that colonizes the xylem of several cultivated and wild plants, where may cause disease. The bacterium is spread by insects known as sharpshooters. Genomes of X. fastidiosa strains isolated from different hosts have been completely or partially sequenced: 9a5c from citrus; Temecula-1 and GB514 from grapevine; Dixon, M12 and M23 from almond tree; Ann-1 from oleander and EB92-1, isolated from elderberry and used as bio-control for Pierce\'s disease of grapevines. Comparative genomics studies associated with approaches from functional genomics and molecular genetics have allowed a detailed study of mechanisms potentially relevant to the colonization of plants and insects by this pathogen as well as to the development of symptoms associated with specific diseases in their respective host plants. Except for 9a5c, all other known genomes are from strains isolated in North America. Here we describe the pyrosequencing of the genomes of strain J1a12, which displays non-virulent phenotype in citrus and of Pr8x and Hib4 strains isolated, respectively, from plum and hibiscus. J1a12 has a main chromosome of 2,788,789 bp and two plasmids, pXF51 and pXF27, respectively of 51,180 bp and 27,268 bp. pXF51 has been described also in the citrus strain 9a5c and pXF27 has similarity with other plasmids found in North American strains isolated from mulberry tree and grapevine. The strain Pr8x has a main chromosome of 2,666,242 bp and one plasmid, pXF39, of 39,580 bp which present similarities with pXF51. Hib4, the strain isolated from hibiscus, has the largest chromosome (2,813,297 bp) and the largest plasmid (pXF64 with 64,251 bp) described for X. fastidiosa. pXF64 shows extensive similarity with the plasmid pBVIE04 of Burkholderia vietnamensis G4 strain and is described for the first time in X. fastidiosa. Comparative analyzes of these genomes have identified several differences that may be correlated with the phenotypes displayed by these strains, in addition to the variety and diversity of regions related to bacteriophages and plasmids that co-exist in different strains of this pathogen.