In this thesis we have focused on the induction of targeted epigenetic changes using modified Potato Virus X vector (PVX, Potato virus X). The gene coding sequence phytoene desaturase (PDS) was isloated from Solanum tuberosum and Nicotiana benthamiana plants using PCR. Primers were specifically designed as universal for both plant species and their Tm temperatures was in the range of 5 °C. The primers also added flanking restriction sites XhoI and ClaI to simplify cloning of PDS PCR products into full length PVX viral vector in AS orientation. The PVX viral vector pGR106 was later introduced into the plants using Agrobacterium tumefaciens. Infection led to the degradation of the target transcript and to efficient gene silencing (PTGS posttranscriptional gene silencing). The process has been manifested by complete or partial fading of a green plant tissue phenotype.. Some of the plants were aslso inoculated in in vitro conditions, for which a novel technique has been implemented.

Orientadores: Dagmar Ruth Stach-Machado, Gisele Abigail Montan Torres / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T19:49:54Z (GMT). No. of bitstreams: 1 Lorenzi_MarcelSalmeron_M.pdf: 4641112 bytes, checksum: 98293f35d81cf7287376bbbf8a203993 (MD5) Previous issue date: 2009 / Resumo: No Brasil a batata é considerada uma das principais hortaliças produzidas, tanto em área plantada quanto em consumo. A produção anual é em torno de 2 milhões de toneladas, ocupando uma área superior a 130 mil hectares, gerando cerca de 500 mil empregos. Nos últimos anos, a bataticultura brasileira vem sofrendo perdas significativas devido ao aumento da incidência de viroses, principalmente provocadas pelo vírus Y da batata (Potato virus Y - PVY). Mundialmente, são conhecidas pelo menos três linhagens distintas do vírus Y: PVYO, PVYC e PVYN, cujos sintomas variam de acordo com o ambiente e a planta hospedeira. A linhagem PVYN apresenta uma variante denominada PVYNTN, que provoca sintomas de mosaico foliar mais intenso e a indução de formação de anéis necróticos nos tubérculos. A presença de focos de infecção de PVY em índices acima de 4% (limite de tolerância regulamentada pelo Ministério de Agricultura, Pecuária e Abastecimento) é suficiente para condenar o uso de determinadas lavouras para a produção de batata-semente, além de representar grandes danos em cultivos comerciais de batata para consumo. Já o PVX pode representar um grande problema quando em associação com o PVY. É um vírus cosmopolita, sendo transmitido mecanicamente e caracterizado por mosaico leve nas folhas, mais visíveis na metade inferior da planta. Atualmente, a análise de fitossanidade e a indexação viral da batata-semente destinada à multiplicação comercial no Brasil são efetuadas através da combinação da utilização de plantas indicadoras, que demanda tempo, e por testes sorológicos como ELISA, os quais representam custos adicionais à cadeia produtiva, uma vez que o Brasil é dependente da importação dos antisoros de detecção. Deste modo, considerando a relevância do estabelecimento de testes de diagnóstico nacionais que permitam a identificação específica dos vírus Y e X, em virtude da grande incidência de infecções do PVY e da grande necessidade de proteção contra o aumento da introdução do PVX, com oagravante de grandes prejuízos na co-infecção, os objetivos específicos deste projeto foram produzir soros policlonais específicos para o PVX e PVY, estabelecer ELISA indireto e desenvolver diagnóstico molecular dos vírus através de primers específicos e do IC-RTPCR. Esse trabalho conseguiu isolar e purificar os vírus X e Y através da indexação biológica e protocolos de extração. Com isso, foi possível produzir e caracterizar os anticorpos policlonais para o PVY e o PVX, com grande especificidade e poder de detecção. Após essas etapas, foi realizado a construção de primers específicos, e o desenvolvimento de técnicas moleculares de detecção baseadas na extração de material genético de folhas infectadas e PCR com os respectivos primers, obtendo-se os respectivos fragmentos esperados, concluindo com sucesso o estabelecimento do teste. Por fim, tentouse estabelecer o IC-RT-PCR, todavia o tamanho reduzido das partículas virais inviabilizou a realização do teste, mas a execução da detecção por ELISA e molecular separadamente foi estabelecido com sucesso, permitindo agregar novas tecnologias nacionais e contribuindo com a redução dos custos para a execução dos testes de fitossanidade, importantes e imprescindíveis para o controle das viroses predominantes na bataticultura nacional. / Abstract: The potato culture (Solanum tuberosum L.) represents the fourth major vegetal production in the world, reaching productivity indexes which overcome that of cerals in 5 times. Potato crop may be affected by several disease. Actually, some references point out near 40 in a total of 70 disorders are caused by viral agents. Among the most important viruses of potato, virus X and Y usually cause major concern, due to their harmfull effects in productivity and comercialization of potato. Nowadays, the viral infecctions have found their way into Brazil's potato production reagions in a very fast maner. The state of São Paulo is the most afeccted. Althought potato crop is among the stocks 10 major crops in the country, its production relies on virus-free imported potato-seeds to mantain its production. This is not only responsable by a considerable part of the production costs, but also represents a dangerous entrance door for a number of exotic pathogens inexistent in our territory. Thus, a fitosanity control of imported seed-potato lots, which enter Brazil via Santos harbor in the State of São Paulo, has fundamental importance. The usage of imunodiagnosis like ELISA, althought being efficient and widely applied, results in an additional cost due to the dependency of Brazil on the importation of policlonal antisera and monoclonal antibodys necessaries for the proper detection of the most important virus afeccting the national potato plantations. It has becoming a must for the seed-potato production a program in Brazil to have our independence of the diagnoses antiserum reagents for ELISA to at least the most important seed-potato viruses. The independence on importation of these biotecnological products and the modernization of imunodiagnosis by applying molecular techniques with specifics primers and like IC-RT-PCR (ImmoCapture - Reverse Transcriptase - Polimerase Chain Reaction), which makes possible to detect the major potato's virus in a fast, efficient and cheap way in order to screen the sanity of the material produced in the country, leading to a increase in production and stating the quality of it. This work isolated and purified the virus X and Y by biological indexing and extraction protocols. Therefore, it was possible to produce and to characterize the polyclonals antiserum to PVX and to PVY, with great specificity and power of detection. After these steps, it was made specifics primers and the development of molecular detection techniques based on extraction of genetic material from infected leaves and PCR with the primers. The expected fragments was obtained, concluding with successful the establishment of the test. Finally, there was the attempt to establish the IC-RT-PCR, however the small size of viral particles prevented the test, but the implementation of detection by ELISA and molecular separately was successful, add new national technologies allowing and contributing to the reduction of costs for implementing of the fitosanitty tests, importants and essentials for the control of viruses prevailing in the national potato culture. / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

De nos jours, les agents pathogènes des plantes, engendrent encore des pertes agricoles importantes. A terme, la solution la plus adaptée semble être la création de variétés de plantes cultivées présentant une résistance génétique à large spectre et durable. Créer de telles plantes nécessite la compréhension des mécanismes de résistance mis en jeu dans des pathosystèmes modèles, tels que celui impliquant le gène de résistance Rx et le virus X de la pomme de terre. Dans ce système, la protéine Rx intervient dans la perception d’un facteur d'avirulence issu du virus: sa protéine de capside (Cp). Cependant, les mécanismes moléculaires déclenchant la résistance chez la plante exprimant Rx demeurent obscurs. Cette étude s’est focalisée sur l’étape de reconnaissance de l’éliciteur viral par Rx. Une première approche a permis la mise en évidence d’un éliciteur minimal de 90 acides amine. L’étude d’un fragment légèrement plus grand par des méthodes de biochimie structurale, tend à exclure un modèle selon lequel, la différence de structuration tertiaire entre Cp élicitrice et Cp non élicitrice déterminerait la reconnaissance par Rx. Une seconde approche, de biologie moléculaire, a mis en évidence deux protéines hôte interagissant avec ce petit fragment éliciteur. L'étude s’est focalisée sur un gène codant un facteur de transcription, nbERF5, et a révélé que cette protéine interagit aussi bien avec les Cp de souches de Potexvirus avirulentes que virulentes dans la résistance liée à Rx, mais également avec la protéine de résistance Rx et l’intéracteur direct: RanGAP2. La poursuite de la caractérisation de cet ERF, permettra de déterminer son importance dans la résistance liée à Rx. / Nowadays, phytopathogenic agents are still causing significant agricultural losses. The most suitable option appears to be the creation of crop species carrying a genetic durable and broad spectrum resistance. In order to create such varieties, we need to understand the mechanisms underlying resistance, involved in model Pathosystems, Such as the one composed of the resistance gene Rx and the potato virus X. In that system, the host gene encodes a protein assimilated to a receptor implicated in the perception of an avirulence factor produced by the virus: its capside protein (Cp). Nevertheless, the molecular mechanisms triggering the resistance remain largely unknown. This study has been focused on the elicitor recognition mediated by Rx. A first approach led to the identification of a minimal elicitor containing 90 amino acids has. The structural characterization of a slightly larger protein fragment using biochemical methods suggested that the difference in the tertiary structuration of both elicitor and non-elicitor Cp would not be the determinant of Rx mediated recognition. Second, a molecular approach led to the discovery of two host proteins interacting with the small elicitor fragment. The work was focused on a transcription factor, nbERF5 and showed that this protein interacts similarly with elicitor or non-elicitor Cps of Rx mediated resistance. Interestingly, this gene product is able to directly interact with the Rx protein, but also with the direct interactor of Rx: RanGAP2, protein required for the Rx mediated resistance efficiency. Further characterization of this ethylen response factor will help us to understand its role in Rx mediated resistance.

Made available in DSpace on 2014-08-20T13:42:06Z (GMT). No. of bitstreams: 1 Dissertacao_Gabrie_Olle_ Dalmazo.pdf: 526235 bytes, checksum: 5e78165179f8f9e3164e0269bd617ab9 (MD5) Previous issue date: 2011-02-28 / The tropane alkaloids (TA) pathway has a branch-point controlled by the enzymes tropinone reductase 1 and 2 (TR1 and TR2). Tropinone is the common substrate for these enzymes and is reduced either by TR1 to form tropine, hyoscyamine and scopolamine or by TR2 to form pseudotropine and calystegines. Hyoscyamine and scopolamine are largely used in medicine as anticholinergic, antiemetic, parasympatholytic and anaesthetic. Calystegines mimic different sugars and are potent and specific inhibitors of glucosidases. The function of hyoscyamine, scopolamine and calystegines in the plants is not fully understood. Recent studies suggest that they are involved in the plant defense against pathogens. Regulation of TA biosynthesis in planta has attracted interest not only in view of its applications in the pharmaceutical industry, but also in respect to human nutrition and plant physiology. In the present study a virus-based transgenic approach devised to induce PTGS of tr1 and tr2 in whole transformed Hyoscyamus muticus plants is evaluated. It was observed that a significant reduction in transcript accumulation for tr2 caused a tremendous increase in transcript accumulation for tr1. / Um ponto de bifurcação na rota metabólica de tropano alcalóides (TA) é controlado pelas enzimas tropinona redutase 1 e 2 (TR1 e TR2). Tropinona, o substrato comum para estas enzimas, é reduzido por TR1 para formar tropina, hiosciamina e escopolamina ou por TR2 para formar pseudotropine e calisteginas. Hiosciamina e escopolamina são largamente utilizadas em medicina como anticolinérgicos, antieméticos, parassimpatolíticos e anestésicos. Calisteginas têm estrutura molecular semelhante a açúcares e são potentes inibidores específicos de glucosidases. A função dos alcalóides hiosciamina, escopolamina e calisteginas em plantas não é completamente entendida. Estudos recentes sugerem o envolvimento destes alcalóides na defesa da planta contra patógenos. A regulação da biossíntese de TA na planta tem atraído interesse não apenas quanto à aplicação na indústria farmacêutica, mas também com respeito à nutrição humana e fisiologia de plantas. No presente estudo avaliou-se uma estratégia transgênica, baseada em vírus, para induzir o silenciamento gênico pós-transcricional de tr1 e tr2 em plantas de Hyoscyamus muticus L. Observou-se que reduções significativas no acúmulo de transcritos para tr2 causaram um tremendo aumento no acúmulo de transcritos para tr1.

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