La cryptogéine, sécrétée par l’oomycète Phytophthora cryptogea, est un éliciteur protéique des réactions de défense qui active chez le tabac un ensemble d’événements de signalisation conduisant à la mise en place d’une mort cellulaire de type réponse hypersensible et d’une résistance systémique acquise. La caractérisation de la modulation de l’activité de kinases cytosoliques, dont SIPK et WIPK, par des événements de phosphorylation en réponse à la cryptogéine, traduit la place majeure que tiennent les modifications post-traductionnelles dans la cascade de signalisation induite dans les cellules de tabac en réponse à la cryptogéine. Il s’avère que la signalisation cellulaire induite par la cryptogéine, et impliquant ces protéines kinases, converge entre autre vers le noyau à travers la modulation de l’activité d’éléments nucléaires par phosphorylation. Dans ce contexte, d’importants travaux de purification/séquencage, visant à identifier les protéines nucléaires cibles de ces activités kinases, ont permis d’identifier deux isoformes redondantes d’histones désacétylases de type 2 nommés NtHD2a et NtHD2b qui sont rapidement phosphorylées en réponse à la cryptogéine dans les cellules de tabac.Ce travail de thèse s’inscrit dans l’étude du rôle de NtHD2a/b dans l’établissement du processus de mort cellulaire des cellules de tabac et de la RH in planta en réponse à la cryptogéine. Par des approches de pharmacologie ainsi que des approches de surexpression ou d’invalidation de l’expression de NtHD2a/b chez le tabac, nous avons d’une part confirmé l’implication de NtHD2a/b en tant que régulateurs négatifs de la mort cellulaire induite par la cryptogéine ou d’autres élicitines, et d’autre part mieux appréhendé les événements de la cascade de signalisation prépondérants dans l’établissement de cette mort cellulaire. Les mécanismes moléculaires sous-jacents à la mise en place de la mort cellulaire apparaissent complexes et semblent notamment impliquer la modulation de l’expression de gènes de défense, la synthèse de novo de protéines ainsi que l’activation de protéines kinases, dont notamment WIPK et SIPK.Des travaux relatifs à l’étude des événements de (dé)/acétylation dans les cellules de tabac traitées par la cryptogéine et invalidées dans l’expression de NtHD2a/b suggèrent le concours de modifications post-traductionnelles de protéines nucléaires telles que l’acétylation dans la mise en place de la mort cellulaire induite par la cryptogéine chez le tabac. / Cryptogein, which is secreted by the oomycete Phytophthora cryptogea, is a proteinaceous elicitor of plant defense reactions that activates a set of signaling events leading to the hypersensitive response and to systemic acquired resistance. Although the early cytosolic signaling events induced by cryptogein are well described, the only nuclear events characterized to date are the variations in free calcium concentrations and defense-related gene expression. The characterization of the activation of cytosolic protein kinases, including WIPK and SIPK, by phosphorylation in response to cryptogein highlights the key-role played by posttranslational modifications in cryptogein-induced signaling events in tobacco cells. In this context, purification/sequencing approaches revealed that two redundant isoforms of type-2 nuclear histone deacetylases, NtHD2a and NtHD2b, were rapidly phosphorylated in cryptogein-treated tobacco cells.This thesis work is part of a comprehensive study of the role of NtHD2a/b in the establishment of the cell death process in tobacco cells and of the hypersensitive response in planta, in response to cryptogein. By using a pharmacological approach and overexpression and RNA interference-based approaches, we confirmed the involvement of NtHD2a/b as negative regulators of elicitin-induced cell death and we achieved a better understanding of cell death signaling events. The molecular events that underly the cell death process appear particularly complex and seem to involve the modulation of defense-related gene expression, de novo protein synthesis and protein kinase activation such as WIPK and SIPK.The study of (de)/acetylation events in tobacco cells treated by cryptogein and invalidated in NtHD2a/b expression suggests a role for posttranslational modifications in cryptogein-induced cell death.

The main objective of this thesis dissertation is functionally characterizing the genes involved in biotic and abiotic stresses of plants at molecular level. Previously, upon pathogen attack Rad6 gene expression was found to be changed in wheat and barley plants. To functionally characterize the Rad6 gene, VIGS (Virus induced gene silencing) system was used. HR (Hypersensitive response) like symptoms was detected in every silenced barley and wheat plants. To figure out, transcriptomes and proteomes of Rad6 silenced plants were analyzed. 2-D PAGE analysis was also performed on silenced and control wheat plants. No pathogen growth was observed in Rad6 silenced barley lines. Additionally, the susceptible wild type Arabidopsis plants showed resistant phenotype when any of the Rad6 gene copies is mutated. This suggests that Rad6 gene has a negative regulatory role in plant disease resistance which was proved for the first time. Yeast two hybrid protein interaction study suggests that RAD6 carrying out its function by interacting with SGT1 protein and regulating resistance related genes. It has been first time reported in this thesis that E2 (Ubiquitin conjugating enzyme) takes role in plant disease resistance. Boron which is the other consideration in the scope of thesis as an abiotic stress factor at a very limited amount is necessary for the normal development of plants. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were tested in the presence of high boron (35 mg/kg) concentrations. The transcriptomes of the plant samples treated with the excess levels of boron to that of the samples grown under normal concentration were compared using differential display PCR method. Thirty bands showing differential expression levels at varying time points were analyzed. 18 of them were confirmed via qRT-PCR.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Interações como a herbivoria há o consumo de partes de tecidos vegetais por insetos herbívoros que promovem impactos no desempenho vegetativo e reprodutivo de plantas hospedeiras. Mecanismos de defesas induzidas como respostas de hipersensibilidade (RH) são observadas em diversas espécies e têm relação direta com a especificidade entre inseto herbívoro e planta hospedeira. No entanto, a capacidade de desenvolver RH varia de acordo com o genótipo e fenótipos de plantas hospedeiras e com distúrbios ambientais como o fogo. Assim, o objetivo geral do nosso estudo foi avaliar o desempenho de Bauhinia brevipes, a capacidade de desenvolver defesas induzidas contra o inseto galhador Schizomyia macrocapillata e as taxas de herbivoria por insetos de vida livre. Avaliamos essas interações no contexto das variações fenotípicas em desenvolver respostas de hipersensibilidade, e de distúrbios ambientais como o fogo. Assim, no primeiro capítulo, avaliamos o desempenho de plantas adultas e da progênie de acordo com os fenótipos de resistência (resistente e susceptível) aos ataques de S. macrocapillata. Para tal, avaliamos o desempenho vegetativo como arquitetura e o desempenho reprodutivo como número de sementes (componente feminino), o número de frutos formados (componente masculino) e a qualidade nutricional (macro e micronutrientes) em indivíduos resistentes e susceptíveis. Além disso, avaliamos o desempenho da prole em três estágios de desenvolvimento: sementes, plântulas e juvenis. Não houve diferenças nos desempenhos vegetativo e reprodutivo das plantas adultas, bem como na relação entre macro e micronutrientes e o fator resistência entre os fenótipos. Além disso, as taxas de germinação e investimentos em porção aérea e subterrânea na prole dos diferentes fenótipos foram similares. Acreditamos que a baixa incidência de S. macrocapillata sobre a população de B. brevipes contribuiu para o desempenho similar entre os fenótipos. Além disso, estratégias como tolerância à herbivoria devem ser analisadas nesse sistema inseto galhador / planta hospedeira. No segundo capítulo, avaliamos como as interações entre herbívoros e plantas hospedeiras, no contexto das defesas induzidas, como RH e defesas mecânicas como o acúmulo de silício em plantas com diferentes respostas ao fogo. Verificamos os indivíduos da população de B. brevipes apresentaram diferentes características quanto à incidência de fogo. As plantas que não rebrotaram apresentaram maiores taxas de herbivoria por insetos mastigadores e menores conteúdos de silício em relação às rebrotas. Por outro lado, as rebrotas foram mais atacadas por S. macrocapillata, mas exibiram maiores quantidades RH que àquelas plantas que não rebrotaram e em relação à estação anterior. Acreditamos que rebrotas de B. brevipes podem ser mais bem defendidas uma vez que alocam mais nutrientes, como nitrogênio para o crescimento e silício que reduz o desempenho de insetos mastigadores. As RH também podem ser aumentadas pela alocação de recursos pós-fogo o que levou às maiores taxas de defesas entre as rebrotas. Este estudo forneceu evidencias sobre o papel do fogo nas interações entre plantas hospedeiras e insetos de diferentes guildas e possíveis alterações no comportamento de insetos mastigadores. / In interactions such as herbivory the consumption of plant tissue by herbivore insects promotes several impacts on vegetative and reproductive performance of host plants. Defense mechanisms induced as hypersensitive response (HR) are observed in several species and is directly related to the specificity of herbivore insects and host plants. However, the ability to develop HR varies according to genotype and phenotype of plant hosts and with environmental disturbances such as fire. Thus, we aimed evaluate the performance of Bauhinia brevipes, the ability to evolve induced defenses against gall insect Schizomyia macrocapillata and herbivory rates by free-living insects. We analyzed these interactions in the context of phenotypic variation of resistance and environmental disturbances as fire. Then, in the first chapter, we evaluated the performance of mature plants and offspring according to the phenotypic resistance (resistant and susceptible) to S. macrocapillata attacks. Thus, we evaluate vegetative performance as architecture and reproductive performance as number of seeds (female component), the number of fruits (male component) and the nutritional quality (macro and micronutrients in leaves) on resistant and susceptible individuals. In addition, we evaluated the performance of the offspring in three stages of development: seeds, seedlings and juveniles. There were no differences in vegetative and reproductive performances of mature plants from resistant and susceptible phenotypes. Besides, there was no relationship between macro and micronutrients and the resistance factor. Germination rates and investments in shoot and root portion in the offspring of different phenotypes were similar. We believe that the low incidence of S. macrocapillata on the population of B. brevipes contributed to the similar performance between phenotypes. Moreover, strategies such as tolerance to herbivory should be analyzed in this gall insect / plant host system. In the second chapter, we evaluate the differences in responses to fire effects in relation to interactions between herbivore insects and host plants in and induced defenses (HR and silicon content). We detected an individual variation in response to fire as resprouting and non-resprouting plants in the population of B. brevipes. Non-resprouting plants showed higher rates of herbivory by chewing insects and lower silicon content in relation to resprouting. Contrasting, resprouting presented greater attack rate by S. macrocapillata, but exhibited larger amounts of HR than non- resprouting. We believe that resprouting of B. brevipes can be better defended, since allocate more nutrients such as nitrogen for growth and silicon which reduces the performance of chewing insects. HR can also be increased by the allocation of resources post-fire, which lead to the greatest defenses rates among resprouting plants. This study provided evidences about the role of fire in the interactions between host plants and insects of different guilds and possible changes in the behavior of chewing insects. / Dissertação (Mestrado)

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