Any interaction of plants with phytopathogens involves the generation of various chemical molecules that are critical for activation of their defense machinery. One of the chemicals, salicylic acid (SA)induces systemic acquired resistance (SAR)in plants. The activation of SAR provides a broad-spectrum resistance against a wide range of related or unrelated pathogens. There has been considerable progress in the biochemical and molecular understanding of SAR activation in various plants. In addition, several chemicals including SA and its analogs are known to provide a direct or indirect defense against pathogens when applied to plants. Molecular mechanism of plant defense induced by synthetic chemical inducers is not very well understood. This review highlights the importance of salicylic acid and its most studied analog, Acibenzolar-S-methyl in inducing SAR and it also provides a description of other major chemical elicitors of plant defenses and their possible molecular mechanism.

A interacao planta-patogeno desencadeia uma serie de sinais que ainda nao estao completamente elucidados. Uma das respostas e a reacao de hipersensibilidade (RH), onde ocorre a morte celular programada no sitio da infeccao, impedindo a proliferacao do patogeno. Acredita que a morte celular e provocada pelo aumento do ERO, principalmente peroxido de hidrogenio (H2O2) e com o acumulo de acido salicilico (AS) que inibe a catalase, enzima responsavel pela transformacao de H2O2 em H2O e O2. Alem disso, ocorre o aumento da sintese e liberacao dos compostos fenolicos e alteracao da parede celular dos vegetais, com o aumento das atividades de diversas enzimas, capazes de degradar a parede celular da planta e do microrganismo invasor, liberando fragmentos que podem atuar como moleculas sinalizadoras, tornando as plantas mais resistentes. Nesse trabalho as celulas de Rubus fruticosus (amora-preta) foram tratadas, separadamente, com tres diferentes moleculas elicitoras, ou seja, moleculas capazes de ativar o mecanismo de defesa das plantas, o acido salicilico (AS), o metil jasmonato (MeJA) e ramnoglucuronogalactana (F-I), na concentracao de 1 Êmol/L durante 1h, para o estudo dos componentes extracelulares liberados e das modificacoes dos monossacarideos da parede celular durante resposta de hipersensibilidade. A concentracao de proteinas totais extracelulares foi aumentada com os indutores F-I e MeJA. A atividade enzimatica de -D-xilosidase nao se alterou na presenca de F-I, AS e MeJA. Entretanto, o MeJA tem a capacidade de aumentar as atividades das enzimas -D-galactosidase, -Dglucosidase, quitinase e laminarinase e inibir as atividades das enzimas galacturonase e -Lfucosidase na concentracao e tempo usado. O AS e F-I provocaram um aumento nas atividades de galacturonase e quitinase e inibiram a laminarinase. A aplicacao exogena de F-I e AS induziram a liberacao de compostos fenolicos para meio extracelular, que provavelmente, foi decorrente da tentativa das celulas de se protegerem de microrganismos invasores, com um decrescimo desses compostos no meio intracelular. O MeJA nao foi capaz de alterar a sintese de compostos fenolicos totais intracelulares e extracelulares e de acucares extracelulares, em tais condicoes. Tambem F-I e AS nao alteraram o teor de acucar redutor extracelular. O MeJA foi mais efetivo na producao de ERO durante 30 minutos de incubacao na concentracao de1 Êmol/L . F-I foi tambem ativador na liberacao de ERO, no entanto, o AS provocou inibicao. Os principais monossacarideos neutros que constitui a parede celular de suspensao de celulas de Rubus fruticosus sao as glucose (55-61%), arabinose (22-29%) e manose (13,8-15%). Ocorrendo em menor concentracoes os monossacarideos de fucose (0,65-1,2%), galactose (0,5-0,8%), xilose (0,5-0,8%) e ramnose (aproximadamente 0,5%).Os monossacarideos ramnose, fucose, xilose e galactose de parede celular tiveram um decrescimo na presenca do AS e um aumento na presenca de MeJA. Entretanto, o AS e o MeJA nao alteraram o percentual de arabinose, manose e glucose. O F-I foi capaz de aumentar o percentual dos monossacarideos ramnose e fucose e diminuir de glucose. Os resultados obtidos demonstram que a via de ativacao dos mecanismos de defesa da celula vegetal, induzida pelo MeJA, difere das vias ativadas pelo AS e F-I, pois o F-I e o AS induziram a liberacao de compostos fenolicos e o MeJA provocou aumento nas atividades enzimaticas, principalmente que atuam na parede celular da propria planta. O AS e o F-I foram mais efetivos no aumento das atividades enzimaticsa relacionadas a defesa da planta, as quais agem nas paredes de diversos fitopatogenos, sendo que as enzimas que podem atuar na parede celular da propria planta foram inibidas ou nao sofreram alteracao. / The plant-pathogen interactions trigger a series of signals that are not yet completely understood. One of the mechanisms is the hypersensitive response (HR), which is characterized by cell death in the infection site in order to prevent pathogen proliferation. Our previous studies with different elicitors demonstrated the correlation between the formation of reactive oxygen species (ROS) and cell wall degradation. Here, the cells were elicited with 1 mol/L salicylic acid (SA), methyl jasmonate (MeJA) or acid polysaccharide (rhamnoglucuronogalactan, F-I) (1mol/L) from characterization the extracellular components released and the modifications of the monosaccharide composition in cell wall during a hypersensitive response in Rubus fruticosus (blackberry-black).The extracellular proteins released to the extracellular were increased with the inducers molecules F-I and MeJA. The -D-xylosidase enzymatic activities didnt change in the presence of F-I, SA and MeJA. The time-course curves for -D-galactosidase, -D-glucosidase activities in fraction E were most effective for MeJA, while F-I and AS inhibited -Dgalactosidase. Also, the MeJA has ability to activate laminarinase and chitinase enzymatic activities and inhibit galacturonase and -L-fucosidase enzymatic activities. After 1h, the SA and F-I caused an increase galacturonase and chitinase activities and inhibited laminarinase enzymatic activity. Also, the time-course curves chitinase in the fraction increased with SA.The F-I and SA increased extracellular phenolic compounds, although they decreased them in the fraction I. MeJA was unable to change the synthesis of either intracellular or extracellular phenolic compounds. The data suggest that F-I and AS modulate the defense responses of plants through a via different that of MeJA. The extracellular reducing sugar didnt change with F-I, SA and MeJA.The MeJA was more effective in the release ROS incubation of 30 minutes at concentration of 1 mol/L. However, the presence of SA caused inhibition and F-I activated of ROS by cells.The main constituents of neutral sugars in the cell wall of Rubus fruticosus were glucose (55-61%), arabinose (22-29%) and mannose (13.8-15%). Minor constituents were fucose (0.65-1.2%), galactose (0.5- 0.8%), xylose (0.5-0.8%) and rhamnose (~0.5%). SA decreased the rhamnose and fucose concentrations; F-I both decreased the percentage of mannose and glucose and increased rhamnose and fucose. MeJA, in turn, increased the percentage of rhamnose, xylose and galactose. The data suggest that F-I and SA modulate the defense responses of plants through a mechanism unrelated to the MeJA via. Since the F-I and the SA induced the release phenolic compounds and the MeJA increased in enzymatic activities, mainly age in the own plant cell wall. The SA and F-I were more effective in the increasing defense enzyme-related activity of the plant that acts on the walls of several phytopathogens, and the enzymes that can act in the cell wall of the plant were inhibited or did not change.

L'utilisation de biofongicides et de molécules stimulatrices des défenses des plantes (SDP) constituent des stratégies de lutte alternative permettant la réduction de l'utilisation des fongicides conventionnels. Toutefois, il n'existe que peu de produits homologués et appliqués sur blé et encore moins contre la septoriose. Dans le cadre de cette étude, nous avons testés et mis en évidence l'efficacité de protection du blé contre Zymoseptoria tritici de plusieurs molécules. En effet, 29 molécules biosourcées dérivées de la fonctionnalisation de l'acide pyroglutamique (PGA), contenu dans les mélasses de la betterave sucrière, ont été évaluées en plus de l'acide y-aminobutyrique GABA, pour la première fois contre Z. tritici. Le GABA et 16 molécules fonctionnalisées ont réduit significativement les pourcentages de la maladie. L'absence d'effets directs sur Z. tritici vérifiée in vitro confirme que ces protections sont dues exclusivement à un effet SDP. Pour ces molécules, l'étude de la relation structure activité a mis en évidence l'importance des différents groupements chimiques impliqués dans les protections observées. D'autre part, la conjugaison du PGA avec l'acide salicylique (SA) a permis l'obtention de 5 nouvelles molécules parmi lesquelles 4 étaient plus efficaces que les molécules mères et cette efficacité semble être également due à un effet SDP. Enfin, la saccharine ainsi que trois lipopeptides cycliques produits par Bacillus subtilis (mycosubtiline, surfactine et fengycine seuls ou en mélange) ont été évalués pour la première fois sur le pathosystème blé - Z. tritici et ont conféré des protections significatives dues à des propriétés SDP, à des activités antifongiques directes ou à des effets combinés. Cette étude a donc permis d'identifier de nouvelles molécules possédant différents modes d'action et qui pourraient être envisageables en lutte alternative contre la septoriose du blé, toutefois, des analyses complémentaires sont requises. / The use of biofungicides and elicitors of plant defenses are alternative control stategies that can reduce the use of conventional fungicides. However, there are few products registered and applied on wheat and even less against Septoria tritici blotch. As part of this study, we tested and demonstrated the protective efficacity of several molecules on wheat against Zymoseptoria tritici. In fact, 29 biosourced molecules derived from the functionalization of pyroglutamic acid (PGA) contained in the molasses of sugar beet were evaluated with y-aminobutyric acid GABA, for the first time against Z. tritici. GABA and 16 functionalized molecules significantly reduced the percentages of the disease. The absence of direct effects on the fungus tested in vitro confirms that these protections are due exclusively to an elicitation of plant defense mechanisms. For these molecules, the study of the structure activity relationship has highlighted the importance of different chemical groups involved in the observed protections. On the other hand, the conjugation of PGA with salicylic acid (SA) allowed the obtention of 5 new molecules among which 4 were more effective than the parent molecules and this efficiency seems to be also due to an eliciting effect. Finally, saccharin and three cyclic lipopeptides produced by Bacillus subtilis (mycosubtilin, surfactin and fengycin alone or as mixtures) were evaluated for the first time on the pathosystem wheat - Z. tritici and conferred significant protection due to eliciting properties, direct antifungal activities or combined effects. This study has thus made it possible to identify new molecules with different modes of action and which could be considered in alternative control against wheat leaf blotch, however, additional analyses are required.

Puncturevine (Tribulus terrestris L.) is an annual plant of the Zygophyllaceae family. Its medicinal properties have long been used in traditional Chinese and Indian medicine to treat various diseases. It has been shown that active substances, among which we count steroid saponins, glycosides, flavonoids, phytosterols and alkaloids, have effects on reproduction, effects aphrodisiac, antibacterial, anti-carcinogenic, anti-oxidant, diuretic, antidiabetic, cardiovascular and many other. The aim of this work was to verify the effect of elicitors on the content of selected active ingredients in Puncturevine. The elicitor in this work was acetylsalicylic acid of three different concentrations (10-3 mol.l-1, 10-4 mol.l-1, 10-5 mol.l-1) applied by spraying. Using high performance liquid chromatography and mass spectrometry were in fruits and stems determined the content of diosgenin, protodioscin, ruscogenin. For all three compounds was observed positive effect of the elicitor on their content.

Milk Thistle (Silybum marianum) has been one of the best known medici herb for a very long time. Seeds contain biologicky aktive substances taxifoiln, silychristin, silydianin, silybin A, silybin B, isosilybin A and isosilybin B, commonly known as silymarin complex. The aim of this thesis was the influence of elicitors, fertilization and growing technology on this product and its utilizaion. In the first part I focused on history, botanic attributes, agrotechnology, chemical composition and substance efficiency method of determinativ and also on pharmacological onpacts of effective substances. In the practical part I conducted twho small parcel experiments to reveal the impal of elicitors on effective substances. I used two elicitors NanoFYTSi? in 1ml/l concentrantion and N-FENOLMIX? in 0.5ml/l concentration. In the second experiment from my supervisor elicitor NanoFYT Si? in 1ml/l concentraiton and N-FENOL MIX? in 0.5ml/l and elicitor ASA were used in three different concentration: low [10-5 mol/l], medium [ 10-4 mol/l] and high [10-3 mol/l]. In the conclusion I did statistical analysis of the above montioned experiments and compared them with other Publisher experiments. Then I propřed the growing technology and utilization of Milk Thistle.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A soja à uma das leguminosas mais utilizadas no mundo, sendo o uso justificado pelo elevado teor nutricional de seus grÃos, constituÃdos, principalmente, por proteÃnas e lipÃdios. A estimativa atual da produÃÃo mundial de soja à de 250.000 toneladas/ano, porÃm hà fatores limitantes dessa produÃÃo, como o ataque de pragas. As doenÃas causadas por fungos, por exemplo, causam na soja perdas em torno de 4%, sendo 20% destas causadas por Septoria glycines e Cercospora kikuchii. Assim, a busca por medidas alternativas de controle à crescente, particularmente em diminuiÃÃo ao uso de agrotÃxicos, mas, para isso, à importante o entendimento dos mecanismos de defesa vegetal. Das sementes de soja foi purificada uma proteÃna, denominada toxina da soja (SBTX), que à ativa contra diversos fungos de plantas e do homem e, tambÃm, à neurotÃxica para ratos e camundongos, razÃo pela qual recebeu o nome de toxina. A SBTX apresenta massa molecular de 44 kDa, constituÃda por duas subunidades (17 e 27 kDa) codificadas por genes distintos e jà foi identificada nas sementes, raÃzes, caules e folhas. O presente trabalho teve como objetivo avaliar o perfil de expressÃo gÃnica da SBTX em plantas cujas folhas primÃrias foram tratadas com elicitores (biÃtico e abiÃtico), usando a tÃcnica de PCR em tempo real, na tentativa de reforÃar o papel fisiolÃgico de defesa proposto para proteÃna. Assim sendo, Ãcido salicÃlico, injÃria mecÃnica e esporos do fungo Cercospora kikuchii foram utilizados como elicitores e os nÃveis de transcritos para as duas subunidades proteicas de SBTX avaliados. Respostas de induÃÃo foram verificadas para ambas as subunidades da SBTX, porÃm os perfis de expressÃo gÃnica foram diferenciados. Para o gene SBTX 27 kDa, o maior nÃvel de transcritos foi detectado quando o tratamento envolveu injÃria mecÃnica associada ao Ãcido salicÃlico, correspondendo a um aumento de cerca de 100 vezes apÃs 12 horas de aplicaÃÃo do tratamento. Jà para o gene SBTX 17 kDa este aumento nÃo foi verificado na mesma intensidade, tendo sido apenas em torno de 10 vezes. Os dados em conjunto mostram que SBTX à uma proteÃna passÃvel de induÃÃo por elicitores biÃticos e abiÃticos, reforÃando o seu papel fisiolÃgico de defesa, podendo vir a ser utilizada como ferramenta biotecnolÃgica no sentido de amenizar as perdas causadas por fungos. / Soybean is a legume most commonly utilized in the world, whose use is justified by the high nutritional content of its grain, consisting mainly of proteins and lipids. The current estimate of global soybean production is 250,000 tons/year, but there are limiting factors of this production, such as the pest attack. The fungal diseases, for example, cause losses in soybeans around 4%, where 20% of these are derived from infection by Septoria glycines and Cercospora kikuchii. Thus, the search for alternative measures is increasing, particularly in reducing the use of pesticides, but for this it is important to understand the plant defense mechanisms. Soybean toxin (SBTX) is a protein purified from soybean seeds with activity against plant and human pathogenic fungi and neurotoxic action to rats and mice, hence the reason it received the name of toxin. SBTX shows a molecular mass of 44 kDa, composed of two subunits (17 and 27 kDa) encoded by distinct genes and it has been detected seeds, roots, stems and leaves. This study aimed to evaluate the gene expression profile of SBTX in soybean plants whose primary leaves were treated with elicitors (biotic and abiotic), using the real-time PCR technique, in an attempt to strength the physiological role of defense proposed for this protein. Therefore, salicylic acid, mechanic injury and Cercospora kikuchii spores were used as elicitors and it was measured the transcript levels of SBTX subunits. Induction responses were observed for both subunits of SBTX, but the gene expression profiles were different. For SBTX 27 kDa gene, the highest transcript level was detected when the treatment involved mechanic injury associated to salicylic acid, an increase of about 100 fold after 12 hours of treatment application. Nevertheless, for SBTX 17 kDa gene the induction response was much smaller, it was only around 10 times. The data together show that SBTX is an inducible protein by biotic and abiotic elicitors, reinforcing its physiological role of defense, which could eventually be used as biotechnological tool in order to mitigate losses caused by fungi.

A interacao planta-patogeno desencadeia uma serie de sinais que ainda nao estao completamente elucidados. Uma das respostas e a reacao de hipersensibilidade (RH), onde ocorre a morte celular programada no sitio da infeccao, impedindo a proliferacao do patogeno. Acredita que a morte celular e provocada pelo aumento do ERO, principalmente peroxido de hidrogenio (H2O2) e com o acumulo de acido salicilico (AS) que inibe a catalase, enzima responsavel pela transformacao de H2O2 em H2O e O2. Alem disso, ocorre o aumento da sintese e liberacao dos compostos fenolicos e alteracao da parede celular dos vegetais, com o aumento das atividades de diversas enzimas, capazes de degradar a parede celular da planta e do microrganismo invasor, liberando fragmentos que podem atuar como moleculas sinalizadoras, tornando as plantas mais resistentes. Nesse trabalho as celulas de Rubus fruticosus (amora-preta) foram tratadas, separadamente, com tres diferentes moleculas elicitoras, ou seja, moleculas capazes de ativar o mecanismo de defesa das plantas, o acido salicilico (AS), o metil jasmonato (MeJA) e ramnoglucuronogalactana (F-I), na concentracao de 1 Êmol/L durante 1h, para o estudo dos componentes extracelulares liberados e das modificacoes dos monossacarideos da parede celular durante resposta de hipersensibilidade. A concentracao de proteinas totais extracelulares foi aumentada com os indutores F-I e MeJA. A atividade enzimatica de -D-xilosidase nao se alterou na presenca de F-I, AS e MeJA. Entretanto, o MeJA tem a capacidade de aumentar as atividades das enzimas -D-galactosidase, -Dglucosidase, quitinase e laminarinase e inibir as atividades das enzimas galacturonase e -Lfucosidase na concentracao e tempo usado. O AS e F-I provocaram um aumento nas atividades de galacturonase e quitinase e inibiram a laminarinase. A aplicacao exogena de F-I e AS induziram a liberacao de compostos fenolicos para meio extracelular, que provavelmente, foi decorrente da tentativa das celulas de se protegerem de microrganismos invasores, com um decrescimo desses compostos no meio intracelular. O MeJA nao foi capaz de alterar a sintese de compostos fenolicos totais intracelulares e extracelulares e de acucares extracelulares, em tais condicoes. Tambem F-I e AS nao alteraram o teor de acucar redutor extracelular. O MeJA foi mais efetivo na producao de ERO durante 30 minutos de incubacao na concentracao de1 Êmol/L . F-I foi tambem ativador na liberacao de ERO, no entanto, o AS provocou inibicao. Os principais monossacarideos neutros que constitui a parede celular de suspensao de celulas de Rubus fruticosus sao as glucose (55-61%), arabinose (22-29%) e manose (13,8-15%). Ocorrendo em menor concentracoes os monossacarideos de fucose (0,65-1,2%), galactose (0,5-0,8%), xilose (0,5-0,8%) e ramnose (aproximadamente 0,5%).Os monossacarideos ramnose, fucose, xilose e galactose de parede celular tiveram um decrescimo na presenca do AS e um aumento na presenca de MeJA. Entretanto, o AS e o MeJA nao alteraram o percentual de arabinose, manose e glucose. O F-I foi capaz de aumentar o percentual dos monossacarideos ramnose e fucose e diminuir de glucose. Os resultados obtidos demonstram que a via de ativacao dos mecanismos de defesa da celula vegetal, induzida pelo MeJA, difere das vias ativadas pelo AS e F-I, pois o F-I e o AS induziram a liberacao de compostos fenolicos e o MeJA provocou aumento nas atividades enzimaticas, principalmente que atuam na parede celular da propria planta. O AS e o F-I foram mais efetivos no aumento das atividades enzimaticsa relacionadas a defesa da planta, as quais agem nas paredes de diversos fitopatogenos, sendo que as enzimas que podem atuar na parede celular da propria planta foram inibidas ou nao sofreram alteracao. / The plant-pathogen interactions trigger a series of signals that are not yet completely understood. One of the mechanisms is the hypersensitive response (HR), which is characterized by cell death in the infection site in order to prevent pathogen proliferation. Our previous studies with different elicitors demonstrated the correlation between the formation of reactive oxygen species (ROS) and cell wall degradation. Here, the cells were elicited with 1 mol/L salicylic acid (SA), methyl jasmonate (MeJA) or acid polysaccharide (rhamnoglucuronogalactan, F-I) (1mol/L) from characterization the extracellular components released and the modifications of the monosaccharide composition in cell wall during a hypersensitive response in Rubus fruticosus (blackberry-black).The extracellular proteins released to the extracellular were increased with the inducers molecules F-I and MeJA. The -D-xylosidase enzymatic activities didnt change in the presence of F-I, SA and MeJA. The time-course curves for -D-galactosidase, -D-glucosidase activities in fraction E were most effective for MeJA, while F-I and AS inhibited -Dgalactosidase. Also, the MeJA has ability to activate laminarinase and chitinase enzymatic activities and inhibit galacturonase and -L-fucosidase enzymatic activities. After 1h, the SA and F-I caused an increase galacturonase and chitinase activities and inhibited laminarinase enzymatic activity. Also, the time-course curves chitinase in the fraction increased with SA.The F-I and SA increased extracellular phenolic compounds, although they decreased them in the fraction I. MeJA was unable to change the synthesis of either intracellular or extracellular phenolic compounds. The data suggest that F-I and AS modulate the defense responses of plants through a via different that of MeJA. The extracellular reducing sugar didnt change with F-I, SA and MeJA.The MeJA was more effective in the release ROS incubation of 30 minutes at concentration of 1 mol/L. However, the presence of SA caused inhibition and F-I activated of ROS by cells.The main constituents of neutral sugars in the cell wall of Rubus fruticosus were glucose (55-61%), arabinose (22-29%) and mannose (13.8-15%). Minor constituents were fucose (0.65-1.2%), galactose (0.5- 0.8%), xylose (0.5-0.8%) and rhamnose (~0.5%). SA decreased the rhamnose and fucose concentrations; F-I both decreased the percentage of mannose and glucose and increased rhamnose and fucose. MeJA, in turn, increased the percentage of rhamnose, xylose and galactose. The data suggest that F-I and SA modulate the defense responses of plants through a mechanism unrelated to the MeJA via. Since the F-I and the SA induced the release phenolic compounds and the MeJA increased in enzymatic activities, mainly age in the own plant cell wall. The SA and F-I were more effective in the increasing defense enzyme-related activity of the plant that acts on the walls of several phytopathogens, and the enzymes that can act in the cell wall of the plant were inhibited or did not change.

Orientadores: Gonçalo Amarante Guimarães Pereira, Sandra Martha Gomes Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T22:03:27Z (GMT). No. of bitstreams: 1 Barsottini_MarioRamosdeOliveira_M.pdf: 16356564 bytes, checksum: 9cf8e738f13f95639e35471c9a7da160 (MD5) Previous issue date: 2013 / Resumo: O fungo Moniliophthora perniciosa desperta grande interesse agroeconômico, pois é o agente etiológico da Vassoura-de-Bruxa do cacau. A cultura do cacaueiro é de grande importância no cenário nacional e na América Latina, sendo o entendimento dos mecanismos moleculares desta doença de grande valia. Durante a interação entre patógeno e hospedeiro, o primeiro produz moléculas para evadir ou alterar as respostas normais de defesa do segundo. O sequenciamento do genoma de M. perniciosa levou à identificação de proteínas-chave potencialmente envolvidas no processo patogênico do fungo, dentre as quais, estão: proteínas pertencentes à família das Cerato-plataninas (MpCPs), bem como proteínas pertencentes à família das Necrosis- and Ethylene-inducing Proteins (MpNEPs). As CPs são amplamente associadas à interação fungo-hospedeiro, agindo como toxinas, indutoras de resposta de defesa ou alergenos. As NEPs induzem morte celular e necrose em plantas dicotiledônes através da permeabilização da membrana celular. O objetivo desse projeto foi caracterizar funcionalmente as MpCPs e as MpNEPs, determinando assim sua relevância durante a Vassoura-de-Bruxa. A partir do transcriptoma de M. perniciosa e da análise filogenética das doze MpCPs encontradas, foi revelada uma correlação entre grupos de MpCPs e sua expressão diferencial ao longo da doença. Quatro MpCPs foram clonadas, expressas em sistema heterólogo e tiveram sua estrutura cristalográfica resolvida. Ensaios bioquímicos e biofísicos confirmaram que as MpCPs presentes em diferentes grupos filogenéticos apresentam capacidades distintas no tocante à interação com o açúcar N-acetilglicosamina e de formar agregados ordenados. Estudos funcionais indicaram que estas características estão potencialmente relacionadas ao bloqueio de resposta de defesa da planta e ao crescimento do fungo, respectivamente. Quanto às MpNEPs, somente a isoforma MpNEP2 foi detectada durante a Vassoura-de-Bruxa. A partir da estrutura cristalográfica dessa proteína e ensaios de mutação sítio-dirigida, foi identificado um hairpin hidrofóbico exposto ao solvente, possivelemte associado à ancoragem da MpNEP2 na membrana celular, o qual é tão importante quanto o sítio ativo da proteína para a atividade biológica da mesma / Abstract: Moniliophthora perniciosa is the causal agent of Witches' Broom Disease of cocoa and a major agroeconomic concern in Brazil and Latin America. In order to efficiently control this disease, it is crucial to understand the molecular basis underlying its progression. During the attack to the plant, a pathogen releases molecules to suppress or alter the regular defense response of the host. Results obtained from the genome sequencing of M. perniciosa lead to the identification putative virulence factors belonging to the Cerato-platanin protein family (MpCPs), and to the Necrosis- and Ethylene-inducing Proteins (MpNEPs). CPs are important to fungus-host interaction process, acting as toxins, defense response-inducing molecules or allergens. NEPs are toxin-like pore-forming proteins, which affect only dicot plants. This project aimed at the functional characterization of the MpCPs and MpNEPs, as well as understanding their importance for the Witches' Broom Disease progression. Twelve MpCP-coding genes were identified, and comprehensive transcriptome and phylogenetic analyses showed a correlation between MpCPs evolutionary clusters and their expression patterns throughout the disease. Four representative MpCPs had their crystal structure resolved. Biophysical and biochemical characterizations showed a correlation between the MpCP clusters, regarding sugar (N-acetylglucosamine) binding and protein self-assembling, which are possibly related to plant defense response suppression and hyphal growth, respectively. As for the MpNEPs, only the isoform MpNEP2 was shown to be expressed during the Whitches' Broom Disease. Its crystalloghaphic structure, along with site-directed mutagenesis and functional assays revealed that, besides the protein's active site, an hydrophobic hairpin exposed to te solvent is important to the necrosis-promoting activity, probably mediating the contact of MpNEP2 with the cell membrane / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Phenolic compounds in plants have attracted considerable attention during the last decade due to their contribution to human health in reduce the risk of chronic diseases. The grape is one of the largest sources of these substances and like other plants, the synthesis of these compounds is usually associated with defense responses against stress factors, also called elicitors. More recently, ultrasound has been studied as possible abiotic elicitors of secondary metabolites due to mechanical stress and microstreaming induced by acoustic cavitation. Thus, this work aimed evaluate the application of different power densities (53 and 113 W cm-2) and exposure times (1, 5 and 10 minutes) to ultrasound in Isabel and Cabernet Sauvignon grapes about the effects on total polyphenols of juices made from this two cultivars. The samples were also subjected to the determination of total anthocyanins, tannins, total soluble solids, total acidity, pH and sensory evaluation. The Isabel cultivar showed more significant results in response to ultrasound, an increase of up to 83% in the levels of total polyphenols of juice, as well as significant increase in the content of total anthocyanins and tannins. These results were also accompanied by an increase in total soluble solids and total acidity of the juices. The application of ultrasound emphasized color, odor and taste of the Isabel grape juice, and the sonicated treatments were preferred by tasters. About Cabernet Sauvignon cultivar, it only showed a little variation in the most parameters. Power density and exposure time to ultrasound influenced the most of parameters evaluated in juices regarding to degree of response to sonication. Treatment with 5 and 10 min of exposure to ultrasound and power density of 113 W cm-2, is the combination that proved more effective for the elicitation of phenolic compounds by activation of plant secondary metabolism, improving the quality of the Isabel grape juice. / Compostos fenólicos em plantas têm atraído grande atenção durante a última década devido à sua contribuição para a saúde humana, podendo reduzir os riscos de doenças crônicas. A uva é uma das maiores fontes dessas substâncias e assim como os demais vegetais, tem a síntese desses compostos geralmente associada a respostas de defesa contra fatores de estresse, também chamados elicitores. Mais recentemente, o ultrassom vem sendo estudado como possível elicitor abiótico de metabólitos secundários devido a estresse mecânico e microagitação induzidos por cavitação acústica. Dessa forma, este trabalho foi conduzido com o intuito de avaliar a aplicação de diferentes densidades de potência (53 e 113 W cm-2) e tempos de exposição (1, 5 e 10 minutos) ao ultrassom em uvas Isabel e Cabernet Sauvignon quanto aos efeitos sobre os polifenóis totais dos sucos elaborados com as duas cultivares. As amostras também foram submetidas à determinação de antocianinas totais, taninos, sólidos solúveis totais, acidez total, pH e análise sensorial. A cultivar Isabel mostrou resultados mais significativos em resposta ao ultrassom, com elevação de até 83% nos níveis de polifenóis totais do suco, bem como, importante aumento no conteúdo de antocianinas totais e taninos. Estes resultados também foram acompanhados por um aumento nos sólidos solúveis totais e acidez total dos sucos. A aplicação do ultrassom acentuou cor, odor e sabor dos sucos de uvas Isabel, sendo os tratamentos sonicados os preferidos pelos provadores. Quanto a cultivar Cabernet Sauvignon, esta somente apresentou pequena variação na maioria dos parâmetros analisados. A densidade de potência e tempo de exposição ao ultrassom influenciaram a maioria dos parâmetros avaliados nos sucos quanto ao grau de resposta à sonicação. Tratamentos com 5 e 10 min de exposição ao ultrassom, com densidade de potência de 113 W cm-2 mostraram ser a combinação mais efetiva para a elicitação de compostos fenólicos mediante ativação do metabolismo secundário da planta, melhorando a qualidade dos sucos de uva Isabel.

Saccharothrix algeriensis est une Actinobactérie rare capable de synthétiser divers dérivés de dithiolopyrrolones (DTPs) aux propriétés phytosanitaires intéressantes. L'objectif de la thèse vise à développer différentes méthodes, alternatives à la synthèse organique, pour optimiser la production de DTPs par la régulation du métabolisme de la bactérie. Deux approches ont été envisagées : * la première fait appel à des éliciteurs chimiques tels que l'éthanol, le butan-1-ol, le diméthylsulfoxyde et des homosérines lactones (C4-HSL, 3-oxo-C12-HSL et 3-OH-C12-HSL). Les effets de la concentration de ces composés et du temps d'addition dans le milieu de culture ont été étudiés et une investigation sur le mécanisme d'action de l'éthanol a été initiée * la deuxième utilise une approche physico-chimique innovante modifiant le métabolisme microbien à l'aide d'une électrode polarisée. Des électrodes en carbone, en acier et en platine de topographies variées ont servi pour l'étude de l'effet du potentiel, par rapport à une électrode au calomel saturée (ECS), sur la croissance de S. algeriensis et la production de dithiolopyrrolones. Les résultats obtenus montrent que l'ajout de l'éthanol à une concentration de 1,74 % v/v ou du diméthysulfoxyde à 3 % v/v au début de la culture, induit une augmentation significative de la production spécifique des DTPs. Celle-ci est multipliée par un facteur 19 avec l'éthanol et par 6 avec le diméthylsulfoxyde. Dans le cas de l'éthanol, cette induction serait médiée par des espèces réactives de l'oxygène. Par ailleurs, l'ajout simultané de la C4-HSL et de la 3-oxo-C12-HSL à des doses successives de 500 µM permet de multiplier la production spécifique des DTPs par un facteur 2,5. L'utilisation d'une électrode polarisée, en platine ou en carbone, permet un bon développement mycélien de S. algeriensis contrairement à une électrode en acier. Sous le potentiel -0.3 V/ECS, des concentrations élevées en dithiolopyrrolones ont été quantifiées. / Saccharothrix algeriensis is a rare Actinobacterium that produces a variety of dithiolopyrrolone derivatives (DTPs). These biocidal compounds are used as plant and crop- protection products. The aim of this thesis is to develop new strategies for DTPs production by regulating bacterial metabolism as an alternative to the chemical synthesis of these compounds. Two strategies were used : * the first strategy consist of using chemical elicitors such as ethanol, butan-1-ol, dimethylsulfoxide and homoserine lactones (C4-HSL, 3-oxo-C12-HSL and 3-OH-C12-HSL). The effects of concentration and time addition of these compounds were studied and ethanol mode of action was investigated. * the second strategy is a physico-chemical innovative approach that modifies bacterial metabolism using a polarized electrod. Platinum, carbon and stainless steel electrodes with varied topographies were used to study the effect of the potential, versus a saturated calomel electrode (SCE), on the growth of S. algeriensis and DTPs production. Our results show that the addition of pure ethanol and dimethylsulfoxide, at the beginning of the culture (t0), in respective concentrations of 1,74 % v/v and 3 % v/v increases DTPs specific production. This one was multiplied by nineteen-fold in presence of ethanol and by six-fold in presence of dimethylsulfoxide. In the case of ethanol, the induction of DTPs biosynthesis is mediated by reactive oxygen species (ROS). Furthermore, simultaneous addition of C4-HSL and 3-oxo-C12-HSL in multiple doses of 500 µM increases DTPs specific production by 2,5 fold. In presence of polarized platinum and carbon electrodes mycelium development of S. algeriensis is thicker than that grown on stainless steel electrode. Although, under - 0,3 V/SCE, high DTPs concentrations were quantified in comparison to other conditions tested.