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Doctor of Philosophy / Department of Grain Science and Industry / Ronald L. Madl / Praveen Vadlani / Producers of whole wheat products are interested in marketing the health-promoting benefits of wheat antioxidants. However, they need a steady crop supply with consistent levels of antioxidants. The variable phenolic content in wheat crops is a problem. The objectives of this research were to 1) identify the factor (s) that contribute the most to the variability in phenolic content, 2) understand the mechanism (s) responsible for phenolic synthesis, and 3) artificially trigger that mechanism (s). Phenolics are hypothesized to be part of the defense response of hard red winter wheat (Triticum aestivum L) to stress. The effect of insect feeding, pathogen infection, and heat stress on phenolics in grains from wheat plants cv. Karl 92 was evaluated. Bird-cherry oat aphid (Rhopalosiphum padi) feeding stress significantly explained the variation in phenolic content. Furthermore, the relative allocation of carbon resources to grain yield/phenolic content was influenced by the stage of the plant at which aphid feeding started to occur. Based on these findings, phenolics were hypothesized to be an active defense response acting through a mechanism known as systemic acquired resistance (SAR). In order to prove this hypothesis, several synthetic elicitors of SAR were tested for their effectiveness at inducing de novo phenolic synthesis in wheat foliage and in mature grains. Elicitors that acted through the salicylic- and jasmonic acid signaling pathways were effective at inducing phenolic synthesis by 49% and 177%, respectively, in the leaves 36 hours post spray application. They also elicited a phenolic response in mature grains of up to 21% induction. Enhancement of the levels of naturally occurring phenolic compounds with antioxidant activity in wheat grains through SAR activation is a value addition strategy that can potentially increase the profitability of hard red winter wheat crops. It can also provide manufacturers of whole wheat with natural antioxidants that can potentially be used to substitute their synthetic counterparts in wheat based products.

Active compounds have been always originated from plants. Plants though, were able to produce only very low amounts of them and that was the reason for trying many alternative ways of production, one of them being plant tissue culture cultivation. This method is any fragment of living tissue or organ taken from an intact plant or an already existing explant culture, with the intention of growing an artificial growth medium. Even this method though, is not able to produce large amounts compared to extraction from field plants. Elicitation is considered a possible way to increase the production of secondary metabolites. This method used the plant's own defense system, in order to increase the production of secondary metabolites in vitro. The compound which is used to produce the effect is called elicitor. During our experimental work I used as an elicitor the compound Ethephon (2-Chloroethylphosphonic acid) upon callus and suspension cultures of Hypericum perforatum, with intention to observe its effect on flavonoid production. This experiment was based on three different concentrations and six different withdrawal times, being 6, 12, 24, 72, 168 hours. The maximum effect of elicitor was reached with concentration c1 (1mg/100ml) after 12 hours and with concentration c3 (100mg/100ml) after 72 hours.

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T08:30:39Z (GMT). No. of bitstreams: 1 Zaparoli_GustavoHenriqueAlcala_M.pdf: 1165845 bytes, checksum: 3216f460da7ecd604e45a6b880dd2d75 (MD5) Previous issue date: 2007 / Resumo: O fungo basidiomiceto hemibiotrófico Monilophthora perniciosa é o agente causador da doença Vassoura-de-Bruxa em cacaueiros. A infecção é caracterizada pela hipertrofia e hiperplasia dos tecidos da planta durante a fase biotrófica do fungo, formando as denominadas vassouras-verdes, que sofrem necrose e completa degradação durante a fase saprotrófica do fungo, formando as vassouras-secas. Encontramos no genoma de M. perniciosa pelo menos cinco seqüências codificando possíveis proteínas similares aos membros da família Cerato-platanina, uma classe de proteínas inicialmente encontrada no fitopatógeno Ceratocystis fimbriata. Um dos genes de M. perniciosa cerato-platanina (MpCP1) foi expresso ¿in vitro¿ e mostrou ter capacidade de induzir necrose em folhas de tabaco e cacaueiro. A proteína apresenta-se em solução como um dímero e é capaz de recuperar a capacidade de necrose após tratamento de denaturação térmica. Análises de transcrição ¿ex planta¿ mostraram que MpCP1 é mais expresso no micélio biotrófico do que no saprotrófico. O perfil de necrose apresentado é diferente do causado pelas MpNEPs (outra família de elicitores presente em M. perniciosa), mas quando aplicadas em conjunto nas plantas, MpCP1 e MpNEP2 apresentam um efeito sinergético muito semelhante ao encontrado em plantas naturalmente infectadas pelo fungo / Abstract: The hemibiotrophic basidiomycete Moniliophthora perniciosa is the causal agent of the Witches¿ Broom Disease of cacao. The infection in pod is characterized by the formation of hypertrophic and hyperplasic tissues in the biotrophic phase, forming the green broom, which is followed by necrosis and complete degradation of the organ, causing the dry broom. We found in the fungus genome at least five sequences encoding putative proteins similar to cerato-platanin-like proteins, a class of proteins initially found in the phytopathogen Ceratocystis fimbriata. One M. perniciosa cerato-platanin gene (MpCP1) was expressed ¿in vitro¿ and proved to have necrosis inducing ability in tobacco and cacao leaves. The protein is present in solution as dimers and is able to recover necrosis activity after heat treatment. Transcription analysis ¿ex planta¿ showed that MpCP1 is more expressed in biotrophic-like mycelia in comparison to saprotrophic mycelia. The necrosis profile presented is different from that caused by MpNEPs (other family of elicitors also found in M. perniciosa). Most remarkable, mixture of MpCP1 with MpNEP2 led to a synergic necrosis effect very similar to that found in naturally infected plants / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Submitted by Jadson Francisco de Jesus SILVA (jadson@uefs.br) on 2017-02-15T01:08:04Z No. of bitstreams: 1 Disserta??o DANIEL DA SILVA DE JESUS - PDF.pdf: 3083183 bytes, checksum: bd6b14e2cc2a38ab54b674866d480292 (MD5) / Made available in DSpace on 2017-02-15T01:08:04Z (GMT). No. of bitstreams: 1 Disserta??o DANIEL DA SILVA DE JESUS - PDF.pdf: 3083183 bytes, checksum: bd6b14e2cc2a38ab54b674866d480292 (MD5) Previous issue date: 2016-04-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Lamiaceae species have been widely studied due to their antioxidant, antimicrobial and antiviral properties. There is scientific interest in developing technologies for the enhancement of production systems of medicinal plants, given that the low concentration of bioactive compounds is a major obstacle to their economic exploitation. Works with some species have shown that traces metals such as Cd, Cu and Zn, act as abiotic elicitors, directly influencing in the production of bioactive substances. Martianthus leucocephalus (Mart. Ex Benth.) J.F.B. Pastore produces rich essential oil in isobornyl formate with high antimicrobial activity. This work aimed to evaluate the effect of Cd, Cu and Zn stress, on the species through growth, minerals accumulation, essential oil content and its major components, phenolic content and antioxidant enzymes. Furthermore, the phytoremediation potential of this species was observed in the presence of the tested metals. When applied in high concentrations all metals caused sharp reduction in the M. leucocephalus growth, however, was observed that Zn stress caused an increase in the content of volatile compounds. Plant exposure of to milder metals concentrations caused less growth reduction and nutritional unbalance M. leucocephalus. In this case, Cd stress stimulate higher production of volatile compounds. No metal treatment alters the essential oils quality. Cd, Cu and Zn was accumulated in higher concentration in the roots, demonstrating the potential of this species to phytostabilization in contaminated environments. The Cd stress caused an increase in total phenolics concentration, while the peroxidases activity and lipid peroxidation was higher in metals stressed plants. / Esp?cies da fam?lia Lamiaceae t?m sido amplamente estudadas, devido as suas propriedades antioxidantes, antimicrobianas e antivirais. Existe interesse cientifico no desenvolvimento de tecnologias para a potencializa??o de sistemas de produ??o das plantas medicinais, tendo em vista que a baixa concentra??o dos compostos bioativos representa um dos maiores obst?culos para a sua explora??o econ?mica. Trabalhos com algumas esp?cies t?m demonstrado que os metais tra?os, como o Cd, Cu e Zn, atuam como eliciadores abi?ticos, influenciando de forma direta na produ??o de subst?ncias bioativas. Martianthus leucocephalus (Mart. ex Benth.) J.F.B. Pastore ? uma esp?cie que produz ?leo essencial rico em formiato de isobornila, com elevada atividade antimicrobiana. Neste trabalho objetivou-se avaliar o efeito do estresse por Cd, Cu e Zn, sobre a esp?cie, quantificando-se o crescimento, ac?mulo de minerais nas diversas partes da planta, teor de ?leo essencial e de seus componentes majorit?rios, teor de compostos fen?licos e de enzimas antioxidantes. Al?m disso, foi verificado o potencial fitorremediador desta esp?cie, na presen?a dos metais testados. Quando aplicados em elevadas concentra??es todos os metais provocaram forte redu??o no crescimento de M. leucocephalus, entretanto, nestas condi??es apenas o Zn provocou incremento no teor dos compostos vol?teis. A exposi??o das plantas desta esp?cie a concentra??es mais amenas dos metais provocou menor redu??o no crescimento e desbalan?o nutricional de M. leucocephalus. Neste caso, apenas o Cd estimulou maior produ??o de compostos vol?teis, mas nenhum tratamento com metal alterou a qualidade dos ?leos essenciais obtidos. As plantas acumularam Cd, Cu e Zn em maior concentra??o nas ra?zes, demonstrando o potencial da esp?cie para a fitoestabiliza??o desses metais em ambientes contaminados. O estresse por Cd provocou aumento da concentra??o de fen?licos totais, enquanto que a atividade das peroxidases e a peroxida??o lip?dica foi maior nas plantas submetidas aos tr?s metais.

In the so-called induced systemic resistance phenomenon (ISR), some non pathogenic rhizobacteria are able to stimulate defence mechanisms in the host plant thereby rendering it less susceptible to subsequent phytopathogen attack. This immunization process is initiated into the plant following the perception of elicitors (or determinants) produced by the beneficial microorganism. Previous studies performed in our laboratory have demonstrated the ability of Pseudomonas putida strain BTP1 and Bacillus subtilis strain S499 (or M4) at triggering ISR. By using these two rhizobacteria, the global objective of this thesis is to contribute to a better understanding of this molecular dialogue between ISR-inducing bacteria and plant cells. Our first researches with BTP1 led to the isolation, as an elicitor, of a new compound produced by Pseudomonas. It was purified from bacterial culture supernatant and identified as a benzylamine core alkylated with two methyl and one tetradecyl groups, conferring its hydrophobic properties. On the basis of this structure, the BTP1 determinant was called NABD, for N-Alkylated Benzylamine Derivative. By testing the pure molecule or mutant strains altered in its production, we have shown that NABD was mainly responsible for ISR-activity of the BTP1 strain on bean and cucumber while in tomato, another unidentified compound could also be involved. In the case of Bacillus subtilis S499, we used similar approaches to demonstrate for the first time that the lipopeptides surfactin and fengycin may act as elicitors to stimulate systemic resistance, thereby attributing them a new role in the biocontrol of plant diseases. Structure/activity studies have revealed the implication of the benzylamine aromatic core in the biological activity of NABD. In the case of surfactin, reduced activity of some homologues indicates that its perception is dictated by structural clues such as the length of the acyl moiety and the presence of charges in the cyclic peptide part. Also in support to their involvement in ISR triggering, significant quantities of NABD and lipopeptides were recovered from the rhizosphere of bacterized plants. The influence of some physiological and physico-chemical factors on NABD production by BTP1 was further investigated in in vitro experiments. Our results show that the molecule is more efficiently produced at low cell growth rate and in the presence of amino acids in the medium but is negatively iron-regulated. Other abiotic factors, such as low oxygen concentration or low pH do not have drastic effects on NABD biosynthesis by the strain. Globally, with regard to specific conditions that the strain undergoes in the rhizosphere environment, it supposes that the nutritional/physiological state of BTP1 cells growing on plant roots is compatible with an effective production of the elicitor. Plant responses induced following root treatment with Bacillus subtilis S499 have also been investigated. Working on whole plants, it first revealed an increase in enzyme activities of the oxylipin pathway (lipoxygenase (LOX) and lipid-hydroperoxydase) after infection. Further investigations were conducted on cultured tobacco cells and we have shown the induction of some early events, such as extracellular pH alkalinization, reactive oxygen species production, defence enzyme stimulation (LOX and phenylalanine ammonia lyase (PAL), and accumulation of some phenolics from the phenylpropanoid pathway. These experimentations performed with Bacillus lipopeptides are the first conducted with ISR-specific elicitors and led to the characterization of early events that can also be triggered by pathogen associated molecules. It is still not clear whether bacterial LPs are recognized by plant cells via specific receptors but the amphiphilic and detergent properties of surfactin strongly suggest that these LPs can interact via a less specific mechanism based on some limited destabilization of the membrane structure. In conclusion, NABD and lipopeptides studied in this thesis enlarge the range of elicitors from non pathogenic bacteria isolated to date, and may be considered as members of a new class of ISR-inducing compounds of amphiphilic nature./ Lors du phénomène appelé « résistance systémique induite » (ISR), des rhizobactéries non pathogènes peuvent conférer à la plante un certain degré de protection à des attaques ultérieures par un phytopathogène via la stimulation de mécanismes de défense systémiques. Cette « immunité » sinitie suite à la perception par la plante de molécules dites élicitrices produites par le microorganisme bénéfique. Lobjectif de cette thèse est de contribuer à la caractérisation de ce dialogue moléculaire en se basant sur des études antérieures poursuivies au laboratoire qui ont mis en évidence la capacité des souches Pseudomonas putida BTP1 et Bacillus subtilis S499 (aussi dénommée M4) à induire lISR chez leur hôte végétal. La première partie des recherches sur BTP1 a permis disoler en tant quéliciteur un nouveau type de composé produit par les Pseudomonas. Cet éliciteur a été isolé à partir de surnageant de culture de la bactérie et est constitué dun noyau benzylamine dont lazote est alkylé par deux groupements méthyle et un groupement tétradécyle, responsables de lhydrophobicité relative de la molécule. Sur cette base, ce métabolite a été dénommé NABD (N-alkylated benzylamine derivative). Grâce au traitement des plantes avec le composé purifié ou via lutilisation de mutants altérés dans sa production, nous avons démontré que le NABD était responsable de lessentiel de lactivité ISR de BTP1 sur le haricot et le concombre. Dans le cas de la tomate par contre, il semble quil ne soit pas le seul éliciteur impliqué. En ce qui concerne Bacillus subtilis S499, en utilisant des approches similaires, nous avons pour la première fois montré limplication de deux lipopeptides, la surfactine et la fengycine, comme agents stimulateurs de lISR, permettant ainsi de leur attribuer un nouveau rôle dans le contrôle biologique des maladies des plantes par Bacillus. Une étude de la relation structure/activité a été entamée et a révélé un rôle du noyau aromatique dans létablissement de la résistance systémique par le NABD. Pour la surfactine, limportance de la longueur de la chaîne alkylée ainsi que la présence des charges sur les acides aminés suggèrent que son activité élicitrice soit liée à ses propriétés amphiphiles. La détection en quantités significatives des deux types déliciteurs dans la rhizosphère des plants traités par les bactéries est également en faveur de leur rôle biologique. Dans le cas de BTP1, nous avons étudié linfluence de certains paramètres physiologiques, nutritionnels et physico-chimiques sur la production du NABD. Les résultats montrent que la production du NABD est dépendante de la présence dacides aminés dans le milieu, quelle est effective à un taux de croissance faible du microorganisme, quelle est négativement influencée par le fer mais quelle nest pas inhibée par une restriction en oxygène dissous disponible ou par une acidification du milieu. Globalement, ces données suggèrent que les conditions physico-chimiques dictant létat physiologique des cellules bactériennes in situ peuvent être propices à une synthèse effective de léliciteur au niveau des racines. Des recherches sur les réponses induites chez lhôte lors de son interaction avec S499 ont été initiées et montrent une nette augmentation dactivités enzymatiques de la voie des oxylipines (activités lipoxygénase (LOX) et lipide hydroperoxydase) dans les feuilles après infection. Une exploration plus approfondie sur suspensions de cellules de tabac en culture avec la surfactine a montré linduction de plusieurs autres mécanismes précoces de défenses, comme une alcalinisation du milieu extracellulaire, la production despèces oxydantes, lactivation denzymes de défense telles que LOX et phenylalanine ammonia lyase (PAL), et une possible réorientation de la voie des phénylpropanoïdes. Ces expérimentations menées avec les lipopeptides de Bacillus sont parmi les premières à être réalisées avec des éliciteurs spécifiques de lISR. Elles ont abouti à la caractérisation de plusieurs réponses précoces dont certaines sont aussi induites par des molécules isolées dagents pathogènes. Les lipopeptides pourraient ainsi agir en stressant les cellules de lhôte via une perturbation temporaire de la membrane externe facilitée par leur propriété amphiphile. Limplication de récepteurs dans la reconnaissance des éliciteurs de BTP1 et S499 ne peut cependant pas être exclue. En conclusion, les lipopeptides et le NABD élargissent la gamme des éliciteurs bactériens de lISR isolés jusquà présent et peuvent être considérés comme appartenant à une nouvelle classe dimmuno-stimulateurs des plantes comprenant des molécules amphiphiles.

La protection des vignes contre les maladies cryptogamiques telles que le mildiou est assurée majoritairement par des fongicides de synthèse dont certains posent des problèmes environnementaux et sanitaires. L’induction de résistance de la vigne par des éliciteurs des réactions de défenses pourrait permettre de réduire leur utilisation. Toutefois, l’efficacité de cette stratégie est avérée en conditions de serres, mais reste plus variable au vignoble. L’efficacité des éliciteurs est conditionnée par le niveau de réponse défensif de la plante, et plus globalement, par son état physiologique. Dans ce contexte et dans le cadre de ce travail intégré au projet FUI IRIS+, l’objectif était de vérifier si des biostimulants, via leurs effets sur la physiologie de la plante, étaient susceptibles d’augmenter son niveau de réponse aux éliciteurs. Ce projet impliquait en amont le criblage d’éliciteurs et de biostimulants efficaces sur vigne. L’activation des défenses par des éliciteurs a un coût métabolique et énergétique que la plante doit assumer. Dans un premier temps, nous avons utilisé le modèle « suspensions cellulaires » afin de comparer l’impact de deux éliciteurs oligosaccharidiques sur le métabolisme primaire de la vigne. Des analyses enzymatiques et métaboliques ont permis de montrer que l’oligogalacturonide, qui a eu la plus forte capacité à activer le métabolisme secondaire associé à la défense, comparé à la laminarine, est également celui qui a eu l’impact le plus marqué sur le métabolisme primaire ; notamment sur certains glucides et acides aminés. Ainsi, l’élicitation mobilise des ressources, impacte le métabolisme primaire de la vigne et son efficacité pourrait être conditionnée par l’état physiologique global de la plante. Dans un second temps, un criblage a été réalisé afin d’identifier de nouveaux éliciteurs potentiels parmi sept fournis par le partenaire industriel. Des tests de protection contre Plasmopara viticola (agent du mildiou) réalisés sur boutures herbacées en conditions de serres, ont permis de retenir un extrait de plante codé SDN3. Les autres ont été écartés car déjà utilisés en laboratoire (l’un d’entre eux), insuffisants en terme d’efficacité, instables ou phytotoxiques. Des études in vitro et in planta ont révélé que l’activité biologique de SDN3 est liée à deux modes d’action : par activation des défenses et par effet direct contre l’agent pathogène. Dans un troisième temps, un criblage de cinq biostimulants potentiellement efficaces sur vigne était à réaliser mais aucune méthodologie de test n’était disponible au laboratoire. Ainsi, quatre dispositifs de complexités différentes ont dû être mis au point, permettant le suivi phénotypique des parties aériennes et/ou racinaires (et dans certains cas physiologique) : systèmes « godets », « rhizotron plan », « tubes » et un autre système appelé « X » (non décrit car lié à une protection industrielle). Seuls deux modèles ont montré un intérêt. En effet, le modèle « tube » a permis de montrer une augmentation du poids frais moyen par racine primaire en réponse à un apport par la voie racinaire de l’un des produits testés, codé BS3. Le modèle « X » a également mis en évidence une accélération de l’ouverture du bourgeon, et une augmentation du nombre de racines primaires en réponse à BS3 appliqué par voie racinaire. La mise au point d’applications d’éliciteurs et d’infection par P. viticola est en cours de finalisation afin de vérifier l’hypothèse de départ à l’aide de la combinaison de BS3 avec SDN3. / The protection of vineyards against cryptogamic diseases such as downy mildew is mainly ensured by synthetic fungicides, which cause serious environmental and health problems. The induction of resistance by elicitors could allow to reduce their use. However, even if their efficacy is demonstrated in greenhouse conditions, it remains quite variable in field conditions. Indeed, the efficacy of an elicitor depends on the plant’s ability to respond, or more generally, on the latter’s physiological status. In this context, part of the FUI project IRIS+, the aim of this present work is to evaluate whether biostimulants, through their effect on the plant’s physiology, would be able to increase their responsiveness to elicitors. The activation of defenses implies a metabolic and energetic cost that the plant must get in charge. First of all, we used a cell suspension model in order to compare two oligosaccharidic elicitors on the primary metabolism of grapevine. Enzymatic and metabolic analyses showed that the oligogalacturonide, which had a stronger impact on secondary metabolism related to defense, compared to laminarin, also showed a more notable impact on primary metabolism, particularly on some sugars and amino acids. The elicitation of grapevine defenses effectively turned out to require resources. Secondly, a screening was carried out to select the most effective elicitor among seven products that were provided by the company. Protection assays against Plasmopara viticola on herbaceous cuttings allowed us to identity SDN3 as the most interesting candidate. In vitro and in planta studies revealed that the mode of action of SDN3 relied on both the activation of defenses and a direct effect against the pathogen. Lastly, as no protocol, nor methodology were available in our laboratory to screen biostimulants, four systems were developed, in order to monitor phenotypic traits of aerial and/or root system: the “pot” model, the “rhizotron” model, the “tubes”, and another termed “X” (no description allowed because of an industrial protection). Only the “X” model showed potential interests, since it allowed to display biostimulating effects such as the acceleration of the bud opening, and the increase of the mean number of primary roots, in response to BS3, applied to the roots. The development of the protocol to apply the elicitor and to infect with P. viticola spores are currently in progress, in order to assess the initial hypothesis by using the combination of BS3 and SDN3.

Echinacea purpurea belongs to adaptogenic plants containig pharmacologically active compounds. The most important parts of the plants for medical use are roots, but also aboveground parts are processed. The plants were grown in triennial precise small-parcel experiment using acetylsalicylic acid as an in vivo elicitor to increase the content of bioactive substances. The contents of cichoric acid, caffeic acid, chlorogenic acid and caftaric acid were assayed in roots, tops and flowers. Repeated application elicitors on leaves were conducted with three different concentrations in fortnightly intervals. Ethanol extracts from the plant samples were analysed by high performance liquid chromatography. The effect of the elicitor on the content of bioactive substances was statistically significant. The optimal concentration of the elicitor considering the content of bioactive substances in roots is the middle one. The effect of elicitor is year-dependent.

Orientadores: Thelma de Aguiar Pertinhez, Celso Eduardo Benedetti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T14:11:48Z (GMT). No. of bitstreams: 1 Oliveira_AlineLimade_D.pdf: 9722926 bytes, checksum: ed4754fa74fbf62fbf6092d5ec8dfd53 (MD5) Previous issue date: 2008 / Resumo: Um dos desafios que envolvem a patologia de plantas é determinar as bases moleculares que relacionam a patogenicidade do fungo na planta hospedeira. Em geral, o mecanismo pelo qual a planta se defende contra uma variedade de patógenos envolve o reconhecimento de moléculas específicas codificadas por esses microorganismos. A ceratoplatanina (CP) é uma proteína moderadamente hidrofóbica que é secretada, porém também está localizada na parede celular do fungo ascomiceto Ceratocystis fimbriata. Esse fungo é o agente causador de severas doenças de cancro que atinge um grande número de plantas como Platanus acerifolia, Theobroma cacao, Coffea Arabic, entre outras. A proteína secretada por culturas de Ceratocystis fimbriata f. sp. platani é capaz de elicitar a síntese de fitoalexinas em folhas de Platanus acerifolia e causar morte celular. De acordo com o banco de dados de família de proteínas CP é indicada como a fundadora da "Família Cerato-Platanina", que inclui outras várias proteínas secretadas por fungos envolvidos em fenômenos fitopatológicos e/ou reações imunológicas. Buscas em banco de dados de proteínas mostram que não existe nenhuma proteína similar a CP com estrutura conhecida. Assim, a elucidação estrutural de moléculas possivelmente envolvidas interação fungohospedeiro é de primária importância para a compreensão dos eventos iniciais que levam às respostas de defesa. A CP foi clonada e expressa em sistema eucariótico e marcada isotópicamente com 13C e 15N. Experimentos de ressonância magnética nuclear multinucleares e muiltidimensionais foram usados para determinar sua estrutura 3D em solução. A estrutura da CP é composta por duas fitas-ß paralelas e cinco fitas-ß antiparalelas, reunidas em uma única folha-ß, e três elementos helicoidais. A dinâmica da CP foi determinada e os dados experimentais mostraram que a proteína apresenta uma estrutura caracterizada por uma limitada mobilidade interna. Além disso, a proteína apresenta alta estabilidade, mantendo a sua conformação mesmo em temperaturas elevadas ou em uma larga escala de pH e de concentração de uréia. Com o objetivo de procurar outras proteínas ou domínios conformacionalmente similares ao da CP, sua estrutura foi submetida a análises estruturais utilizando-se a ferramenta ProFunc. Os resultados de alinhamento estrutural mostram que a CP não possui similaridade conformacional a nenhuma proteína conhecida envolvida em avirulência de microorganismos ou a proteínas elicitoras de fungos ou bactérias patógenas. Porém, surpreendentemente, o enovelamento da CP mostrou-se similar a moléculas pertencentes às famílias das quimiocinas e do complexo de histocompatibilidade principal de classe I (MHC-I). De fato, a estrutura de menor energia da CP se sobrepõe melhor a forma dimérica da interleucina-8 (IL-8) e do peptídeo ativador de neutrófilos-2 (NAP-2), da família de quimiomicinas, e aos domínios a1, a2 da proteína MHC-I. Interessante notar que ambas famílias de proteínas que possuem enovelamento similar ao da CP estão relacionadas com respostas de defesa a patógenos em animais. A semelhança estrutural entre CP e essas moléculas sinalizadoras de defesa sugere que, na célula do hospedeiro, o alvo da CP pode ter similaridade estrutural aos receptores das proteínas IL-8/NAP-2 ou MHC-I. Buscas no genoma de Arabdopsis usando a sequência desses receptores forneceram alguns potenciais alvos. A maioria desses alvos são proteínas intracelulares ou de membrana que estão envolvidos na percepção de proteínas de avirulência e de padrões moleculares associados a patógenos, corroborando com a hipótese de que a CP agiria como um elicitor de respostas de defesa e poderia interagir com essas classes de receptores. Embora a função da CP ainda seja desconhecida, com a elucidação da sua estrutura 3D surgiram novas hipóteses sobre o mecanismo molecular pelo qual a CP interagiria com seus hospedeiros. / Abstract: One of the most challenging problems facing plant pathology is to understand the molecular basis of the interaction between fungal plant pathogens and their host. Often, the mechanisms by which plants defend themselves against a variety of pathogens involve the recognition of specific molecules encoded by these microorganisms. Cerato-platanin (CP) is a moderately hydrophobic protein that is secreted and localized in the cell wall of Ceratocystis fimbriata produced by the ascomycete fungus Ceratocystis fimbriata, which is the causative agent of canker stain, a severe disease with incidence in a great number of plants, such as Platanus acerifolia, Theobroma cacao, Coffea Arabic, among others. The protein secreted by culture of Ceratocystis fimbriata f. sp. platani in the medium is able to elicit phytoalexin synthesis in Platanus acerifolia leaves and to enhance cell death. According to protein family database CP has been indicated as the founder of the "Cerato-Platanin Family" that includes other secreted fungal proteins involved in a variety of phytopathological phenomena and/or immunological reactions. The search in the Protein Data Bank revealed this protein family is not structurally characterized. In this way, the resolution of 3D structure of molecules involved in the host-fungus interaction is important to the comprehension of the initial events in the plant defence response. CP has been cloned and expressed in eukaryotic systems in its 13C, 15N isotopically labelled form. Multinuclear and multidimensional nuclear magnetic resonance has been used to determine its 3D solution structure. The CP structure is composed of two ß-strand parallel and five ß-strands antiparallel, assembled in a seven-stranded ß-sheet, facing three helical elements. The CP dynamics has been determined and the experimental data reveal that CP shows a structure characterized by limited internal mobility. Moreover, the protein has a good stability, keeping its conformation even at high temperature or in a wide rage of both pH and urea concentration. With the aim to search for other protein or domains conformationally similar to the CP, its 3D structure was submitted to structural analysis using ProFunc Server. The results of structure alignment show that CP does not share 3D structural similarity with any known avirulence proteins or elicitors from bacterial or fungal pathogens. However, surprisingly, the core of CP has a fold similar to molecules belonging to the chemokines and to the major histocompatibility complex class I (MHC-I) families. Indeed, the minimum energy CP structure superposes better to the dimeric form of interleunkin-8 (IL-8) and of the neutrophil activating peptide-2 (NAP-2), from the chemokines family, and to the a1, a2- domains of MHC-I protein. Interestingly, both families of proteins which have a similar fold to CP are related with defense response to pathogens in animals. The structural resemblance between CP and these defense signaling molecules suggests that, in the host cells, CP targets might have structural similarities to the human IL-8/NAP-2 or MHC-I proteins receptors and a search in the Arabidopsis genome with them produced a few hits. Not surprisingly, most of these proteins are surface or intracellular receptors implicated in the recognition of molecular patterns associated to pathogens or avirulence, a finding consistent with the hypothesis that CP, acting as an elicitor, should interact with such class of receptor proteins. In summary, although the biological function of the CP is still unknown, the elucidation of its 3D structure provides some insights about the molecular mechanism by which CP interact with their hosts. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-19T11:06:39Z No. of bitstreams: 2 Dissertação - Jacqueline Santos Marques - 2014.pdf: 4633133 bytes, checksum: 8058e7f850a7ea713f7c77b0ad35cc32 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-19T11:08:30Z (GMT) No. of bitstreams: 2 Dissertação - Jacqueline Santos Marques - 2014.pdf: 4633133 bytes, checksum: 8058e7f850a7ea713f7c77b0ad35cc32 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-05-19T11:08:30Z (GMT). No. of bitstreams: 2 Dissertação - Jacqueline Santos Marques - 2014.pdf: 4633133 bytes, checksum: 8058e7f850a7ea713f7c77b0ad35cc32 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The extracts and essential oils from plants are bioactive molecules of interest to resistance induction in plants and the control of plant pathogens. The aim this study was to evaluate the effect of the ethanol extract and essential oil from leaves of Eugenia uniflora and two commercial flavonoid fractions, quercetin and rutin, as bioactive compounds for the control of white mold, caused by the fungus Sclerotinia sclerotiorum in common bean (Phaseolus vulgaris L.). The compounds were tested against pathogen through the mycelial growth, cell viability, sclerotia formation, cell morphology and biochemical aspects. And on the pathossystem Phaseolus vulgaris-Sclerotinia sclerotiorum was evaluated biochemical and molecular aspects (RT-qPCR) involved in the mechanism of plant defense, into a kinetic treatment and infection (12, 24 e 48 hours). The data show that the tested compounds were significantly relevant (P<0.01) to growth inhibition pathogen’s mycelium and cell viability in vitro and in the formation of sclerotia of the pathogen a positive correlation between percent inhibition and the concentration of active compounds applied was observed. The minimum inhibitory concentration (MIC) for S. sclerotiorum was 0.6% (v/v) ethanol extract of leaves from E. uniflora and the 100 ppm of the essential oil. In Bioassays in plants, the treatment with ethanol extract or essential oil from Eugenia uniflora and rutin inhibited the formation of necrotic area in common bean leaves 48 hours after inoculation. Biochemical analysis showed that the compounds induced 12 hours after treatment the activation of key proteins related to plant defense, chitinases, β-1,3- glucanase, phenylalanine ammonia lyase, peroxidases and polyphenoloxidases. Molecular analysis showed that treatment with ethanolic extract, quercetin and rutin in healthy plants activated pathogenesis-related genes (PRs) at different times into the kinetic treatment, and that these genes are highly expressed in plants infected compared to the pretreated and infected plants. The results of this research suggest that the effects of the ethanol extract, flavonoids and oil from Eugenia uniflora are potential bioactive molecules to reduce the severity of disease white mold in bean leaves. / Os extratos e óleos essenciais de plantas são moléculas bioativas de grande interesse para a indução de resistência em plantas e no controle de fitopatógenos. O objetivo deste trabalho foi avaliar o efeito do extrato etanólico e óleo essencial de folhas de E. uniflora e duas frações flavonoides comerciais, quercetina e rutina como compostos bioativos para o controle de mofo branco, causado pelo fungo Sclerotinia sclerotiorum, em feijoeiro comum (Phaseolus vulgaris L.). Os compostos foram testados sobre patógeno através do crescimento micelial, viabilidade celular, esclerogênese, morfologia celular e aspectos bioquímicos. E sobre o patossistema Phaseolus vulgaris-Sclerotinia sclerotiorum foi avaliado aspectos bioquímicos e moleculares (RT-qPCR) envolvidos no mecanismo de defesa da planta, dentro de uma cinética de tratamento e infecção (12, 24 e 48 horas). Os dados mostram que os compostos testados foram significantemente (P<0.01) relevantes na inibição do desenvolvimento e viabilidade celular in vitro do micélio e na formação de escleródios do patógeno, sendo observada uma correlação positiva entre percentual de inibição e as concentrações dos compostos avaliadas. A concentração inibitória mínima (MIC) para o S. sclerotiorum foi de 0.6% (v/v) do extrato etanólico de folhas de E. uniflora e de 100 ppm do óleo essencial. Nos bioensaios em plantas, o tratamento com extrato etanólico ou óleo essencial de folhas de Eugenia uniflora e rutina inibiram a formação de necrose em folhas de feijoeiro comum com 48 horas ápos inoculação. A análise bioquímica mostrou que os compostos induzem com 12 horas após tratamento a ativação das principais proteínas relacionadas à defesa da planta, quitinases, β-1,3- glucanase, fenilalanina amônia-liase, peroxidases, e polifenoloxidases. A análise molecular mostrou que o tratamento com extrato etanólico, quercetina e rutina em plantas sadias ativaram genes relacionados à patogênese (PRs) em diferentes tempos dentro da cinética de tratamento, e que tais genes são altamente expressos em plantas infectadas em comparação as plantas pré tratadas e infectadas. Os resultados obtidos neste trabalho indicam que o extrato etanólico, flavonoides e o óleo essencial de folhas de Eugenia uniflora são moléculas bioativas potenciais para reduzir a severidade da doença mofo branco em plantas de feijoeiro comum.