Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-04-19T16:19:33Z No. of bitstreams: 1 texto completo.pdf: 2766887 bytes, checksum: 6f8f01cd7e538b366f3e29fab1e9c82c (MD5) / Made available in DSpace on 2018-04-19T16:19:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2766887 bytes, checksum: 6f8f01cd7e538b366f3e29fab1e9c82c (MD5) Previous issue date: 2017-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Brasil é o segundo produtor mundial de soja. Porém, estresses bióticos e abióticos tem limitado a expansão da produtividade. Nosso grupo de pesquisa do LBMP, BIOAGRO-UFV, tem observado que plantas transgênicas superexpressando BiP (Binding protein), chaperona com atividade associada à via UPR (unfolded protein response) e à modulação de eventos de PCD (programmed cell death), são mais tolerantes à seca pela manutenção da homeostase celular e retardo do acionamento da PCD. Dos fitopatógenos, a Pseudomonas syringae pv. tomato provoca reação de hipersensibilidade na soja, uma vez que a interação planta-bactéria é incompatível. Neste trabalho, foi caracterizado o perfil metabólico dos genótipos transgênico superexpressando BiP (C9) e selvagem (WT) por GC-MS. Foram também avaliados por LC-MS, a abundância de fito-hormônios e alguns metabólitos secundários alvos em resposta à interação de soja com P. s. pv. tomato para determinar alterações metabólicas nestes genótipos relacionadas com o fenótipo de morte celular e reações de hipersensibilidade. O acúmulo de aminoácidos, açúcares e ácidos orgânicos pode estar relacionado ao controle de danos ocasionado pelo estresse biótico, sendo menor em C9 devido à capacidade de BiP em manter a homeostase celular. Compostos como di- hidroesfingosina (DHS) e gama-aminobutirato (GABA) podem estar envolvidos com mecanismos de PCD em células visando restringir a colonização do tecido foliar por bactérias. Além disso, observou-se uma tendência para o aumento de ácido salicílico (SA) e ácido jasmônico (JA), embora os níveis de ácido abscísico (ABA) em C9 tenham sido menores, indicando o papel antagonista do ABA na via de sinalização mediada por SA/JA e síntese de fitoalexinas ao longo da infecção bacteriana. A considerável importância do SA e JA, que disparam vias de sinalização de controle microbiano, em C9 possivelmente está no controle negativo da PCD por BiP, que impede a contenção do patógeno por esta via. As concentrações de metabólitos secundários, por sua vez, sofreram o maior incremento ao longo do tempo, sobretudo daidzeína e genisteína, sendo maior em C9. Este acréscimo pode estar associado com as propriedades antimicrobianas das isoflavonas. Por isso, em plantas C9, a superexpressão de BiP atrasa a via de PCD, impedindo a contenção da colonização bacteriana em tempo hábil, sendo a ação antimicrobiana o mecanismo de resposta predominante em C9, ao passo que em WT predomina a via de PCD. / Brazil is the world's second largest soybean's producer. However, biotic and abiotic stresses have limited productivity expansion. Our research group at the LBMP, BIOAGRO-UFV, has observed that transgenic plants overexpressing BiP (Binding protein), chaperone with activity associated with pathway UPR (unfolded protein response) and modulation of PCD (programmed cell death) events, are more tolerant to drought by the maintenance of cellular homeostasis and retarding the activation of PCD. Plant pathogens, Pseudomonas syringae pv. tomato provokes hypersensitivity reaction in soybean, since it is an incompatible interaction. In this work, the metabolic profile of the transgenic genotypes overexpressing BiP (C9) and wild type (WT) by GC-MS. We also evaluated by LC-MS, the abundance of phytohormones and some target secondary metabolites in response to the interaction of soybean with P. s. pv. tomato to determine metabolic changes in these genotypes related to cell death phenotype and hypersensitivity reactions. The accumulation of amino acids, sugars and organic acids may be related to damage control caused by biotic stress, being lower in C9 due to BiP's ability to maintain cellular homeostasis. Compounds such as dihydrosphingosine (DHS) and gamma aminobutyric acid (GABA) may be involved with PCD mechanisms in cells with the aim of to restrain the colonization of foliar tissue by bacteria. Furthermore, there was a tendency for increased salicylic acid (SA) and jasmonic acid (JA), although abscisic acid (ABA) levels have been lower in C9 plants, indicating the ABA antagonist role in the SA-mediated or JA-mediated signaling pathway and phytoalexin synthesis throughout the bacterial infection. The considerable importance of SA and JA, which trigger pathways of microbial control signaling, in C9 is possibly in the negative PCD control by BiP, which prevents the containment of the pathogen for this pathway. Concentrations of secondary metabolites suffered the greatest increase over time, especially daidzein and genistein, being higher in C9 plants. This increase may be associated with the antimicrobial properties of isoflavones. Therefore, in C9 plants, the overexpression of BiP delays the PCD pathway, preventing bacterial colonization in a timely manner, and the antimicrobial action is the predominant response mechanism in C9, whereas in WT, the PCD pathway predominates.

L'induction du métabolisme secondaire fait partie du système de défense des plantes lors d’une attaque par un microorganisme pathogène. Les propriétés antimicrobiennes ou de signalisation des métabolites secondaires (MS) peuvent être régulées par un processus majeur et efficace, la glycosylation. Cette réaction est catalysée par les glycosyltransférases (UGTs) qui assurent le transfert d’une molécule de sucre sur un MS. Mon travail de thèse a porté sur l’étude de trois UGTs d’Arabidopsis thaliana (Arabidopsis), UGT76E12 d’une part, et UGT73B3 et UGT73B5 d’autre part, et a montré que 1) UGT76E12 est impliquée dans la mise en place de la résistance basale à la souche virulente de la bactérie Pseudomonas syringae pv. tomato, Pst DC3000, et participe au dialogue entre les voies de signalisation hormonale de l’acide jasmonique (JA) et de l’acide salicylique (SA). Des analyses par clustering hiérarchique ont révélé une co-expression forte entre le gène UGT76E12, et deux gènes codant une terpène synthase (TPS4) et un cytochrome P450 (CYP82G1), impliqués dans la biosynthèse d’un diterpène, le TMTT ((E,E)-4,8,12-triméthyletridéca-1,3,7,11-tetraène), suggérant un rôle pour UGT76E12 dans la régulation de la voie de biosynthèse des terpènes ; 2) UGT73B3 et UGT73B5 sont impliquées dans la mise en place de la résistance spécifique d’Arabidopsis à la souche avirulente Pst DC3000-AvrRpm1, notamment par la régulation de MS intervenant dans le maintien de l’état redox cellulaire au cours de la réaction d’hypersensibilité (HR). Ces deux gènes appartiennent à la classe des « SA-early induced-genes » et présentent un profil co-expression avec le gène TOLB-related et deux gènes codant des glutathion S-transférases (GSTU7 et GSTU24) impliqués dans la réponse au stress oxydant et la détoxication des MS. Nos résultats suggèrent un rôle pour UGT73B3 et UGT73B5 dans la glycosylation de métabolites oxydants associés à la mise en place de la HR. / Plant secondary metabolism induction is part of an integrated defense system after pathogen infection. Signalling and antimicrobial properties of secondary metabolites (SM) can be regulated by an efficient process named, glycosylation. This reaction is ensured by glycosyltransferases (UGTs) which catalyze the transfer of a sugar moiety on a SM. My PhD work was divided in two parts, the first on UGT76E12 and the second part on UGT73B3 and UGT73B5, three UGTs of Arabidopsis thaliana (Arabidopsis) and it demonstrated that 1) UGT76E12 is involved in basal resistance establishment during plant challenge with the virulent strain of Pseudomonas syringae pv. tomato (Pst DC3000), and participates to the hormonal crosstalk between jasmonic acid (JA) and salicylic acid (SA). Clustering analyses revealed a strong co-expression between UGT76E12 and two genes encoding a terpene synthase (TPS4) and a cytochrome P450 (CYP82G1), both involved in the biosynthesis of a diterpene named TMTT ((E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene), which suggests a role for UGT76E12 in terpene biosynthesis pathway regulation. 2) UGT73B5 and UGT73B3 are involved in the establishment of Arabidopsis specific resistance against the avirulent strain of Pst DC3000-AvrRpm1, especially in the regulation of MS playing a role in redox homeostasis status during the hypersensitive response (HR). These two genes are part of SA-early induced-genes and showed co-expression patterns with the TOLB-related gene and two genes encoding glutathione S-transferases (GSTU7 and GSTU24) involved in oxidative stress responses and in MS detoxification. Our results suggest a role for UGT73B3 and UGT73B5 in the glycosylation of oxidant MS associated with HR establishment.

Atualmente, o HLB é considerado a principal doença que acomete as plantas cítricas. Diante desse fator, pesquisas por cultivares resistentes a esta doença são necessárias. A transformação genética via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verificação do funcionamento dos genes em resposta a patógenos, isto porque as plantas modelos possuem como característica ciclo de vida curto e alto poder de regeneração. Assim sendo, objetivou-se com o presente trabalho, a transformação genética via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as proteínas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotilédones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transformação genética com os genes PR-8 e CDR-1. Esses explantes foram subcultivados até o aparecimento de brotos regenerantes e posteriormente plantas transgênicas, as quais foram aclimatizadas e levadas a casa-de-vegetação. A transgenia foi confirmada por PCR e o número de inserções do gene por Southern blot. As plantas foram cultivadas até a obtenção da geração T1. Simultaneamente, foram realizados experimentos de transformação genética em segmentos de epicótilo, provenientes de sementes de laranja ‘Hamlin’ germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transgênicas e sua caracterização. Paralelamente, foi realizada a construção da curva padrão pela análise de qPCR para identificação de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transgênicas de tomate Micro-Tom com o gene PR-8 e três com o gene CDR-1. As eficiências de transformação foram em torno de 0,38 a 1,98%. Três plantas de tomate Micro-Tom transgênicas com o gene PR-8 foram caracterizadas por Southern blot e o número de inserções variou de 1 a 3. Dezenove plantas transgênicas de laranja ‘Hamlin’ com o gene CDR-1 foram obtidas através dos experimentos de transformação genética. A eficiência de transformação foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 número de cópia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

Atualmente, o HLB é considerado a principal doença que acomete as plantas cítricas. Diante desse fator, pesquisas por cultivares resistentes a esta doença são necessárias. A transformação genética via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verificação do funcionamento dos genes em resposta a patógenos, isto porque as plantas modelos possuem como característica ciclo de vida curto e alto poder de regeneração. Assim sendo, objetivou-se com o presente trabalho, a transformação genética via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as proteínas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotilédones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transformação genética com os genes PR-8 e CDR-1. Esses explantes foram subcultivados até o aparecimento de brotos regenerantes e posteriormente plantas transgênicas, as quais foram aclimatizadas e levadas a casa-de-vegetação. A transgenia foi confirmada por PCR e o número de inserções do gene por Southern blot. As plantas foram cultivadas até a obtenção da geração T1. Simultaneamente, foram realizados experimentos de transformação genética em segmentos de epicótilo, provenientes de sementes de laranja ‘Hamlin’ germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transgênicas e sua caracterização. Paralelamente, foi realizada a construção da curva padrão pela análise de qPCR para identificação de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transgênicas de tomate Micro-Tom com o gene PR-8 e três com o gene CDR-1. As eficiências de transformação foram em torno de 0,38 a 1,98%. Três plantas de tomate Micro-Tom transgênicas com o gene PR-8 foram caracterizadas por Southern blot e o número de inserções variou de 1 a 3. Dezenove plantas transgênicas de laranja ‘Hamlin’ com o gene CDR-1 foram obtidas através dos experimentos de transformação genética. A eficiência de transformação foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 número de cópia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-20T13:00:17Z No. of bitstreams: 1 texto completo.pdf: 327861 bytes, checksum: 10b3c4ac9c1fb002cc94c4da4da28d0e (MD5) / Made available in DSpace on 2017-04-20T13:00:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 327861 bytes, checksum: 10b3c4ac9c1fb002cc94c4da4da28d0e (MD5) Previous issue date: 2002-12-03 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A busca de alimentos produzidos sob um sistema de manejo menos agressivo ao meio ambiente vem sendo adotado por um número cada vez maior de produtores. Entretanto, apesar de existirem diversos benefícios na redução ou até eliminação do uso de defensivos, a grande diversidade de doenças em tomateiro (Lycopersicon esculentum Mill.), capazes de limitar a produção, torna necessária a busca por alternativas viáveis, eficientes e tecnicamente comprovadas. Dentre os organismos mais estudados, bactérias têm sido relatadas como agentes de biocontrole capazes de atuar por meio de mecanismos como antibiose, parasitismo, competição e indução de resistência. O presente trabalho teve como objetivos selecionar bactérias do filoplano do tomateiro, baseado em uma estratégia de seleção in vivo, verificando se há um método de isolamento que permita obter antagonistas eficientes no controle da pinta-preta, causada por Alternaria solani, requeima por Phytophthora infestans, mancha-bacteriana pequena por Pseudomonas syringae pv. tomato e mancha-bacteriana por Xanthomonas vesicatoria. Objetivou ainda estudar a possibilidade dos mecanismos de antibiose e indução de resistência serem responsáveis pelo controle destas doenças e se testes de antibiose in vitro são adequados como critério de seleção. Caracterizar aspectos biológicos dos antagonistas que podem otimizar sua aplicação como agente de biocontrole. Determinar a quais produtos antimicrobianos os isolados são insensíveis, visando fornecer subsídios para o desenvolvimento de meios semi-seletivos e estudos de dinâmica populacional. Verificar sua compatibilidade com antibióticos e fungicidas registrados para o controle de enfermidades do tomateiro, a fim de inseri-lo no sistema de manejo integrado e estudar a eficiência de antagonistas selecionados em condições de campo. Os resultados demonstram que, em folíolos mais jovens, os métodos de isolamento que visam obter bactérias da população total e da superfície do filoplano, foram os que permitiram obter a maioria dos antagonistas. O único obtido de folíolos mais velhos foi proveniente da população capaz de habitar sítios protegidos do filoplano e/ou resistir a fatores de estresse. Não se observou relação entre características biológicas dos antagonistas e dos patógenos testados. Nos testes de antibiose com os antagonistas selecionados, o isolado UFV-STB 6 foi capaz de produzir compostos voláteis e inibir a germinação de cistos de Phytophthora infestans, o que possivelmente deve estar envolvido no controle da requeima. O isolado UFV-IEA 6 produziu quitinase, havendo uma tendência em reduzir a taxa de crescimento de Alternaria solani por compostos voláteis. Ficou demonstrado que os testes de antibiose in vitro são inadequados como critério para seleção de agentes de biocontrole do filoplano de tomateiro. A caracterização dos melhores antagonistas demonstrou que três são bactérias Gram-positivas, em forma de bastonete, e uma Gram-negativa, pleiomórfica. Dentre as Gram-positivas todas são anaeróbias facultativas e uma forma endósporos. Nenhum antagonista foi capaz de causar reação de hipersensibilidade (HR) em fumo e produzir pigmento fluorescente in vitro. Os períodos de geração calculados a partir das curvas de crescimento revelaram que três isolados são capazes de se multiplicar rapidamente em meio de cultura, o que é uma característica desejável. Os resultados obtidos a partir dos antibiogramas, mostraram que existem antibióticos que podem ser utilizados para elaboração de meios semi-seletivos, adequados a cada antagonista e os testes de compatibilidade com antibióticos e fungicidas utilizados na cultura do tomateiro revelaram que os antagonistas podem ser expostos aos fungicidas benomyl, enxofre, dimetomorph e tiofanato-metílico. Verificou-se também a inadequação de se utilizarem compostos antimicrobianos em meio de cultura para isolamento de agentes bacterianos de controle biológico, uma vez que os antagonistas selecionados foram sensíveis à maioria dos produtos testados. Os testes com as enzimas indicadoras do estado de indução de resistência, β-1,3-glucanases, Fenilalanina amônia-liase (PAL), Peroxidases (PO), Polifenoloxidases (PPO) e Lipoxigenases (LOX), indicaram que o isolado UFV-IEA 6 foi capaz de promover aumento significativo na atividade das PO, evidenciando a possibilidade do antagonista agir como indutor de resistência. Esse parece ser o primeiro caso que se tem conhecimento de uma bactéria não fitopatogênica do filoplano induzindo resistência na mesma cultura de onde foi obtida. Os testes com os dois antagonistas em condições de campo demonstraram que UFV-STB 6 foi o mais eficiente em reduzir a severidade da requeima no terços médio e superior das plantas, enquanto UFV-IEA 6, somente no terço superior. Houve tendência na redução do progresso da septoriose por UFV-STB 6 e capacidade em diminuir o número de frutos com sintomas de requeima. Os resultados demonstram o potencial de uso dos agentes de biocontrole selecionados para as doenças da parte aérea de tomateiro estudadas. / Farmers are increasingly adapting environmentally less aggressive management systems for food production and there are many benefits in reducing or even eliminating pesticide use. Due to a large number of production limiting diseases on tomato (Lycopersicon esculentum), it is desirable to find technically viable and proven alternatives for their control. Among the microorganisms, bacteria have been reported as biocontrol agents capable of acting through antibiosis, parasitism, competition and induced resistance. The present study aimed at selecting the tomato phylloplane bacteria, based on in vivo isolation strategy, and to determine if this method permits obtaining efficient antagonists to control Alternaria solani leaf spot, Phytophthora infestans blight, small bacterial leaf spot caused by Pseudomonas syringe pv tomato, and bacterial leaf spot caused by Xanthomonas vesicatoria. The study also aimed at determining mechanisms of action, such as antibiosis and induced resistance, involved in disease control, and to determine if the antibiosis tests are sufficient selection criteria. The study also included biological characterization of the antagonists that may optimize their use. To help develop selective or semi-selective media for population dynamic studies, insensitivity of selected isolates to some antimicrobial compounds was also determined. The compatibility of select antagonists with antibiotics and fungicides registered for control of tomato diseases was elucidated so that the antagonist can be inserted in the integrated management. Field studies were done to determine the efficiency of select antagonists. The isolation method that obtain total bacterial population from the phylloplane of the young leaflets permitted obtaining maximum number of antagonists. The only one isolate obtained from the older leaflets originated from the population capable of inhabiting protected sites of phylloplane and/or that resist stress factors. There was no relation between biological characteristics of the antagonists and of the pathogen tested. In the antibiosis testes, the isolate UFV-STB 6 produced volatile compounds that inhibited germination of P. infestance cyst and may be involved in the control of blight. The isolate UFV-IEA 6 produced chitinase and showed a tendency to reduce A. solani growth by the volatile compounds. In vitro antibiosis testes were inadequate criteria to select biocontrol agents from tomato phylloplane. The characterization of promising antagonists showed that three were Gram positive bacilli and one was gram negative pleiomorphic bacteria. Among the Gram positives all were facultative anaerobes and the one formed endospores. None of the antagonists caused hypersensitive reaction (HR) in tobacco, and did not produce fluorescent pigment in vitro. The generation period calculated from the growth curve revealed that three isolates are capable of multiplying rapidly in the culture media, which is a desirable characteristic. The antibiograms showed that there are antibiotics that can be used for elaboration of semi-selective media for each of antagonists and the compatibility testes with antibiotics and fungicides used on tomato crop revealed that the antagonists can be exposed to fungicides such as benomyl, sulfur, dimethomorph and thiophante-methyl. Many antimicrobial compounds were inhibitory to the selecte antagonists in culture media used for isolation of bacterial biocontrol agents, therefore were inadequate for use in selective media. The analysis of enzymes involved in induced resistance, like -1,3-gluconase, phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and lipoxigenase (LOX), showed that the isolate UFV-IEA 6 was capable of increasing PO activity, showing the possibility of being a resistance inducer . This appears to be the first case of a non-pathogenic phylloplane bacterium inducing resistance in a plant of origin. The field testes with two antagonists, UFV-STB 6 was more efficient in reducing the blight severity in the middle and upper third of the plant, while UFV-IEA 6 only in the upper third. The latter isolate also showed a tendency for reducing the Septoria leaf spot progress and the number of fruits with the blight symptoms. The results showed these isolates have the potential of use to control tomato diseases of aerial parts. / Tese importada do Alexandria

The aim of this diploma thesis is determination of the influence of deficient nutrition in combination of Pseudomonas syringae pv. tomato infection on tomato (Solanum lycopersicum L.) growth and development. In hydroponic cultivation experiment, tomato variety Darinka was cultivated in solutions without N, P, K, Ca, Mg and in variant with all nutrition (control variant). The dry matter of root and stem was weighed and root length, density and electrical capacitance were set. The content of nutrition in plants was also assessed. The level of infection was determined in 1st term, the highest level had plants with insufficient of Ca and P. High reduction of root and stem dry matter was found in Ca, N, P and Mg deficient plants. The low root electrical capacitance, length and density was determined in plants with Ca, N a P deficiency within infected group and in Ca and N deficient plants within noninfected group. Bacterial infection caused the reduction of all observing parameters in contrast to the plants from noninfected group. The antagonism and synergism between the elements were also showed.

<p>Systemic Acquired Resistance (SAR) is a plant defense response induced by an initial infection in one part of the plant that leads to broad-spectrum resistance to normally virulent pathogens in distant naïve leaves. As part of the Cameron research team, I contributed to demonstrating that the lipid transfer protein, DIR1 is required for SAR long distance signaling in <em>Arabidopsis</em> and travels from induced to distant tissues during SAR. A highly similar<em> Arabidopsis</em> protein DIR1-like was identified and is thought to be responsible for the occasional SAR-competent phenotype observed in the <em>dir1-1</em> mutant. This work provides evidence for the idea that DIR1 and DIR1-like are paralogs created by a recent duplication event and that similar to DIR1, DIR1-like may travel to distant tissues during SAR. To better understand DIR1 and DIR1-like contribution during SAR, <em>dir1-1dir1-like</em> double mutant transgenic plants were created as well as transgenic plants expressing epitope- (HA and FLAG) and fluorescent- (iLOV and phiLOV) tagged DIR1 and DIR1-like to facilitate visualization of movement during SAR. Several putative DIR1 orthologs were identified in crop plants and cucumber CucDIR1 was shown to be functionally equivalent to AtDIR1 in <em>dir1-1</em> complementation studies providing further evidence that DIR1 plays an important role in SAR across plant species. By analyzing conservation between DIR1, DIR1-like and the putative DIR1 orthologs, several protein residues were identified that may be important for DIR1 function during SAR. DIR1 proteins were modified at these sites and the importance of these residues was supported by the reduced binding of the TNS hydrophobic probe in these DIR1 variants. Taken together, this thesis suggests that DIR1 and DIR1-like both participate in SAR in <em>Arabidopsis</em>, that DIR1 crop orthologs are also important for the SAR response and that DIR1 possesses several sites that are critical for its function in long distance SAR signaling.</p> / Doctor of Philosophy (PhD)

La défense pré-invasive ou stomatique est un mécanisme qui consiste en la fermeture des pores stomatiques présents sur les organes aériens des plantes lorsque celles-ci sont en contact avec certains agents pathogènes. Cette fermeture empêche ces derniers de pénétrer dans l’hôte et de le coloniser. Ce mécanisme s’active chez Arabidopsis inoculée par la bactérie Pseudomonas syringae pv tomato (Pst) DC3000. Des travaux préliminaires de notre groupe avaient montré que la carbonylation de protéines cibles par des espèces réactives électrophiles (EREs) représentait une étape cruciale de la signalisation cellulaire nécessaire à la mise en place de cette défense. Par des approches de marquage ciblé et de purifications couplées à des identifications par spectrométrie de masse en tandem (nanoLC-MS/MS), nous avons pu caractériser une sérine-thréonine protéine kinase qui joue un rôle déterminant dans ce mécanisme de défense. En effet, des plantes mutées sur le gène codant cette protéine ont perdu la capacité à induire la fermeture de leurs stomates et à déployer la défense stomatique vis-à-vis de la bactérie. De plus, l’introduction de la chimie click (cycloaddition alcyne-azide catalysée par le cuivre), dans nos approches de marquage, nous a permis d’identifier un ensemble de protéines très probablement carbonylées et susceptibles de jouer un rôle crucial dans ces évènements cellulaires qui contribuent à une part de l’immunité végétale. Enfin, les EREs étant capables d’induire la fermeture des stomates, nous avons cherché à savoir, dans le cadre de l’établissement d’une preuve de concept, si leur application sur des plantes permettrait la protection de ces dernières contre Pst. / Pre-invasive or stomatal defense is a mechanism which consists of closing the stomata present at surface of aerial organs of plants when they are in contact with certain pathogens. This closure prevents them from entering and colonizing the host. This mechanism is activated in Arabidopsis inoculated by the bacterium Pseudomonas syringae pv tomato (Pst) DC3000. Preliminary work by our group had shown that carbonylation of target proteins by reactive electrophile species (RES) was a crucial step of the cell signaling required to set up this defense. Through targeted tagging and purifications approaches coupled with tandem mass spectrometry identifications (nanoLC-MS/MS), we have been able to characterize a serine-threonine protein kinase that plays a crucial role in this defense mechanism. Indeed, plants mutated on the gene encoding this protein have lost their ability to trigger stomatal closure and to deploy the stomatal defense against the bacteria. In addition, the use of the click chemistry and notably, the copper-catalyzed alkyne-azide cycloaddition, in our tagging approaches has enabled us to identify a set of proteins that are most likely carbonylated and likely to play a significant role in these cell events that contribute to part of plant immunity. Finally, since RES are able to induce stomatal closure we sought to find out, in the context of establishing a proof-of-concept, whether their application to plants would enable them to be protected against the Pst.